Unlocking the Mysteries of Chromatin Biology through Functional Epigenomics.

In the ever evolving field of functional genomics, CRISPR-based screening technologies have become pivotal tools for elucidating gene function across various cell types. A recent study by Gilan and colleagues advances this technological frontier by introducing CRISPR-ChIP, a platform designed to investigate the complex dynamics of epigenetic regulation of chromatin. In proof-of-concept experiments, the authors demonstrate the potential of this tool to identify key molecular regulators of two major histone modifications associated with active transcription, H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 79 dimethylation (H3K79me2). They further unveiled a previously unknown functional partitioning of the H3K79-specific methyltransferase DOT1L into an oncogenic complex with MLL-AF9 and a native complex with MLLT10, which cooperatively regulate mixed lineage leukemia fusion protein (MLL-FP) target gene expression. This novel epigenomic approach integrates high-throughput CRISPR screening with chromatin immunoprecipitation-based direct readout of chromatin modifications in situ, offering a powerful tool to investigate the epigenetic regulatory layers across a diverse spectrum of biological processes and disease states.

Altered chromatin occupancy of patient-associated H4 mutants misregulate neuronal differentiation.

Chromatin is a crucial regulator of gene expression and tightly controls development across species. Mutations in only one copy of multiple histone genes were identified in children with developmental disorders characterized by microcephaly, but their mechanistic roles in development remain unclear. Here we focus on dominant mutations affecting histone H4 lysine 91. These H4K91 mutants form aberrant nuclear puncta at specific heterochromatin regions. Mechanistically, H4K91 mutants demonstrate enhanced binding to the histone variant H3.3, and ablation of H3.3 or the H3.3-specific chaperone DAXX diminishes the mutant localization to chromatin. Our functional studies demonstrate that H4K91 mutant expression increases chromatin accessibility, alters developmental gene expression through accelerating pro-neural differentiation, and causes reduced mouse brain size in vivo, reminiscent of the microcephaly phenotypes of patients. Together, our studies unveil a distinct molecular pathogenic mechanism from other known histone mutants, where H4K91 mutants misregulate cell fate during development through abnormal genomic localization.

A De Novo Sequence Variant in Barrier-to-Autointegration Factor Is Associated with Dominant Motor Neuronopathy.

Barrier-to-autointegration factor (BAF) is an essential component of the nuclear lamina. Encoded by BANF1, this DNA binding protein contributes to the regulation of gene expression, cell cycle progression, and nuclear integrity. A rare recessive BAF variant, Ala12Thr, causes the premature aging syndrome, Néstor-Guillermo progeria syndrome (NGPS). Here, we report the first dominant pathogenic BAF variant, Gly16Arg, identified in a patient presenting with progressive neuromuscular weakness. Although disease variants carry nearby amino acid substitutions, cellular and biochemical properties are distinct. In contrast to NGPS, Gly16Arg patient fibroblasts show modest changes in nuclear lamina structure and increases in repressive marks associated with heterochromatin. Structural studies reveal that the Gly16Arg substitution introduces a salt bridge between BAF monomers, reducing the conformation ensemble available to BAF. We show that this structural change increases the double-stranded DNA binding affinity of BAF Gly16Arg. Together, our findings suggest that BAF Gly16Arg has an increased chromatin occupancy that leads to epigenetic changes and impacts nuclear functions. These observations provide a new example of how a missense mutation can change a protein conformational equilibrium to cause a dominant disease and extend our understanding of mechanisms by which BAF function impacts human health.

A Molecular Switch between Mammalian MLL Complexes Dictates Response to Menin-MLL Inhibition.

