Regulation Of Heterochromatin Research Articles

Prevalent Fast Evolution of Genes Involved in Heterochromatin Functions.

Heterochromatin is a gene-poor and repeat-rich genomic compartment universally found in eukaryotes. Despite its low transcriptional activity, heterochromatin plays important roles in maintaining genome stability, organizing chromosomes, and suppressing transposable elements. Given the importance of these functions, it is expected that genes involved in heterochromatin regulation would be highly conserved. Yet, a handful of these genes were found to evolve rapidly. To investigate whether these previous findings are anecdotal or general to genes modulating heterochromatin, we compile an exhaustive list of 106 candidate genes involved in heterochromatin functions and investigate their evolution over short and long evolutionary time scales in Drosophila. Our analyses find that these genes exhibit significantly more frequent evolutionary changes, both in the forms of amino acid substitutions and gene copy number change, when compared to genes involved in Polycomb-based repressive chromatin. While positive selection drives amino acid changes within both structured domains with diverse functions and intrinsically disordered regions, purifying selection may have maintained the proportions of intrinsically disordered regions of these proteins. Together with the observed negative associations between the evolutionary rate of these genes and the genomic abundance of transposable elements, we propose an evolutionary model where the fast evolution of genes involved in heterochromatin functions is an inevitable outcome of the unique functional roles of heterochromatin, while the rapid evolution of transposable elements may be an effect rather than cause. Our study provides an important global view of the evolution of genes involved in this critical cellular domain and provides insights into the factors driving the distinctive evolution of heterochromatin.

Prevalent fast evolution of genes involved in heterochromatin functions.

Heterochromatin is a gene-poor and repeat-rich genomic compartment universally found in eukaryotes. Despite its low transcriptional activity, heterochromatin plays important roles in maintaining genome stability, organizing chromosomes, and suppressing transposable elements (TEs). Given the importance of these functions, it is expected that the genes involved in heterochromatin regulation would be highly conserved. Yet, a handful of these genes were found to evolve rapidly. To investigate whether these previous findings are anecdotal or general to genes modulating heterochromatin, we compile an exhaustive list of 106 candidate genes involved in heterochromatin functions and investigate their evolution over short and long evolutionary time scales in Drosophila. Our analyses find that these genes exhibit significantly more frequent evolutionary changes, both in the forms of amino acid substitutions and gene copy number change, when compared to genes involved in Polycomb-based repressive chromatin. While positive selection drives amino acid changes within both structured domains with diverse functions and intrinsically disordered regions (IDRs), purifying selection may have maintained the proportions of IDRs of these proteins. Together with the observed negative associations between evolutionary rates of these genes and genomic TE abundance, we propose an evolutionary model where the fast evolution of genes involved in heterochromatin functions is an inevitable outcome of the unique functional roles of heterochromatin, while the rapid evolution of TEs may be an effect rather than cause. Our study provides an important global view of the evolution of genes involved in this critical cellular domain and provides insights into the factors driving the distinctive evolution of heterochromatin.

The SUN-family protein Sad1 mediates heterochromatin spatial organization through interaction with histone H2A-H2B

Heterochromatin is generally associated with the nuclear periphery, but how the spatial organization of heterochromatin is regulated to ensure epigenetic silencing remains unclear. Here we found that Sad1, an inner nuclear membrane SUN-family protein in fission yeast, interacts with histone H2A-H2B but not H3-H4. We solved the crystal structure of the histone binding motif (HBM) of Sad1 in complex with H2A-H2B, revealing the intimate contacts between Sad1HBM and H2A-H2B. Structure-based mutagenesis studies revealed that the H2A-H2B-binding activity of Sad1 is required for the dynamic distribution of Sad1 throughout the nuclear envelope (NE). The Sad1-H2A-H2B complex mediates tethering telomeres and the mating-type locus to the NE. This complex is also important for heterochromatin silencing. Mechanistically, H2A-H2B enhances the interaction between Sad1 and HDACs, including Clr3 and Sir2, to maintain epigenetic identity of heterochromatin. Interestingly, our results suggest that Sad1 exhibits the histone-enhanced liquid-liquid phase separation property, which helps recruit heterochromatin factors to the NE. Our results uncover an unexpected role of SUN-family proteins in heterochromatin regulation and suggest a nucleosome-independent role of H2A-H2B in regulating Sad1’s functionality.

