BIOLOGICAL OVERVIEW

Nuclear lamins belong to the intermediate filament (IF) superfamily of proteins that includes type I and II IFs called keratins (a component of wool fibers), type III IF (such a vimentin, desmin and peripherin), type IV IF (expressed in axons, dendrites and perikarya), and type V IF, proteins making up nuclear lamina (Fuchs, 1994). Drosophila Lamin is one of two nuclear lamins. Lamin is expressed constitutively, in contrast to the second Drosophila lamin, Lamin C, which is developmentally regulated (Riemer, 1995). Lamins are the major structural proteins of the nuclear lamina, a structure that lines the nucleoplasmic surface of the inner nuclear membrane in higher eukaryotic cells. The nuclear lamina is composed of a meshwork of 10 nm filaments that are thought to provide a skeletal support for the nuclear envelope and to mediate the attachment of the nuclear envelope to interphase chromatin. Additional functions of the nuclear lamina may include the proper organization and anchoring of nuclear pore complexes. During mitosis the lamins also play a crucial role in the disassembly and reassembly of the nuclear envelope (Lenz-Bohme, 1997 and references).

Before describing the biological properties of Drosophila Lamin, the general properties of the IF superfamily members will be described. IF proteins are all predicted to share a common secondary structure. All IF proteins have a central alpha-helical domain, the rod, which is flanked by nonhelical head (amino-end) and tail (carboxy-end) domains. The rods of two polypeptide chains intertwine in a coiled-coil fashion. Throughout the alpha-helical sequences are repeats of hydrophobic amino acids, such that the first and fourth repeats of every seven residues are frequently apolar. This provides a hydrophobic seal on the helical surface, enabling the coiling between two IF polypeptides. The IF alpha-helical rod is subdivided by three short nonhelical linker segments, which often contain proline or multiple glycine residues (Fuchs, 1994).

For all IFs, the first step in assembly is the formation of parallel, in-register dimers. Upon assembly, lamin dimers align in a head-to-tail fashion to form linear polymers. Most IF proteins can form functional homodimers. Each 10-nm IF filament is composed of smaller protofibrils. IFs appear to have approximately four protofibrils per unit width. It is thought that the conserved amino ends of IF proteins play an important role in assembly of the 10-Nm filament structure. One role of IF tails may be to control lateral associations at the protofilament and protofibril level, thereby influencing filament diameter. In addition, the IF head might promote lateral associations of protofilaments and protofibrils. It is known that headless lamins cannot form a linear array of dimers typical of tail-less and wild-type lamins, suggesting that the lamin head might function in tetramer elongation. Phosphorylation is known to negatively regulate IF assembly. It is thought that the archetypal IF gene had a laminlike structure (Fuchs, 1994 and references).

Lamin Dm0, the precursor form of Lamin, has an apparent molecular mass of 76 kDa and is rapidly processed proteolytically in the cytoplasm into a form migrating at 74 kDa (Lamin Dm1). Lamin Dm1 is imported into the nucleus, where about 50% is posttranslationally modified into a slower migrating form (75 kD) called Lamin Dm2. In vivo pulse-chase studies indicated that lamins Dm1 and Dm2 are in equilibrium. Treatment of lamins Dm1 and Dm2 with phosphatase results in a single form that comigrates with Lamin Dm1 (Smith, 1987 and Smith, 1989). Lamins Dm1 and Dm2 are present as a random mixture of homo- and heterodimers. It is thought that serine 25 is the Lamin Dm2-specific phosphorylation site (Stuurman, 1995).

Lamin is known to interact directly with highly conserved sequences of DNA. Lamin binds with high affinity to scaffold/matrix-associated regions (M/SARs). These DNA sequences are held responsible for mediating the interaction between the nuclear matrix and chromatin. M/SARs are several hundred base pairs long and contain stretches of AT-rich sequences that are likely to form an open chromatin configuration. Indeed, the binding of M/SARs to lamin polymers involves single-stranded regions. In addition, this binding is saturable and requires the minor groove. Lamin polymers also bind to Drosophila centromeric and telomeric sequences. The polymerized alpha-helical rod domain of Lamin, on its own, provides for specific binding to the fushi tarazu M/SAR (Zhao, 1996). The ftz M/SAR functions as an autonomously replicating sequence (ARS) in the budding yeast S. cerevisiae. This M/SAR is found in a 2.57 kb ftz upstream regulatory element. A 189 base pair minimal fragment has ARS function. However, based on growth rates and mitotic stability, its activity is lower than that of the entire SAR. The addition of flanking sequences, including as little as 100 bp of AT-rich DNA to the left of the minimal sequence, can enhance the replicative ability of the ARS. These results implicate lamins in initiation of DNA replication (Amati, 1990 a and b)

Several proteins are associated with the nuclear lamina, and specifically with Lamin proteins. The interaction of lamins with the inner nuclear membrane may be supported by integral membrane proteins, e.g., the putative lamin receptor p54 (Bailer, 1991) or LAPs, the lamina-associate proteins (Foisner, 1993).

One protein associated with Drosophila Lamin is Otefin. Otefin is the corruption and transliteration of the Hebrew word "otef," meaning envelope. Otefin is a peripheral protein of the inner nuclear membrane in Drosophila. During nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. Otefin colocalizes with Lamin derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. It is suggested that Otefin plays a role in the assembly of the Drosophila nuclear envelope (Ashery-Padan, 1997b).

