InteractiveFly: GeneBrief

ballchen: Biological Overview | References

BIOLOGICAL OVERVIEW

The apical Par complex, which contains atypical protein kinase C (aPKC), Bazooka (Par-3), and Par-6, is required for establishing polarity during asymmetric division of neuroblasts in Drosophila, and its activity depends on L(2)gl. This study shows that loss of Ankle2, a protein associated with microcephaly in humans and known to interact with Zika protein NS4A, reduces brain volume in flies and impacts the function of the Par complex. Reducing Ankle2 levels disrupts endoplasmic reticulum (ER) and nuclear envelope morphology, releasing the kinase Ballchen-VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par-complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen or l(2)gl suppresses Ankle2 mutant phenotypes and restores viability and brain size. Human mutational studies implicate the above-mentioned genes in microcephaly and motor neuron disease. It is suggested that NS4A, ANKLE2, VRK1, and LLGL1 define a pathway impinging on asymmetric determinants of neural stem cell division (Link, 2019).

Proper development of the human brain requires an exquisitely coordinated series of steps and is disrupted in disorders associated with congenital microcephaly. Congenital microcephaly in humans is characterized by reduced brain size (using occipital frontal circumference [OFC] as a surrogate measure) more than two standard deviations below the mean (Z score < -2) at birth. It is associated with neurodevelopmental disorders such as developmental delay and intellectual disability and can be caused by external exposures to toxins, in utero infections, or gene mutations. Pathogenic gene variants for microcephaly have been identified through targeted genetic testing, genomic copy number studies, and exome sequencing (ES), identifying 18 primary microcephaly loci. Many syndromes significantly overlap with classic microcephaly phenotypes, and together, these disorders can be caused by defects in a wide variety of biological processes, including centriole biogenesis, DNA replication, DNA repair, cell cycle and cytokinesis, genome stability, and multiple cell signaling pathways. In flies, microcephalic phenotypes are referred to when the third instar brain lobes are reduced in size or when adult flies exhibit small heads relative to the their body size. As in humans, microcephaly in flies can be a result of mutations that affect cell division and centrosome biology as demonstrated with mutations in WDR62 and ASPM or ASP and neuroblast (NB) proliferation (Link, 2019).

A forward, mosaic screen for neurodevelopmental and neurodegenerative phenotypes associated with lethal mutations on the X chromosome in Drosophila identified 165 loci, many with corresponding human genetic disease trait phenotypes. Among them, a mutation in Ankryin repeat and LEM domain containing 2 (Ankle2) causes loss of peripheral nervous system (PNS) organs in adult mutant clones and severely reduced brain size in hemizygous third instar larvae. To identify patients with pathogenic variants in ANKLE2, the exome database of the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) was surveyed; compound heterozygous mutations were identified in ANKLE2 in two siblings. Both infants exhibited severe microcephaly, and the surviving patient displayed cognitive and neurological deficits alongside extensive intellectual and developmental disabilities. Mutations in Ankle2 led to cell loss of NBs and affected NB division in the developing third instar larval brain. Remarkably, expression of the wild-type human ANKLE2 in flies rescued the observed mutant phenotypes. This study explored the molecular pathways and proteins that are affected by Ankle2 loss (Link, 2019).

ANKLE2 belongs to a family of proteins containing LEM (LAP2, Emerin, and MAN1) domains that typically associate with the inner nuclear membrane. Conventional LEM proteins have been shown to interact with barrier to autointegration factor (BAF), which binds to both DNA and the nuclear lamina to organize nuclear and chromatin structure. However, the LEM domain in Drosophila and C. elegans Ankle2 is not obviously conserved. Studies in C. elegans indicate that a homolog of ANKLE2 regulates nuclear envelope morphology and functions in mitosis to promote reassembly of the nuclear envelope upon mitotic exit. During this process, ANKLE2 modulates the activities of Vaccina-Related Kinase 1 (VRK1) and protein phosphatase 2A (PP2A). However, all experiments in worms were performed at the embryonic two-cell stage and no other phenotypes were reported except early lethality. While mutations in ANKLE2 have been associated with severe microcephaly, human VRK1 pathogenic variant alleles can cause a neurological disease trait consisting of complex motor and sensory axonal neuropathy and microcephaly (Link, 2019).

Mutations in both Ankle2 and the fly homolog of VRK1, ballchen, cause a loss of NBs in 3rd instar larval brains in Drosophila (Yamamoto, 2014, Yakulov, 2014). NBs divide asymmetrically and are often used as a model to investigate stem cell biology and asymmetric cell division. Most NBs in the larval central brain give rise to another NB and a smaller ganglion mother cell (GMC), which then divides once again to produce neurons or glia. Proper NB maintenance and regulation is essential for precise development of the adult nervous system, and misregulation of NB number or function can lead to defects in brain size (Link, 2019).

Congenital Zika virus infection in humans during pregnancy has been associated with severe microcephaly that can be as dramatic as certain genetic forms of microcephaly including phenotypes associated with biallelic mutations in MCPH16/ANKLE2. Recently, it has been showed that a Zika virus protein, NS4A, physically interacts with ANKLE2 in human cells. Expression of NS4A in larval brains causes microcephaly, induces apoptosis, and reduces proliferation. Importantly, expression of human ANKLE2 in flies that express NS4A suppresses the associated phenotypes, demonstrating that NS4A interacts with the ANKLE2 protein and inhibits its function (Shah, 2018). Interestingly, Zika virus crosses the blood brain barrier and targets radial glial cells, the neural progenitors in the vertebrate cortex (Link, 2019).

This study shows that Ankle2 is localized to the endoplasmic reticulum and nuclear envelope, similar to NS4A, and genetically interacts with ball-VRK1 to regulate brain size in flies. An allelic series at the ANKLE2 and VRK1 loci shows that perturbation of this pathway results in neurological disease including microcephaly. The data indicate that the Ankle2-Ball (VRK1) pathway is required for proper localization of asymmetric proteins and spindle alignment during NB cell division by affecting two proteins, atypical protein kinase C (aPKC) and L(2)gl, which play critical roles in the asymmetric segregation of cell fate determinants. In addition, NS4A expression in NBs mimics phenotypes seen in Ankle2 mutants, and NS4A induced microcephaly is suppressed by removing a single copy of ball or l(2)gl. Human genomics variant data and disease trait correlations extend this asymmetric cell division pathway from proteins identified in flies and reveal insights into neurological disease. In summary, NS4A hijacks the Ankle2-Ball (VRK1) pathway, which regulates progenitor stem cell asymmetric division during brain development and defines a human microcephaly pathway (Link, 2019).

This study reports six additional patients with microcephaly that carry mutations in ANKLE2 and shows that three variants identified in probands cause a loss of ANKLE2 function when tested in flies, providing compelling evidence that its loss causes reduced brain size in flies and severe microcephaly in humans. Ankle2 is a dosage-sensitive locus whose product is inhibited by the Zika virus protein NS4A. Ankle2, similar to NS4A, is localized to the ER and it targets the nuclear envelope during mitosis. Loss of Ankle2 affects the nuclear envelope and ER distribution and results in a redistribution of Ball or VRK1, a kinase that is normally localized to the nucleus except when the nuclear envelope breaks down during mitosis. Loss of Ankle2 disrupts the localization of NB apical-basal polarity determinants such as aPKC, Par-6, Baz, and Mir, and aPKC phosphorylation is reduced by Ankle2 mutations. Importantly, loss of one copy of ball or l(2)gl suppresses the reduced brain volume associated with a partial loss of Ankle2, suggesting that much of the biological function of Ankle2 is modulated by aPKC and L(2)gl. Finally, the negative influence of NS4A on the activity of ANKLE2 can also be suppressed by removal of one copy of ball or l(2)gl, suggesting the following pathway: NS4A -| ANKLE2 -| Ball-VRK1 -> L(2)gl-LLGL1 -| aPKC. This pathway, regulated by ANKLE2, plays an important role in NB stem cell divisions in flies and microcephaly and potentially other neurological disease phenotypes in humans (Link, 2019).

Interestingly, the above pathway links environmental cues with several genetic causes of sporadic and autosomal recessive microcephaly in humans; moreover, it implicates this pathway in microcephaly accompanying congenital infection. As one example of the latter, the Zika virus has been shown to cross the infant blood brain barrier and has been identified in radial glial cells as well as intermediate progenitor cells and neurons. It is proposed that NS4A affects the function of Ankle2 leading to the release of Ball-VRK1 from the nucleus. It is speculated that this in turn affects the phosphorylation of aPKC and L(2)gl directly by masking phosphorylation sites or indirectly by promoting the activity of one or more phosphatases. Loss of VRK1 has been shown to cause microcephaly and some variant alleles are also associated with pontocerebellar hypoplasia (PCH) in humans (Gonzaga-Jauregui, 2013, Renbaum, 2009), consistent with the loss of ball in flies that causes a severe reduction in brain size (Yakulov, 2014). Note that ANKLE2, VRK1, LLGL1, and aPKC as well as other components of the apical complex such as PARD3 are all present in radial glial cells during cortical development. These data suggest that ANKLE2 and its partners such as LLGL1 and asymmetric determinants are important proteins during neural cell proliferation and that the proper levels and relative amounts of these proteins determine how many neurons will eventually be formed in vertebrates. These data also indicate that variant alleles at either ANKLE2 or VRK1 are responsible for some causes of embryonic lethality and severe congenital microcephaly (Link, 2019).

LLGL1 has recently been shown to play an important role in radial glia in mice during neurogenesis, and its loss in clones increases the number of divisions. In addition, aPKCζ or λ localizes at the apical membrane of proliferating neural stem cells in chicken embryos during division and has been shown to provide an instructive signal for apical assembly of adherens junctions. Mouse knockouts of aPKCλ and aPKCι are embryonic lethal; however, aPKCζ knockouts are viable, perhaps suggesting redundant functions within the aPKC family. These proteins have not been linked to microcephaly in mice, but conditional removal of an apical complex protein Pals1 in cortical progenitors resulted in complete cortex loss. Finally, Numb is asymmetrically localized by the Par complex protein in Drosophila, segregated to the daughter cell during asymmetric cell division, and essential for daughter cells to adopt distinct fates. In mice, Numb localization is also asymmetric and null mutations exhibit embryonic lethality, neural tube closure defects, and premature neuron development. These data indicate that asymmetric division may be important for vertebrate neuronal development, but microcephaly is not a phenotype that typically associates with loss of the mice homologs of asymmetric-localized determinants identified in Drosophila. However, the observations reported in this study indicate that the ANKLE2-PAR complex pathway is evolutionarily conserved from flies to humans, although the precise mechanisms remain to be determined as different cells may use this pathway in different contexts (Link, 2019).

