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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. Mutations in histone H1 induce the remodelling of chromatin architecture to a more relaxed state, which leads to malignant transformation through changes in histone modifications and the expression of stem cell genes.

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.

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., Otefin is a nuclear lamina protein required for survival of Drosophila germ stem cells. Here the authors show that nuclear lamina dysfunction resulting from loss of Otefin activates a DNA damage-independent germ stem cell-specific checkpoint, mediated by the ATR and Chk2 kinases, which ensures that healthy gametes are passed on to the next generation.

International audience; 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.

Somatic missense mutations in histone 1 genes occur in ~30% of follicular lymphomas and DLBCL and 85% of Hodgkin's lymphomas, with significant mutual co-occurrence among these alleles, most frequently involving H1C and H1E. We crossed constitutive H1C+/-H1E+/- mice with VavP-Bcl2 transgenic mice and observed significant acceleration of lymphomagenesis (p=0.0001). Lymphoma H1 mutations affect the H1 globular domain or C-terminus. We found that the globular domain mutants fail to insert into chromatin whereas C-ter mutants fail to compact chromatin as shown by atomic force microscopy, in vitro assembled nucleosome arrays, and FRET assays in live cells. Hence both types of mutation confer loss of function. Constitutive H1C/E knockout mice are healthy and have no overt phenotype. However, immunization with T-cell dependent antigen caused significant GC hyperplasia (p=0.013) and disruption of polarity due to expansion in the number of centrocytes. Notably, H1C/EDKO GC B-cells readily outcompeted WT GC B-cells in mixed chimera experiments indicating that they have superior fitness (p=0.0086). To understand the mechanism through which this occurs we performed RNA-seq in H1C/EDKO GC B-cells which revealed an aberrant gene expression signature composed almost entirely of transcriptional activation (n=721 upregulated and n=61 downregulated q=0.05, LFC=log(1.5)). Strikingly, these same genes are upregulated during induced pluripotency (iPS cell) reprogramming, and are normally silenced during early development by the PRC2 complex (p This prompted us to extensively characterize the epigenome of purified H1C/EDKO vs WT GC B-cells using High-throughput chromosome conformation capture (Hi-C), ATAC-seq, and ChIP-seq for multiple histone marks. From the topological standpoint the genome is distributed in two compartments: Compartment A, consisting of unpacked chromatin available for gene regulatory processes, and compartment B, which is highly packed, silenced and inaccessible. Beyond this organization, sets of genes are organized into boundary delimited domains called TADs that share regulatory information. Remarkably, the primary effect of H1 loss of function was the shifting of approximately 256 TADs from compartment B to compartment A (there was no A to B shift). These TADs manifested highly significant gain of chromatin accessibility by ATACs-seq and featured reciprocal loss of H3K27me3 and gain of H3K36me2. These "B to A" TADs yielded increased looping connectivity and contained the primitive stem cell genes that were upregulated. Indeed, B to A shifting enabled critical stem cell enhancers to interact with and activate various stem cell genes as shown by v4C (e.g. KLF5, PRDM5, MEIS1, etc). Most remarkably, the 3D architecture of H1C/H1EDKO GC B-cells was similar to that of intermediate stages of iPS cell reprogramming. Consistent with this finding, H1C/EDKO cells exhibited 4-fold greater reprogramming to the iPS state than WT cells after OKSM transduction, and these iPS cells exhibited reduced H3K27me3, stronger pluripotency potential, and impaired differentiation. This effect was rescued by transduction of WT H1, but not globular or C terminal domain mutant H1. Strikingly, the H1C/EDKO primitive stem cell gene expression signature was highly significantly enriched (NES 1.24, FDR Collectively, we find that H1 isoforms are bona fide lymphoma tumor suppressors. We speculate that H1 mutations are limited to GC derived lymphomas due to the stoichiometric need for H1 proteins in these rapidly dividing cells. To the best of our knowledge these are the first data to implicate disruption of topological packing order of chromatin as a cancer driver mechanism, as well as the first data to provide a mechanism whereby mature B-cells can acquire cancer stem cell-like characteristics. Disclosures Melnick: Janssen: Research Funding; Epizyme: Consultancy; Constellation: Consultancy.

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.

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 DrosophilaTopoisomerase 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.

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.