The fs(1)Ya protein (Ya stands for "young arrest") is an essential, maternally encoded, nuclear lamina protein that is under both developmental and cell cycle control. A strong Ya mutation results in early arrest of embryos. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that Ya function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Ya might bind to chromatin and organize the chromatin structure in early embryos in a way that permits DNA replication. Whether Ya functions in conjunction with lamin is unknown (Liu, 1995).

A Drosophila Lamin mutant shows a severe phenotype: this includes retardation in development, reduced viability, sterility, and impaired locomotion. Mutant adult flies die within 2 weeks after eclosion. Late stages of oogenesis are rarely detected in mutant ovaries, and the egg chambers present show an abnormal morphology. In heads from homozygous mutant flies, Lamin is significantly decreased in nuclei of the densely packed cell bodies of the central nervous system. Reduced Lamin expression causes an enrichment of nuclear pore complexes in cytoplasmic annulate lamellae and in nuclear envelope clusters. Annulate lamellae are stacked sheets of membranes in the cytoplasm that contain pore complexes in high densities and are often continuous with rough endoplasmic reticulum in several cells, particularly the densely packed somata of the central nervous system. Defective nuclear envelopes are also observed. The lack of full lethality at early developmental stages may be due to maternal transmission from heterozygous mothers. Indeed, Lamin protein is highly enriched inside the oocyte nucleus, which may serve as a storage compartment for lamin required during the early nuclear divisions in the embryo. These data constitute the first genetic proof that lamins are essential for the structural organization of the cell nucleus (Lenz-Bohme, 1997).

The B-type lamin is required for somatic repression of testis-specific gene clusters

Large clusters of coexpressed tissue-specific genes are abundant on chromosomes of diverse species. The genes coordinately misexpressed in diverse diseases are also found in similar clusters, suggesting that evolutionarily conserved mechanisms regulate expression of large multigenic regions both in normal development and in its pathological disruptions. Studies on individual loci suggest that silent clusters of coregulated genes are embedded in repressed chromatin domains, often localized to the nuclear periphery. To test this model at the genome-wide scale, transcriptional regulation of large testis-specific gene clusters was studied in somatic tissues of Drosophila. These gene clusters showed a drastic paucity of known expressed transgene insertions, indicating that they indeed are embedded in repressed chromatin. Bioinformatics analysis suggested the major role for the B-type lamin, LamDm(o), in repression of large testis-specific gene clusters, showing that in somatic cells as many as three-quarters of these clusters interact with LamDm(o). Ablation of LamDm(o) by using mutants and RNAi led to detachment of testis-specific clusters from nuclear envelope and to their selective transcriptional up-regulation in somatic cells, thus providing the first direct evidence for involvement of the B-type lamin in tissue-specific gene repression. Finally, it was found that transcriptional activation of the lamina-bound testis-specific gene cluster in male germ line is coupled with its translocation away from the nuclear envelope. These studies, which directly link nuclear architecture with coordinated regulation of tissue-specific genes, advance understanding of the mechanisms underlying both normal cell differentiation and developmental disorders caused by lesions in the B-type lamins and interacting proteins (Shevelyov, 2009).

It was hypothesized that, in addition to the somatic silencing of testis-specific gene clusters, LamDm₀ is also required for attachment of these clusters to the nuclear envelope. To test this hypothesis, intranuclear positions of the 60D1 and 22A1 regions was determined in the interphase nuclei of cultured S2 cells in which LamDm₀ was ablated by RNAi. Fluorescence in situ hybridization (FISH) combined with immunostaining for LamDm₀ confirmed RNAi-induced ablation of LamDm₀, and showed approximately 2-fold decrease in frequency of the 60D1 and 22A1 loci bound to lamina. Next, association of the 60D1 gene cluster with nuclear envelope wa analyzed during male germ-line differentiation. The whole-mount testes dissected from the third instar larvae were analyzed for intranuclear localization of the 60D1 region by FISH combined with immunostaining for LamDm₀. Morphologically, a group of small cells is located at the end of testis and includes spermatogonia, cyst cells and stem cells, in which the 60D1 locus is silent. In these cells, the 60D1 region is associated with nuclear lamina in 76% of nuclei, similarly to the cultured somatic S2 cells. On the contrary, in spermatocytes (identified by characteristic large nuclei), in which testis-specific genes in the 60D1 region are expressed, this region is found at the nuclear envelope in only 6% of the nuclei. Thus, transcriptional activation of the testis-specific gene cluster in male germ line is coupled to its dissociation from the nuclear envelope. Similarly, detachment from the nuclear lamina has been associated with transcriptional activation of other lamina-bound loci both in Drosophila and in mammals. These observations strongly suggest a model in which gene repression is controlled in a cell type-specific manner through regulated tethering of chromatin to the nuclear lamina. Further dissection of the mechanisms that mediate repression of lamina-bound multigenic regions and control localization of these regions at nuclear envelope will provide new insights into coordinated regulation of tissue-specific genes, thus advancing understanding of cell differentiation both in normal development and in disease (Shevelyov, 2009).

Role of histone deacetylases in gene regulation at nuclear lamina

Theoretical models suggest that gene silencing at the nuclear periphery may involve 'closing' of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). This study provides experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila S2 cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. The data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development (Milon, 2012).