Menin interacts with oncogenic MLL1-fusion proteins, and small molecules that disrupt these associations are in clinical trials for leukemia treatment. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacologic, and biochemical approaches, we discovered a conserved molecular switch between the MLL1-Menin and MLL3/4-UTX chromatin-modifying complexes that dictates response to Menin-MLL inhibitors. MLL1-Menin safeguards leukemia survival by impeding the binding of the MLL3/4-UTX complex at a subset of target gene promoters. Disrupting the Menin-MLL1 interaction triggers UTX-dependent transcriptional activation of a tumor-suppressive program that dictates therapeutic responses in murine and human leukemia. Therapeutic reactivation of this program using CDK4/6 inhibitors mitigates treatment resistance in leukemia cells that are insensitive to Menin inhibitors. These findings shed light on novel functions of evolutionarily conserved epigenetic mediators like MLL1-Menin and MLL3/4-UTX and are relevant to understand and target molecular pathways determining therapeutic responses in ongoing clinical trials. SIGNIFICANCE: Menin-MLL inhibitors silence a canonical HOX- and MEIS1-dependent oncogenic gene expression program in leukemia. We discovered a parallel, noncanonical transcriptional program involving tumor suppressor genes that are repressed in Menin-MLL inhibitor-resistant leukemia cells but that can be reactivated upon combinatorial treatment with CDK4/6 inhibitors to augment therapy responses. This article is highlighted in the In This Issue feature, p. 1.

Histone H1 Mutations in Lymphoma: A Link(er) between Chromatin Organization, Developmental Reprogramming, and Cancer.

Aberrant cell fate decisions due to transcriptional misregulation are central to malignant transformation. Histones are the major constituents of chromatin, and mutations in histone-encoding genes are increasingly recognized as drivers of oncogenic transformation. Mutations in linker histone H1 genes were recently identified as drivers of peripheral lymphoid malignancy. Loss of H1 in germinal center B cells results in widespread chromatin decompaction, redistribution of core histone modifications, and reactivation of stem cell-specific transcriptional programs. This review explores how linker histones and mutations therein regulate chromatin structure, highlighting reciprocal relationships between epigenetic circuits, and discusses the emerging role of aberrant three-dimensional chromatin architecture in malignancy.

Histone H1 loss drives lymphoma by disrupting 3D chromatin architecture.

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction1, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B-E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.

Epigenomic Reprogramming as a Driver of Malignant Glioma.

Malignant gliomas are central nervous system tumors and remain among the most treatment-resistant cancers. Exome sequencing has revealed significant heterogeneity and important insights into the molecular pathogenesis of gliomas. Mutations in chromatin modifiers-proteins that shape the epigenomic landscape through remodeling and regulation of post-translational modifications on chromatin-are very frequent and often define specific glioma subtypes. This suggests that epigenomic reprogramming may be a fundamental driver of glioma. Here, we describe the key chromatin regulatory pathways disrupted in gliomas, delineating their physiological function and our current understanding of how their dysregulation may contribute to gliomagenesis.

Mutant EZH2 Induces a Pre-malignant Lymphoma Niche by Reprogramming the Immune Response.

Follicular lymphomas (FLs) are slow-growing, indolent tumors containing extensive follicular dendritic cell (FDC) networks and recurrent EZH2 gain-of-function mutations. Paradoxically, FLs originate from highly proliferative germinal center (GC) B cells with proliferation strictly dependent on interactions with T follicular helper cells. Herein, we show that EZH2 mutations initiate FL by attenuating GC B cell requirement for T cell help and driving slow expansion of GC centrocytes that become enmeshed with and dependent on FDCs. By impairing T cell help, mutant EZH2 prevents induction of proliferative MYC programs. Thus, EZH2 mutation fosters malignant transformation by epigenetically reprograming B cells to form an aberrant immunological niche that reflects characteristic features of human FLs, explaining how indolent tumors arise from GC B cells.

The expanding landscape of 'oncohistone' mutations in human cancers.

