InteractiveFly: GeneBrief

Haspin: Biological Overview | References

Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. This study reports that Haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. The data reveal that Haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. Haspin is a suppressor of position-effect variegation involved in heterochromatin organization. These results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for Haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation (Fresan, 2020).

Drosophila Haspin modulates key aspects of chromatin organization during interphase: insulator activity, heterochromatin-induced position-effect variegation, nuclear morphology and PRE-dependent pairing-sensitive silencing. Some of these aspects could be influenced by mitotic events and, therefore, be regulated by Haspin functionality in chromosome organization during mitosis. However, the results also show that Haspin phosphorylates histone H3 in interphase and associates with the cohesin complex mediating Pds5 binding to chromatin in interphase cells, strongly suggesting that this kinase modulates chromatin organization not only during mitosis but also in interphase (Fresan, 2020).

Thr3 in histone H3 is located immediately adjacent to Lys4, which has been shown to be tri-methylated at active promoter sites. TFIID binding to H3K4me3, which is involved in transcription machinery recruitment, is severely reduced in mitosis as a result of H3T3 phosphorylation. On the other hand, in vitro studies have shown that tri-methylation of H3K4 reduces substrate recognition by Haspin suggesting antagonism between H3K4 methylation and H3T3 phosphorylation (Eswaran, 2009). In agreement with these studies, the current results show that H3T3ph localizes at heterochromatin and lamin-enriched regions, while it does not colocalize with active RNA polymerase II. Even though, future investigations will be needed to decipher whether or not H3T3ph never colocalizes with H3K4me and active transcription, altogether these studies suggest antagonism between phosphorylated H3T3 and active transcription. Moreover, Haspin is preferentially found at the nuclear lamina, like CP190 insulator protein and polycomb proteins. While proteins that are associated with transcriptionally active chromatin are easily solubilized in subcellular fractionation assays, Haspin, insulator and polycomb proteins are tightly bound to the nuclear matrix where they colocalize with laminas suggesting that nuclear organization of these proteins might contribute to their functionality (Fresan, 2020).

This study reports that haspin is a strong suppressor of position-effect variegation playing a role in heterochromatin organization. HP1 proteins, which are key components of heterochromatin, have been reported to promote haspin localization at mitotic centromeres to protect centromeric cohesion in mammals (Yi, 2018). Whether HP1 proteins promote Haspin localization at centromeric heterochromatin in interphase and whether phosphorylation of H3T3 is involved in heterochromatin organization remain to be determined (Fresan, 2020).

This study has also shown that haspin is required for robust Polycomb-dependent homeotic gene silencing based on the following observations in the absence of haspin: (1) derepression of Abd-B transcription; (2) reduction of Pc binding at several PRE-containing regulatory elements and (3) enhanced homeotic transformations in Pc mutant sensitized backgrounds. Moreover, depletion of haspin has a strong impact in pairing-sensitive silencing which involves long-range chromatin organization. The current results also show that Haspin cooperates with Pds5-cohesin to enhance Polycomb-dependent homeotic transformations. Thus, Haspin might regulate homeotic gene silencing by directly affecting the binding of PcG proteins to chromatin or by affecting Pds5-cohesin dynamics modulating chromatin organization of Polycomb domains. Recent results point to an important role for cohesin complexes in the establishment and/or maintenance of Polycomb-repressed domains in mammalian cells but also to restrict their aggregation. This study has shown that Haspin mediates Pds5-binding to chromatin in interphase and modulates cohesin association with chromatin along the cell cycle. Pds5 proteins have both positive and negative effects on cohesin association with chromatin, they cooperate with Wapl in releasing cohesin from DNA but they are also required to maintain sister-chromatid cohesion in G2/M. Pds5 interacts with Wapl, Dalmatian/Sororin, Eco/Eso acetyltransferase and Haspin through the same conserved protein-protein module [Goto, 2017; Nishiyama, 2010]. Wapl-Pds5 interaction has been shown to be counteracted by Eco and Sororin in S phase antagonizing Wapl's ability to dissociate cohesin from DNA. On the other hand, Haspin has been shown to phosphorylate Wapl (Liang, 2018) and to antagonize Wapl-Pds5 interaction to protect proper centromeric cohesion in mitosis (Goto, 2017; Liang, 2018; Zhou, 2017). Although relationship between Haspin and the cohesin complex needs to be further characterized, this work points to an important role of Haspin in the complex regulation of Pds5-cohesin dynamics along the entire cell cycle (Fresan, 2020).

It has been suggested that inhibition of Haspin could have potent anti-tumoral effects with fewer adverse effects compared with other anti-cancer agents. The current results show mitotic defects in Drosophila haspin mutants in agreement with previous reported studies in yeast and mammals (Dai, 2006; Goto, 2017). However, haspin mutants have been reported not lethal in budding yeast and in fission yeast and the current data show that they are also viable in Drosophila, even though life span and fertility are affected. Besides, these findings demonstrate that haspin is controlling genome organization of interphase cells raising concerns with respect to the use of Haspin inhibitors as potent mitosis-specific anticancer drugs (Fresan, 2020).