In order to determine whether predicted deleterious biallelic variants in PAR-complex-encoding genes or their paralogs associated with a neurologic disease trait, The BHCMG database was searched for mutations associated with neurological disease. Homozygous predicted deleterious missense variants in were found PARD3B (c.1222G>A, p.G408S) in a patient that has microcephaly and compound heterozygous mutations in PARD3B (c.1654G>A, p.A552T) that are associated with other neurological defects. The human ortholog of L(2)gl, LLGL1, is deleted in Smith-Magenis syndrome (SMS), and 86%-89% of the SMS patients have brachycephaly. These observations extend the mutational load beyond ANKLE2 and VRK1 and suggest an association between congenital disease and variants within the PAR complex, potentially by a compound inheritance gene dosage model (Link, 2019).

The Aurora A (AurA) kinase has been shown to phosphorylate the Par complex as well as L(2)gl and regulates cortical polarity and spindle orientation in NBs. The aberrant localization of Ball-VRK1 in Ankle2 mutants may lead to gain-of-function phenotypes that are highly dosage sensitive, as they can be repressed by removing a single copy of Ball-VRK1 in Ankle2A. Mislocalized Ball-VRK1 may mask or interfere with the function of AurA in NB asymmetric division as they share similar kinase substrate consensus sequences. Future studies are needed to assess Ball-VRK1 redundancy or interference with AurA function (Link, 2019).

Another possible evolutionarily parallel with implications in multicellular organismal development is the genetic interaction between the C. elegans homolog of VRK1 and an ANKLE2-like protein at the two-cell stage. Whereas VRK1 in both Drosophila and humans is localized to the nucleus, except during mitosis when the nuclear envelope is broken down, the worm VRK1 protein is localized to the nuclear envelope. The worm ANKLE2-like protein, Lem-4L, also interacts with the phosphatase PP2A (Asencio, 2012), and the fly PP2A regulates NB asymmetric division by interacting with aPKC and excluding it from the basal cortex. PP2A also antagonizes the phosphorylation of Baz by PAR-1 to control apical-basal polarity in dividing embryonic NBs and regulates Baz localization in other cells such as neurons. This raises the possibility that the Ankle2 pathway also acts with PP2A in NB asymmetric division (Link, 2019).

This study identified a pathway that plays a significant role in NB asymmetric division. By combining functional studies in Drosophila together with human subject data, this study has linked several microcephaly-associated genes and congenital infection to a single genetic pathway. These studies allowed the highlighting of conserved functions of the ANKLE2 pathway and provide mechanistic insight into how a Zika infection might affect asymmetric division. This ANKLE2-VRK1 gene dosage-sensitive pathway can be perturbed by genetic variants that disturb biological homeostasis resulting in neurological disease traits or by environmental insults such as Zika virus impinging on neurodevelopment. Hence, lessons learned from the study of rare diseases can provide insights into more common diseases and potential gene- environment interactions (Link, 2019).

Recruitment of BAF to the nuclear envelope couples the LINC complex to endoreplication

DNA endoreplication has been implicated as a cell strategy to grow in size and in tissue injury. This study demonstrates that barrier to autointegration factor (BAF), represses endoreplication in Drosophila myofibers. This study shows that BAF localization at the nuclear envelope was eliminated either in mutants of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, in which the LEM-domain protein Otefin was similarly excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope required the activity of the BAF kinase VRK1/Ball, and consistently non-phosphorytable BAF-GFP was excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlated with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication was found to overlap BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope resulted with increased E2F1 levels in the nucleoplasm, and subsequent elevated DNA content. It is suggested that LINC-dependent, and phospho-sensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation at the nucleoplasm (Unnikannan, 2020).

Endoreplication emerges as an important strategy of differentiated cells, enabling them to grow in size or rescue tissue integrity following injury, in a wide range of non-dividing cell types. Recent experimental studies have proposed a functional link between mechanical inputs and endoreplication events in various cell types. Moreover, mechanical signals transmitted across the nuclear membrane have been implicated in the regulation of cell cycle, epigenetic events and gene transcription. As part of the mechanism linking cell cycle events with mechanical inputs, the translocation of specific essential factors into the nucleus has been proposed. However, the molecular link between nuclear translocation of such factors, mechanical inputs on the nuclear envelope and endoreplication is still elusive (Unnikannan, 2020).

The linker of nucleoskeleton and cytoskeleton (LINC) complex has been suggested to mediate mechanically induced nuclear entry of essential factors (Driscoll, 2015; Horn, 2014; Osmanagic-Myers, 2015). It physically connects the cytoskeleton and the nucleoskeleton at the interface of the nuclear envelope and has been associated with various human myopathies. The LINC complex is composed of Nesprin protein family members, which associate at their cytoplasmic N-terminal end with distinct cytoskeletal components, and on their nuclear C-terminal end with SUN domain proteins at the perinuclear space. SUN domain proteins bind to various nuclear lamina components, resulting in a physical link between the cytoskeleton and the nucleoskeleton. Recent results indicate that, in Drosophila larval muscles, the LINC complex is essential for arresting endoreplication in the muscle nuclei (myonuclei) and that LINC mutants exhibit additional rounds of DNA replication, resulting in elevated polyploidy. The molecular nature of this process is currently elusive (Unnikannan, 2020).

In an attempt to reveal the components downstream of the LINC-dependent arrest of DNA endoreplication, a screen was performed for genes whose transcription changes in Drosophila Nesprin/klar mutant muscles. One of the identified genes was barrier-to-autointegration factor (baf), shown to be significantly reduced at the transcription level. BAF is a small protein of 89 amino acids that binds dsDNA as well as the nuclear envelope, and in addition forms homodimers. Furthermore, BAF binds to the inner components of the nuclear membrane, including the Lap-2, Emerin, MAN1 (LEM) domain proteins, as well as to lamins A/C and B. Thus, BAF dimers might bridge between dsDNA and the nuclear envelope. Proteomic analysis of BAF partners indicate its potential association with additional proteins, including transcription factors, damage-specific DNA binding proteins and histones. Furthermore, the binding of BAF to its potential partners might be regulated by its phosphorylation state. For example, phosphorylated BAF associates with LEM-domain proteins, whereas de-phosphorylated BAF favors binding to dsDNA. One kinase that has been implicated in BAF phosphorylation is the threonine-serine VRK1 kinase, whose homolog in Drosophila is Ballchen (Ball, also known as NHK-1) (Unnikannan, 2020).

BAF has a crucial role in the condensation and assembly of post-mitotic DNA. Its interaction with both dsDNA and the nuclear lamina enables DNA compaction through cross-bridges between chromosomes and the nuclear envelope, a process essential for the assembly of DNA within a single nucleus following mitosis. Likewise, BAF is recruited to the sites of ruptured nuclear membrane, where it is essential for resealing the ruptured nuclear membrane. Interestingly, in humans a single amino acid substitution of BAF causes Nestor-Guillermo progeria syndrome (NGPS); however, the molecular basis for the disease awaits further investigation (Unnikannan, 2020).

Previous studies demonstrated that in Drosophila, muscle-specific knockdown of BAF increases the levels of DNA endoreplication, phenocopying the LINC mutant outcome. This led to the hypothesis that BAF acts downstream of the LINC complex-dependent mechanotransduction in promoting the arrest of DNA endoreplication in muscle. This study demonstrates that BAF localization at the nuclear envelope is crucial for that process, and that it is downstream of the LINC complex, depends on nucleus-sarcomere connections, and is phosphosensitive. Importantly, elimination of BAF from the nuclear envelope correlates with increased DNA content in the myonuclei and a concomitant increase in E2F1 levels in the nucleoplasm. Taken together, these findings suggest a model in which a LINC-dependent localization of BAF at the nuclear envelope promotes E2F1 tethering to the nuclear envelope to inhibit its accumulation in the nucleoplasm (Unnikannan, 2020).

This study demonstrates the contribution of a novel mechanosensitive component, BAF, in controlling the nuclear accumulation of E2F1, a crucial transcription factor required for the regulation of endoreplication. Whereas previous reports implicated BAF in promoting the condensation and assembly of post-mitotic dsDNA into single nuclei, this study demonstrates that BAF is also essential for the arrest of DNA endoreplication in fully differentiated muscle fibers. Importantly, only BAF that localizes to the nuclear envelope appears to be relevant for this function in post-mitotic differentiated cells. The contribution of BAF to larval muscle functionality is unclear, as baf mutants did not survive up to third instar stage and BAF knockdown in muscles by using RNAi did not eliminate BAF very efficiently (Unnikannan, 2020).

In Drosophila muscle fibers, it was found that BAF was detected in various subcellular sites, including the cytoplasm, nuclear envelope, nucleoplasm and at the nucleolus borders. Yet, only the portion of BAF localized at the nuclear envelope was found to change following elimination of a functional LINC complex. It is well accepted that the LINC complex transmits cytoplasmic mechanical inputs from the cytoskeleton to the nucleoskeleton in various cell types. Moreover, nuclear deformations (from oval into spheroid shape) observed both in larval muscles of LINC complex mutants and in conditions where nuclei detach from the sarcomeres or following Sls knockdown are indicative of changes in the mechanical inputs applied on the nuclear envelope. Because BAF localization at the nuclear envelope was specifically impaired in both conditions, it is proposed that maintenance of BAF at the nuclear envelope is mechanically sensitive (Unnikannan, 2020).

