Mrc1 regulates parental histone segregation and heterochromatin inheritance.

Chromatin-based epigenetic memory relies on the symmetric distribution of parental histones to newly synthesized daughter DNA strands, aided by histone chaperones within the DNA replication machinery. However, the mechanism of parental histone transfer remains elusive. Here, we reveal that in fission yeast, the replisome protein Mrc1 plays a crucial role in promoting the transfer of parental histone H3-H4 to the lagging strand, ensuring proper heterochromatin inheritance. In addition, Mrc1 facilitates the interaction between Mcm2 and DNA polymerase alpha, two histone-binding proteins critical for parental histone transfer. Furthermore, Mrc1's involvement in parental histone transfer and epigenetic inheritance is independent of its known functions in DNA replication checkpoint activation and replisome speed control. Instead, Mrc1 interacts with Mcm2 outside of its histone-binding region, creating a physical barrier to separate parental histone transfer pathways. These findings unveil Mrc1 as a key player within the replisome, coordinating parental histone segregation to regulate epigenetic inheritance.

A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance.

Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone-binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.

The PCNA-Pol δ complex couples lagging strand DNA synthesis to parental histone transfer for epigenetic inheritance.

Inheritance of epigenetic information is critical for maintaining cell identity. The transfer of parental histone H3-H4 tetramers, the primary carrier of epigenetic modifications on histone proteins, represents a crucial yet poorly understood step in the inheritance of epigenetic information. Here, we show the lagging strand DNA polymerase, Pol δ, interacts directly with H3-H4 and that the interaction between Pol δ and the sliding clamp PCNA regulates parental histone transfer to lagging strands, most likely independent of their roles in DNA synthesis. When combined, mutations at Pol δ and Mcm2 that compromise parental histone transfer result in a greater reduction in nucleosome occupancy at nascent chromatin than mutations in either alone. Last, PCNA contributes to nucleosome positioning on nascent chromatin. On the basis of these results, we suggest that the PCNA-Pol δ complex couples lagging strand DNA synthesis to parental H3-H4 transfer, facilitating epigenetic inheritance.

Coordination of histone chaperones for parental histone segregation and epigenetic inheritance.

Chromatin-based epigenetic memory relies on the accurate distribution of parental histone H3-H4 tetramers to newly replicated DNA strands. Mcm2, a subunit of the replicative helicase, and Dpb3/4, subunits of DNA polymerase ε, govern parental histone H3-H4 deposition to the lagging and leading strands, respectively. However, their contribution to epigenetic inheritance remains controversial. Here, using fission yeast heterochromatin inheritance systems that eliminate interference from initiation pathways, we show that a Mcm2 histone binding mutation severely disrupts heterochromatin inheritance, while mutations in Dpb3/4 cause only moderate defects. Surprisingly, simultaneous mutations of Mcm2 and Dpb3/4 stabilize heterochromatin inheritance. eSPAN (enrichment and sequencing of protein-associated nascent DNA) analyses confirmed the conservation of Mcm2 and Dpb3/4 functions in parental histone H3-H4 segregation, with their combined absence showing a more symmetric distribution of parental histone H3-H4 than either single mutation alone. Furthermore, the FACT histone chaperone regulates parental histone transfer to both strands and collaborates with Mcm2 and Dpb3/4 to maintain parental histone H3-H4 density and faithful heterochromatin inheritance. These results underscore the importance of both symmetric distribution of parental histones and their density at daughter strands for epigenetic inheritance and unveil distinctive properties of parental histone chaperones during DNA replication.

Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

Effectiveness of the treatment of depression associated with cancer and neuroimaging changes in depression-related brain regions in patients treated with the mediator-deuterium acupuncture method.