This study provides direct experimental evidence for the long-persisting assumptions that HDACs are involved in gene silencing at the nuclear lamina, by identifying Class I enzymes HDAC1 and HDAC3 as the major players in this mechanism. Likewise gene silencing, histone hypoacetylation and chromatin compaction in the multigenic chromatin domain are lamin-dependent. Moreover, HDAC1 was identified as the key factor required for silencing and specifically responsible for histone H4 deacetylation, and the data implicate HDAC3 as an auxiliary factor specifically responsible for localization of the repressed chromatin at the lamina. The 'closed' chromatin configuration of the repressed domain also depends on HDAC1 and thus probably mediates the major repressive action of this enzyme at the nuclear periphery. Published data indicate that the 60D1 cluster interacts with HDAC1, in particular in the Crtp and Pros28.1B regions, supporting direct involvement of this enzyme in histone deacetylation. A model is proposed in which Class I HDACs participate in lamina-dependent gene silencing through diverse pathways: HDAC1, tethered to the lamin scaffold by LEM domain proteins, is involved in deacetylation of histones H3 and H4 and 'closing' of lamina-bound chromatin while HDAC3 contributes to histone H3 deacetylation and retention of the repressed chromatin at the lamina. Interestingly, a recent study showed that HDAC3 is also involved in peripheral localization of the lamina-interacting chromatin in mammals (Zullo, 2012) indicating that this mechanism is conserved between diverse animals (Milon, 2012).

Lamina-associated chromatin domains harbor numerous cell type-specific genes that must be precisely regulated to orchestrate cell differentiation and development. Genetic defects in the lamina components result in severe and currently incurable tissue degenerative disorders known as laminopathies. Identification of the key role of Class I HDACs, and particularly HDAC1, in lamina-associated gene silencing implies that modulation of this enzyme may help to restore gene expression disrupted by nuclear lamina defects, and may be instrumental in establishing new expression patterns in pluripotent cells to guide their differentiation (Milon, 2012).

Age-associated loss of lamin-B leads to systemic inflammation and gut hyperplasia

Aging of immune organs (see Drosophila as a Model for Human Diseases: Aging and Lifespan), termed as immunosenescence, is suspected to promote systemic inflammation and age-associated disease. The cause of immunosenescence and how it promotes disease, however, has remained unclear. This study reports that the Drosophila fat body, a major immune organ, undergoes immunosenescence and mounts strong systemic inflammation that leads to deregulation of immune deficiency (IMD) signaling in the midgut of old animals. Inflamed old fat bodies secrete circulating peptidoglycan recognition proteins that repress IMD activity in the midgut, thereby promoting gut hyperplasia. Further, fat body immunosenecence is caused by age-associated lamin-B reduction specifically in fat body cells, which then contributes to heterochromatin loss and derepression of genes involved in immune responses. As lamin-associated heterochromatin domains are enriched for genes involved in immune response in both Drosophila and mammalian cells, these findings may provide insights into the cause and consequence of immunosenescence during mammalian aging (Chen, 2014).

By analyzing gene expression changes upon aging in fat bodies and midguts, it was shown that an increase of immune response in the fat body is accompanied by a striking reduction in the midgut. Specifically, it was demonstrate that the age-associated increase in Immune deficiency (IMD) signaling in fat bodies leads to reduction of IMD activity in the midgut, which in turn contributes to midgut hyperplasia. This fat body to midgut effect requires peptidoglycan recognition proteins (PGRPs) secreted from fat body cells and is mediated by both bacteria dependent and independent pathways. Therefore, fat body aging contributes to systemic inflammation, which contributes to the disruption of gut homeostasis. Importantly, it was shown that the age-associated lamin-B loss in fat body cells causes the derepression of a large number of immune responsive genes, thereby resulting in fat body-based systemic inflammation (Chen, 2014).

B-type lamins have long been suggested to have a role in maintaining heterochromatin and gene repression. Consistently, this study's global analyses of fat body depleted of lamin-B revealed a loss of heterochromatin and derepression of a large number of immune responsive genes. This is further supported by ChIP-qPCR analyses of H3K9me3 on specific IMD regulators. Recent studies in different cell types show that tethering genes to nuclear lamins do not always lead to their repression. Deleting B-type lamins or all lamins in mouse ES cells or trophectdoderm cells does not result in derepression of all genes in LADs. In light of these studies, it is suggested that the transcriptional repression function of lamin-B could be gene and cell type dependent. Interestingly, GO analyses revealed a significant enrichment of immune responsive genes in Lamin-associated domains (LADs) in four different mammalian cell types and Drosophila Kc cells. Since the large-scale pattern of LADs is conserved in different cell types in mammals, it is possible that the immune-responsive genes are also enriched in LADs in the fly fat body cells. Supporting this notion, the IKKγ, key, which is one of the two derepressed IMD regulators and was found to exhibit H3K9me3 reduction and gene activation, is localized to LADs in Kc cells. It is speculated that lamin-B might play an evolutionarily conserved role in repressing a subset of inflammatory genes in certain tissues, such as the immune organs, in the absence of infection or injury. Consistently, senescence-associated lamin-B1 loss in mammalian fibroblasts is correlated with senescence-associated secretory phenotype senescence-associated secretory phenotype (SASP). Although the in vivo relevance of fibroblast SASP in chronic inflammation and aging-associated diseases in mammals remains to be established, the findings in Drosophila provide insights and impetus to investigate the role of lamins in immunosenescence and systemic inflammation in mammals (Chen, 2014).