In control myofibers, BAF exhibited a relatively broad distribution along the outlines of the nuclear envelope, often extending beyond the Lamin C expression domain towards the cytoplasm, overlapping with the nucleus-associated microtubules. This suggested that, in addition to its association with the inner aspects of the nuclear membrane through binding to LEM-domain proteins and Lamin A/C, BAF associates with the outer aspects of the nuclear membrane. Previous experiments indicate that despite its small size BAF does not diffuse passively from the cytoplasm to the nucleus. Furthermore, photobleaching experiments with GFP-BAF indicate that BAF-dependent repair of nuclear ruptures occurs when cytoplasmic BAF, but not nuclear BAF, rapidly associates with the ruptured sites and further recruits LEM-domain proteins to establish membrane sealing. The authors suggest that their findings are consistent with a dynamic exchange of BAF between cytoplasmic and nuclear pools, where BAF in the cytoplasm primarily responds to mechanical signals. Because the current experiments indicate that BAF phosphorylation is crucial for its maintenance at the nuclear membrane, it is possible that the exchange of BAF localization between the cytoplasm and the nucleus is stabilized by its phosphorylation. The contribution of the LINC complex to BAF association with the nuclear envelope could be either direct (e.g. by binding to components of the LINC complex) or indirect (e.g. through an effect of the LINC complex on the distribution of LEM proteins at the nuclear envelope). The results support the latter model, in which the LINC complex maintains the localization of the LEM protein Otefin at the nuclear envelope to mediate BAF association with the nuclear envelope. Hence, a model is suggested in which the contribution of the LINC complex to BAF localization at the nuclear envelope is through an effect on Otefin localization at the nuclear envelope (Unnikannan, 2020).

Endoreplication has been implicated in a wide variety of differentiated cells in a broad range of species, including human tissues. A link between mechanical tension and endoreplication has been recently suggested. However, the molecular mechanism coupling mechanical tension with the endoreplication process is still elusive. This study found that a key regulator of endoreplication, E2F1, exhibits a specific distribution at the nuclear envelope in fully differentiated myofibers, where it probably resides non-actively. Changes in the mechanical environment of the nuclear envelope correlate with the localization of E2F1 and promote its accumulation within the nucleoplasm, where it is expected to promote DNA synthesis. It will be of interest to find which proteins associate directly with E2F1 at the nuclear envelope. Attempts to co-immunoprecipitate BAF with Msp300 or E2F1 failed to show a specific protein interaction between these proteins. From a physiological point of view, no detectable changes in muscle size or movement were observed in the baf knockdown muscles, and the larvae developed up to adult stage. The baf homozygous mutant did not develop up to the third instar larval stage, so the full physiological contribution of BAF to muscle growth awaits experiments in which a more efficient reduction in BAF levels is induced in muscle tissue (Unnikannan, 2020).

In summary, these results reveal a novel insight into the role of the LINC complex in coupling endoreplication with changes in the nuclear envelope composition in mature muscle fibers. In particular, the mechanosensitive component, BAF, whose localization at the nuclear envelope is tightly regulated by the LINC complex, is shown to negatively control the nuclear accumulation of the cell cycle regulator E2F1 at the level of the nuclear envelope. The localization of Otefin in the nuclear envelope and BAF phosphorylation by Ball kinase are both crucial in this context. This process might be part of a mechanosensitive pathway that regulates polyploidy in a wide variety of differentiated cells (Unnikannan, 2020).

Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle

Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. This study has identified proteins binding to the kinase domains. Kinase domain 1 bound Ballchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle (Katzemich, 2015).

A stable lattice of thick and thin filaments in striated muscle is needed to maintain the optimum register of the filaments as the fibres contract. Thin filaments from neighbouring sarcomeres are anchored in the Z-disc by α-actinin and other cross-linking proteins, and thick filaments are held in position by cross-links at the M-line in the middle of the sarcomere. The register of thick filaments is also maintained by elastic links between the ends of the filaments and the Z-disc. Large modular proteins of the titin family, associated with thick filaments, contribute to both the stability and the stiffness of the sarcomere. These proteins are made up of tandem immunoglobulin (Ig) and fibronectin-like (Fn3) domains and can have one, or sometimes two, kinase domains near the C-terminus, and there can also be signalling domains (Katzemich, 2015).

The M-line protein, obscurin, has a similar modular structure in invertebrates and vertebrates, although the number of modules in different isoforms and the position of the signalling domains vary. Both Unc-89 (the obscurin homologue in Caenorhabditis elegans; note that this protein is also known as Unc-89 in Drosophila) and obscurin in Drosophila have SH3 and Rho-GEF signalling domains near the N-terminus and two kinase domains near the C-terminus. In vertebrate obscurin, the signalling domains are near the C-terminus; the isoform obscurin A has an ankyrin-binding domain instead of the two C-terminal kinase domains in obscurin B. Both these isoforms are at the periphery of myofibrils in the M-line region of mature skeletal fibres. Binding of obscurin A to ankyrins creates a link between the sarcoplasmic reticulum (SR) and the myofibril. By contrast, Drosophila obscurin is found throughout the M-line and there is no ankyrin-binding domain, so direct binding to the SR is unlikely. However, in the nematode, loss-of-function mutations in unc89 result in displaced ryanodine receptor and SERCA, as well as abnormal Ca2+ signalling. This suggests that there is a function for Unc-89 in Ca2+ regulation involving the SR. So far, five large isoforms of obscurin have been identified in Drosophila muscles: one expressed in the larva, and four expressed in the pupa and adult. All these isoforms have Ig domains in the tandem Ig region, and at least the first of the kinase domains (denoted Kin1). The indirect flight muscle (IFM) has two isoforms: a major isoform of 475 kDa and a minor isoform that is somewhat smaller. The two remaining isoforms are in other thoracic muscles. Drosophila obscurin is essential for the formation of an M-line, and for the correct assembly of thick and thin filaments in the sarcomere: lack of obscurin in the IFM results in asymmetrical thick filaments and thin filaments of abnormal length and polarity. Paradoxically, vertebrate obscurin is not necessary for normal sarcomere structure, given that obscurin knockout in the mouse had no serious effect on sarcomere assembly or maintenance (Katzemich, 2015).

The kinase domains of titin-like proteins often function as scaffolds binding other proteins, and might or might not be active kinases. In C. elegans, the Unc-89 kinase 1 domain (PK1) is predicted to be inactive because the ATP-binding site lacks essential residues. The Unc-89 kinase 2 domain (PK2) might be active, although a motif contributing to ATP-binding is atypical. Both Unc-89 kinase domains interact with the protein small, C-terminal domain, phosphatase-like 1 (SCPL-1), which is thought to be involved in muscle-specific signalling. Unc-89 PK1 also interacts with the LIM-domain protein, LIM-9; the complex of PK1, SCPL-1 and LIM-9 links Unc-89 to integrin adhesion sites at the cell surface. In Drosophila, both Obscurin kinase domains (denoted Kin1 and Kin2) are predicted to be inactive because the catalytic site lacks the catalytic aspartate and other crucial residues. Both kinase domains in vertebrate obscurin B are predicted to be catalytically active, and can apparently be auto-phosphorylated. The kinase domains are reported to interact with membrane associated proteins: kinase domain 1 (SK1) with the extracellular domain of a Na+/K+-ATPase at adherens junctions, which is not a substrate, and kinase domain 2 (SK2) with the cell-adhesion molecule, N-cadherin, which is an in vitro substrate (Katzemich, 2015).

The kinase domains in titin-like proteins have sequences at the C-terminus that sterically block the active site (the C-terminal regulatory domain). This sequence can inhibit an active kinase, or regulate ligand binding; it can also be part of the structure of the kinase domain, and necessary to maintain the stability of the domain. Titin-like kinases are linked to stretch-activated signalling pathways in muscle. Mechano-sensing by the kinase can result in changes in the C-terminal regulatory domain and transient binding of ligands to the kinase scaffold. The precise mechanism of regulation varies in different species (Katzemich, 2015).

The aim of this study was to identify proteins binding to the two kinase domains in Drosophila obscurin, and to determine the effect of the proteins on the assembly of an ordered sarcomere in IFM. Ball (a protein kinase) was shown to bind to Kin1, and MASK (an ankyrin repeat protein) binds to both Kin1 and Kin2. The kinase ligands are essential for the formation of an intact M-line and Z-disc in the IFM sarcomere (Katzemich, 2015).

The kinase domains of Drosophila Obscurin differ from those of the C. elegans homologue, Unc89, and the vertebrate protein. Both domains are predicted to be inactive as kinases. However, some pseudokinases can become catalytically active by replacing missing residues with residues from neighbouring domains, or from associated ligands. Pseudokinases commonly act as scaffolds for binding proteins involved in signal transduction, and are often tethered to other domains, including Ig and Fn3 domains, which contribute to the binding site. Titin kinase in the M-line region of vertebrate skeletal muscle forms part of a binding site for the autophagy and kinase scaffold proteins, Nbr1 and SQSTM1, and the ubiquitin ligase, MuRF1 (also known as TRIM63). In the case of MuRF1, the site includes the preceding Ig and Fn3 domains, which will also bind MuRF1 without the kinase domain (Katzemich, 2015).

This study has found that Kin1 in Drosophila Obscurin binds Ball, which has the hallmarks of an active serine-threonine kinase. Ball differs from other kinase molecules in having a long extension C-terminal to the kinase sequence. This extension binds to Kin1 of Obscurin, with or without the flanking Ig and regulatory domains. Given that Ball also binds to the Ig domain alone, it is likely the molecule spans a region of Obscurin that includes the Ig domain as well as the kinase. Binding to Kin1 with the regulatory domain was unexpected. However, it is not clear how much of the sequence downstream of the kinase is included in a possible regulatory domain. In pseudokinases that are part of a larger molecule, the association of the regulatory domain with a ligand-binding site can be altered by force applied to the whole molecule. The regulatory domain of Kin1, taken out of its usual context, might not associate with the active site in the same way as it would in vivo. Alternatively, the regulatory domain might be required to stabilise the kinase structure when the muscle is stretched, as suggested for twitchin kinase (Katzemich, 2015).

Ball is found in the Z-disc of IFM, as well as being diffusely distributed in the sarcomere and in some samples, Ball is also detected at the M-line. Ball might migrate to bind to Kin1 in the M-line when the kinase activity of Ball is needed. There are other examples of protein migration in the muscle sarcomere. A transient translocation from the M-line to the Z-disc and cytoplasm has been observed for titin kinase ligands in cardiac muscle. In zebrafish, the myosin chaperone, Unc-45, is associated with myosin during myofibrillogenesis; in the adult, Unc-45 is in the Z-disc in normal fibres and it migrates to the A-band under conditions of stress, where it transiently associates with myosin again. Similarly, Ball might migrate from the Z-disc to the M-line under some conditions. Although this study has shown that Ball is capable of binding to Kin1 in vivo, the conditions necessary for the association are not yet known. Ball is still present in the IFM of obscurin-knockdown flies, though with a less-ordered distribution in the sarcomere, which suggests there are likely to be other binding partners for Ball (Katzemich, 2015).

