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Histone phosphorylation is cell cycle specific in isolated chromatin and chromosomes. A, schematic drawing of the experiment. To detect proteins phosphorylated by chromatin or chromosome-associated protein kinases, chromatin is assembled first in cytosol containing no label, isolated, and then incubated in buffer containing [-32 P]ATP (see "Experimental Procedures"). B, cell cycle-dependent histone phosphorylation in isolated interphase chromatin and mitotic chromosomes. Interphase chromatin and mitotic chromosomes were assembled in vitro, isolated, incubated with [-32 P]ATP, and then acid extracted to isolate histones. These were run on 15% Triton X-100/acetic acid/urea gel. The gel was stained with Coomassie Blue (lanes 1 and 2) and autoradiographed (lanes 3 and 4). The migration of standard histone markers is indicated. C, phosphorylation of Ser 10 histone H3 in isolated chromatin and chromosomes. Isolated chromatin and chromosomes were either boiled immediately after isolation in gel sample buffer (first and second lanes) or incubated at 22 °C for 10 in the absence (third and fourth lanes) or the presence of ATP (fifth and sixth lanes). Isolated interphase chromatin and mitotic chromosomes were separated by SDS-PAGE, transferred to nitrocellulose, stained with Sypro Orange (Total Histones), and then immunoblotted with anti-Ser(P) 10 histone H3 (Phospho H3). In the gel system used, H2A, H3, and H2B comigrate in the upper, brighter band observed by Sypro Orange. Lanes I, interphase chromatin; lanes M, mitotic chromosomes. D, interphase chromatin and mitotic chromosomes contain PP1. Interphase (I) and mitotic (M) cytosol (Cytosol) and chromatin isolated from interphase and mitotic cytosols (Chromatin) were separated by SDS-PAGE and immunoblotted with an antibody specific for PP1 (41).
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Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. We have used an
in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts to study mitotic histone H3 phosphorylation. We identified a histone H3 Ser10 kinase activity associated with isolated mitotic chromosomes. The histon...
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... kinase activities that phosphorylate his- tones H1 and H3. To determine whether a similar activity copurified with interphase chromatin and mitotic chromosomes assembled in vitro, we added sperm nuclei to interphase and mitotic extracts, allowed chromatin and chromosomes to form, and then purified the reaction products through a sucrose cushion (Fig. 1A). [-32 P]ATP was added to the purified inter- phase chromatin and mitotic chromosomes to detect any copu- rifying kinase activity. Histones were then purified by acid extraction and separated using a Triton X-100/acetic acid/urea gel system (54). This method resolves the histones on the basis of charge and hydrophobicity. Comparison of ...
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... any copu- rifying kinase activity. Histones were then purified by acid extraction and separated using a Triton X-100/acetic acid/urea gel system (54). This method resolves the histones on the basis of charge and hydrophobicity. Comparison of the Coomassie- stained gel and the autoradiogram of a representative Triton X-100/acetic acid/urea gel (Fig. 1B) shows that all histones are present in equal amounts in interphase chromatin and mitotic chromosomes, but histones H2A and H4 are phosphorylated in isolated interphase chromatin, whereas histones H3 and H1 are phosphorylated in isolated mitotic chromosomes. The core histones and histone B4, the Xenopus egg linker histone (55), are ...
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... characterize the site of phosphorylation on histone H3, we immunoblotted interphase chromatin and mitotic chromo- somes with an anti-Ser(P) 10 histone H3 antibody (3). A signif- icantly higher level of phosphorylation at Ser 10 was detected in isolated mitotic chromosomes than in interphase chromatin (Fig. 1C, lanes 1 and 2). Incubation of isolated chromatin and chromosomes at 22 °C in the absence of ATP caused the loss of the mitotic-specific phosphorylation, suggesting the presence of a protein phosphatase in isolated mitotic chromosomes (Fig. 1B, lanes 3 and 4). Incubation in the presence of ATP increased the amount of anti-Ser(P) 10 signal in ...
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... of phosphorylation at Ser 10 was detected in isolated mitotic chromosomes than in interphase chromatin (Fig. 1C, lanes 1 and 2). Incubation of isolated chromatin and chromosomes at 22 °C in the absence of ATP caused the loss of the mitotic-specific phosphorylation, suggesting the presence of a protein phosphatase in isolated mitotic chromosomes (Fig. 1B, lanes 3 and 4). Incubation in the presence of ATP increased the amount of anti-Ser(P) 10 signal in interphase chromatin but gave a much stronger increase in mitotic chromosomes (Fig. 1B, lanes 5 and 6). A small amount of interphase H3 phospho- rylation was also observed by 32 P labeling (Fig. 1B). Neither the interphase chromatin-or mitotic ...