Lamin-B gradually decreases in fat body cells of aging flies, whereas lamin-C amount remains the same. Since it has been recently shown that the assembly of an even and dense nuclear lamina is dependent on the total lamin concentration, the age-associated appearance of lamin-B and lamin-C gaps around the nuclear periphery of fat body cells is likely caused by the drop of the lamin-B level. How aging triggers lamin-B loss is unknown, but it appears to be posttranscriptional, because lamin-B transcripts in fat bodies remain unchanged upon aging. Interestingly, among the tissues examined, no changes of lamin-B and lamin-C proteins were found in cells in the heart tube, oenocytes, or gut epithelia in old flies. Therefore, the age-associated lamin-B loss does not occur in all cell types in vivo. A systematic survey to establish the cell/tissue types that undergo age-associated reduction of lamins in both flies and mammals should provide clues to the cause of loss. Deciphering how advanced age leads to lamin loss should open the door to further investigate the cellular mechanism that contributes to chronic systemic inflammation and how it in turn promotes age-associated diseases in humans (Chen, 2014).

Old Drosophila gut is known to exhibit increased microbial load, which would cause increased stress response and activation of tissue repair, thereby leading to midgut hyperplasia. Systemic inflammation caused by lamin-B loss in fat body leads to repression of local midgut IMD signaling. The upregulation of targets of IMD in the aged whole gut has been recently reported, while a downregulation of target genes was observed in the current analyses of the midgut. However, the previous study found a similar upregulation of the genes when performing RNA-seq of the whole gut (Chen, 2014).

These studies reveal an involvement of bacteria in the repression of midgut IMD signaling by the PGRPs secreted from the fat body. How PGRPs from the fat body repress midgut IMD is still unknown. One possibility is that the body cavity bacteria contribute to the maintenance of midgut IMD activity, and the increased circulating PGRPs limit these bacteria. The circulating PGRPs may also reduce midgut IMD activity indirectly by affecting other tissues. The evidence suggests that lamin-B loss could also contribute to midgut hyperplasia independent of the IMD pathway. While it will be important to further address these possibilities, the findings have revealed a fat body mediated inflammatory pathway that can lead to reduced migut IMD, increased gut microbial accumulation, and midgut hyperplasia upon aging (Chen, 2014).

Interestingly, microbiota changes also occur in aging human intestine and have been linked to altered intestinal inflammatory states and diseases. Although, much effort has been devoted to understand how local changes in aging mammalian intestines affect gut microbial community, the cause remains unclear. The findings in Drosophila reveal the importance of understanding the impact of immunosenescence and systemic inflammation on gut microbial homeostasis. Indeed, if increased circulating inflammatory cytokines perturb the ability of local intestine epithelium and the gut-associated lymphoid tissue to maintain a balanced microbial community, the unfavorable microbiota in the old intestine would cause chronic stress response and tissue repair, thereby leading to uncontrolled cell growth as observed in age-associated cancers (Chen, 2014).

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. This study identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, this study identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, these findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression (Nazer, 2018).

Argonaute proteins correspond to an evolutionarily conserved protein family engaged in gene silencing. The well-studied RNA interference (RNAi) pathway effector protein Argonaute2 (AGO2) interacts with microRNAs (miRNAs) or short interfering RNAs (siRNAs) to regulate post-transcriptional gene silencing in the cytoplasm. In addition, several reports have shown that AGO2 is not restricted to the cytoplasm and can also function in the nucleus. In Drosophila, ChIP-seq analysis of AGO2 revealed association with active promoters, enhancers, and chromatin insulator sites. At the Hox gene Abd-B, AGO2 interacts with insulator proteins and exerts a positive role in gene expression. Consistent with a role in transcription, AGO2 was found to interact with the RNA Polymerase II (Pol II) core complex as well as Negative elongation factor (NELF), a key factor involved in transcriptional pausing. Finally, transcriptional profiling of AGO2 null but not catalytic mutants suggested that AGO2 functions primarily in transcriptional repression. These results suggest that AGO2 may harbor both positive and negative roles in transcriptional regulation (Nazer, 2018).

AGO2 has also been shown to affect chromatin topology and has been implicated in chromosome pairing. At Abd-B, AGO2 is required for proper looping between the promoter and its enhancer, resulting in activation of transcription. Moreover, AGO2 promotes long-range insulator-dependent pairing interactions within the nucleus. In addition to the aforementioned transcriptional profiling, all these studies showed that the function of AGO2 in controlling chromatin topology and transcription occurs independently of the RNAi pathway (Nazer, 2018 and references therein).

Transcription is not only regulated through interactions between promoter and cis-regulatory factors, but also through chromatin accessibility and interactions with distant regulatory elements. High-throughput chromosome conformation capture techniques have revealed that chromatin contacts take place within functional and structural domains termed topologically-associated domains (TADs. These structures consist of highly self-interacting genomic regions within the domain, each separated by adjacent genomic regions called domain partition sites (DPSs). TADs can be sub-classified as active or inactive depending on their protein content and transcriptional activity. A subset of inactive TADs are associated with the nuclear lamina, and these regions are referred to as Lamin-associated domains (LADs). LADs are broad regions defined by their interaction with the LaminB protein (associated with the BLACK chromatin state, and on average, they are 90 kb in length and tend to be gene poor. Less transcriptionally active in nature, LADs are enriched in repressive chromatin marks, such as Polycomb Group (BLUE) and heterochromatin factors (GREEN). Nevertheless, some hallmarks of active chromatin (RED and YELLOW) are also present within LADs (Nazer, 2018).