Kin2 binds MASK, which has two regions with ankyrin repeats, and a relatively long sequence C-terminal to a KH domain. A peptide near the end of the molecule binds to Kin2 with or without the Fn3 domain preceding the kinase. MASK does not bind to the Fn3 domain alone, nor does it bind to Kin2 with sequence C-terminal to the kinase. It is not clear at present whether the C-terminal sequence acts as a regulatory domain (Katzemich, 2015).

The dual position of MASK in both the Z-disc and the M-line of IFM is unlikely to be due to migration of a protein of 400 kDa. The RNA coding for ankyrin-repeat proteins undergoes extensive alternative splicing, which can alter the binding sites of the different ankyrin isoforms. The independence of the binding sites for MASK in the M-line and Z-disc is confirmed by the finding that in obscurin-knockdown flies, MASK is almost eliminated from the M-line but still present in the Z-disc. Thus, Obscurin is needed for MASK to bind in the M-line, but not to the Z-disc. The presence of Obscurin in the M-line of MASK-knockdown flies is consistent with an Obscurin scaffold that binds MASK (Katzemich, 2015).

In addition to MASK, Tropomyosin-1 was identified as a ligand associated with Kin2 expressed in vivo. As tropomyosin is a thin filament protein, the significance of an association with Obscurin, which is at the midline of the thick filament, is not clear. Smaller isoforms of Obscurin have been detected in IFM, and it is possible that Tropomyosin-1 could bind, outside the M-line, to a small isoform containing a Kin2 domain (Katzemich, 2015).

The effect of downregulating Ball or MASK on the structure of the M-line and Z-disc in IFM shows the importance of these proteins in the development of a regular filament lattice. In the IFM of Ball-knockdown flies, the shifted position of the H-zone and M-line is associated with fragmented Z-discs; where the Z-disc is normal within a sarcomere, the H-zone and M-line are at the midline. The aggregates of multiple Z-discs in the IFM of MASK-knockdown flies dominate the sarcomere and there is no regular H-zone. This phenotype differs from the effect of reducing the expression of obscurin in IFM, where the H-zone and M-line are often shifted from the midline, without a corresponding anomaly in the Z-disc. The difference might be due to the presence of Ball and MASK in the Z-disc, whereas Obscurin is solely in the M-line. The high mortality rate at the larval and pupal stages of flies when either Ball or MASK is reduced differs from the survival of flies with reduced obscurin, which is unaffected in RNAi lines. Evidently, the crucial function of Ball and MASK is in binding to the Z-disc. The association of the proteins with Obscurin in the M-line is not necessary for the performance of most muscles, although it is essential for the development of the precise filament lattice that is needed for the performance of the IFM. The function of Obscurin in the assembly of the IFM sarcomere is not seen in vertebrates. Knockouts of obscurin in the mouse have no effect on the assembly of myosin filaments, Z-disc or M-line, but they do impair the assembly of the SR. Whether or not invertebrate and vertebrate obscurin-like proteins are true homologues (with the same functions, rather than just having a similar patterns of domains) is at present uncertain (Katzemich, 2015).

Ball and MASK are reported to be involved in cell proliferation, growth and differentiation in Drosophila. Ball regulates the proliferation and differentiation of germline stem cells and neuroblasts (Herzig, 2014; Yakulov, 2014). However, it is not clear whether this has any relevance to the function of Ball in Drosophila muscle. The VRK family of kinases (of which Ball is a member) phosphorylate the barrier-to-autointegration factor (BAF), which is necessary for the correct assembly of chromatin. Comparison of domains in the sequences of Drosophila Ball (also called NHK-1) and VRK in human, mouse, Xenopus and C. elegans, shows that C. elegans and Drosophila are the only homologues with a long C-terminal sequence extension; the other species have a relatively short stretch of sequence following the kinase domain. This suggests that Ball has a different function in the invertebrates; in Drosophila, the C-terminal sequence binds to Kin1 (Katzemich, 2015).

MASK was first identified in the developing eye of Drosophila, where it is required for proliferation and differentiation of photoreceptors. MASK genetically interacts with Corkscrew (CSW), a protein phosphatase that acts downstream of the epidermal growth factor receptor (EGFR) in a signalling pathway involved in myogenesis in Drosophila. Through these interactions, Obscurin can potentially be linked to a receptor tyrosine kinase (RTK) pathway involved in myogenesis. Obscurin and Kettin are present at an early stage in the development of pupal IFM. Both are large titin-like proteins with tandem Ig domains, which have a dual function in myogenesis and in the mature muscle. Obscurin kinase domains appear to be scaffolds for binding MASK. Ankyrin repeats act as adaptor modules, binding cytoskeletal proteins and signalling molecules. The repeats stabilise protein networks, often together with large structural proteins. Therefore, an interaction between obscurin kinases and MASK could provide a platform for the assembly of signalling proteins, and this could be affected by force on the obscurin molecule. Ankyrin-repeat proteins in vertebrate skeletal muscle (MARPs) interact with the N2A elastic region of titin; in the case of Ankrd2 (also known as Arpp), expression is induced by stretch, and MARPs are thought to be involved in stretch-induced signalling pathways. There are two smaller homologues of MASK in human cells: MASK1 (Ankhd1) and MASK2 (Ankrd17), which have the same domain structure as Drosophila MASK. The Drosophila protein (~400 kDa) is larger than the human proteins (~280 kDa), mainly due to a longer stretch of sequence between the ankyrin repeat domains. MASK is a cofactor of Drosophila Yorkie (Yki) and mammalian Yes-associated protein (YAP) in the Hippo signalling pathway, which controls tissue growth. The signalling function in this pathway is similar for Drosophila and human MASK; however, MASK isoforms have not been found it human muscle. There is a sequence in the C. elegans genome that codes for a protein with homology to Drosophila, mouse and human MASK. The protein, ankyrin repeat and KH domain-containing protein is predicted to be 287 kDa, so similar in size to the human protein. The function is unknown (Katzemich, 2015).

The presence of Ball and MASK in mature IFM suggests the proteins have a signalling function in the adult fly. There is turnover of contractile proteins in Drosophila muscles, including the IFM, throughout the life of a fly. The function of obscurin kinase domains as scaffolds for the assembly of signalling proteins is likely to be important in the continual remodelling of the muscle. During contraction, the M-line experiences shearing stress, due to unbalanced forces in the two half sarcomeres, and the M-line is thought to act as a strain sensor. Ball and MASK might be recruited to the M-line in response to mechanical stress sensed by obscurin. Importantly, mutations in human titin kinase lead to a phenotype (Z-disc streaming) similar to that of Ball and MASK knockdowns, possibly by disrupting protein turnover, which supports the finding that Z-disc abnormalities can be an indirect consequence of mutations in proteins associated with the M-line (Katzemich, 2015).

In summary, this study has identified two proteins, Ball and MASK, that are essential for the assembly of an ordered IFM. The pseudokinase domains of obscurin act as scaffolds binding the proteins. This raises the possibility of investigating the regulation of signalling pathways involved in assembly and maintenance of IFM through interaction with obscurin (Katzemich, 2015).

The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes

Chromosome condensation during cell division is one of the most dramatic events in the cell cycle. Condensin and topoisomerase II are the most studied factors in chromosome condensation. However, their inactivation leads to only mild defects and little is known about the roles of other factors. This study took advantage of Drosophila oocytes to elucidate the roles of potential condensation factors by performing RNA interference (RNAi). Consistent with previous studies, depletion of condensin I subunits or topoisomerase II in oocytes only mildly affected chromosome condensation. In contrast, severe undercondensation of chromosomes was found after depletion of the Mi-2-containing NuRD nucleosome remodelling complex or the protein kinase NHK-1 (also known as Ballchen in Drosophila). The further phenotypic analysis suggests that Mi-2 and NHK-1 are involved in different pathways of chromosome condensation. The main role of NHK-1 in chromosome condensation is to phosphorylate Barrier-to-autointegration factor (BAF) and suppress its activity in linking chromosomes to nuclear envelope proteins. It was further shown that NHK-1 is important for chromosome condensation during mitosis as well as in oocytes (Nikalayevich, 2015).

This report is the first to use Drosophila oocytes to study chromosome condensation. It is argued that the Drosophila oocyte combined with RNAi is an excellent system for research of chromosome condensation, which complements commonly used mitotic systems. Firstly, Drosophila oocytes grow enormously in volume between completion of pre-meiotic mitosis and recombination and chromosome condensation. shRNA expression can be induced after the protein executes its role in the previous mitosis and/or recombination but prior to oocyte growth. Even if the target protein is stable, it becomes sufficiently diluted before chromosome condensation in oocytes. This is in contrast to mitotic cycles where cells only double in size between divisions. Secondly, Drosophila oocytes arrest in metaphase of the first meiotic division. This allows chromosome defects to be studied in the first division after the target protein is depleted, rather than as a mixture of defects accumulated through multiple divisions caused by a gradual decrease of the protein. Finally, as oocytes are large, the condensation state of chromosomes can be clearly observed without mechanical treatment such as squashing or spreading. Therefore, RNAi in Drosophila oocytes could be a powerful system to study chromosome condensation, although negative results should be interpreted with caution as they might be caused by insufficient depletion, genetic redundancy or cell-type-specific function (Nikalayevich, 2015).

Indeed, in this study, a small-scale survey of chromosomal proteins, new chromosome condensation factors were identified in addition to well-known ones, demonstrating the effectiveness of Drosophila oocytes as a research system. Well-known factors, including condensin I subunits, topoisomerase II and Aurora B, showed milder chromosome condensation defects. Knockdown of topoisomerase II or condensin I showed similar condensation defects, and appeared to affect mainly centromeric and/or pericentromeric regions. The previous reports in mitosis are consistent with this result, suggesting that these two factors are not the main condensation factors in mitosis or in meiosis (Nikalayevich, 2015).