Asymmetric assembly of centromeres epigenetically regulates stem cell fate

Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. This study shows that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. This work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis (Dattoli, 2020).

Stem cells are fundamental for the generation of all tissues during embryogenesis and replace lost or damaged cells throughout the life of an organism. At division, stem cells generate two cells with distinct fates: (1) a cell that is an exact copy of its precursor, maintaining the 'stemness,' and (2) a daughter cell that will subsequently differentiate. Epigenetic mechanisms, heritable chemical modifications of the DNA/nucleosome that do not alter the primary genomic nucleotide sequence, regulate the process of self-renewal and differentiation of stem cells. In Drosophila male germline stem cells (GSCs), before division, phosphorylation at threonine 3 of histone H3 (H3T3P) preferentially associates with chromosomes that are inherited by the future stem cell (Xie, 2015). Furthermore, centromeric proteins seem to be asymmetrically distributed between stem and daughter cells in the Drosophila intestine and germline. These findings support the 'silent sister hypothesis', according to which epigenetic variations differentially mark sister chromatids driving selective chromosome segregation during stem cell mitosis. Centromeres, the primary constriction of chromosomes, are crucial for cell division, providing the chromatin surface where the kinetochore assembles. In turn, the kinetochore ensures the correct attachment of spindle microtubules and faithful chromosome partition into the two daughter cells upon division. Centromeric chromatin contains different kinds of DNA repeats (satellite and centromeric retrotransposons) wrapped around nucleosomes containing the histone H3 variant centromere protein A (CENP-A). Centromeres are not specified by a particular DNA sequence. Rather, they are specified epigenetically by CENP-A. Centromere assembly, classically measured as CENP-A deposition to generate centromeric nucleosomes, occurs at the end of mitosis (between telophase and G1) in humans. Additional cell cycle timings for centromere assembly have been reported in flies. Interestingly, Drosophila spermatocytes and starfish oocytes are the only cells known to date to assemble centromeres before chromosome segregation, during prophase of meiosis I. These examples show that centromere assembly dynamics can differ among metazoans and also among different cell types in the same organism (Dattoli, 2020).

A key player in centromere assembly in vertebrates is HJURP (holliday junction recognition protein), which localizes at centromeres during the cell cycle window of CENP-A deposition. Furthermore, centromere assembly is regulated by the cell cycle machinery. In flies, deposition of CID (the homologue of CENP-A) requires activation of the anaphase promoting complex/cyclosome (APC/C) and degradation of CYCLIN A (CYCA). In humans, centromere assembly is antagonized by Cdk1 activity, while the kinase Plk1 promotes assembly. Additionally, the CYCLIN B (CYCB)/Cdk1 complex inhibits the binding of CENP-A to HJURP, preventing CENP-A loading at centromeres. To date, little is known about centromere assembly dynamics and functions in stem cell asymmetric divisions. Drosophila melanogaster ovaries provide an excellent model to study stem cells in their native niche. In this tissue, germline stem cells (GSCs) are easily accessible and can be manipulated genetically. Moreover, centromere assembly mechanisms in GSCs and their differentiated cells, cystoblasts (CBs), could be used to epigenetically discriminate between these two cell types. In Drosophila, CID binds to CAL1 (fly functional homologue of HJURP) in a prenucleosomal complex, and its localization to centromeres requires CAL1 and CENP-C (Dattoli, 2020).

This study investigated the dynamics of CENP-A deposition in Drosophila GSCs. GSC centromeres are assembled after replication, but before chromosome segregation, with neural stem cells following the same trend. Centromere assembly in GSCs is tightly linked to the G2/M transition. Indeed, CYCA localizes at centromeres, and its knockdown is responsible for a marked reduction of centromeric CID and CENP-C, but not CAL1. Surprisingly, excessive CID deposition is prevented by CYCB, through the kinase HASPIN. Superresolution microscopy analysis of GSCs at prometaphase and metaphase shows that CID incorporation on sister chromatids occurs asymmetrically, and chromosomes that will be inherited by the stem cell are loaded with more CID. Moreover, GSC chromosomes make stronger kinetochores, which anchor more spindle fibers. This asymmetric distribution of CID between GSC and CB is maintained also at later stages of the cell cycle, while it is not observed in differentiated cells outside of the niche. This study also found that the depletion of CAL1 at centromeres blocks GSC proliferation and differentiation. Notably, overexpression of both CID and CAL1, as well as HASPIN knockdown, promotes stem cell self-renewal and disrupts the asymmetric inheritance of CID. Conversely, overexpression of CAL1 causes GSC-like tumors. Finally, CAL1 and CID knockdown at later stages of egg development have no obvious effect on cell division, suggesting that these cells inherit CID from GSCs. Taken together, these findings establish centromere assembly as a new epigenetic pathway that regulates stem cell fate (Dattoli, 2020).