Cancer patients should be concerned about depression, which can negatively impact their mental health. To develop efficient therapies, it is essential to comprehend the connection between cancer and depression. This study used neuroimaging to investigate the use of mediator-deuterium acupuncture (MDA) for people with cancer-induced depression and its effects on brain regions associated with depression. Resting-state functional magnetic resonance imaging and neurocognitive testing were conducted on the participants, and statistical package for the social sciences was utilized to analyze the behavioral data. Clinical and theoretical data were analyzed to evaluate acupuncture's effectiveness against gynecological cancer. In the research, there were 40 participants, 20 in each group. Except for psychomotor speed, there was no discernible difference in pre-chemotherapy cognitive test results between patients and healthy controls (HCs). However, there were substantial differences in post-treatment cognition test results, showing that the patient group had progressed. According to longitudinal graph analysis, the patient group's local and global brain efficiency significantly declined, and lower local efficiency was associated with lower raw Trail Making Test-A results. Furthermore, poorer verbal memory scores were associated with lower overall performance in the sick group but not in the HC group. According to the research, MDA has potential as a supplemental therapy since it may improve brain function and address depression-related neurological abnormalities in cancer patients. More research is required to fully comprehend the variations between cancer and depression-related brain areas during patient therapy, maybe incorporating MDA.

Leaving histone unturned for epigenetic inheritance.

Post-translational modifications in histones play important roles in regulating chromatin structure and gene expression programs, and the modified histones can be passed on to subsequent generations as an epigenetic memory. The fission yeast has been a great model organism for studying histone modifications in heterochromatin assembly and epigenetic inheritance. Here, we review findings in this organism that cemented the idea of chromatin-based inheritance and highlight recent studies that reveal the role of histone turnover in regulating this process.

Mechanisms of chromatin-based epigenetic inheritance.

Multi-cellular organisms such as humans contain hundreds of cell types that share the same genetic information (DNA sequences), and yet have different cellular traits and functions. While how genetic information is passed through generations has been extensively characterized, it remains largely obscure how epigenetic information encoded by chromatin regulates the passage of certain traits, gene expression states and cell identity during mitotic cell divisions, and even through meiosis. In this review, we will summarize the recent advances on molecular mechanisms of epigenetic inheritance, discuss the potential impacts of epigenetic inheritance during normal development and in some disease conditions, and outline future research directions for this challenging, but exciting field.

The cAMP signaling pathway regulates Epe1 protein levels and heterochromatin assembly.

The epigenetic landscape of a cell frequently changes in response to fluctuations in nutrient levels, but the mechanistic link is not well understood. In fission yeast, the JmjC domain protein Epe1 is critical for maintaining the heterochromatin landscape. While loss of Epe1 results in heterochromatin expansion, overexpression of Epe1 leads to defective heterochromatin. Through a genetic screen, we found that mutations in genes of the cAMP signaling pathway suppress the heterochromatin defects associated with Epe1 overexpression. We further demonstrated that the activation of Pka1, the downstream effector of cAMP signaling, is required for the efficient translation of epe1+ mRNA to maintain Epe1 overexpression. Moreover, inactivation of the cAMP-signaling pathway, either through genetic mutations or glucose deprivation, leads to the reduction of endogenous Epe1 and corresponding heterochromatin changes. These results reveal the mechanism by which the cAMP signaling pathway regulates heterochromatin landscape in fission yeast.

[Effect of acupoint application of Zhanjin Huoxue formula combined with local cold compress on swelling and pain after knee arthroscopy in patients with knee osteoarthritis].