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... in the absence of ATP caused the loss of the mitotic-specific phosphorylation, suggesting the presence of a protein phosphatase in isolated mitotic chromosomes (Fig. 1B, lanes 3 and 4). Incubation in the presence of ATP increased the amount of anti-Ser(P) 10 signal in interphase chromatin but gave a much stronger increase in mitotic chromosomes (Fig. 1B, lanes 5 and 6). A small amount of interphase H3 phospho- rylation was also observed by 32 P labeling (Fig. 1B). Neither the interphase chromatin-or mitotic chromosome-associated kinase activities were sensitive to the CDK inhibitors roscovi- tine or butyrolactone I, wortmannin, H-89, or rapamycin (data not shown). None of these treatments resulted in ...
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... phosphatase in isolated mitotic chromosomes (Fig. 1B, lanes 3 and 4). Incubation in the presence of ATP increased the amount of anti-Ser(P) 10 signal in interphase chromatin but gave a much stronger increase in mitotic chromosomes (Fig. 1B, lanes 5 and 6). A small amount of interphase H3 phospho- rylation was also observed by 32 P labeling (Fig. 1B). Neither the interphase chromatin-or mitotic chromosome-associated kinase activities were sensitive to the CDK inhibitors roscovi- tine or butyrolactone I, wortmannin, H-89, or rapamycin (data not shown). None of these treatments resulted in changes in the total amount of histone protein (Fig. 1C). These data con- firm the specificity ...
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... rylation was also observed by 32 P labeling (Fig. 1B). Neither the interphase chromatin-or mitotic chromosome-associated kinase activities were sensitive to the CDK inhibitors roscovi- tine or butyrolactone I, wortmannin, H-89, or rapamycin (data not shown). None of these treatments resulted in changes in the total amount of histone protein (Fig. 1C). These data con- firm the specificity of histone phosphorylation in isolated chro- mosomes; not only is histone H3 phosphorylated in a cell cycle- dependent fashion in isolated mitotic chromosomes, but phosphorylation occurred at a site known to be phosphorylated in mitotic chromosomes in vivo. Most importantly, they indi- cate that ...
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... with all isoforms of the human enzyme (41). The identity of the antigen and Xenopus PP1 is 98%, suggesting that the antibody might recognize the Xenopus PP1. A protein of 36 kDa, the correct size for PP1, cross-reacted with the anti-PP1 antibody in both interphase and mitotic cytosols and isolated interphase chromatin and mitotic chro- mosomes (Fig. 1D). One additional uncharacterized polypep- tide of slower mobility was recognized in cytosol. Further in- hibitor studies confirmed this localization (see below). In summary, these results show that a cell cycle-specific histone H3 kinase and a phosphatase known to modulate histone H3 phosphorylation are components of isolated mitotic ...
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... and characterize its regulation, we developed a method for solubilizing chromatin-associated kinase activities compatible with biochemical fractionation and quantitative kinase activity assays. Fig. 2A shows that washing isolated chromosomes with 0.4 M NaCl and 0.1% Triton X-100 diminished the phosphoryl- ation of chromosomal proteins by over 90% (Fig. 2A, lanes 1 and 2). Strikingly, most of the proteins detected by Coomassie staining are retained on the chromosomes (Fig. 2A, lanes 3 and 4). There are two notable exceptions that are eluted from iso- lated mitotic chromosomes, histone B4 ( Fig. 2A, bracket), and DNA topoisomerase II ( Fig. 2A, dot; see Ref. 40 for gel band identification). These ...
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... of the Mitotic Histone H3 Kinase by PP1-Bear- ing in mind that isolated interphase chromatin contains a weak histone H3 kinase activity (Fig. 1) and having identified PP1 as a component of isolated chromatin assembled in vitro (Fig. 1), we tested whether PP1 activity regulated the ability of the chromatin-associated kinase activity to efficiently phosphoryl- ate histone H3. We designed an assay that could determine whether PP1 modulates histone H3 phosphorylation by regu- ...