In this study a detailed characterization of centromere dynamics was performed throughout the cell cycle in Drosophila female GSCs. This analysis reveals that GSCs initiate CID incorporation after replication and that its deposition continues until at least prophase. Drosophila neural stem cells follow the same trend. Notably, this timing is different from existing studies in other metazoans. It was also found that CYCA, CYCB, and HASPIN are critically involved in CID (and CENP-C) loading at centromeres. According to the model, CYCA promotes centromere assembly, while CYCB prevents excessive deposition of CID, through the HASPIN kinase. Moreover, chromosomes that will be inherited by GSCs are labeled with a higher amount of CID and capture more spindle microtubules. Importantly, this study shows that overexpression of CAL1 and CID together, as well as HASPIN knockdown, promotes stem cell self-renewal, disrupting the asymmetric inheritance of CID. Depletion of CAL1 in stem cells blocks cell division, while CAL1 overexpression causes GSC-like tumors, highlighting its crucial role in cell proliferation. Three main points of discussion are raised: (1) the biological significance of centromere assembly in G2-M phase; (2) CAL1 is a cell proliferation marker; and (3) CID incorporation into centromeric chromatin occurs before meiosis (Dattoli, 2020).

According to the data, CID deposition occupies a wide window of time from after replication and early G2 phase to prophase. The assembly of GSC centromeres during the G2/M transition could be due to the contraction of the G1 phase, a characteristic of stem cells. Yet, in fly embryonic divisions, G1 phase is missing, and instead CID loading occurs at anaphase. Therefore, G2/M assembly might be a unique property of stem cells. This timing is also similar to the one found for Drosophila spermatocytes, which assemble centromeres in prophase of meiosis I. These spermatocytes undergo an arrest in prophase I for days, indicating a gradual loading of CID over a long period of time. Intriguingly, a similar phenomenon has been recently observed in G0-arrested human tissue culture cells and starfish oocytes. Given that GSCs are mostly in G2 phase, Drosophila stem cells might show similar properties to quiescent cells. According to the most recent models, there is a dual mechanism for CENP-A deposition: (a) a rapid pulse during G1 in mitotically dividing cells; and (b) a slow but constant CENP-A deposition in nondividing cells to actively maintain centromeres. Indeed, while previous studies in Drosophila NBs show a rapid pulse of CENP-A incorporation at telophase/G1, the majority of the loading could occur between G2 and prophase. The new results also support this model (Dattoli, 2020).

Incorporation of CID before chromosome segregation might reflect a different CYCLIN-CDK activity in these cells. For instance, it has been already shown that in Drosophila GSCs CYCLIN E, a canonical G1/S cyclin, exists in its active form (in combination with Cdk2) throughout the cell cycle, indicating that some of the biological process commonly occurring in G1 phase might actually take place in G2 phase. This is in line with the current functional findings, where depletion of CYCA causes a decreased efficiency in CID and CENP-C assembly. This study also found that this loss might be independent from CAL1. Surprisingly, correct CID deposition in GSCs also requires CYCB and HASPIN. Indeed, an inhibitory mechanism for CID deposition through CYCB has already been proposed in mammals (Stankovic, 2017). Interestingly, in Drosophila male GSCs, centromeric CAL1 is reduced between G2 and prometaphase (Ranjan, 2019), further suggesting a role for additional regulators of CID assembly, such as CYCA/B or HASPIN, at this time (Dattoli, 2020).

According to the current results, asymmetric cell division of GSCs is epigenetically regulated by differential amounts of centromeric proteins deposited at sister chromatids, which in turn can influence the attachment of spindle microtubules and can ultimately bias chromosome segregation. It is interesting to speculate on the temporal sequence of these events. Two scenarios can be proposed: (a) the nucleation of microtubules from the GSC centrosome requires bigger kinetochores; or (b) bigger kinetochores require a higher amount of spindle fibers to attach. The current results together with recent studies support the latter scenario. In fact, in Drosophila male GSCs, asymmetric distribution of centromeric proteins is established before microtubule attachment. Furthermore, microtubule disruption leaves asymmetric loading of CID intact, while it disrupts the asymmetric segregation of sister chromatids (Ranjan, 2019). The current data confirm this model, symmetric segregation of CID was observed upon HASPIN knockdown. Indeed, in vertebrates HASPIN knockdown causes spindle defects. Specifically, it was observed that a 1.2-fold difference in CID and CENP-C levels between GSC and CB chromosomes can bias segregation. While this difference is small, it fits with the observation that small changes in CENP-A level (on the order of 2-10% per day) impact on centromere functionality in the long run. In Drosophila male GSCs, an asymmetric distribution of CID on sister chromatids >1.4-fold was reported. This higher value might reflect distinct systems in males and females or the quantitation methods used. Importantly, CID asymmetry in males is established in G2/prophase, in line with the time window this study defines for CID assembly. Further support for unexpected CID loading dynamics comes from the finding that GSCs in G2/prophase contain ~30% more CID on average compared with S phase, indicating that CID is not replenished to 100% each cell cycle. Interestingly, the time course of H3T3P appearance during the GSC cell cycle closely follows the timing of CID incorporation, suggesting that the asymmetric deposition of CID might drive the differential phosphorylation of the histone H3 on sister chromatids. Finally, the results are in line with findings that the long-term retention of CENP-A in mouse oocytes has a role in establishing asymmetric centromere inheritance in meiosis (Dattoli, 2020).