A previous study of Mi-2 in Drosophila suggested that it promotes decondensation of chromosomes because overexpression of wild-type Mi-2 results in chromosome decondensation in polytene or mitotic cells and overexpression of dominant-negative Mi-2 results in overcondensation. In the current study, Mi-2 RNAi in oocytes showed chromosome decondensation, whereas in a preliminary study in neuroblasts Mi-2 RNAi did not show chromosome decondensation. The difference from the previous study might be due to the method of disrupting the Mi-2 function or cell types used for the studies. It is argued that the phenotype caused by RNAi in oocytes is a better reflection of the in vivo function. RNAi of other NuRD subunits indicated that the NuRD complex is important for chromosome condensation (Nikalayevich, 2015).

How does the NuRD complex promote chromosome condensation? It is possible that nucleosome remodelling is directly required during chromosome condensation. For example, proper positioning of nucleosomes might be important for full chromosome condensation. Indeed, other nucleosome remodelling complexes have been suggested to be involved in chromosome condensation in fission yeast. Alternatively, histone deacetylase acivity of the NuRD complex might be important for chromosome condensation, as histone modifications are a major way to regulate chromosome structure. The possibility cannot be excluded that NuRD acts through transcription of other chromosome condensation factors, as it is known to regulate gene transcription. Further studies using more sophisticated mutations would help to distinguish these possibilities (Nikalayevich, 2015).

This study found that knockdown of NHK-1 resulted in severe chromosome condensation defects in nearly all oocytes. Previously, involvement of NHK-1 or its orthologues in metaphase chromosome condensation has not been reported, although overexpression of the human orthologue disrupts chromatin organisation in interphase. None of the three female sterile nhk-1 mutants showed chromosome condensation defects in metaphase I in oocytes. This might be because the minimal NHK-1 activity required for producing viable adults is sufficient to allow chromosome condensation in oocytes. Female-germline-specific RNAi is likely to have achieved greater depletion of NHK-1 in oocytes. This study showed that phosphorylation of BAF, thus inactivating its linking of DNA to LEM-domain-containing inner nuclear membrane proteins, is the major role of NHK-1 in chromosome condensation in oocytes. However, NHK-1 might regulate multiple pathways during condensation, for example, it has been shown that it is required for histone 2A phosphorylation and condensin recruitment in prophase I oocytes (Nikalayevich, 2015).

A crucial question is whether the chromosome condensation defect is a direct consequence of NHK-1 loss or a secondary consequence of a karyosome defect in prophase I oocytes. Evidence indicates that the compact karyosome in the prophase I nucleus and chromosome condensation in metaphase I are at least partly independent. In female-sterile hypomophic nhk-1 mutants, chromatin organisation in prophase I oocytes is defective, but metaphase I chromosomes are properly condensed in mature oocytes (Cullen, 2005; Ivanovska, 2005). By contrast, in Mi-2 RNAi oocytes, the karyosome is normal in prophase I, but chromosomes become undercondensed after nuclear envelope breakdown in some metaphase I oocytes. Furthermore, as chromosome condensation in mitosis is also defective in nhk-1 mutants, the role for NHK-1 in chromosome condensation must be at least partly independent from meiosis-specific chromatin organisation. Therefore, release of LEM-containing nuclear envelope proteins from chromosomes might be a prerequisite for proper chromosome condensation (Nikalayevich, 2015).

In conclusion, this targeted survey using RNAi in Drosophila oocytes has already identified new factors required for chromosome condensation. Further analysis provided new insights into the molecular mechanism of condensation including the release of nuclear envelope proteins from chromosomes and nucleosome remodelling and/or histone deacetylation as essential steps for condensation. In future, a larger scale screen of putative chromosomal proteins might prove to be fruitful (Nikalayevich, 2015).

Ballchen participates in proliferation control and prevents the differentiation of Drosophila melanogaster neuronal stem cells

Stem cells continuously generate differentiating daughter cells and are essential for tissue homeostasis and development. Their capacity to self-renew as undifferentiated and actively dividing cells is controlled by either external signals from a cellular environment, the stem cell niche, or asymmetric distribution of cell fate determinants during cell division. This study reports that the protein kinase Ballchen (Ball) is required to prevent differentiation as well as to maintain normal proliferation of neuronal stem cells of Drosophila melanogaster, called neuroblasts. These results show that the brains of ball mutant larvae are severely reduced in size, which is caused by a reduced proliferation rate of the neuroblasts. Moreover, ball mutant neuroblasts gradually lose the expression of the neuroblast determinants Miranda and aPKC, suggesting their premature differentiation. These results indicate that Ball represents a novel cell intrinsic factor with a dual function regulating the proliferative capacity and the differentiation status of neuronal stem cells during development (Yakulov, 2014).

These results establish that BALL is essential to maintain the proliferation rate as well as the undifferentiated state of postembryonic neuroblasts (pNBs) and therefore interlink these two aspects of stem cell self-renewal. The proliferation rate of ball² mutant pNBs was reduced already at 72 h ALH, a time point when approximately half of the pNBs continued to express the stem cell determinants MIRA and aPKC. Therefore, it is plausible that the primary function of BALL is to control the proliferation rate of pNBs as a prerequisite for continuous self-renewal of neuroblastst (Yakulov, 2014).

The effects of a reduced proliferation rate were previously studied in epithelial tissue such as wing imaginal discs, which led to the discovery of a phenomenon termed cellular competition. It describes that cells with reduced cellular fitness proliferate at a lower rate and are eventually eliminated by apoptosis. This phenomenon was observed after generating ball² mutant cells by MARCM in wing imaginal discs, showing that the mutant cells are capable to proliferate and to form cell clones. However, these cell clones fail to compete with wild type cells and subsequently undergo apoptosis. Maintenance of the stem cell character of pNBs is unlikely to be regulated through a competitive mechanism, since the pNB lineages contain only a single stem cell. The data suggest that the same process that determines competitiveness of wing disc epithelial cells is a prerequisite to maintain the self-renewal of pNBst (Yakulov, 2014).

Recent work has shown that BALL is required to sustain self-renewal of niche-controlled stem cells (Herzig, 2014). This work shows that this function of BALL is not restricted to niche-controlled stem cells but is also required in pNBs, which depend on asymmetric distribution of cell fate determinants for self-renewal. Thus, the function of BALL for stem cell self-renewal is not limited by the factors and mechanisms that mediate cell fate decisions in the different stem cell systems. This study therefore suggests that Drosophila stem cells employ cell intrinsic mechanisms to ensure stem cell self-renewal that are independent of the tissue specific modes of stem cell fate decisions and shared by diverse stem cell populations. The molecular basis of these mechanisms and how BALL is integrated in these processes remains to be established by future studiest (Yakulov, 2014).

Ballchen is required for self-renewal of germline stem cells in Drosophila melanogaster

Self-renewing stem cells are pools of undifferentiated cells, which are maintained in cellular niche environments by distinct tissue-specific signalling pathways. In Drosophila melanogaster, female germline stem cells (GSCs) are maintained in a somatic niche of the gonads by BMP signalling. This study reports a novel function of the Drosophila kinase Ballchen (BALL), showing that its cell autonomous role is to maintain the self-renewing capacity of female GSCs independent of BMP signalling. ball mutant GSCs are eliminated from the niche and subsequently differentiate into mature eggs, indicating that BALL is largely dispensable for differentiation. Similar to female GSCs, BALL is required to maintain self-renewal of male GSCs, suggesting a tissue independent requirement of BALL for self-renewal of germline stem cells (Herzig, 2014).

This study shows that the VRK-1 kinase BALL is required for self-renewal of germline stem cells in Drosophila, including the symmetrically amplifying PGCs of larvae and both male and female GSCs. These stem cells are actively maintained undifferentiated and they require BMP signalling for self-renewal that emanates from their cellular niche environments. In ball² mutant female GSCs, where the requirement of BMP signalling for self-renewal is most pronounced, known targets of BMP signalling are regulated as in wild type GSCs. This indicates that BALL participates neither in the transmission nor the regulation of BMP signalling, and that it is needed to maintain stem cell character in a cell autonomous manner, irrespective of the tissue-specific maintenance signals that emanate from the niches (Herzig, 2014).

The loss of self-renewing stem cells could be caused by the induction of ectopic differentiation in these cells. The differentiation pathway was blocked in ball mutant female GSCs by removing also the central differentiation factor BAM. The results suggest that ball mutant GSCs are not eliminated from the stem cell niche because they initiate germline differentiation but that the GSCs differentiate because they lost the capacity for self-renewal (Herzig, 2014).

It is unclear by which mechanism BALL mediates the ability for GSC self-renewal. In ovaries, GSCs and FSCs undergo a regular turnover and are continuously replaced in the niche either by their own daughter cells or by symmetric divisions of the neighbouring stem cells. The replacement of GSCs involves competition between stem cells. Cells lacking BAM for instance, successfully displace less competitive wild type stem cells in the niche. However, if BALL is additionally removed from bam mutant cells, they appear to loose their competitive advantage. The molecular basis of stem cell competiveness is still poorly understood. However, it has been shown that overexpression of the Drosophila dMyc transcription factor diminutive enhances the competitiveness of GSCs and causes significantly enlarged nucleoli and increased rRNA expression in epithelial cells. These observations suggest a correlation between ribosome biogenesis and GSC competitiveness. Downregulation of ribosome biogenesis appears in fact to be directly required for germ cell differentiation, since BAM activates the Mei-P26 protein which downregulates the expression of dMyc. Furthermore, when overexpressed from a transgene, dMyc abrogates the tumour growth phenotype and the size increase of nucleoli in bam mutant cells. Additional support for the proposal that increased ribosome biogenesis in stem cells is crucial for their competitiveness and for maintaining their undifferentiated state derives from studies on wicked. Wicked is an essential component of the U3 snoRNP pre-rRNA processing, which is required to maintain the self-renewal of GSC. The findings that BALL is enriched in stem cell nucleoli and required for the structural integrity of nucleoli in tumourous GSCs provide a plausible link between ribosome biogenesis and BALL-dependent competitiveness of GSCs (Herzig, 2014).