Mutations in epigenetic pathways are common oncogenic drivers. Histones, the fundamental substrates for chromatin-modifying and remodelling enzymes, are mutated in tumours including gliomas, sarcomas, head and neck cancers, and carcinosarcomas. Classical 'oncohistone' mutations occur in the N-terminal tail of histone H3 and affect the function of polycomb repressor complexes 1 and 2 (PRC1 and PRC2). However, the prevalence and function of histone mutations in other tumour contexts is unknown. Here we show that somatic histone mutations occur in approximately 4% (at a conservative estimate) of diverse tumour types and in crucial regions of histone proteins. Mutations occur in all four core histones, in both the N-terminal tails and globular histone fold domains, and at or near residues that contain important post-translational modifications. Many globular domain mutations are homologous to yeast mutants that abrogate the need for SWI/SNF function, occur in the key regulatory 'acidic patch' of histones H2A and H2B, or are predicted to disrupt the H2B-H4 interface. The histone mutation dataset and the hypotheses presented here on the effect of the mutations on important chromatin functions should serve as a resource and starting point for the chromatin and cancer biology fields in exploring an expanding role of histone mutations in cancer.

Nuclear lamina dysfunction triggers a germline stem cell checkpoint.

LEM domain (LEM-D) proteins are conserved components of the nuclear lamina (NL) that contribute to stem cell maintenance through poorly understood mechanisms. The Drosophila emerin homolog Otefin (Ote) is required for maintenance of germline stem cells (GSCs) and gametogenesis. Here, we show that ote mutants carry germ cell-specific changes in nuclear architecture that are linked to GSC loss. Strikingly, we found that both GSC death and gametogenesis are rescued by inactivation of the DNA damage response (DDR) kinases, ATR and Chk2. Whereas the germline checkpoint draws from components of the DDR pathway, genetic and cytological features of the GSC checkpoint differ from the canonical pathway. Instead, structural deformation of the NL correlates with checkpoint activation. Despite remarkably normal oogenesis, rescued oocytes do not support embryogenesis. Taken together, these data suggest that NL dysfunction caused by Otefin loss triggers a GSC-specific checkpoint that contributes to maintenance of gamete quality.

Diabetes-linked transcription factor HNF4α regulates metabolism of endogenous methylarginines and ß-aminoisobutyric acid by controlling expression of alanine-glyoxylate aminotransferase 2.

Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1-6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.

Greater Than the Sum of Parts: Complexity of the Dynamic Epigenome.

Information encoded in DNA is interpreted, modified, and propagated as chromatin. The diversity of inputs encountered by eukaryotic genomes demands a matching capacity for transcriptional outcomes provided by the combinatorial and dynamic nature of epigenetic processes. Advances in genome editing, visualization technology, and genome-wide analyses have revealed unprecedented complexity of chromatin pathways, offering explanations to long-standing questions and presenting new challenges. Here, we review recent findings, exemplified by the emerging understanding of crossregulatory interactions within chromatin, and emphasize the pathologic outcomes of epigenetic misregulation in cancer.

Critical Role of Histone Turnover in Neuronal Transcription and Plasticity.

Turnover and exchange of nucleosomal histones and their variants, a process long believed to be static in post-replicative cells, remains largely unexplored in brain. Here, we describe a novel mechanistic role for HIRA (histone cell cycle regulator) and proteasomal degradation-associated histone dynamics in the regulation of activity-dependent transcription, synaptic connectivity, and behavior. We uncover a dramatic developmental profile of nucleosome occupancy across the lifespan of both rodents and humans, with the histone variant H3.3 accumulating to near-saturating levels throughout the neuronal genome by mid-adolescence. Despite such accumulation, H3.3-containing nucleosomes remain highly dynamic-in a modification-independent manner-to control neuronal- and glial-specific gene expression patterns throughout life. Manipulating H3.3 dynamics in both embryonic and adult neurons confirmed its essential role in neuronal plasticity and cognition. Our findings establish histone turnover as a critical and previously undocumented regulator of cell type-specific transcription and plasticity in mammalian brain.

Networking in the nucleus: a spotlight on LEM-domain proteins.