OBJECTIVE: To compare the effect between acupoint application of Zhanjin Huoxue formula combined with local cold compress and simple local cold compress on swelling and pain after knee arthroscopy in patients with knee osteoarthritis (KOA). METHODS: A total of 62 KOA patients with knee swelling after knee arthroscopy were randomly divided into an observation group and a control group, 31 cases in each group. In the control group, cold compress was adopted after surgery, 3 times a day. On the basis of the treatment as the control group, acupoint application of Zhanjin Huoxue formula (angelicae sinensis radix, chuanxiong rhizome, cinnamon twig, poria, etc.) was applied at Liangqiu (ST 34), Xuehai (SP 10), Zusanli (ST 36), Fenglong (ST 40), Sanyinjiao (SP 6), Yinlingquan (SP 9), Yanglingquan (GB 34), Xuanzhong (GB 39) on the affected side in the observation group, 4 h each time, 2 times a day. The treatment was given 7 days in both groups. Before treatment and 1,3,5 and 7 days into treatment, the pain visual analogue scale (VAS) score and swelling value of knee joint (2 cm above the patella upper pole, patella midline, 5 cm below the patella lower pole) were compared in the two groups. RESULTS: The VAS scores 3, 5 and 7 days into treatment were lower than those before treatment in the two groups (P<0.05), and those in the observation group were lower than the control group (P<0.05). The swelling values of 2 cm above the patella upper pole 3, 5 and 7 days into treatment were lower those before treatment in the two groups (P<0.05), and those in the observation group were lower than the control group (P<0.05). The swelling values of patella midline 1, 3, 5 and 7 days into treatment were lower than those before treatment in the two groups (P<0.05), and except for 1 day into treatment, those in the observation group were lower than the control group (P<0.05). The swelling values of 5 cm below the patella lower pole 1 day into treatment in the observation group and 3, 5 and 7 days into treatment in the two groups were lower those before treatment (P<0.05), and except for 1 day into treatment, those in the observation group were lower than the control group (P<0.05). The total effective rate in the observation group was 93.5% (29/31), which was higher than 74.2% (23/31) in the control group (P<0.05). CONCLUSION: Acupoint application of Zhanjin Huoxue formula combined with cold compress could effectively improve the knee joint swelling and pain after arthroscopy in KOA patients, and the curative effect is better than simple cold compress.

The cooperative assembly of shelterin bridge provides a kinetic gateway that controls telomere length homeostasis.

Shelterin is a six-protein complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1 and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.

The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin.

Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild-type histones. One attractive model is that these mutations sequester histone methyltransferases, but genome-wide studies show that mutant histones and histone methyltransferases often do not colocalize. Using chromatin immunoprecipitation sequencing (ChIP-seq), here, we show that, in fission yeast, even though H3K9M-containing nucleosomes are broadly distributed across the genome, the histone H3K9 methyltransferase Clr4 is mainly sequestered at pericentric repeats. This selective sequestration of Clr4 depends not only on H3K9M but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4-associated E3 ubiquitin ligase complex. In vitro, H3K14ub synergizes with H3K9M to interact with Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Moreover, binding kinetics show that H3K14ub overcomes the Clr4 aversion to H3K9M and reduces its dissociation. The selective sequestration model reconciles previous discrepancies and demonstrates the importance of protein-interaction kinetics in regulating biological processes.

The INO80 Complex Regulates Epigenetic Inheritance of Heterochromatin.

One key aspect of epigenetic inheritance is that chromatin structures can be stably inherited through generations after the removal of the signals that establish such structures. In fission yeast, the RNA interference (RNAi) pathway is critical for the targeting of histone methyltransferase Clr4 to pericentric repeats to establish heterochromatin. However, pericentric heterochromatin cannot be properly inherited in the absence of RNAi, suggesting the existence of mechanisms that counteract chromatin structure inheritance. Here, we show that mutations of components of the INO80 chromatin-remodeling complex allow pericentric heterochromatin inheritance in RNAi mutants. The ability of INO80 to counter heterochromatin inheritance is attributed to one subunit, Iec5, which promotes histone turnover at heterochromatin but has little effects on nucleosome positioning at heterochromatin, gene expression, or the DNA damage response. These analyses demonstrate the importance of the INO80 chromatin-remodeling complex in controlling heterochromatin inheritance and maintaining the proper heterochromatin landscape of the genome.

Effects of the microtubule nucleator Mto1 on chromosomal movement, DNA repair, and sister chromatid cohesion in fission yeast.

Although the function of microtubules (MTs) in chromosomal segregation during mitosis is well characterized, much less is known about the role of MTs in chromosomal functions during interphase. In the fission yeast Schizosaccharomyces pombe, dynamic cytoplasmic MT bundles move chromosomes in an oscillatory manner during interphase via linkages through the nuclear envelope (NE) at the spindle pole body (SPB) and other sites. Mto1 is a cytoplasmic factor that mediates the nucleation and attachment of cytoplasmic MTs to the nucleus. Here, we test the function of these cytoplasmic MTs and Mto1 on DNA repair and recombination during interphase. We find that mto1Δ cells exhibit defects in DNA repair and homologous recombination (HR) and abnormal DNA repair factory dynamics. In these cells, sister chromatids are not properly paired, and binding of Rad21 cohesin subunit along chromosomal arms is reduced. Our findings suggest a model in which cytoplasmic MTs and Mto1 facilitate efficient DNA repair and HR by promoting dynamic chromosomal organization and cohesion in the nucleus.