This study has elucidated genome-wide mechanisms of AGO2 transcriptional control in concert with LaminB. LaminB and Poll II as AGO2-associated factors in nuclear extracts. Nascent euRNA-seq (neuRNA-seq) experiments showed that depletion of AGO2 or LaminB produce highly similar transcriptome profiles, with both factors affecting transcription of genes located in active chromatin both inside and outside LADs. First revealed by transcriptional profiling of AGO2 mutants, it was found that both AGO2 and LaminB prevent transcription of nht, a master regulator of the spermatogenesis gene program, in somatic cells. Finally, 4C-seq analysis showed that AGO2 and LaminB modulate the chromatin topology of the TAD/LAD in which nht is located, thus contributing to transcriptional silencing of this key developmental regulator (Nazer, 2018).

This study has employ proteomics, neuRNA-seq, ChIP-seq, and 4C-seq to elucidate a novel genome-wide relationship between Drosophila AGO2 and LaminB to function in genome organization and thereby affect gene regulation. By examining AGO2 null mutants, focus was placed on a single somatic target of AGO2 repression, nht, which is a key activator of spermatogenesis genes. The nht locus is immersed in a LAD located within a repressive TAD, which is flanked by AGO2 binding sites. Finally, it was found that depletion of either AGO2 or LaminB results in a significant decrease in frequency of interactions within the TAD as well as increases in inter-TAD interactions. It is concluded that AGO2 and LaminB can work in concert to regulate gene expression by orchestrating overall genome organization (Nazer, 2018).

The majority of genes for which transcription is altered in AGO2- or LaminB-knockdowns are normally actively transcribed, but specificity of this effect was observed. In particular, up-regulated genes are enriched in RED compared to YELLOW chromatin, while down-regulated genes show the opposite pattern. Although AGO2 is present in both types of active chromatin, one major difference between these two active chromatin states is the absence of histone H3K36me3, a hallmark of elongation, in RED chromatin. Therefore, depletion of AGO2 may preferentially relieve attenuation of RED versus YELLOW chromatin. Furthermore, YELLOW chromatin generally corresponds to constitutively expressed genes, whereas RED is characteristic of developmentally regulated genes. Surprisingly, it was found that a substantial number of co-up-regulated genes within RED chromatin were distributed equally between LADs and non-LADs. Perhaps these RED domains are located within a subclass of LADs that allow gene expression at a certain level, likely to more efficiently respond to stimulus or developmental signals. Further analysis would be required in order to establish sub-classification of LADs in Drosophila and analyze their potential function, as has been recently performed for the mouse genome. Overall, the presence of RED chromatin within a LAD could correspond to an additional layer of regulation of developmentally controlled gene expression (Nazer, 2018).

Another non-exclusive possibility is that topological constraints imposed by AGO2 differentially affect the two chromatin types. For example, restriction of tissue-specific enhancer-promoter interactions present in RED chromatin might be relaxed by changes in topology while housekeeping genes could become ectopically subject to repression by surrounding chromatin from which it is normally insulated. The results suggest that AGO2 can exert either positive or negative effects on transcription depending on the chromatin context (Nazer, 2018).

While transcription changes observed in AGO2 and LaminB knockdowns genome-wide are most enriched for actively transcribed regions, some normally inactive or potentially active regions including within LADs, are also up-regulated. One such gene that is subject to tight tissue-specific regulation and repressed by AGO2 and LaminB is nht, which normally activates spermatogenesis specifically in primary spermatocytes. Since mRNA-seq profiling of AGO2 mutants was performed on whole female larvae, detection of up-regulation of normally silent genes was favored. Previous work showed that certain spermatogenesis gene clusters are associated with the nuclear periphery in somatic cells and become de-repressed and repositioned toward the interior upon depletion of LaminB. However, no significant detachment of nht from the nuclear periphery was observed upon depletion of AGO2 or LaminB. These results are in agreement with recent work showing that LaminB is not required to a large extent for LAD structure in mES cells. In AGO2- or LaminB-depleted somatic cells, nht partially escapes from repression, although it is not fully activated likely due to its presence in a silent BLACK domain and the absence of other required factors. It is important to note that despite being able to detect higher levels of a variety of spermatogenesis transcripts in AGO2- and LaminB-depleted somatic cells, increased protein levels were not observed. Strict regulation of spermatogenesis gene expression also occurs on the post-transcriptional level and helps ensure no phenotypic consequences when transcription becomes dysregulated (Nazer, 2018).