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... of the Mitotic Histone H3 Kinase by PP1-Bear- ing in mind that isolated interphase chromatin contains a weak histone H3 kinase activity (Fig. 1) and having identified PP1 as a component of isolated chromatin assembled in vitro (Fig. 1), we tested whether PP1 activity regulated the ability of the chromatin-associated kinase activity to efficiently phosphoryl- ate histone H3. We designed an assay that could determine whether PP1 modulates histone H3 phosphorylation by regu- lating a chromatin-associated H3 kinase as opposed to simply dephosphorylating H3 and ...
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... histone phosphorylation is mediated by a protein kinase and regulated by a phosphatase that are stably associated with chromatin and chromosomes. Consistent with events in living cells, histone H3 Ser 10 and the linker histone B4 are phosphorylated in mitotic chromosomes, whereas histones H2A and H4 are phosphorylated in inter- phase chromatin (Fig. 1). Eluates of isolated mitotic chromo- somes contain the mitotic histone H3 kinase, and this activity is associated with the X aurora-B kinase (Figs. 2 and 3). The X aurora-B-associated activity is inactivated by treatment with phosphatase, showing that it requires phosphorylation for its activity. Furthermore, the X aurora-B-associated ...
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Summary The centromere is the chromosomal region in which the kinetochore is assembled to orchestrate chromosome segregation. It is defined by the presence of a histone H3 variant called Centromere Protein A (CENP-A) or CenH3. Propagation of centromere identity entails deposition of new CENP-A upon exit from mitosis in vertebrate cells. A group of...
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... Consistently, OGA inhibitor also decreases H3 phosphorylation [153]. Phosphorylation at H3-S10, S28 and S32 is known to be mutually regulated by Aurora B kinase and protein phosphatase 1 (PP1) [156][157][158]. Interestingly, OGT, OGA, Aurora B and PP1 form a complex in mitosis and localize to midbody, indicating this complex may orchestrate histone o-GlcNAcylation and phosphorylation during mitosis [84]. ...
Prevalent dysregulation of epigenetic modifications plays a pivotal role in cancer. Targeting epigenetic abnormality is a new strategy for cancer therapy. Understanding how conventional oncogenic factors cause epigenetic abnormality is of great basic and translational value. O-GlcNAcylation is a protein modification which affects physiology and pathophysiology. In mammals, O-GlcNAcylation is catalyzed by one single enzyme OGT and removed by one single enzyme OGA. O-GlcNAcylation is affected by the availability of the donor, UDP-GlcNAc, generated by the serial enzymatic reactions in the hexoamine biogenesis pathway (HBP). O-GlcNAcylation regulates a wide spectrum of substrates including many proteins involved in epigenetic modification. Like epigenetic modifications, abnormality of O-GlcNAcylation is also common in cancer. Studies have revealed substantial impact on HBP enzymes and OGT/OGA by oncogenic signals. In this review, we will first summarize how oncogenic signals regulate HBP enzymes, OGT and OGA in cancer. We will then integrate this knowledge with the up to date understanding how O-GlcNAcylation regulates epigenetic machinery. With this, we propose a signal axis from oncogenic signals through O-GlcNAcylation dysregulation to epigenetic abnormality in cancer. Further elucidation of this axis will not only advance our understanding of cancer biology but also provide new revenues towards cancer therapy.
... This places a layer of PPP specificity at the same level as motifs of kinase and pSer/pThr-binding proteins and makes these amino acid sequences crossroads for signaling and motif strength between enzyme classes. In fact, several cases are known for such a scenario: PP1/Repo-Man regulates the highly disordered N-terminus of Histone 3 (H3) [69,70] by dephosphorylating T3 [60] and S10 [71,72] to regulate mitosis, which in turn regulates the association of 14-3-3 proteins [73]. Similarly, the intrinsically disordered region around S455 of IRSp53 is a well-established substrate of the PP1/Phactr complex [74], which is also bound by 14-3-3 proteins [75]. ...