These functional studies support a role for CAL1 in cell proliferation, with no apparent role in asymmetric cell division. Indeed, centromeric proteins have been already proposed as biomarkers for cell proliferation. Specifically, functional analysis of centromeric proteins, as well as the HASPIN kinase, allowed discrimination between the classic role of centromeres in cell division and a role in asymmetric cell division. In a favorite scenario, CAL1 is needed to make functional centromeres crucial for cell division, while the asymmetric distribution of CID sister chromatids regulates asymmetric cell division and might depend on other factors, such as HASPIN. However, it cannot be rule out that the effects on cell fate observed with the functional analysis might reflect alternative CAL1 functions outside of the centromere, for example due to changes in chromosome structure or gene expression (Dattoli, 2020).

Centromeres are crucially assembled in GSCs and therefore before meiosis of the oocyte takes place. Thus, it is possible that the 16-cell cysts inherit centromeric proteins synthesized and deposited in the GSCs, and the rate of new CID loading is reduced. This would explain why CAL1 function at centromeres is dispensable at this developmental stage (Dattoli, 2020).

Ultimately, the results provide the first functional evidence that centromeres have a role in the epigenetic pathway that specifies stem cell identity. Furthermore, these data support the silent sister hypothesis (Lansdorp, 2007), according to which centromeres can drive asymmetric division in stem cells (Dattoli, 2020).

Asymmetric division of Drosophila male germline stem cell shows asymmetric histone distribution

A long-standing question concerns how stem cells maintain their identity through multiple divisions. It has been reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. This study shows that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity (Xie, 2015).

Epigenetic phenomena are heritable changes in gene expression or function that can persist throughout many cell divisions without alterations in primary DNA sequences. By regulating differential gene expression, epigenetic processes are able to direct cells with identical genomes to become distinct cell types in humans and other multicellular organisms. However, with the exception of DNA methylation, little is known about the molecular pathways leading to epigenetic inheritance (Xie, 2015).

Prior research has shown that epigenetic events play particularly important roles in ensuring both proper maintenance and differentiation of several stem cell populations. Many types of adult stem cells undergo asymmetric cell division to generate a self-renewed stem cell and a daughter cell that will subsequently differentiate. Mis-regulation of this balance leads to many human diseases, ranging from cancer to tissue dystrophy to infertility. However, the mechanisms of stem cell epigenetic memory maintenance as well as how loss of this memory contributes to disease remain unknown (Xie, 2015).

During the asymmetric division of the Drosophila male germline stem cell (GSC), the pre-existing histone 3 (H3) is selectively segregated to the self-renewed GSC daughter cell whereas newly synthesized H3 is enriched in the differentiating daughter cell known as a gonialblast (GB). In contrast, the histone variant H3.3, which is incorporated in a replication-independent manner, does not exhibit such an asymmetric pattern. Furthermore, asymmetric H3 inheritance occurs specifically in asymmetrically dividing GSCs, but not in the symmetrically dividing progenitor cells. These findings demonstrate that global asymmetric H3 histone inheritance possesses both molecular and cellular specificity. The following model is proposed to explain these findings (Xie, 2015).

First, the cellular specificity exhibited by the H3 histone suggests that global asymmetric histone inheritance occurs uniquely in a cell-type (GSC) where the mother cell must divide to produce two daughter cells each with a unique cell fate. Because this asymmetry is not observed in symmetrically dividing GB cells, asymmetric histone inheritance is proposed to be a phenomenon specifically employed by GSCs to establish unique epigenetic identities in each of the two daughter cells. Second, as stated previously, a major difference between H3 and H3.3 is that H3 is incorporated to chromatin during DNA replication, while H3.3 variant is incorporated in a replication-independent manner. Because this asymmetric inheritance mode is specific to H3, a two-step model is proposed to explain asymmetric H3 inheritance: (1) prior to mitosis, pre-existing and newly synthesized H3 are differentially distributed on the two sets of sister chromatids, and (2) during mitosis, the set of sister chromatids containing pre-existing H3 is segregated to GSCs, while the set of sister chromatids enriched with newly synthesized H3 is segregated to the GB that differentiates (Tran, 2012; Tran, 2013; Xie, 2015 and references therein)

This study reports that a mitosis-enriched H3T3P mark acts as a transient landmark that distinguishes sister chromatids with identical genetic code but different epigenetic information, shown as pre-existing H3-GFP and newly synthesized H3-mKO. By distinguishing sister chromatids containing different epigenetic information, H3T3P functions to allow these molecularly distinct sisters to be segregated and inherited differentially to the two daughter cells derived from one asymmetric cell division. The selective segregation of different populations of histones likely allows these two cells to assume distinct fates: self-renewal versus differentiation. Consequently, loss of proper epigenetic inheritance might lead to defects in both GSC maintenance and GB differentiation, suggesting that both cells need this active partitioning process to either 'remember' or 'reset' their molecular properties (Xie, 2015).