AGO2 and LaminB control the overall chromatin topology of the LAD and TAD in which nht is located, and these changes could be a key driver of observed transcriptional effects in AGO2- and LaminB-depleted cells. In the case of Abd-B, which is located outside of a LAD, AGO2 functions in concert with the insulator proteins CTCF and CP190 to promote or stabilize looping between the Abd-B promoter and iab-8 enhancer region, thus stimulating transcription. However, AGO2 itself does not associate with the nht promoter, so it is unlikely that AGO2 directly prevents the nht promoter from looping to nearby enhancers. Rather, AGO2 localizes to borders of the LAD/TAD encompassing nht, suggesting that it may play a larger role in constraining topology of the entire region. In fact, 3C and 4C-seq analyses show that interactions between nht and other sites within the TAD decrease in interaction frequency in both AGO2- and LaminB-depleted cells. At the same time, increased interactions are observed between the nht promoter and sequences located in other chromatin domains beyond 1 Mb away. In this structural view, AGO2 appears to function in a manner hypothesized for insulator proteins at TAD borders; however, no single insulator protein flanks this LAD/TAD. Large alterations in topology in AGO2- and LaminB-depleted cells could explain the resultant transcriptional increase of nht by allowing interaction with inappropriate enhancers or otherwise creating a more permissive transcriptional environment. The results are in agreement with recent work performed in mouse showing that the absence of lamins decreases inter-TAD interactions within constitutive LADs and increases inter-TAD interactions between TADs inside LADs and TADs outside LADs. Importantly, this topology remodelling correlates with changes in gene expression, overall suggesting an evolutionary conserved role of lamins to regulate transcription by controlling chromatin topology (Nazer, 2018).

Drosophila Wash and the Wash regulatory complex function in nuclear envelope budding

Nuclear envelope (NE) budding is a recently described phenomenon wherein large macromolecular complexes are packaged inside the nucleus and extruded through the nuclear membranes. Although a general outline of the cellular events occurring during NE budding is now in place, little is yet known about the molecular machinery and mechanisms underlying the physical aspects of NE bud formation. Using a multidisciplinary approach, this study identified Wash, its regulatory complex (SHRC), capping protein and Arp2/3 as new molecular components involved in the physical aspects of NE bud formation in a Drosophila model system. Interestingly, Wash affects NE budding in two ways: indirectly through general nuclear lamina disruption via an SHRC-independent interaction with Lamin B leading to inefficient NE bud formation, and directly by blocking NE bud formation along with its SHRC, capping protein and Arp2/3. In addition to NE budding emerging as an important cellular process, it shares many similarities with herpesvirus nuclear egress mechanisms, suggesting new avenues for exploration in both normal and disease biology (Verboon, 2020).

Transport of macromolecules from the nucleus to the cytoplasm is essential for all developmental processes, including the regulation of differentiation and aging, and, when mis-regulated, is associated with diseases and cancer. This indispensable process has been thought to occur exclusively through nuclear pore complexes (NPCs), channels that regulate what exits (and enters) the nucleus. Recently, nuclear envelope (NE) budding was identified as an alternative pathway for nuclear exit, particularly for large developmentally required ribonucleoprotein (megaRNP) complexes that would otherwise need to unfold/remodel to fit through the NPCs. In this pathway, large macromolecule complexes, such as megaRNPs, are encircled by the nuclear lamina (type-A and -B lamins) and the inner nuclear membrane (INM), pinched off from the INM, fuse with the outer nuclear membrane and release the megaRNPs into the cytoplasm. Strikingly, NE budding shares many features with the nuclear egress mechanism used by herpesviruses. As viruses often utilize pre-existing host pathways, the parallel between nuclear exit of herpesvirus nucleocapsids and that of megaRNPs and/or other large cargoes suggests that NE budding may be a general cellular mechanism. Indeed, this pathway has also been implicated in the removal of obsolete macromolecular complexes or other material (i.e. large protein aggregates or poly-ubiquitylated proteins) from the nucleus (Verboon, 2020).

NE budding was first demonstrated in Drosophila synapse development, proving to be essential for neuromuscular junction (NMJ) integrity. In this context, a C-terminal fragment of the Wingless receptor Fz2, Fz2C, was shown to associate with megaRNPs that formed foci at the nuclear periphery and exited the nucleus by budding through the nuclear envelope (Speese, 2012). Failure of this process resulted in aberrant synapse differentiation and impaired NMJ integrity (Speese, 2012). In a subsequent study, the NE budding pathway was shown to be necessary for the nuclear export of megaRNPs containing mitochondrial RNAs: disruption of NE budding led to deterioration of mitochondrial integrity and premature aging phenotypes that were similar to those associated with lamin mutations (i.e., laminopathies). Similar endogenous perinuclear foci/buds have been observed in plants and vertebrates, as well as other Drosophila tissues (i.e., larval salivary gland nuclei), suggesting that cellular NE budding is a widely conserved process (Verboon, 2020).

The spectrum of processes requiring this non-canonical nuclear exit pathway and the molecular machineries needed for this process, which encompasses membrane deformations, traversal across a membrane bilayer and nuclear envelope remodeling for a return to homeostasis, are largely unknown. One class of proteins that are involved in membrane-cytoskeletal interactions and organization is the Wiskott-Aldrich Syndrome (WAS) protein family. WAS protein subfamilies are involved in a wide variety of essential cellular and developmental processes, as well as in pathogen infection and disease. WAS family proteins polymerize branched actin through the Arp2/3 complex, and often function as downstream effectors of Rho family GTPases. This study identified Wash as a new WAS subfamily that is regulated in a context-dependent manner: Wash can bind directly to Rho1 GTPase (in Drosophila) or it can function along with the multi-protein WASH regulatory complex [SHRC; comprised of SWIP, Strumpellin, FAM21 and CCDC53 (also known as WASHC4, WASHC5, WASHC2 and WASHC3, respectively, in mammals)]. Wash regulation by Rho family GTPases outside of Drosophila has not yet been described; instead its regulation has been characterized in the context of its SHRC. WASH and its SHRC are evolutionarily conserved and their mis-regulation is linked to cancers and neurodegenerative disorders. Importantly, it has been shown that Wash is present in the nucleus where it interacts directly with B-type lamins and, when mutant, affects global nuclear organization/functions, as well as causing an abnormal wrinkled nucleus morphology reminiscent of that observed in diverse laminopathies (Verboon, 2015a and 2015b). Mammalian WASH proteins have also been shown to localize to the nucleus in developmental and cell-type specific manners (Verboon, 2020).