Phosphorylation of the hydroxyl group of the amino acids serine and threonine is among the most prevalent post-translational modifications in mammalian cells. Phospho-serine (pSer) and -threonine (pThr) represent a central cornerstone in the cell's toolbox for adaptation to signal input. The true power for the fast modulation of the regulatory pSer/pThr sites arises from the timely attachment, binding and removal of the phosphate. The phosphorylation of serine and threonine by kinases and the binding of pSer/pThr by phosphorylation-dependent scaffold proteins is largely determined by the sequence motif surrounding the phosphorylation site (p-site). The removal of the phosphate is regulated by pSer/pThr-specific phosphatases with the two most prominent ones being PP1 and PP2A. For this family, recent advances brought forward a more complex mechanism for p-site selection. The interaction of regulatory proteins with the substrate protein constitutes a first layer for substrate recognition, but also interactions of the catalytic subunit with the amino acids in close proximity to pSer/pThr contribute to p-site selection. Here, we review the current pieces of evidence for this multi-layered, complex mechanism and hypothesize that, depending on the degree of higher structure surrounding the substrate site, recognition is more strongly influenced by regulatory subunits away from the active site for structured substrate regions, whereas the motif context is of strong relevance with p-sites in disordered regions. The latter makes these amino acid sequences crossroads for signaling and motif strength between kinases, pSer/pThr-binding proteins and phosphatases.
... Specific histone phosphorylation patterns are regulated during different stages of the cell cycle through different sets of kinases and phosphatases that are stage specific. For instance, proteins phosphatase 1 (PP1) can rapidly neutralize the action of Aurora B kinase, which causes large scale, genome-wide, phosphorylation events at H3S10ph and H3S28ph during mitosis [44]. However, it is currently unclear how the kinases are accurately recruited to phosphorylation sites on histones in chromatin. ...
Liquid-liquid phase separation is increasingly recognized as a phenomenon that affects cell behavior. For example, phase separation of transcription factors and coactivators has been shown to drive efficient transcription. For many years, phase separation of intracellular components has been observed; however, only recently have researchers been able to garner functional significance from such events. Inspired from recent literature that describes phase separation of chromatin in a histone-dependent manner, we review the role and effect of phase separation and histone epigenetics in regulating the genome and discuss how these phenomena can be leveraged to control cell behavior.
... A hypothesis that emerges from these data suggests that histone hyperacetylation-associated chromatin recruitment of PP1α could regulate activity of the kinase(s). While hypo-acetylated histone tails were found to be preferred substrates for AURKB mediated mitotic H3S10ph(50), it was also reported that presence of PP1 negatively regulates the kinase activity of AURKB in Xenopus interphase chromatin extracts (51). Most interestingly, our data suggests PP1α chromatin recruitment to occur irrespective of radiation exposure. ...
Background - Histone Post Translational Modifications (PTMs) change in a cell cycle dependent manner and also orchestrate the DNA repair process for radiation induced DNA damage. Mitosis is the most radiosensitive phase of the cell cycle but the epigenetic events that regulate its radiosensitivity remain elusive.
Results - This study explored the dynamics between histone marks H3S10/S28ph, H3K9ac and γH2AX during mitotic DNA damage response. The presence of a mononucleosome level association between γH2AX and H3S10ph was observed only during mitosis. This association was abrogated upon cell cycle progression and chromatin de-condensation, concomitant with chromatin recruitment of DNA repair proteins Ku70 and Rad51. Moreover, the levels of H3S10/28ph remained unchanged upon DNA damage during mitosis, but decreased in a cell cycle dependent manner upon mitotic exit. However, the population that arose after mitotic progression of damaged cells comprised of binucleated tetraploid cells. This population was epigenetically distinct from interphase cells, characterized by reduced H3S10/S28ph, increased H3K9ac and more open chromatin configuration. These epigenetic features correlated with decreased survival potential of this population. The low levels of H3S10/28ph were attributed to decreased protein translation and chromatin recruitment of histone kinase Mitogen and Stress-activated Kinase 1 (MSK1) along with persistent levels of Protein phosphatase1 catalytic subunit α (PP1α).
Conclusions – This study suggests that a unique epigenetic landscape attained during and after mitotic DNA damage collectively contributed to mitotic radiosensitivity. The findings of this study have potential clinical significance in terms of tackling resistance against anti-mitotic chemotherapeutic agents.
... Aurora B also stimulates Haspin to phosphorylate histone H3 at the chromosome arms for chromatin condensation (9)(10)(11)(12). In contrast, the chromatin-associated protein phosphatase PP1g dephosphorylates histone H3 as well as other phosphorylated chromatin-associated proteins to facilitate chromosome decondensation (13)(14)(15)(16). A recent report analyzed primarily in vitro the global RV(S/T)F motif-containing proteins including Repo-Man that can be regulated by Aurora B and PP1 and revealed that phosphorylation of these proteins by Aurora B may disrupt the binding between PP1 and the RV(S/T)F motif-containing proteins (17). ...