Phosphorylation regulates tau’s phase separation behavior and interactions with chromatin

Tau is a microtubule-associated protein often found in neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer’s disease. Beyond this context, mounting evidence suggests that tau localizes into the nucleus, where it may play a role in DNA protection and heterochromatin regulation. The molecular mechanisms behind these observations are currently unclear. Using in vitro biophysical experiments, here we demonstrate that tau can undergo liquid-liquid phase separation (LLPS) with DNA, mononucleosomes, and reconstituted nucleosome arrays under low salt conditions. Low concentrations of tau promote chromatin compaction and protect DNA from digestion. While the material state of samples at physiological salt is dominated by chromatin oligomerization, tau can still associate strongly and reversibly with nucleosome arrays. These properties are driven by tau’s strong interactions with linker and nucleosomal DNA. In addition, tau co-localizes into droplets formed by nucleosome arrays and phosphorylated HP1α, a key heterochromatin constituent thought to function through an LLPS mechanism. Importantly, LLPS and chromatin interactions are disrupted by aberrant tau hyperphosphorylation. These biophysical properties suggest that tau may directly impact DNA and chromatin accessibility and that loss of these interactions could contribute to the aberrant nuclear effects seen in tau pathology.

Baf-mediated transcriptional regulation of teashirt is essential for the development of neural progenitor cell lineages

Accumulating evidence hints heterochromatin anchoring to the inner nuclear membrane as an upstream regulatory process of gene expression. Given that the formation of neural progenitor cell lineages and the subsequent maintenance of postmitotic neuronal cell identity critically rely on transcriptional regulation, it seems possible that the development of neuronal cells is influenced by cell type-specific and/or context-dependent programmed regulation of heterochromatin anchoring. Here, we explored this possibility by genetically disrupting the evolutionarily conserved barrier-to-autointegration factor (Baf) in the Drosophila nervous system. Through single-cell RNA sequencing, we demonstrated that Baf knockdown induces prominent transcriptomic changes, particularly in type I neuroblasts. Among the differentially expressed genes, our genetic analyses identified teashirt (tsh), a transcription factor that interacts with beta-catenin, to be closely associated with Baf knockdown-induced phenotypes that were suppressed by the overexpression of tsh or beta-catenin. We also found that Baf and tsh colocalized in a region adjacent to heterochromatin in type I NBs. Notably, the subnuclear localization pattern remained unchanged when one of these two proteins was knocked down, indicating that both proteins contribute to the anchoring of heterochromatin to the inner nuclear membrane. Overall, this study reveals that the Baf-mediated transcriptional regulation of teashirt is a novel molecular mechanism that regulates the development of neural progenitor cell lineages.