Once displaced from the niche, ball² mutant female GSCs differentiate according to their germline fate with only minor defects. This study did not address whether the differentiation of ball mutant cells is fully completed like in the respective wild type lineages in systems other than the adult female germline, but it was found, irrespective of the system examined, that BALL is not strictly required as proliferation factor. Especially the analysis of dividing follicle cells showed that BALL is not a cell cycle regulator. With two remarkable exceptions, BALL is also not essential for cellular survival. These exceptions, i.e., ball mutant PGCs at late larval stages and ball² bamΔ86 double mutant germline cells outside the ovarian niche, represent conditions in which differentiation is either not supported by the tissue or not possible due to the lack of a differentiation factor. Therefore, it is tempting to speculate that BALL becomes only essential for cellular survival, when the ball mutant stem cells are unable to 'escape' from self-renewal into differentiation. Although ball clearly has multiple functions, e.g., oocyte chromatin organization or modulation of female PGC proliferation rate, the common defect observed in all systems examined so far is a failure in maintaining pools of undifferentiated cells. Since BALL is not an essential proliferation factor, these data suggest that also the additional defects in ball mutant larvae, i.e., lacking imaginal discs and degenerate brains, could be due to premature loss of undifferentiated progenitor or stem cells. Analysis of these systems will eventually show whether BALL is broadly required to maintain the undifferentiated state of cells during development (Herzig, 2014).

The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila

The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. This study shows that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, unrepaired DNA breaks formed during recombination were found to suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore it is proposed that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired (Lancaster, 2010).

Based on the evidence, a model is proposed in which DSBs formed during recombination suppress the activity of the conserved kinase NHK-1 through the meiotic recombination checkpoint to delay oocyte nuclear reorganisation from a recombination to a post-recombination phase. Although the evidence is mostly genetic or cytological, all data are so far consistent with this model. Nevertheless, the model is likely to be too simplistic and to represent only a part of the whole picture. For example, the possibility was not excluded that other checkpoint effectors are also involved in delaying meiotic progression. It is hopeed that the proposed model will prompt further investigation to fully uncover how the meiotic checkpoint is linked to meiotic progression (Lancaster, 2010).

How does NHK-1 kinase control this critical transition in meiosis? A previous study showed that NHK-1 directly controls karyosome formation through phosphorylation of BAF, a linker between the nuclear envelope and chromatin (Lancaster, 2007). Phosphorylation of BAF by NHK-1 releases meiotic chromosomes from tethering at the nuclear envelope to allow karyosome formation. Expression of non-phosphorylatable BAF disrupts karyosome formation, but not synaptonemal complex disassembly or condensin loading. Therefore, NHK-1 appears to control two independent pathways during nuclear reorganisation. This is consistent with a recent study showing that condensin is required for synaptonemal complex disassembly but not for karyosome formation. Karyosome formation and condensin loading are therefore likely to be two primary targets of NHK-1 activity (Lancaster, 2010).

Finally, this study in Drosophila is likely to have significant implications for understanding of meiotic regulation at a molecular level in other organisms, since the processes analyzed in this study are conserved among eukaryotes. The meiotic checkpoint that coordinates recombination events with meiotic progression is universally found across eukaryotes. Furthermore, NHK-1 is well conserved among animals, and karyosome-like clustering of meiotic chromosomes, as well as synaptonemal complex disassembly and condensin loading, is widely found in oocytes of various species including humans. In addition, this study has also suggested an involvement of NHK-1 in the DNA damage response during the mitotic cell cycle (Lancaster, 2010).

NHK-1 phosphorylates BAF to allow karyosome formation in the Drosophila oocyte nucleus

Accurate chromosome segregation in meiosis requires dynamic changes in chromatin organization. In Drosophila melanogaster, upon completion of recombination, meiotic chromosomes form a single, compact cluster called the karyosome in an enlarged oocyte nucleus. This clustering is also found in humans; however, the mechanisms underlying karyosome formation are not understood. This study reports that phosphorylation of barrier to autointegration factor (BAF) by the conserved kinase nucleosomal histone kinase-1 (NHK-1; Drosophila Vrk1) has a critical function in karyosome formation. The noncatalytic domain of NHK-1 is crucial for its kinase activity toward BAF, a protein that acts as a linker between chromatin and the nuclear envelope. A reduction of NHK-1 or expression of nonphosphorylatable BAF results in ectopic association of chromosomes with the nuclear envelope in oocytes. It is proposed that BAF phosphorylation by NHK-1 disrupts anchorage of chromosomes to the nuclear envelope, allowing karyosome formation in oocytes. These data provide the first mechanistic insight into how the karyosome forms (Lancaster, 2007).

Concerted action of Aurora B, Polo and NHK-1 kinases in centromere-specific histone 2A phosphorylation

The spatial and temporal control of histone modifications is crucial for precise regulation of chromatin structure and function. This study reports that phosphorylation of H2A at threonine 119 (T119) is enriched at centromere regions in Drosophila mitosis. The Aurora B kinase complex is essential for this phosphorylation at centromeres, while Polo kinase is required to down-regulate H2A phosphorylation on chromosome arms in mitosis. Cyclin B degradation triggers loss of centromeric H2A phosphorylation at anaphase onset. Epistasis analysis indicated that Polo functions upstream of the H2A kinase NHK-1 but parallel to Aurora B. Therefore, multiple mitotic kinases work together to specify the spatial and temporal pattern of H2A T119 phosphorylation (Brittle, 2007).

A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes

To promote faithful propagation of the genetic material during sexual reproduction, meiotic chromosomes undergo specialized morphological changes that ensure accurate segregation of homologous chromosomes. The molecular mechanisms that establish the meiotic chromosomal structures are largely unknown. This study describes a mutation in a recently identified Histone H2A kinase, nhk-1, in Drosophila that leads to female sterility due to defects in the formation of the meiotic chromosomal structures. The metaphase I arrest and the karyosome, a critical prophase I chromosomal structure, require nucleosomal histone kinase-1 (NHK-1) function. The defects are a result of failure to disassemble the synaptonemal complex and to load condensin onto the mutant chromosomes. Embryos laid by nhk-1^-/- mutant females arrest with aberrant polar bodies and mitotic spindles, revealing that mitosis is affected as well. This study analyzed the role of Histone H2A phosphorylation with respect to the histone code hypothesis and found that it is required for acetylation of Histone H3 and Histone H4 in meiosis. These studies reveal a critical role for histone modifications in chromosome dynamics in meiosis and mitosis (Ivanovska, 2005).

This study explored the functional requirements for a newly identified histone kinase in meiosis and found that NHK-1 functions in meiotic progression. The phenotypes of the female-sterile nhk-1^Z3-0437 mutant showed that NHK-1 is required for the establishment of several meiosis-specific chromosomal configurations, including the prophase I karyosome, the metaphase I spindle, and the polar body. Histone H2AT119ph, as well as Histone H3K14ac and Histone H4K5ac, were reduced in the mutant oocytes, whereas the other histone modifications examined were unaffected. Strikingly, disassembly of the SC and loading of condensin failed in the mutant. Therefore, it is suggested that NHK-1 and Histone H2AT119ph, the C-terminus of H2A, are required specifically for proper chromosome architecture in meiosis (Ivanovska, 2005).

The histone code hypothesis conjectures functional interactions among histone modifications and between modifications and proteins that bind to them. It has been postulated that histones have signature modification profiles in meiosis to accommodate the meiosis-specific chromosomal events. However, most research in vertebrates has been limited to identifying the modifications and has not provided extensive insight into their function (Ivanovska, 2005).

The finding that NHK-1 is required for meiosis, presumably via its phosphorylation of Histone H2AT119 (Aihara 2004), prompted an examination of the presence of other histone modifications in Drosophila oocytes. It was found that Histone H1 is phosphorylated, HP1 is bound (indicative of histone H3 methylation), and Histone H3 and H4 are acetylated during prophase I of meiosis in Drosophila. The presence of these histone modifications in oocyte nuclei suggests that they play a role in chromosome dynamics during meiosis. It is of interest to note that Histone H4 and H3 were shown to be acetylated in mouse oocytes in prophase I, suggesting an evolutionary conservation. Future analysis of mutations in histone modifying enzymes may shed light on the functions of these modifications in meiosis (Ivanovska, 2005).

One stipulation of the histone code is that histone modifications affect each other. To test this hypothesis with respect to Histone H2AT119ph, the panel of histone modifications was examined in the nhk-1^Z3-0437 mutant ovaries. Histone H1ph, HP1 binding, and Histone H4K12ac were unaffected in the mutant oocytes, consistent with Histone H2AT119ph being downstream or independent of them in Drosophila meiosis. In contrast, Histone H3K14ac and Histone H4K5ac were absent specifically from the chromosomes in the nhk-1^Z3-0437 mutant oocytes, indicating that Histone H2AT119ph is a prerequisite for acetylation of these residues. Although the significance of these acetylations is unclear at present, it is intriguing to speculate that they play an important role in meiosis (Ivanovska, 2005).

Another stipulation of the histone code is that modifications recruit or exclude proteins from binding to chromatin. One possibility is that Histone H2AT119ph is required for function of the histone acetyltransferases for Histone H3K14 and Histone H4K5, the residues that lack acetylation in the mutant oocytes. There is a precedent for such a cascade of dependencies in transcription: Phosphorylation of H3 Ser 10 leads to acetylation of H3 Lys 14. Histone H2AT119 phosphorylation may also be a prerequisite for proper chromosomal associations of SMC4 and for dissociation of the SC (Ivanovska, 2005).

In conclusion, Histone H2AT119 phosphorylation appears to affect both other histone modifications and the binding of proteins to chromatin as predicted by the histone code hypothesis. Histone H2AT119 phosphorylation by NHK-1 may, therefore, be a key component of a meiotic histone code in oocytes (Ivanovska, 2005).

The Drosophila karyosome is a subnuclear organelle comprised of the prophase I chromosomes. It is the best studied example of a family of similar structures found throughout evolution. Although the exact function of the karyosome is unclear, several Drosophila mutants that disrupt karyosome structure lead to female sterility, suggesting that karyosome formation is required for fertility. In addition, it has been postulated that retaining the oocyte chromosomes in close proximity within the large oocyte nucleus (the germinal vesicle) facilitates proper chromosome segregation during the meiotic divisions. Therefore, understanding the molecular mechanisms that regulate karyosome formation is of great interest (Ivanovska, 2005).

The spindle mutants in Drosophila (spindle-B, spindle-D, and okra) are characterized by defects in karyosome formation, in establishment of the dorsal/ventral polarity of the oocyte and the egg, and defects in repair of the DSBs following recombination. The nhk-1^Z3-0437 mutant does not show either of the latter phenotypes, consistent with the NHK-1 histone kinase having a primary role in karyosome formation, whereas the spindle mutants may affect karyosome function as a secondary consequence of other defects. The specific requirement for NHK-1 implicates Histone H2AT119ph in formation of the karyosome. The conservation of the NHK-1 kinase and Thr 119 in Histone H2A among diverse species suggests that this histone modification plays a conserved role in chromosomal dynamics in meiosis (Ivanovska, 2005).