The mitotic kinase Aurora B regulates the condensation of chromatin into chromosomes by phosphorylating chromatin proteins during early mitosis, whereas the phosphatase PP1γ performs the opposite function. The roles of Aurora B and PP1γ must be tightly coordinated to maintain chromosomes at a high phosphorylation state, but the precise mechanisms regulating their function remain largely unclear. Here, mainly through immunofluorescence microscopy and co-immunoprecipitation assays, we find that dissociation of PP1γ from chromosomes is essential for maintaining chromosome phosphorylation. We uncover that PP1γ is recruited to mitotic chromosomes by its regulatory subunit Repo-Man in the absence of Aurora B activity, and that Aurora B regulates dissociation of PP1γ by phosphorylating and disrupting PP1γ-Repo-Man interactions on chromatin. Overexpression of Repo-Man mutants that cannot be phosphorylated or inhibition of Aurora B kinase activity resulted in the retention of PP1γ on chromatin and prolonged the chromatin condensation process; a similar outcome was caused by the ectopic targeting of PP1γ to chromatin. Together, our findings reveal a novel regulation mechanism of chromatin condensation in which Aurora B counteracts PP1γ activity by releasing PP1γ from Repo-Man and provide important implications for understanding the regulations of dynamic structural changes of the chromosomes in mitosis.
... Thus, although PP1γ-KNL1 activity seems to be dispensable for initial microtubule binding, it does contribute to preserve attachment stability following biorientation. In that respect, PP1γ-KNL1 antagonizes the activating T-loop autophosphorylation of Aurora B [385,387,388] and enables the recruitment of the SKA complex to the KMN network [389,390] (Figure 6C,D). The SKA complex at the kinetochore-microtubule interface is proposed to enhance the microtubule-binding affinity and end-tracking ability of the Ndc80 complex [391]. ...
Mitosis requires extensive rearrangement of cellular architecture and of subcellular structures so that replicated chromosomes can bind correctly to spindle microtubules and segregate towards opposite poles. This process originates two new daughter nuclei with equal genetic content and relies on highly-dynamic and tightly regulated phosphorylation of numerous cell cycle proteins. A burst in protein phosphorylation orchestrated by several conserved kinases occurs as cells go into and progress through mitosis. The opposing dephosphorylation events are catalyzed by a small set of protein phosphatases, whose importance for the accuracy of mitosis is becoming increasingly appreciated. This review will focus on the established and emerging roles of mitotic phosphatases, describe their structural and biochemical properties, and discuss recent advances in understanding the regulation of phosphatase activity and function.
... In addition to ␥-H2AX, another important histone phosphorylation event is H3S10p, which is coupled to mitotic chromosome condensation (105). The kinases and phosphatases that regulate this modification have also been identified (106). In addition, histone H3 can be phosphorylated at tyrosine 41 as well as threonine 45, and a recent study in JBC reported that these modifications regulate DNA accessibility in the nucleosome (107). ...
During Herbert Tabor’s tenure as Editor-in-Chief from 1971 to 2010, JBC has published many seminal papers in the fields of chromatin structure, epigenetics, and regulation of transcription in eukaryotes. As of this writing, more than 21,000 studies on gene transcription at the molecular level have been published in JBC since 1971. This brief review will attempt to highlight some of these ground-breaking discoveries and show how early studies published in JBC have influenced current research. Papers published in the Journal have reported the initial discovery of multiple forms of RNA polymerase in eukaryotes, identification and purification of essential components of the transcription machinery, and identification and mechanistic characterization of various transcriptional activators and repressors and include studies on chromatin structure and post-translational modifications of the histone proteins. The large body of literature published in the Journal has inspired current research on how chromatin organization and epigenetics impact regulation of gene expression.
... Since Aurora kinases are phosphorylated, they are obviously targeted by phosphatases. T288 in Aurora-A and T232 in Aurora-B for instance are dephosphorylated and inactivated by type 1 protein phosphatase [101,102]. T288 is also dephosphorylated by PPP6 that specifically target Aurora-A when bound to TPX2 [103]. These dephosphorylations inactivated Aurora kinase activity. ...