This study shows that Wash, its SHRC, capping protein and Arp2/3 are also involved in the NE budding pathway, as mutants for any of these components lack Fz2C foci/lamin buds and display the NMJ integrity and premature aging phenotypes previously associated with the loss of NE budding. In addition, this study found that CCDC53 and SWIP (SHRC subunits) colocalize with Fz2C foci/lamin buds. Wash is shown to be present in several independent nuclear complexes. The nuclear interactions of Wash with its SHRC are separate from those with B-type Lamin, leading to effects on different subsets of nuclear Wash functions. This study also found that Wash-dependent Arp2/3 actin nucleation activity is required for proper NE budding. It is proposed that Wash and its SHRC play a physical and/or regulatory role in the process of NE budding (Verboon, 2020).

NE budding is an increasingly appreciated pathway for nuclear export of large macromolecular machineries, such as megaRNPs involved in the co-regulation of major developmental pathways or unwanted RNA or protein aggregates. Previous work has shown that Drosophila Wash is present in the nucleus where it is likely involved in a number of different nuclear processes (Verboon, 2015a). This study shows that all members of the four-subunit Wash regulatory complex (SHRC), as well as the heterodimeric capping protein, are also present within the nucleus and, along with Wash, are necessary for NE budding. Wash and SHRC act early in the NE budding pathway, and this process requires actin nucleation activity of Wash achieved through its interaction with Arp2/3. Loss of Wash, any of its SHRC subunits or Arp2/3 leads to the loss of Fz2C foci/NE buds, and mutants for these factors exhibit phenotypes associated with the two cellular processes shown to require NE budding: aberrant synaptic 'ghost' bouton formation leading to disrupted NMJ integrity, and mitochondrial degeneration associated with premature aging phenotypes. This study also shows that the interaction of Wash with Lamin B results in a general disruption of the nuclear lamina and separation of the Lamin B/Lamin C homotypic meshes, leading to inefficient, rather than loss of, NE bud formation (Verboon, 2020).

While the spectrum of processes that require this alternate nuclear egress mechanism is not yet known, SHRC components are linked to neurodegenerative disorders, including hereditary spastic paraplegias, Parkinson disease, amyotrophic lateral sclerosis (ALS) and Hermansky-Pudlak syndrome. As an increasing number of neurodegenerative diseases and myopathies have been associated with the accumulation of RNA-protein aggregates in the nucleus, NE budding may be part of the endogenous cellular pathway for removing such aggregates/megaRNPs from the nucleus in normal cells (Verboon, 2020).

The parallels between the mechanism of NE budding and herpesvirus nuclear egress, as well as the presence of similar endogenous perinuclear foci/buds in other plant and animal nuclei, has suggested that NE budding is a conserved endogenous cellular pathway for nuclear export. Indeed, INM-encapsulated electron-dense granules have been identified in yeast and Torsin-deficient HeLa cells, and these show similarities to the Fz2C foci/NE buds observed in Drosophila muscle and salivary gland nuclei. While the full relationship between NPCs and NE buds is not yet known, one important difference is that the yeast and HeLa nuclear granules observed are much smaller (∼120 nm) than Fz2C foci/NE buds (∼500 nm). This identification of Wash and SHRC, proteins with the capability of remodeling cortical cytoskeleton and/or membranes, in the physical aspects of NE budding lend support for NE budding being an alternative endogenous nuclear exit pathway (Verboon, 2020).

NE budding has been proposed to occur at sites along the INM where the nuclear lamina is modified by aPKC phosphorylation. Both A- and B-type lamins play a role in NE budding and are thought to be the target of aPKC phosphorylation within the nuclear lamina, similar to the PKC-mediated phosphorylation of lamins that precedes lamina disassembly in mitotic NE breakdown (Guttinger, 2009), apoptosis (Cross, 2000) or during viral capsid nuclear egress. Viral NE budding requires a virus-encoded nuclear egress complex (NEC), which has been implicated in the recruitment of kinases to the INM. Cellular counterparts for these virally encoded NEC proteins have not yet been identified. It is also not yet known how this kinase activity is restricted to specific sites along the nuclear lamina or how those specific sites are selected (Verboon, 2020).

Previous work has shown that Wash interacts directly with Lamin B and that loss of nuclear Wash results in a wrinkled nuclear morphology reminiscent of that observed in laminopathies (Verboon, 2015a). It was reasoned that Wash-mediated disruption of the nuclear lamina may account for its NE-budding phenotypes. Consistent with this idea, it was found that Lamin B knockdown nuclei and nuclei from a wash point mutant that disrupts the interaction of Wash with Lamin B (washΔΔLamB) exhibit a wrinkled nuclear morphology, reduced Fz2C foci/NE buds and NE-budding-associated phenotypes, albeit not as strong as those observed in Wash or SHRC mutants (Verboon, 2020).