The temporal and spatial specificities of H3T3P make it a great candidate to regulate asymmetric sister chromatid segregation. First, H3T3P is only detectable from prophase to metaphase, the window of time during which the mitotic spindle actively tries to attach to chromatids through microtubule-kinetochore interactions. Second, the H3T3P signal is enriched at the peri-centromeric region, where kinetochore components robustly crosstalk with chromatin-associate factors. Third, H3T3 shows a sequential order of phosphorylation, first appearing primarily on sister chromatids enriched with pre-existing H3 and then subsequently appearing on sister chromatids enriched with newly synthesized H3 as the GSC nears metaphase. The distinct temporal patterns shown by H3T3P are unique to GSCs and would allow the mitotic machinery to differentially recognize sister chromatids bearing distinct epigenetic information; an essential step necessary for proper segregation during asymmetric GSC division. Furthermore, the tight temporal control of H3T3 phosphorylation suggests that rather than serving as an inherited epigenetic signature, H3T3P may act as transient signaling mark to allow for the proper partitioning of H3. It is hypothesized that H3T3P needs to be under tight temporal control in order to ensure proper H3 inheritance and germline activity (Xie, 2015).

These studies have shown that H3T3P is indeed subject to stringent temporal controls during mitosis. The H3T3P mark is undetectable during G2 phase. Upon entry to mitosis, sister chromatids enriched with pre-existing H3-GFP histone begin to show H3T3 phosphorylation prior to sister chromatids enriched with newly synthesized H3-mKO. As the cell continues to progress toward metaphase, H3T3P signal begins to appear on sister chromatids enriched with newly synthesized H3-mKO. Such a tight regulation of H3T3P is compromised when levels of H3T3P are altered due to the incorporation of mutant H3T3A or H3T3D. Incorporation of the H3T3A mutant results in a significant decrease in the levels of H3T3P on sister chromatids throughout mitosis, such that neither sister becomes enriched with H3T3P as the GSC progresses toward metaphase. Conversely, incorporation of the H3T3D mutant would result in seemingly elevated levels of H3T3P early in mitosis. Although H3T3A and H3T3D act in different ways, both mutations significantly disrupt the highly regulated temporal patterns associated with H3T3 phosphorylation, the result of which is randomized H3 inheritance patterns and germ cell defects in testes expressing either H3T3A or H3T3D (Xie, 2015).

To further evaluate the extent of H3T3A and H3T3D roles in the segregation of sister chromatids enriched with different populations of H3 during mitosis, all possible segregation patterns were modeled in male GSCs, and these estimates were compared to the experimental results. To simplify the calculations, two important assumptions were made: first, nucleosomal density was assumed to be even throughout the genome. This assumption allows the inference that the overall fluorescent signal contributed by each chromosome is proportional to their respective number of DNA base pairs. Second, by quantifying pre-existing H3-GFP asymmetry in anaphase and telophase GSCs, it was estimated that the establishment of H3-GFP asymmetry is ∼4-fold biased, i.e., 80% on one set of sister chromatids and 20% on the other set of sister chromatids, based on quantification of GFP signal in anaphase and telophase GSCs. With these two simplifying assumptions, both GFP and mKO ratios were caculated among all 64 possible combinations. If asymmetry is designed as a greater than 1.5-fold difference in fluorescence intensity, then based on a model of randomized sister chromatid segregation, it is estimated that a symmetric pattern should appear for 53.1% (34/64) of GSC-GB pairs whereas both conventional and inverted asymmetric patterns should occur with equal frequencies and account for 18.7% (12/64) of total GSC-GB pairs. The remaining 9.4% (6/64) of GSC-GB pairs should produce histone inheritance patterns with a 1.45- to 1.55-fold difference in signal intensity (Xie, 2015).

This estimation is close to the experimental data in both H3T3A- and H3T3D-expressing testes. Of the 64 quantified post-mitotic GSC-GB pairs in nos>H3T3A testes, ~71.9% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 9.4% and 12.5%, respectively, and 6.3% at the borderline. Similarly, of the 57 quantified post-mitotic GSC-GB pairs in nos>H3T3D testes, ∼79.0% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 7.0% and 10.5%, respectively with 3.5% of pairs at the borderline. Some differences between predicted ratios and the experimental data could be due to the simplified assumptions, the limited sensitivity of the measurement, and/or some coordinated chromatid segregation modes that bias the eventual read-out. In summary, comparison between the modeling ratios and the experimental data suggest that loss of the tight control of H3T3 phosphorylation in GSCs randomizes segregation of sister chromatids enriched with different populations of H3 (Xie, 2015).

If the temporal separation in the phosphorylation of H3T3 on epigenetically distinct sister chromatids facilitates their proper segregation and inheritance during asymmetric cell division, it is likely that mutations of the Haspin kinase will also affect the temporal control of H3T3 phosphorylation. In the context of H3T3A, where the levels of H3T3P are already reduced, a further decrease in H3T3P by reducing Haspin levels should limit the GSC's ability to distinguish between sister chromatids enriched with distinct H3. Indeed, haspin mutants enhance the phenotypes in nos>H3T3A testes. A different situation appears in the context of H3T3D where sister chromatids experience seemingly elevated levels of H3T3P at the start of mitosis. These elevated H3T3P levels may be exacerbated by the phosphorylation activity of the Haspin kinase. Therefore, it is conceivable that by halving the levels of the Haspin kinase, H3T3 phosphorylation should be reduced to a level more closely resembling wild-type. In this way, some of the temporal specificity that is lost in the H3T3D mutant is restored, resulting in suppression of the phenotypes observed in nos>H3T3D testes. An exciting topic for future study would be to further explore how exactly Haspin phosphorylates H3T3 in the context of chromatin and whether H3T3A and H3T3D mutations act synergistically or antagonistically in regulating asymmetric sister chromatids segregation through differential phosphorylation of a key histone residue (Xie, 2015).