Proteins resident in the inner nuclear membrane and underlying nuclear lamina form a network that regulates nuclear functions. This review highlights a prominent family of nuclear lamina proteins that carries the LAP2-emerin-MAN1-domain (LEM-D). LEM-D proteins share an ability to bind lamins and tether repressive chromatin at the nuclear periphery. The importance of this family is underscored by findings that loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Diverse functions of LEM-D proteins are linked to interactions with unique and overlapping partners including signal transduction effectors, transcription factors and architectural proteins. Recent investigations suggest that LEM-D proteins form hubs within the nuclear lamina that integrate external signals important for tissue homeostasis and maintenance of progenitor cell populations.

Every amino acid matters: essential contributions of histone variants to mammalian development and disease.

Despite a conserved role for histones as general DNA packaging agents, it is now clear that another key function of these proteins is to confer variations in chromatin structure to ensure dynamic patterns of transcriptional regulation in eukaryotes. The incorporation of histone variants is particularly important to this process. Recent knockdown and knockout studies in various cellular systems, as well as direct mutational evidence from human cancers, now suggest a crucial role for histone variant regulation in processes as diverse as differentiation and proliferation, meiosis and nuclear reprogramming. In this Review, we provide an overview of histone variants in the context of their unique functions during mammalian germ cell and embryonic development, and examine the consequences of aberrant histone variant regulation in human disease.

The insulator protein Suppressor of Hairy-wing is an essential transcriptional repressor in the Drosophila ovary.

Suppressor of Hairy-wing [Su(Hw)] is a DNA-binding factor required for gypsy insulator function and female germline development in Drosophila. The insulator function of the gypsy retrotransposon depends on Su(Hw) binding to clustered Su(Hw) binding sites (SBSs) and recruitment of the insulator proteins Centrosomal Protein 190 kD (CP190) and Modifier of mdg4 67.2 kD (Mod67.2). By contrast, the Su(Hw) germline function involves binding to non-clustered SBSs and does not require CP190 or Mod67.2. Here, we identify Su(Hw) target genes, using genome-wide analyses in the ovary to uncover genes with an ovary-bound SBS that are misregulated upon Su(Hw) loss. Most Su(Hw) target genes demonstrate enriched expression in the wild-type CNS. Loss of Su(Hw) leads to increased expression of these CNS-enriched target genes in the ovary and other tissues, suggesting that Su(Hw) is a repressor of neural genes in non-neural tissues. Among the Su(Hw) target genes is RNA-binding protein 9 (Rbp9), a member of the ELAV/Hu gene family. Su(Hw) regulation of Rbp9 appears to be insulator independent, as Rbp9 expression is unchanged in a genetic background that compromises the functions of the CP190 and Mod67.2 insulator proteins, even though both localize to Rbp9 SBSs. Rbp9 misregulation is central to su(Hw)(-/-) sterility, as Rbp9(+/-), su(Hw)(-/-) females are fertile. Eggs produced by Rbp9(+/-), su(Hw)(-/-) females show patterning defects, revealing a somatic requirement for Su(Hw) in the ovary. Our studies demonstrate that Su(Hw) is a versatile transcriptional regulatory protein with an essential developmental function involving transcriptional repression.

Restoration of topoisomerase 2 function by complementation of defective monomers in Drosophila.