Lamin A/C and Lamin B isoforms form homotypic meshworks that interact among themselves (in as yet unknown ways), and that are somehow linked to integral membrane proteins of the INM and to the chromatin adjoining the INM. Intriguingly, the current data suggests that these lamin homotypic meshes are likely layered, rather than interwoven, and that Wash affects the anchoring of these lamin homotypic meshes to each other and/or the INM. Lamin knockdown or disruption of the Wash-Lamin B interaction leads to separated lamin isoform meshes and wrinkled nuclear morphology that are not observed in SHRC and Arp2/3 knockdown nuclei, suggesting that Wash can also affect NE budding by a means independent of disrupted global nuclear lamina integrity. Interestingly, the functions of Wash mediated with the SHRC and with Lamin B involve separate nuclear complexes. Consistent with this, it was shown previously that Drosophila Wash encodes several independent biochemical activities (actin nucleation, actin bundling, MT bundling and actin-MT crosslinking) and that the use of these activities is context dependent. In particular, when Wash interacts with Lamin, it does not require an association with SHRC or Arp2/3. It is suggested that the interaction of Wash with Lamin B is required for organizing the nuclear lamina and likely requires the actin bundling and/or cross-linking activities of Wash rather than its actin nucleation activity, such that wash mutants that cannot bind Lamin result in separation of the Lamin isoform meshes from each other. Taken together, the data suggest that loss of the interaction between Wash and Lamin B makes NE budding inefficient by generally disrupting the nuclear lamina, rather than directly disrupting NE bud formation. The role of aPKC in NE budding may also be somewhat indirect by generally disrupting the nuclear lamina thereby reducing the efficiency of NE bud formation. Alternatively, aPKC may target Wash: WASH phosphorylation by Src kinases has been shown to be necessary for regulating NK cell cytotoxicity (Verboon, 2020).

For bud formation/envelopment of a megaRNP or macromolecular cargo to occur, the INM must interact with its underlying cortical nucleoskeleton to allow the INM deformation/curvature necessary to form the physical NE bud. Force must also be generated that allows the bud to extend into the perinuclear space, as well as for the scission of the nascent bud. In the cytoplasm, WAS family proteins are often involved in membrane-cortical cytoskeleton-coupled processes, including both 'inward' membrane deformations (i.e., endocytosis) and 'outward' membrane deformations (i.e., exocytosis and cell protrusions), that are required for signal/environment sensing and cell movement during normal development, as well as during pathological conditions. Mammalian WASH, in particular, has been implicated in endosome biogenesis and/or sorting in the cytoplasm, where it, along with its SHRC, drives Arp2/3-dependent actin assembly to influence endosome trafficking, remodels membrane, and facilitates membrane scission. Thus, Wash encodes the biochemical properties needed to regulate the membrane deformation/curvature necessary to form the NE bud and/or play a role in generating the forces necessary to pinch off the NE bud from the INM. Consistent with Wash playing a role in the physical production of a NE bud, it was found that Wash acts prior to Torsin, a protein that is implicated in NE bud scission from the INM, and it requires its actin nucleation activity (Verboon, 2020).

This study has identified Wash and its SHRC as new players in the cellular machinery required for the newly described endogenous NE budding pathway. The data suggest that Wash is involved in two nuclear functions that can affect NE budding. (1) Wash is required to maintain the organization of Lamin isoforms relative to each other and the INM through its direct physical interaction with Lamin B. This Wash activity is SHRC and Arp2/3 independent, and is likely a non-specific mechanism because global disruption of the nuclear lamina/nuclear envelope would indirectly affect many nuclear processes, including NE budding. (2) Wash is specifically required for NE bud formation. This Wash activity is SHRC and Arp2/3 dependent. While the focus of NE budding research to date has centered on the composition of the megaRNPs and the spectrum of cellular/developmental processes requiring NE budding, Wash and the SHRC are likely involved in the physical aspects of NE budding. Thus, Wash and SHRC provide a molecular entry into the physical machinery that underlies NE budding. In the future, it will be exciting to further explore the roles of Wash in NE budding, and to determine how it functions to get macromolecular complexes through the INM, and how closely these nuclear roles parallel those in the cytoplasm (Verboon, 2020).

GENE STRUCTURE

The Drosophila Lamin gene is developmentally regulated, giving rise to a 2.8 kb maternal transcript and a 3.0 kb zygotic transcript. The different transcripts are generated by utilizing different polyadenylation sites. None of the putative lamin polyadenylation signals contains the consensus AAUAA sequence. The choice between the different polyadenylation signals might depend on maternal fators that more efficiently recognize the polyadenylation signal of the 2.8 kb transcript (Osman, 1990 and Gruenbaum, 1988)

Exons - 4

Bases in 3' UTR - 710 (maternal) and 921 (zygotic)

PROTEIN STRUCTURE

Amino Acids - 621

Structural Domains

Highly specific features of lamins include a nuclear localization signal, a C-terminal CaaX sequence (where C=cysteine; a=aliphatic amino acid; X=any amino acid) and characteristic phosphorylation sites in the N-terminal head and C-terminal tail domains. The nuclear localization signal is responsible for rapid transport of lamins into the nucleus, thus preventing cytoplasmic assembly. Modification by isoprenylation and carboxymethylation at the CaaX motif targets lamins to the inner nuclear membrane (Lenz-Bohme, 1997 and references).

date revised: 10 June 2023 

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