Noncatalytic Function of a JmjC Domain Protein Disrupts Heterochromatin.

Chromatin-modifying enzymes are frequently overexpressed in cancer cells, and their enzymatic activities play important roles in changing the epigenetic landscape responsible for tumorigenesis. However, many of these proteins also execute noncatalytic functions, which are poorly understood. In fission yeast, overexpression of Epe1, a histone demethylase homolog, causes heterochromatin defects. Interestingly, in our recent work, we discovered that overexpressed Epe1 recruits SAGA, a histone acetyltransferase complex important for transcriptional regulation, to disrupt heterochromatin, independent of its demethylase activity. Our findings suggest that overexpressed chromatin-modifying enzymes can alter the epigenetic landscape through changing their proteomic environments, an area that needs to be further explored in dissecting disease etiology associated with overexpression of chromatin regulators.

Anti-silencing factor Epe1 associates with SAGA to regulate transcription within heterochromatin.

Heterochromatin is a highly condensed form of chromatin that silences gene transcription. Although high levels of transcriptional activities disrupt heterochromatin, transcription of repetitive DNA elements and subsequent processing of the transcripts by the RNAi machinery are required for heterochromatin assembly. In fission yeast, a JmjC domain protein, Epe1, promotes transcription of DNA repeats to facilitate heterochromatin formation, but overexpression of Epe1 leads to heterochromatin defects. However, the molecular function of Epe1 is not well understood. By screening the fission yeast deletion library, we found that heterochromatin defects associated with Epe1 overexpression are alleviated by mutations of the SAGA histone acetyltransferase complex. Overexpressed Epe1 associates with SAGA and recruits SAGA to heterochromatin regions, which leads to increased histone acetylation, transcription of repeats, and the disruption of heterochromatin. At its normal expression levels, Epe1 also associates with SAGA, albeit weakly. Such interaction regulates histone acetylation levels at heterochromatin and promotes transcription of repeats for heterochromatin assembly. Our results also suggest that increases of certain chromatin protein levels, which frequently occur in cancer cells, might strengthen relatively weak interactions to affect the epigenetic landscape.

Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance.

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome.

Molecular basis for the role of oncogenic histone mutations in modulating H3K36 methylation.

Histone H3 lysine 36 methylation (H3K36me) is critical for epigenetic regulation and mutations at or near H3K36 are associated with distinct types of cancers. H3K36M dominantly inhibits H3K36me on wild-type histones, whereas H3G34R/V selectively affects H3K36me on the same histone tail. Here we report the crystal structures of SETD2 SET domain in complex with an H3K36M peptide and SAM or SAH. There are large conformational changes in the substrate binding regions of the SET domain, and the K36M residue interacts with the catalytic pocket of SETD2. H3G34 is surrounded by a very narrow tunnel, which excludes larger amino acid side chains. H3P38 is in the trans configuration, and the cis configuration is incompatible with SETD2 binding. Finally, mutations of H3G34 or H3P38 alleviate the inhibitory effects of H3K36M on H3K36me, demonstrating that the stable interaction of H3K36M with SETD2 is critical for its inhibitory effects.

A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading.

Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.

New Insights into the Regulation of Heterochromatin.

All living organisms are constantly exposed to stresses from internal biological processes and surrounding environments, which induce many adaptive changes in cellular physiology and gene expression programs. Unexpectedly, constitutive heterochromatin, which is generally associated with the stable maintenance of gene silencing, is also dynamically regulated in response to stimuli. In this review we discuss the mechanism of constitutive heterochromatin assembly, its dynamic nature, and its responses to environmental changes.