Investigating the Role of the SETDB1 Target TANGO6 (TMCO7) in Leukemia

Despite advancements in AML therapies over the last 2 decades, 1 out of 3 patients diagnosed with AML succumb to their disease. The mainstay of treatment remains systemic chemotherapy, however better understanding of the genetic drivers in AML has generated new therapeutic opportunities. Targeted therapies have emerged to more precisely tailor leukemic treatment based on genetic and epigenetic drivers. We previously demonstrated that SETDB1, a H3K9 methyltransferase involved in heterochromatin regulation, negatively regulates the expression of proleukemic genes Hoxa9 and Meis1. Further, overexpression of SETDB1 correlates with improved overall AML patient survival and extends leukemic disease latency in mouse models (Ropa et al. Haematologica 2020). Here we describe a novel SETDB1 target, TANGO6, and aim to explore its role in leukemogenesis. We mined our genome wide expression and epigenetic datasets generated upon overexpression of SETDB1 to identify targets that may be important for leukemic progression (Ropa et al. Haematologica 2020). TANGO6 was found to be transcriptionally and epigenetically regulated by SETDB1, with little known about its role in leukemia. We performed a targeted CRISPR-Cas9 mediated knock out of TANGO6 to investigate its importance in several human leukemic cell lines: MV4;11, K562 and KOPN8. We established the above cell lines with Cas9 constitutively expressed and introduced sgRNAs targeting TANGO6 by lentivirus. We then performed a competitive growth assay using flow cytometry to assess cell fitness on days 3, 14, 10, 21. We observed that loss of TANGO6 resulted in decreased fitness across all three leukemic cell lines compared to the negative control. This phenotype was more striking in the K562 and KOPN8 cell lines. In lieu of having a reliable antibody to visualize changes in protein expression, we assessed mutational changes in the DNA sequence to evaluate efficiency of our CRISPR/Cas9 system. Genomic DNA was prepared from sg TANGO6 transduced K562 cells on days 5 and 10. The targeted region of TANGO6 was PCR amplified and TOPO cloned for direct Sanger sequencing. 92% (12 out of 13 samples) showed a mutation involving the target sequence. Of these mutations, ~67% resulted in frameshift mutations leading to early truncation of the protein. The remaining mutations involved deletions ranging from 1 to 28 amino acids, with one sample resulting in two single point mutations conferring a change of leucine to serine and lysine to arginine. These mutations may or may not affect the folding of the protein. This data demonstrates we are successfully targeting TANGO6, which produces the biological phenotype in our competitive growth assay. To further understand the potential importance of TANGO6 in human leukemia, we performed qPCR on 5 leukemic cell lines (2 MLL-rearranged AML cell lines, 2 B-lymphoblastic cell lines, and 1 CML cell line). Our data showed that TANGO6 was expressed at higher levels across all leukemic cell lines compared to normal CD34+ hematopoietic stem/progenitor cells, consistent with a role in leukemogenesis. Our preliminary data suggests that TANGO6 plays an important role in leukemogenesis. Ongoing work focuses on validating this hypothesis using in vivo leukemic mouse models and protein localization studies. Given the lack of available information on human TANGO6, characterizing its location and mechanism is essential to understanding its role in disease. Our work has established the need for further exploration of TANGO6 as a potential therapeutic target for AML.

HHCDB: a database of human heterochromatin regions.

Heterochromatin plays essential roles in eukaryotic genomes, such as regulating genes, maintaining genome integrityand silencing repetitive DNA elements. Identifying genome-wide heterochromatin regions is crucial for studying transcriptional regulation. We propose the Human Heterochromatin Chromatin Database (HHCDB) for archiving heterochromatin regions defined by specific or combined histone modifications (H3K27me3, H3K9me2, H3K9me3) according to a unified pipeline. 42839743 heterochromatin regions were identified from 578 samples derived from 241 cell-types/cell lines and 92 tissue types. Genomic information is provided in HHCDB, including chromatin location, gene structure, transcripts, distance from transcription start site, neighboring genes, CpG islands, transposable elements, 3D genomic structureand functional annotations. Furthermore, transcriptome data from 73 single cells were analyzed and integrated to explore cell type-specific heterochromatin-related genes. HHCDB affords rich visualization through the UCSC Genome Browser and our self-developed tools. We have also developed a specialized online analysis platform to mine differential heterochromatin regions in cancers. We performed several analyses to explore the function of cancer-specific heterochromatin-related genes, including clinical feature analysis, immune cell infiltration analysis and the construction of drug-target networks. HHCDB is a valuable resource for studying epigenetic regulation, 3D genomicsand heterochromatin regulation in development and disease. HHCDB is freely accessible at http://hhcdb.edbc.org/.

Alpha Satellite DNA in Targeted Drug Therapy for Prostate Cancer.