It would also be interesting to understand the potential connection between asymmetric histone inheritance and another phenomenon reported by several investigators: selective DNA strand segregation. Recent development of the chromosome orientation fluorescence in situ hybridization (CO-FISH) technique allows study of selective chromatid segregation at single-chromosome resolution. Using this technique in mouse satellite cells, it has been demonstrated that all chromosomes are segregated in a biased manner, such that pre-existing template DNA strands are preferentially retained in the daughter cell that retains stem cell identity. Interestingly, this biased segregation becomes randomized in progenitor non-stem cells. Using CO-FISH in Drosophila male GSCs, sex chromosomes have been shown to segregate in a biased manner. Remarkably, sister chromatids from homologous autosomes have been shown to co-segregate independent of any specific strand preference. Such findings hint at a possible epigenetic source guiding the coordinated inheritance of Drosophila homologous autosomes. In many cases of biased inheritance, researchers have speculated about the existence of a molecular signature that would allow the cell to recognize and segregate sister chromatids bearing differential epigenetic information. However, the identity of such a signature has remained elusive. The work represented in this paper provides experimental evidence demonstrating that a tightly-controlled histone modification, H3T3P, is able to distinguish sister chromatids and coordinate their segregation (Xie, 2015).

Epigenetic processes play important roles in regulating stem cell identity and activity. Failure to appropriately regulate epigenetic information may lead to abnormalities in stem cell behaviors, which underlie early progress toward diseases such as cancer and tissue degeneration. Due to the crucial role that such processes play in regulating cell identity and behavior, the field has long sought to understand whether and how stem cells maintain their epigenetic memory through many cell divisions. Yhe results of this study suggest that the asymmetric segregation of pre-existing and newly synthesized H3-enriched chromosomes may function to determine distinct cell fates of GSCs versus differentiating daughter cells (Xie, 2015).

HP1 links centromeric heterochromatin to centromere cohesion in mammals

A kinase-dependent role for Haspin in antagonizing Wapl and protecting mitotic centromere cohesion

Sister-chromatid cohesion mediated by the cohesin complex is fundamental for precise chromosome segregation in mitosis. Through binding the cohesin subunit Pds5, Wapl releases the bulk of cohesin from chromosome arms in prophase, whereas centromeric cohesin is protected from Wapl until anaphase onset. Strong centromere cohesion requires centromeric localization of the mitotic histone kinase Haspin, which is dependent on the interaction of its non-catalytic N-terminus with Pds5B. It remains unclear how Haspin fully blocks the Wapl-Pds5B interaction at centromeres. This study shows that the C-terminal kinase domain of Haspin (Haspin-KD) binds and phosphorylates the YSR motif of Wapl (Wapl-YSR), thereby directly inhibiting the YSR motif-dependent interaction of Wapl with Pds5B. Cells expressing a Wapl-binding-deficient mutant of Haspin or treated with Haspin inhibitors show centromeric cohesion defects. Phospho-mimetic mutation in Wapl-YSR prevents Wapl from binding Pds5B and releasing cohesin. Forced targeting Haspin-KD to centromeres partly bypasses the need for Haspin-Pds5B interaction in cohesion protection. Taken together, these results indicate a kinase-dependent role for Haspin in antagonizing Wapl and protecting centromeric cohesion in mitosis (Liang, 2018).

Heterochromatin protein-1 (HP1) is a key component of heterochromatin. Reminiscent of the cohesin complex which mediates sister-chromatid cohesion, most HP1 proteins in mammalian cells are displaced from chromosome arms during mitotic entry, whereas a pool remains at the heterochromatic centromere region. The function of HP1 at mitotic centromeres remains largely elusive. This study shows that double knockout (DKO) of HP1alpha and HP1gamma causes defective mitosis progression and weakened centromeric cohesion. While mutating the chromoshadow domain (CSD) prevents HP1alpha from protecting sister-chromatid cohesion, centromeric targeting of HP1alpha CSD alone is sufficient to rescue the cohesion defects in HP1 DKO cells. Interestingly, HP1-dependent cohesion protection requires Haspin, an antagonist of the cohesin-releasing factor Wapl. Moreover, HP1alpha CSD directly binds the N-terminal region of Haspin and facilitates its centromeric localization. The need for HP1 in cohesion protection can be bypassed by centromeric targeting of Haspin or inhibiting Wapl activity. Taken together, these results reveal a redundant role for HP1alpha and HP1gamma in the protection of centromeric cohesion through promoting Haspin localization at mitotic centromeres in mammalian cells (Yi, 2018).