Type II topoisomerases are essential ATP-dependent homodimeric enzymes required for transcription, replication, and chromosome segregation. These proteins alter DNA topology by generating transient enzyme-linked double-strand breaks for passage of one DNA strand through another. The central role of type II topoisomerases in DNA metabolism has made these enzymes targets for anticancer drugs. Here, we describe a genetic screen that generated novel alleles of Drosophila Topoisomerase 2 (Top2). Fifteen alleles were obtained, resulting from nonsense and missense mutations. Among these, 14 demonstrated recessive lethality, with one displaying temperature-sensitive lethality. Several newly generated missense alleles carry amino acid substitutions in conserved residues within the ATPase, Topoisomerase/Primase, and Winged helix domains, including four that encode proteins with alterations in residues associated with resistance to cancer chemotherapeutics. Animals lacking zygotic Top2 function can survive to pupation and display reduced cell division and altered polytene chromosome structure. Inter se crosses between six strains carrying Top2 missense alleles generated morphologically normal trans-heterozygous adults, which showed delayed development and were female sterile. Complementation occurred between alleles encoding Top2 proteins with amino acid substitutions in the same functional domain and between alleles encoding proteins with substitutions in different functional domains. Two complementing alleles encode proteins with amino acid substitutions associated with drug resistance. These observations suggest that dimerization of mutant Top2 monomers can restore enzymatic function. Our studies establish the first series of Top2 alleles in a multicellular organism. Future analyses of these alleles will enhance our knowledge about the contributions made by type II topoisomerases to development.

Genome-wide studies of the multi-zinc finger Drosophila Suppressor of Hairy-wing protein in the ovary.

The Drosophila Suppressor of Hairy-wing [Su(Hw)] protein is a globally expressed, multi-zinc finger (ZnF) DNA-binding protein. Su(Hw) forms a classic insulator when bound to the gypsy retrotransposon and is essential for female germline development. These functions are genetically separable, as exemplified by Su(Hw)(f) that carries a defective ZnF10, causing a loss of insulator but not germline function. Here, we completed the first genome-wide analysis of Su(Hw)-binding sites (SBSs) in the ovary, showing that tissue-specific binding is not responsible for the restricted developmental requirements for Su(Hw). Mapping of ovary Su(Hw)(f) SBSs revealed that female fertility requires binding to only one third of the wild-type sites. We demonstrate that Su(Hw)(f) retention correlates with binding site affinity and partnership with Modifier of (mdg4) 67.2 protein. Finally, we identify clusters of co-regulated ovary genes flanked by Su(Hw)(f) bound sites and show that loss of Su(Hw) has limited effects on transcription of these genes. These data imply that the fertility function of Su(Hw) may not depend upon the demarcation of transcriptional domains. Our studies establish a framework for understanding the germline Su(Hw) function and provide insights into how chromatin occupancy is achieved by multi-ZnF proteins, the most common transcription factor class in metazoans.

A conserved long noncoding RNA affects sleep behavior in Drosophila.

Metazoan genomes encode an abundant collection of mRNA-like, long noncoding (lnc)RNAs. Although lncRNAs greatly expand the transcriptional repertoire, we have a limited understanding of how these RNAs contribute to developmental regulation. Here, we investigate the function of the Drosophila lncRNA called yellow-achaete intergenic RNA (yar). Comparative sequence analyses show that the yar gene is conserved in Drosophila species representing 40-60 million years of evolution, with one of the conserved sequence motifs encompassing the yar promoter. Further, the timing of yar expression in Drosophila virilis parallels that in D. melanogaster, suggesting that transcriptional regulation of yar is conserved. The function of yar was defined by generating null alleles. Flies lacking yar RNAs are viable and show no overt morphological defects, consistent with maintained transcriptional regulation of the adjacent yellow (y) and achaete (ac) genes. The location of yar within a neural gene cluster led to the investigation of effects of yar in behavioral assays. These studies demonstrated that loss of yar alters sleep regulation in the context of a normal circadian rhythm. Nighttime sleep was reduced and fragmented, with yar mutants displaying diminished sleep rebound following sleep deprivation. Importantly, these defects were rescued by a yar transgene. These data provide the first example of a lncRNA gene involved in Drosophila sleep regulation. We find that yar is a cytoplasmic lncRNA, suggesting that yar may regulate sleep by affecting stabilization or translational regulation of mRNAs. Such functions of lncRNAs may extend to vertebrates, as lncRNAs are abundant in neural tissues.