Prostate cancer is the most common solid cancer in men and, despite the development of many new therapies, metastatic castration-resistant prostate cancer still remains a deadly disease. Therefore, novel concepts for the treatment of metastatic prostate cancer are needed. In our opinion, the role of the non-coding part of the genome, satellite DNA in particular, has been underestimated in relation to diseases such as cancer. Here, we hypothesise that this part of the genome should be considered as a potential target for the development of new drugs. Specifically, we propose a novel concept directed at the possible treatment of metastatic prostate cancer that is mostly based on epigenetics. Namely, metastatic prostate cancer is characterized by the strongly induced transcription of alpha satellite DNA located in pericentromeric heterochromatin and, according to our hypothesis, the stable controlled transcription of satellite DNA might be important in terms of the control of disease development. This can be primarily achieved through the epigenetic regulation of pericentromeric heterochromatin by using specific enzymes as well as their activators/inhibitors that could act as potential anti-prostate cancer drugs. We believe that our concept is innovative and should be considered in the potential treatment of prostate cancer in combination with other more conventional therapies.

Pervasive SUMOylation of heterochromatin and piRNA pathway proteins

Genome regulation involves complex protein interactions that are often mediated through post-translational modifications (PTMs). SUMOylation-modification by the small ubiquitin-like modifier (SUMO)-has been implicated in numerous essential processes in eukaryotes. In Drosophila, SUMO is required for viability and fertility, with its depletion from ovaries leading to heterochromatin loss and ectopic transposon andgene activation. Here, we developed a proteomics-based strategy to uncover the Drosophila ovarian "SUMOylome," which revealed that SUMOylation is widespread among proteins involved in heterochromatin regulation and different aspects of the Piwi-interacting small RNA (piRNA) pathway that represses transposons. Furthermore, we show that SUMOylation of several piRNA pathway proteins occurs in a Piwi-dependent manner. Together, these data highlight broad implications of protein SUMOylation in epigenetic regulation and indicate novel roles of this modification in the cellular defense against genomic parasites. Finally, this work provides a resource for the study of SUMOylation in other biological contexts in the Drosophila model.

Understanding HAT1: A Comprehensive Review of Noncanonical Roles and Connection with Disease.

Histone acetylation plays a vital role in organizing chromatin, regulating gene expression and controlling the cell cycle. The first histone acetyltransferase to be identified was histone acetyltransferase 1 (HAT1), but it remains one of the least understood acetyltransferases. HAT1 catalyzes the acetylation of newly synthesized H4 and, to a lesser extent, H2A in the cytoplasm. However, 20 min after assembly, histones lose acetylation marks. Moreover, new noncanonical functions have been described for HAT1, revealing its complexity and complicating the understanding of its functions. Recently discovered roles include facilitating the translocation of the H3H4 dimer into the nucleus, increasing the stability of the DNA replication fork, replication-coupled chromatin assembly, coordination of histone production, DNA damage repair, telomeric silencing, epigenetic regulation of nuclear lamina-associated heterochromatin, regulation of the NF-κB response, succinyl transferase activity and mitochondrial protein acetylation. In addition, the functions and expression levels of HAT1 have been linked to many diseases, such as many types of cancer, viral infections (hepatitis B virus, human immunodeficiency virus and viperin synthesis) and inflammatory diseases (chronic obstructive pulmonary disease, atherosclerosis and ischemic stroke). The collective data reveal that HAT1 is a promising therapeutic target, and novel therapeutic approaches, such as RNA interference and the use of aptamers, bisubstrate inhibitors and small-molecule inhibitors, are being evaluated at the preclinical level.

Canonical and non-canonical functions of STAT in germline stem cell maintenance.

Maintenance of the Drosophila male germline stem cells (GSCs) requires activation of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway by niche signals. The precise role of JAK/STAT signaling in GSC maintenance, however, remains incompletely understood. Here, we show that, GSC maintenance requires both canonical and non-canonical JAK/STAT signaling, in which unphosphorylated STAT (uSTAT) maintains heterochromatin stability by binding to heterochromatin protein 1 (HP1). We found that GSC-specific overexpressing STAT, or even the transcriptionally inactive mutant STAT, increases GSC number and partially rescues the GSC-loss mutant phenotype due to reduced JAK activity. Furthermore, we found that both HP1 and STAT are transcriptional targets of the canonical JAK/STAT pathway in GSCs, and that GSCs exhibit higher heterochromatin content. These results suggest that persistent JAK/STAT activation by niche signals leads to the accumulation of HP1 and uSTAT in GSCs, which promote heterochromatin formation important for maintaining GSC identity. Thus, the maintenance of Drosophila GSCs requires both canonical and non-canonical STAT functions within GSCs for heterochromatin regulation.