The N-terminal non-kinase-domain-mediated binding of Haspin to Pds5B protects centromeric cohesion in mitosis

Sister-chromatid cohesion, mediated by the multi-subunit cohesin complex, must be precisely regulated to prevent chromosome mis-segregation. In prophase and prometaphase, whereas the bulk of cohesin on chromosome arms is removed by its antagonist Wapl, cohesin at centromeres is retained to ensure chromosome biorientation until anaphase onset. It remains incompletely understood how centromeric cohesin is protected against Wapl in mitosis. This study shows that the mitotic histone kinase Haspin binds to the cohesin regulatory subunit Pds5B through a conserved YGA/R motif in its non-catalytic N terminus, which is similar to the recently reported YSR-motif-dependent binding of Wapl to Pds5B. Knockout of Haspin or disruption of Haspin-Pds5B interaction causes weakened centromeric cohesion and premature chromatid separation, which can be reverted by centromeric targeting of a N-terminal short fragment of Haspin containing the Pds5B-binding motif or by prevention of Wapl-dependent cohesin removal. Conversely, excessive Haspin capable of binding Pds5B displaces Wapl from Pds5B and suppresses Wapl activity, and it largely bypasses the Wapl antagonist Sgo1 for cohesion protection. Taken together, these data indicate that the Haspin-Pds5B interaction is required to ensure proper sister-chromatid cohesion, most likely through antagonizing Wapl-mediated cohesin release from mitotic centromeres (Zhou, 2017).

DNA damage induces a kinetochore-based ATM/ATR-independent SAC arrest unique to the first meiotic division in mouse oocytes

Mouse oocytes carrying DNA damage arrest in meiosis I, thereby preventing creation of embryos with deleterious mutations. The arrest is dependent on activation of the spindle assembly checkpoint, which results in anaphase-promoting complex (APC) inhibition. However, little is understood about how this checkpoint is engaged following DNA damage. This study found that within minutes of DNA damage checkpoint proteins are assembled at the kinetochore, not at damage sites along chromosome arms, such that the APC is fully inhibited within 30 min. Despite this robust response, there is no measurable loss in k-fibres, or tension across the bivalent. Through pharmacological inhibition this study observed that the response is dependent on Mps1 kinase (see Drosophila Mps1), aurora kinase (see Drosophila Aurora B) and Haspin (see Drosophila Haspin). Using oocyte-specific knockouts this study found the response does not require the DNA damage response kinases ATM or ATR. Furthermore, checkpoint activation does not occur in response to DNA damage in fully mature eggs during meiosis II, despite the divisions being separated by just a few hours. Therefore, mouse oocytes have a unique ability to sense DNA damage rapidly by activating the checkpoint at their kinetochores (Lane, 2017).

Sororin mediates sister chromatid cohesion by antagonizing Wapl

Pds5 proteins are also required for proper maintenance of heterochromatin and participate in chromatin loop formation. It has been suggested that they may be required for the boundary function of CTCF, since cells depleted of Pds5 proteins contain many fewer loops than control cells, which is similar to the effect of CTCF depletion. On the other hand, it has been shown that chromatin becomes more compact after reducing levels of CTCF and Rad21 and the analysis of the molecular basis for this counter-intuitive behavior suggested that compaction could be the consequence of changes in chromatin loops. The data show that haspin is required for insulator activity, nuclear compaction, heterochromatin-induced position-effect variegation and PcG-mediated pairing-sensitive silencing strongly suggesting that haspin could be involved in the organization of the genome in chromatin domains and loops by modulating Pds5-cohesin association with chromatin (Fresan, 2010).

Structure and functional characterization of the atypical human kinase haspin

The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. This study reports structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP gamma-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity (Eswaran, 2009).

Regulation of mitotic chromosome cohesion by Haspin and Aurora B

In vertebrate mitosis, cohesion between sister chromatids is lost in two stages. In prophase and prometaphase, cohesin release from chromosome arms occurs under the control of Polo-like kinase 1 and Aurora B, while Shugoshin is thought to prevent removal of centromeric cohesin until anaphase. The regulatory enzymes that act to sustain centromeric cohesion are incompletely described, however. Haspin/Gsg2, a positive regulator of centromeric cohesion, is a histone H3 threonine-3 kinase required for normal mitosis. Both H3 threonine-3 phosphorylation and cohesin are located at inner centromeres. Haspin depletion disrupts cohesin binding and sister chromatid association in mitosis, preventing normal chromosome alignment and activating the spindle assembly checkpoint, leading to arrest in a prometaphase-like state. Overexpression of Haspin hinders cohesin release and stabilizes arm cohesion. It is concluded that Haspin is required to maintain centromeric cohesion during mitosis. It is also suggested that Aurora B regulates cohesin removal through its effect on the localization of Shugoshin (Dai, 2006).