The nhk-1^Z3-0437 mutant showed several interesting phenotypes in the oocyte. First, all embryos laid by mutant females showed aberrant polar bodies and an arrest at metaphase of the first mitotic division. Second, a defect was observed in the metaphase I arrest in 50% of late-stage oocytes. Finally, the karyosome failed to form in all early oocytes. It is not clear how the dispersed chromosomes in the mutant karyosomes later form three masses at metaphase I, but it suggests that the chromosomes are not fragmented and that bivalent associations are retained (Ivanovska, 2005).

These results can be interpreted in two ways. First, NHK-1 may be required continuously throughout meiosis for each of the events affected by the Z3-0437 mutation. Continuous requirement may be due to multiple substrates or due to dynamic Histone H2A phosphorylation and dephosphorylation. Second, NHK-1 function may be required specifically for karyosome formation, whereas the metaphase I and the embryonic phenotypes may be a consequence of the karyosome defect. This model suggests that Histone H2AT119ph serves as an epigenetic mark that contributes to proper completion of later events, such as establishment of the metaphase I arrest and progression through the first mitotic division. To test whether the early mitotic defects in the nhk-1 mutant were due to a failure to load condensin in mitosis, the dCAP-D2 condensin subunit was examined and it was found to localize onto chromosomes in embryos from nhk-1 mutant females. Nevertheless, it is thought likely that NHK-1 plays essential roles in mitosis that may be revealed by additional alleles of nhk-1 (Ivanovska, 2005).

Histone H2AT119 is phosphorylated in the follicle cells, the nurse cells, and the oocyte, suggesting that NHK-1 functions in all three ovarian cell types. However, mutant phenotypes were observed specifically in the oocyte. There are two reasons why Histone H2A phosphorylation persists in the mutant nurse and follicle cells and mutant defects were not observed. First, Histone H2A may be phosphorylated by another kinase in the nurse and follicle cells. Second, NHK-1 may phosphorylate Histone H2A in all cell types, but the oocyte may require higher levels of kinase activity for phosphorylation. The levels of Histone H2AT119ph may be reduced in the mutant nurse and follicle cells, but the concentration on these polyploid chromosomes could still permit detection by the antibody. The second idea is favored because partial loss-of-function alleles of essential genes often lead to female sterility. The observation that the nhk-1^Z3-0437/Df(3R)Tl-I allelic combination has a stronger mutant phenotype than the nhk-1^{Z3-0437/Z3-0437} allelic combination is consistent with nhk-1^Z3-0437 being a partial loss-of-function allele. Stronger alleles of nhk-1 may, therefore, reveal additional requirements for this kinase in other cell types, including in male meiosis (Ivanovska, 2005).

In conclusion, by using a female-sterile Drosophila mutant, a key step was uncovered in the formation of the karyosome, a conserved structure with an elusive function. Based on the analysis of the mutant phenotype, the following model for karyosome formation is proposed: Following DSB repair and dephosphorylation of Histone H2AvS139, phosphorylation of Histone H2AT119 by NHK-1 leads to disassembly of the SC, loading of condensin onto the chromosomes, and subsequent condensation into a karyosome. In addition to providing insight into karyosome formation, the nhk-1^Z3-0437 mutant is an excellent tool for elucidating the interactions of a specific histone modification within the histone code (Ivanovska, 2005).

Origination of an X-linked testes chimeric gene by illegitimate recombination in Drosophila

The formation of chimeric gene structures provides important routes by which novel proteins and functions are introduced into genomes. Signatures of these events have been identified in organisms from wide phylogenic distributions. However, the ability to characterize the early phases of these evolutionary processes has been difficult due to the ancient age of the genes or to the limitations of strictly computational approaches. While examples involving retrotransposition exist, understanding of chimeric genes originating via illegitimate recombination is limited to speculations based on ancient genes or transfection experiments. This study reports a case of a young chimeric gene that has originated by illegitimate recombination in Drosophila. This gene was created within the last 2-3 million years, prior to the speciation of Drosophila simulans, Drosophila sechellia, and Drosophila mauritiana. The duplication, which involved the Ballchen gene on Chromosome 3R, was partial, removing substantial 3' coding sequence. Subsequent to the duplication onto the X chromosome, intergenic sequence was recruited into the protein-coding region creating a chimeric peptide with approximately 33 new amino acid residues. In addition, a novel intron-containing 5' UTR and novel 3' UTR evolved. That this new X-linked gene has evolved testes-specific expression. Following speciation of the D. simulans complex, this novel gene evolved lineage-specifically with evidence for positive selection acting along the a branch (Arguello, 2006).

The conserved kinase NHK-1 is essential for mitotic progression and unifying acentrosomal meiotic spindles in Drosophila melanogaster

Conventional centrosomes are absent from the spindle in female meiosis in many species, but it is not clear how multiple chromosomes form one shared bipolar spindle without centrosomes. This study identified a female sterile mutant in which each bivalent chromosome often forms a separate bipolar metaphase I spindle. Unlike wild type, prophase I chromosomes fail to form a single compact structure within the oocyte nucleus, although the integrity of metaphase I chromosomes appears to be normal. Molecular analysis indicates that the mutant is defective in the conserved kinase nucleosomal histone kinase-1 (NHK-1). Isolation of further alleles and RNA interference in S2 cells demonstrated that NHK-1 is also required for mitotic progression. NHK-1 itself is phosphorylated in mitosis and female meiosis, suggesting that this kinase is part of the regulatory system coordinating progression of mitosis and meiosis (Cullen, 2005).

Nucleosomal histone kinase-1 phosphorylates H2A Thr 119 during mitosis in the early Drosophila embryo

Posttranslational histone modifications are important for the regulation of many biological phenomena. This study shows the purification and characterization of nucleosomal histone kinase-1 (NHK-1). NHK-1 has a high affinity for chromatin and phosphorylates a novel site, Thr 119, at the C terminus of H2A. Notably, NHK-1 specifically phosphorylates nucleosomal H2A, but not free H2A in solution. In Drosophila embryos, phosphorylated H2A Thr 119 is found in chromatin, but not in the soluble core histone pool. Immunostaining of NHK-1 revealed that it goes to chromatin during mitosis and is excluded from chromatin during S phase. Consistent with the shuttling of NHK-1 between chromatin and cytoplasm, H2A Thr 119 is phosphorylated during mitosis but not in S phase. These studies reveal that NHK-1-catalyzed phosphorylation of a conserved serine/threonine residue in H2A is a new component of the histone code that might be related to cell cycle progression (Aihara, 2004).

Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation

Sox2 is a pluripotency transcription factor that as an oncogene can also regulate cell proliferation. Therefore, genes implicated in several different aspects of cell proliferation, such as the VRK1 chromatin-kinase, are candidates to be targets of Sox2. Sox2 and VRK1 colocalize in nuclei of proliferating cells forming a stable complex. Sox2 knockdown abrogates VRK1 gene expression. Depletion of either Sox2 or VRK1 caused a reduction of cell proliferation. Sox2 up-regulates VRK1 expression and both proteins cooperate in the activation of CCND1. The accumulation of VRK1 protein downregulates SOX2 expression and both proteins are lost in terminally differentiated cells. Induction of neural differentiation with retinoic acid resulted in downregulation of Sox2 and VRK1 that inversely correlated with the expression of differentiation markers such as N-cadherin, Pax6, mH2A1.2 and mH2A2. Differentiation-associated macro histones mH2A1.2 and mH2A2 inhibit CCND1 and VRK1 expression and also block the activation of the VRK1 promoter by Sox2. VRK1 is a downstream target of Sox2 and both form an autoregulatory loop in epithelial cell differentiation (Moura, 2016).

VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage

NBS1 (see Drosophila Nbs) is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. This work examined whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (-/-) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (-/-) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (-/-) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. It is concluded that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR (Monsalve, 2016).

Histone H2A T120 Phosphorylation Promotes Oncogenic Transformation via Upregulation of Cyclin D1

How deregulation of chromatin modifiers causes malignancies is of general interest. This study shows that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrates that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation (Aihara, 2016).

The role of acquired epigenetic changes in cancer development has been a focus of interest in recent years. Alterations of histone-modifying enzymes have been described as playing a role in carcinogenesis. However, it is difficult to prove the hypothesis that histone modifications alone are oncogenic, because modifications such as acetylation, methylation, phosphorylation, and ubiquitylation are dynamic, and many histone modifiers have substrates other than histones. The finding that inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma (Khuong-Quang, 2012; Lewis, 2013) is one piece of evidence that histone modification is involved in carcinogenesis. The finding that histone phosphorylation and its modification crosstalk cause transformation provides further support for the role of histone modification in cancer development (Aihara, 2016).

VRK1 has been shown to phosphorylate CREB, leading to its recruitment to the CRE of CCND1 to activate its expression (Kang, 2008). The finding that VRK1 phosphorylates histone H2A T120 in the promoter region of CCND1 (Cyclin D) and activates its transcription suggests that expression of cyclin D1 is regulated by dual mechanisms involving phosphorylation of both CREB and H2A T120 in the CCND1 promoter region, resulting in its activated transcription. This observation, together with previous reports, suggests that dual substrates of VRK1, CREB, and H2A T120 play a role in cell proliferation and carcinogenesis by their phosphorylation (Aihara, 2016).

VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage

All types of DNA damage cause a local alteration and relaxation of chromatin structure. Sensing and reacting to this initial chromatin alteration is a necessary trigger for any type of DNA damage response (DDR). In this context, chromatin kinases are likely candidates to participate in detection and reaction to a locally altered chromatin as a consequence of DNA damage and, thus, initiate the appropriate cellular response. This work demonstrates that VRK1 is a nucleosomal chromatin kinase and that its depletion causes loss of histones H3 and H4 acetylation, which are required for chromatin relaxation, both in basal conditions and after DNA damage, independently of ATM. Moreover, VRK1 directly and stably interacts with histones H2AX and H3 in basal conditions. In response to DNA damage induced by ionizing radiation, histone H2AX is phosphorylated in Ser139 by VRK1. The phosphorylation of H2AX and the formation of gammaH2AX foci induced by ionizing radiation (IR), are prevented by VRK1 depletion and are rescued by kinase-active, but not kinase-dead, VRK1. In conclusion, This study found that VRK1 is a novel chromatin component that reacts to its alterations and participates very early in DDR, functioning by itself or in cooperation with ATM (Salzano, 2015).