Understanding the effect of tau protein phosphorylation on nucleosome array compaction.

Tau is an intrinsically disordered, microtubule associated protein that is mainly expressed in neurons and responsible for stabilizing microtubules in axons. The hyperphosphorylation of tau causes it to dissociate from microtubules which promotes its aggregation into neurofibrillary tangles (NFTs). This can significantly affect brain function. Tau is also found in the nucleus and plays an important role in DNA protection, chromosome stability, and heterochromatin regulation. Recent work has found a correlation between disease-relevant mutations of tau with heterochromatin relaxation, destabilization, and aberrant gene expression. Understanding how tau interacts with nucleosome arrays would give us insight into tau's role in gene expression and regulation. Here, we investigate the question of how the concentration and phosphorylation state of tau affects interactions with nucleosome arrays and their compaction state? We explored this question by using micrococcal nuclease digestion assays to determine the extent of tau's protection on nucleosome arrays. We found that an increase in wild type tau concentration leads to a dramatic increase in array protection but phosphorylated tau shows less dramatic protection patterns. These results provide insights into how tau might be involved in DNA protection and how this process might be disrupted in disease.

Investigations into the phase separation and nuclear role of tau.

Tau is a microtubule-associated protein and pathological hallmark of Alzheimer's disease (AD), most infamous for becoming hyperphosphorylated and fibrilizing into neurofibrillary tangles (NFTs). Beyond this role, mounting evidence suggests that tau localizes into the nucleus and plays unknown roles in DNA protection and heterochromatin regulation. Intriguingly, frontotemporal dementia mutants (P301L) of tau show loss of genetically silent heterochromatin clusters. This has been associated with aberrant expression of heterochromatic genes and in other studies, of transposable element activation in AD patient brain tissue. Similar effects of heterochromatin relaxation and gene dysregulation have been observed in tau knockouts, suggesting that loss of tau is pathologically relevant. Recent literature showed that tau undergoes liquid-liquid phase separation (LLPS). Studies have not fully described the functional role of nuclear tau in gene regulation, and whether this involves LLPS. My work demonstrates that tau has an intrinsic ability to phase separate with, compact, and oligomerize chromatin, likely through its DNA-binding domain, and this is regulated by phosphorylation. In addition, tau phase separates with heterochromatin protein 1α (HP1α), an essential heterochromatin constituent, which may promote its partitioning into heterochromatin despite its lack of DNA-sequence specificity. These data imply a potential role for tau LLPS in the maintenance of chromatin compaction with aging.

Molecular features underlying phase separation of protein-nucleic acid condensates revealed by a new coarse-grained DNA model.

Recent advances in residue-level coarse-grained (CG) computational models for proteins have enabled molecular-level insights into the properties of protein condensates. However, most of the existing CG nucleic acid models include anisotropic potentials between bases that are computationally demanding and are not fully compatible with the existing CG protein models. Here, we present a new CG DNA model that utilizes isotropic potentials between bases and can be used for investigating protein-DNA complexes and nucleosome arrays, thereby enabling a mechanistic understanding of how molecular information is propagated up at the genome level in a computationally tractable manner. To demonstrate the suitability of our model to facilitate large-scale simulations with a molecular resolution, we simulate nucleosomes (mono, di, etc.) to generate equilibrium conformational ensembles. The simulation data provide essential insights into the role of chromatin in the liquid-liquid phase separation of HP1α protein. We find an extensive network of interactions between HP1α, DNA, and flexible histone tails in the condensates formed by these different biomolecules. The findings of this work illuminate the complex molecular framework that contributes to heterochromatin regulation and function.