The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment

Post-translational modifications of conserved N-terminal tail residues in histones regulate many aspects of chromosome activity. Thr 3 of histone H3 is highly conserved, but the significance of its phosphorylation is unclear, and the identity of the corresponding kinase unknown. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation of H3 Thr 3 in prophase and its dephosphorylation during anaphase. Haspin, a member of a distinctive group of protein kinases present in diverse eukaryotes, phosphorylates H3 at Thr 3 in vitro. Importantly, depletion of haspin by RNA interference reveals that this kinase is required for H3 Thr 3 phosphorylation in mitotic cells. In addition to its chromosomal association, haspin is found at the centrosomes and spindle during mitosis. Haspin RNA interference causes misalignment of metaphase chromosomes, and overexpression delays progression through early mitosis. This work reveals a new kinase involved in composing the histone code and adds haspin to the select group of kinases that integrate regulation of chromosome and spindle function during mitosis and meiosis (Dai, 2005 ).

Search PubMed for articles about Drosophila Haspin

Dai, J., Sultan, S., Taylor, S. S. and Higgins, J. M. (2005). The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment. Genes Dev 19(4): 472-488. PubMed ID: 15681610

Dai, J., Sullivan, B. A. and Higgins, J. M. (2006). Regulation of mitotic chromosome cohesion by Haspin and Aurora B. Dev Cell 11(5): 741-750. PubMed ID: 17084365

Dattoli, A. A., Carty, B. L., Kochendoerfer, A. M., Morgan, C., Walshe, A. E. and Dunleavy, E. M. (2020). Asymmetric assembly of centromeres epigenetically regulates stem cell fate. J Cell Biol 219(4). PubMed ID: 32328637

Eswaran, J., Patnaik, D., Filippakopoulos, P., Wang, F., Stein, R. L., Murray, J. W., Higgins, J. M. and Knapp, S. (2009). Structure and functional characterization of the atypical human kinase haspin. Proc Natl Acad Sci U S A 106(48): 20198-20203. PubMed ID: 19918057

Fresan, U., Rodriguez-Sanchez, M. A., Reina, O., Corces, V. G. and Espinas, M. L. (2020). Haspin kinase modulates nuclear architecture and Polycomb-dependent gene silencing. PLoS Genet 16(8): e1008962. PubMed ID: 32750047

Goto, Y., Yamagishi, Y., Shintomi-Kawamura, M., Abe, M., Tanno, Y. and Watanabe, Y. (2017). Pds5 Regulates Sister-Chromatid Cohesion and Chromosome Bi-orientation through a Conserved Protein Interaction Module. Curr Biol 27(7): 1005-1012. PubMed ID: 28343969

Lane, S. I. R., Morgan, S. L., Wu, T., Collins, J. K., Merriman, J. A., ElInati, E., Turner, J. M. and Jones, K. T. (2017). DNA damage induces a kinetochore-based ATM/ATR-independent SAC arrest unique to the first meiotic division in mouse oocytes. Development 144(19): 3475-3486. PubMed ID: 28851706

Lansdorp, P. M. (2007). Immortal strands? Give me a break. Cell 129(7): 1244-1247. PubMed ID: 17604711

Liang, C., Chen, Q., Yi, Q., Zhang, M., Yan, H., Zhang, B., Zhou, L., Zhang, Z., Qi, F., Ye, S. and Wang, F. (2018). A kinase-dependent role for Haspin in antagonizing Wapl and protecting mitotic centromere cohesion. EMBO Rep 19(1): 43-56. PubMed ID: 29138236

Nishiyama T, Ladurner R, Schmitz J, Kreidl E, Schleiffer A, et al. (2010) Sororin mediates sister chromatid cohesion by antagonizing Wapl. Cell 143: 737-749. PubMed ID: 21111234

Ranjan, R., Snedeker, J. and Chen, X. (2019). Asymmetric centromeres differentially coordinate with mitotic machinery to ensure biased sister chromatid segregation in germline stem cells. Cell Stem Cell. PubMed ID: 31564548

Stankovic, A., Guo, L. Y., Mata, J. F., Bodor, D. L., Cao, X. J., Bailey, A. O., Shabanowitz, J., Hunt, D. F., Garcia, B. A., Black, B. E. and Jansen, L. E. T. (2017). A dual inhibitory mechanism sufficient to maintain cell-cycle-restricted CENP-A assembly. Mol Cell 65(2): 231-246. PubMed ID: 28017591

Xie, J., Wooten, M., Tran, V., Chen, B. C., Pozmanter, C., Simbolon, C., Betzig, E. and Chen, X. (2015). Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the Drosophila male germline. Cell 163(4): 920-933. PubMed ID: 26522592

Yi, Q., Chen, Q., Liang, C., Yan, H., Zhang, Z., Xiang, X., Zhang, M., Qi, F., Zhou, L. and Wang, F. (2018). HP1 links centromeric heterochromatin to centromere cohesion in mammals. EMBO Rep 19(4). PubMed ID: 29491004

Zhou, L., Liang, C., Chen, Q., Zhang, Z., Zhang, B., Yan, H., Qi, F., Zhang, M., Yi, Q., Guan, Y., Xiang, X., Zhang, X., Ye, S. and Wang, F. (2017). The N-terminal non-kinase-domain-mediated binding of Haspin to Pds5B protects centromeric cohesion in mitosis. Curr Biol 27(7): 992-1004. PubMed ID: 28343965

date revised: 14 December 2020

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