Aurora-A is a serine/threonine kinase known to regulate mitotic events in cells. Its over-expression in tumours promote cell survival by modulating cell proliferation and overcoming apoptosis. Previously, it has been shown that Aurora-A is important for the stability of a serine/arginine (SR)-rich protein, ASF/SF2, thereby altering the alternative splicing of apoptosis-related genes. In consistence with this role in splicing, we have discovered that the interactome of Aurora-A contains a large number of spliceosome components - both core and non-core spliceosomal proteins. My PhD work is aimed at characterizing the function of Aurora-A in pre-mRNA splicing and to investigate its function at the molecular level. I show that Aurora-A interacts directly with splicing factors such as SR proteins and hnRNP proteins and also phosphorylates few of them in vitro. I demonstrate that Aurora-A is important for the stability of ASF/SF2 and many other SR proteins. Using immunofluorescence microscopy, I further demonstrate that Aurora-A localizes to nuclear speckles, the sites where splicing proteins are stored, as well as to the periphery of nuclear speckles where a majority of pre-mRNA splicing is known to take place. Notably, addition of recombinant Aurora-A enhances the splicing efficiency of beta-globin pre-mRNA in vitro. Through RNA-seq analysis using the VAST tool, I identified 414 alternative splicing events that are altered upon inhibition of Aurora-A. Surprisingly the important SR protein kinases, CLK1 and CLK4 are also identified to be alternatively spliced in an Aurora-A dependent manner. Altogether my work reveals a broad and novel role of Aurora-A in pre-mRNA splicing and suggests molecular means through which Aurora-A could modulate the spliceosome and splicing factors.
... Aside from HATs and HDACs, further research into chemical modulation of histone kinases, phosphorylases, methyltransferases, and demethylases is needed. For example, inhibitors of protein phosphatase 1 and 2, responsible for removal of H3S10ph, 136,137 as well as inhibitors of JMJD6, a Jumonji-domain-containing protein reported to demethylate H4R3me2, 138 may be useful therapeutic targets for ALS. ...
Amyotrophic lateral sclerosis (ALS) is the third most common adult onset neurodegenerative disorder worldwide. It is generally characterized by progressive paralysis starting at the limbs ultimately leading to death caused by respiratory failure. There is no cure and current treatments fail to slow the progression of the disease. As such, new treatment options are desperately needed. Epigenetic targets are an attractive possibility because they are reversible. Epigenetics refers to heritable changes in gene expression unrelated to changes in DNA sequence. Three main epigenetic mechanisms include the methylation of DNA, microRNAs and the post-translational modification of histone proteins. Histone modifications occur in many amino acid residues and include phosphorylation, acetylation, methylation as well as other chemical moieties. Recent evidence points to a possible role for epigenetic mechanisms in the etiology of ALS. Here, we review recent advances linking ALS and epigenetics, with a strong focus on histone modifications. Both local and global changes in histone modification profiles are associated with ALS drawing attention to potential targets for future diagnostic and treatment approaches.
... Aurora B phosphorylates histone 3 at serine 10 (pH3S10), which occurs during mitosis and is required for proper chromosome condensation and segregation. Mitotic histone H3S10 phosphorylation begins at prophase, and initiates at centromeres and progresses along the whole chromosome arm 18,46,47 . We investigated if SB has any effect on pH3S10 levels. ...
Abstract The mitotic checkpoint ensures proper chromosome segregation; defects in this checkpoint can lead to aneuploidy, a hallmark of cancer. The mitotic checkpoint blocks progression through mitosis as long as chromosomes remain unattached to spindle microtubules. Unattached kinetochores induce the formation of a mitotic checkpoint complex (MCC) composed of Mad2, BubR1, Bub1 and Bub3 which inhibits anaphase onset. Spindle toxins induce prolonged mitotic arrest by creating persistently unattached kinetochores which trigger MCC formation. We find that the multifunctional ser/thr kinase, glycogen synthase kinase 3 (GSK3) is required for a strong mitotic checkpoint. Spindle toxin-induced mitotic arrest is relieved by GSK3 inhibitors SB 415286 (SB), RO 318220 (RO) and lithium chloride. Similarly, targeting GSK3β with knockout or RNAi reduced mitotic arrest in the presence of Taxol. GSK3 was required for optimal localization of Mad2, BubR1, and Bub1 at kinetochores and for optimal assembly of the MCC in spindle toxin-arrested cells. The WNT- and PI3K/Akt signaling pathways negatively regulate GSK3β activity. Inhibition of WNT and PI3K/Akt signaling, in the presence of Taxol, induced a longer mitotic arrest compared to Taxol alone. Our observations provide novel insight into the regulation of the mitotic checkpoint and its connection to growth-signaling pathways.