Tuning the phase separation properties of HP1α through ligand interactions.

Heterochromatin protein 1α (HP1α) is a crucial component for gene repression and the structural integrity of heterochromatin. It is proposed that HP1α functions through a liquid-liquid phase separation (LLPS) mechanism where HP1α compacts nucleosome arrays into a liquid compartment and prevents transcription factors from accessing chromatin. HP1α can undergo LLPS via phosphorylation of the N-terminus extension (NTE) or through interactions with DNA/nucleosomes. The multidomain architecture of HP1α allows for simultaneous multivalent interactions with other HP1α proteins and multiple binding partners. These multivalent interactions are believed to be important for forming and maintaining heterochromatin structure but their exact role in HP1α LLPS is not well understood. In our study, we used experimental and computational approaches to investigate how a series of peptides from HP1α binding partners can fine tune the ability of the protein to undergo phase separation. Our results showed that in phosphorylation-driven LLPS, positively charged peptides enhance phase separation, while negatively or near neutral charged peptides disrupt the process. DNA-driven LLPS can also be inhibited by negatively or near neutral charged ligands. However, in this case, positively charged peptides compete with HP1α for DNA interactions, which also disrupts LLPS. Our results suggest that phosphorylation-driven LLPS has a larger tunability range compared to phase separation with DNA, and that the electrostatic properties of HP1α ligands play an important role in modulating this process. Our study provides insights into the potential mechanisms of heterochromatin regulation where binding partners may modulate the biophysical properties of HP1α to control access to chromatin and the genetic information in the cell.

Chromatin remodelers HELLS, WDHD1 and BAZ1A are dynamically expressed during mouse spermatogenesis.

Proper regulation of heterochromatin is critical for spermatogenesis. This study reveals the dynamic localization patterns of distinct chromatin regulators during spermatogenesis and disrupted sex chromatin status in spermatocytes in the absence of DICER. Heterochromatin is dynamically formed and organized in differentiating male germ cells, and its proper regulation is a prerequisite for normal spermatogenesis. While heterochromatin is generally transcriptionally silent, we have previously shown that major satellite repeat (MSR) DNA in the pericentric heterochromatin (PCH) is transcribed during spermatogenesis. We have also shown that DICER associates with PCH and is involved in the regulation of MSR-derived transcripts. To shed light on the heterochromatin regulation in the male germline, we studied the expression, localization and heterochromatin association of selected testis-enriched chromatin regulators in the mouse testis. Our results show that HELLS, WDHD1 and BAZ1A are dynamically expressed during spermatogenesis. They display limited overlap in expression, suggesting involvement in distinct heterochromatin-associated processes at different steps of differentiation. We also show that HELLS and BAZ1A interact with DICER and MSR chromatin. Interestingly, deletion of Dicer1 affects the sex chromosome heterochromatin status in late pachytene spermatocytes, as demonstrated by mislocalization of Polycomb protein family member SCML1 to the sex body. These data substantiate the importance of dynamic heterochromatin regulation during spermatogenesis and emphasize the key role of DICER in the maintenance of chromatin status in meiotic male germ cells.

Molecular interactions underlying the phase separation of HP1α: role of phosphorylation, ligandand nucleic acid binding.

Heterochromatin protein 1α (HP1α) is a crucial element of chromatin organization. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can undergo phase separation upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. Here, we combine computational and experimental approaches to elucidate the molecular interactions that drive these processes. In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged HP1α peptide ligands and disrupted by the addition of negatively charged or neutral peptides. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in HP1α LLPS where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine-tune the properties of HP1α and that can contribute to heterochromatin regulation and function.