VRK1 interacts with p53 forming a basal complex that is activated by UV-induced DNA damage

DNA damage immediate cellular response requires the activation of p53 by kinases. This study found that p53 forms a basal stable complex with VRK1, a Ser-Thr kinase that responds to UV-induced DNA damage by specifically phosphorylating p53. This interaction takes place through the p53 DNA binding domain, and frequent DNA-contact mutants of p53, such as R273H, R248H or R280K, do not disrupt the complex. UV-induced DNA damage activates VRK1, and is accompanied by phosphorylation of p53 at Thr-18 before it accumulates. It is proposed that the VRK1-p53 basal complex is an early-warning system for immediate cellular responses to DNA damage (Lopez-Sanchez, 2014).

VRK1 phosphorylates CREB and mediates CCND1 expression

Vaccinia virus B1 kinase plays a key role in viral DNA replication. The homologous mammalian vaccinia-related kinases (VRKs) are also implicated in the regulation of DNA replication, although direct evidence remains elusive. This study shows that VRK1 regulates cell cycle progression in the DNA replication period by inducing cyclin D1 (CCND1) expression. Furthermore, depletion of VRK1 in human cancer cells reduces the fraction of cells in S phase at a given time. VRK1 specifically enhances activity of the cAMP-response element (CRE) in the CCND1 promoter by facilitating the recruitment of phospho-CREB to this locus. VRK1 phosphorylates CREB at Ser133 in vitro and the expression of a kinase-dead mutant of VRK1 or knockdown of VRK1 using siRNA fails to activate CREB and subsequently activate CRE. Finally, this study shows that VRK1 is a critical link in the CCND1 gene expression pathway stimulated by Myc overexpression. These results indicate that VRK1 is a novel regulator of CCND1 expression (Kang, 2008).

Search PubMed for articles about Drosophila Ballchen

Aihara, H., Nakagawa, T., Yasui, K., Ohta, T., Hirose, S., Dhomae, N., Takio, K., Kaneko, M., Takeshima, Y., Muramatsu, M. and Ito, T. (2004). Nucleosomal histone kinase-1 phosphorylates H2A Thr 119 during mitosis in the early Drosophila embryo. Genes Dev 18(8): 877-888. PubMed ID: 15078818

Aihara, H., Nakagawa, T., Mizusaki, H., Yoneda, M., Kato, M., Doiguchi, M., Imamura, Y., Higashi, M., Ikura, T., Hayashi, T., Kodama, Y., Oki, M., Nakayama, T., Cheung, E., Aburatani, H., Takayama, K. I., Koseki, H., Inoue, S., Takeshima, Y. and Ito, T. (2016). Histone H2A T120 phosphorylation promotes oncogenic transformation via upregulation of Cyclin D1. Mol Cell 64(1): 176-188. PubMed ID: 27716482

Arguello, J. R., Chen, Y., Yang, S., Wang, W. and Long, M. (2006). Origination of an X-linked testes chimeric gene by illegitimate recombination in Drosophila. PLoS Genet 2(5): e77. PubMed ID: 16715176

Asencio, C., Davidson, I. F., Santarella-Mellwig, R., Ly-Hartig, T. B., Mall, M., Wallenfang, M. R., Mattaj, I. W. and Gorjanacz, M. (2012). Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis. Cell 150(1): 122-135. PubMed ID: 22770216

Brittle, A. L., Nanba, Y., Ito, T. and Ohkura, H. (2007). Concerted action of Aurora B, Polo and NHK-1 kinases in centromere-specific histone 2A phosphorylation. Exp Cell Res 313(13): 2780-2785. PubMed ID: 17586492

Cullen, C. F., Brittle, A. L., Ito, T. and Ohkura, H. (2005). The conserved kinase NHK-1 is essential for mitotic progression and unifying acentrosomal meiotic spindles in Drosophila melanogaster. J Cell Biol 171(4): 593-602. PubMed ID: 16301329

Gonzaga-Jauregui, C., Lotze, T., Jamal, L., Penney, S., Campbell, I. M., Pehlivan, D., Hunter, J. V., Woodbury, S. L., Raymond, G., Adesina, A. M., Jhangiani, S. N., Reid, J. G., Muzny, D. M., Boerwinkle, E., Lupski, J. R., Gibbs, R. A. and Wiszniewski, W. (2013). Mutations in VRK1 associated with complex motor and sensory axonal neuropathy plus microcephaly. JAMA Neurol 70(12): 1491-1498. PubMed ID: 24126608

Herzig, B., Yakulov, T. A., Klinge, K., Gunesdogan, U., Jackle, H. and Herzig, A. (2014). Bällchen is required for self-renewal of germline stem cells in Drosophila melanogaster. Biol Open 29;3(6):510-21. PubMed ID: 24876388

Ivanovska, I., Khandan, T., Ito, T. and Orr-Weaver, T. L. (2005). A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes. Genes Dev 19(21): 2571-2582. PubMed ID: 16230526

Kang, T. H., Park, D. Y., Kim, W. and Kim, K. T. (2008). VRK1 phosphorylates CREB and mediates CCND1 expression. J Cell Sci 121(Pt 18): 3035-3041. PubMed ID: 18713830

Katzemich, A., West, R. J., Fukuzawa, A., Sweeney, S. T., Gautel, M., Sparrow, J. and Bullard, B. (2015). Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle. J Cell Sci 128(18): 3386-3397. PubMed ID: 26251439

Lancaster, O. M., Cullen, C. F. and Ohkura, H. (2007). NHK-1 phosphorylates BAF to allow karyosome formation in the Drosophila oocyte nucleus. J Cell Biol 179(5): 817-824. PubMed ID: 18039935

Lancaster, O. M., Breuer, M., Cullen, C. F., Ito, T. and Ohkura, H. (2010). The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila. PLoS Genet 6(10): e1001179. PubMed ID: 21060809

Link, N., Chung, H., Jolly, A., Withers, M., Tepe, B., Arenkiel, B. R., Shah, P. S., Krogan, N. J., Aydin, H., Geckinli, B. B., Tos, T., Isikay, S., Tuysuz, B., Mochida, G. H., Thomas, A. X., Clark, R. D., Mirzaa, G. M., Lupski, J. R. and Bellen, H. J. (2019). Mutations in ANKLE2, a ZIKA virus target, disrupt an asymmetric cell division pathway in Drosophila neuroblasts to cause microcephaly. Dev Cell. PubMed ID: 31735666

Lopez-Sanchez, I., Valbuena, A., Vazquez-Cedeira, M., Khadake, J., Sanz-Garcia, M., Carrillo-Jimenez, A. and Lazo, P. A. (2014). VRK1 interacts with p53 forming a basal complex that is activated by UV-induced DNA damage. FEBS Lett 588(5): 692-700. PubMed ID: 24492002

Monsalve, D. M., Campillo-Marcos, I., Salzano, M., Sanz-Garcia, M., Cantarero, L. and Lazo, P. A. (2016). VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage. Biochim Biophys Acta 1863(4): 760-769. PubMed ID: 26869104

Moura, D. S., Fernandez, I. F., Marin-Royo, G., Lopez-Sanchez, I., Martin-Doncel, E., Vega, F. M. and Lazo, P. A. (2016). Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation. Sci Rep 6: 28532. PubMed ID: 27334688

Nikalayevich, E. and Ohkura, H. (2015). The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes. J Cell Sci 128(3): 566-575. PubMed ID: 25501812

Renbaum, P., Kellerman, E., Jaron, R., Geiger, D., Segel, R., Lee, M., King, M. C. and Levy-Lahad, E. (2009). Spinal muscular atrophy with pontocerebellar hypoplasia is caused by a mutation in the VRK1 gene. Am J Hum Genet 85(2): 281-289. PubMed ID: 19646678

Salzano, M., Sanz-Garcia, M., Monsalve, D. M., Moura, D. S. and Lazo, P. A. (2015). VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage. Epigenetics 10(5): 373-383. PubMed ID: 25923214

Shah, P. S., Link, N., Jang, G. M., Sharp, P. P., Zhu, T., Swaney, D. L., Johnson, J. R., Von Dollen, J., Ramage, H. R., Satkamp, L., Newton, B., Huttenhain, R., Petit, M. J., Baum, T., Everitt, A., Laufman, O., Tassetto, M., Shales, M., Stevenson, E., Iglesias, G. N., Shokat, L., Tripathi, S., Balasubramaniam, V., Webb, L. G., Aguirre, S., Willsey, A. J., Garcia-Sastre, A., Pollard, K. S., Cherry, S., Gamarnik, A. V., Marazzi, I., Taunton, J., Fernandez-Sesma, A., Bellen, H. J., Andino, R. and Krogan, N. J. (2018). Comparative flavivirus-host protein interaction mapping reveals mechanisms of Dengue and Zika virus Pathogenesis. Cell 175(7): 1931-1945 e1918. PubMed ID: 33168584

Yakulov, T., Gunesdogan, U., Jackle, H. and Herzig, A. (2014). Ballchen participates in proliferation control and prevents the differentiation of Drosophila melanogaster neuronal stem cells. Biol Open 3(10): 881-886. PubMed ID: 25190057

Yamamoto, S., Jaiswal, M., Charng, W. L., Gambin, T., Karaca, E., Mirzaa, G., Wiszniewski, W., Sandoval, H., Haelterman, N. A., Xiong, B., Zhang, K., Bayat, V., David, G., Li, T., Chen, K., Gala, U., Harel, T., Pehlivan, D., Penney, S., Vissers, L., de Ligt, J., Jhangiani, S. N., Xie, Y., Tsang, S. H., Parman, Y., Sivaci, M., Battaloglu, E., Muzny, D., Wan, Y. W., Liu, Z., Lin-Moore, A. T., Clark, R. D., Curry, C. J., Link, N., Schulze, K. L., Boerwinkle, E., Dobyns, W. B., Allikmets, R., Gibbs, R. A., Chen, R., Lupski, J. R., Wangler, M. F. and Bellen, H. J. (2014). A Drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases. Cell 159(1): 200-214. PubMed ID: 25259927

date revised: 12 January 2022

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.