SESAME-catalyzed H3T11 phosphorylation inhibits Dot1-catalyzed H3K79me3 to regulate autophagy and telomere silencing

The glycolytic enzyme, pyruvate kinase Pyk1 maintains telomere heterochromatin by phosphorylating histone H3T11 (H3pT11), which promotes SIR (silent information regulator) complex binding at telomeres and prevents autophagy-mediated Sir2 degradation. However, the exact mechanism of action for H3pT11 is poorly understood. Here, we report that H3pT11 directly inhibits Dot1-catalyzed H3K79 tri-methylation (H3K79me3) and uncover how this histone crosstalk regulates autophagy and telomere silencing. Mechanistically, Pyk1-catalyzed H3pT11 directly reduces the binding of Dot1 to chromatin and inhibits Dot1-catalyzed H3K79me3, which leads to transcriptional repression of autophagy genes and reduced autophagy. Despite the antagonism between H3pT11 and H3K79me3, they work together to promote the binding of SIR complex at telomeres to maintain telomere silencing. Furthermore, we identify Reb1 as a telomere-associated factor that recruits Pyk1-containing SESAME (Serine-responsive SAM-containing Metabolic Enzyme) complex to telomere regions to phosphorylate H3T11 and prevent the invasion of H3K79me3 from euchromatin into heterochromatin to maintain telomere silencing. Together, these results uncover a histone crosstalk and provide insights into dynamic regulation of silent heterochromatin and autophagy in response to cell metabolism.

Redistribution of lamina-associated domains reshapes binding of pioneer factor FOXA2 in development of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in type 2 diabetes mellitus and the elderly, impacting 40% of individuals over 70. Regulation of heterochromatin at the nuclear lamina has been associated with aging and age-dependent metabolic changes. We previously showed that changes at the lamina in aged hepatocytes and laminopathy models lead to redistribution of lamina-associated domains (LADs), opening of repressed chromatin, and up-regulation of genes regulating lipid synthesis and storage, culminating in fatty liver. Here, we test the hypothesis that change in the expression of lamina-associated proteins and nuclear shape leads to redistribution of LADs, followed by altered binding of pioneer factor FOXA2 and by up-regulation of lipid synthesis and storage, culminating in steatosis in younger NAFLD patients (aged 21-51). Changes in nuclear morphology alter LAD partitioning and reduced lamin B1 signal correlate with increased FOXA2 binding before severe steatosis in young mice placed on a western diet. Nuclear shape is also changed in younger NAFLD patients. LADs are redistrubted and lamin B1 signal decreases similarly in mild and severe steatosis. In contrast, FOXA2 binding is similar in normal and NAFLD patients with moderate steatosis and is repositioned only in NAFLD patients with more severe lipid accumulation. Hence, changes at the nuclear lamina reshape FOXA2 binding with progression of the disease. Our results suggest a role for nuclear lamina in etiology of NAFLD, irrespective of aging, with potential for improved stratification of patients and novel treatments aimed at restoring nuclear lamina function.

Chromatin structure can introduce systematic biases in genome-wide analyses of Plasmodium falciparum.

Background: The maintenance, regulation, and dynamics of heterochromatin in the human malaria parasite, Plasmodium falciparum, has drawn increasing attention due to its regulatory role in mutually exclusive virulence gene expression and the silencing of key developmental regulators. The advent of genome-wide analyses such as chromatin-immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental in understanding chromatin composition; however, even in model organisms, ChIP-seq experiments are susceptible to intrinsic experimental biases arising from underlying chromatin structure. Methods: We performed a control ChIP-seq experiment, re-analyzed previously published ChIP-seq datasets and compared different analysis approaches to characterize biases of genome-wide analyses in P. falciparum. Results: We found that heterochromatic regions in input control samples used for ChIP-seq normalization are systematically underrepresented in regard to sequencing coverage across the P. falciparum genome. This underrepresentation, in combination with a non-specific or inefficient immunoprecipitation, can lead to the identification of false enrichment and peaks across these regions. We observed that such biases can also be seen at background levels in specific and efficient ChIP-seq experiments. We further report on how different read mapping approaches can also skew sequencing coverage within highly similar subtelomeric regions and virulence gene families. To ameliorate these issues, we discuss orthogonal methods that can be used to characterize bona fide chromatin-associated proteins. Conclusions: Our results highlight the impact of chromatin structure on genome-wide analyses in the parasite and the need for caution when characterizing chromatin-associated proteins and features.