3D Chromatin Architecture Research Articles

SMCHD1 has separable roles in chromatin architecture and gene silencing that could be targeted in disease

The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1’s role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.

Accessible gene borders establish a core structural unit for chromatin architecture in Arabidopsis.

Three-dimensional (3D) chromatin structure is linked to transcriptional regulation in multicellular eukaryotes including plants. Taking advantage of high-resolution Hi-C (high-throughput chromatin conformation capture), we detected a small structural unit with 3D chromatin architecture in the Arabidopsis genome, which lacks topologically associating domains, and also in the genomes of tomato, maize, and Marchantia polymorpha. The 3D folding domain unit was usually established around an individual gene and was dependent on chromatin accessibility at the transcription start site (TSS) and transcription end site (TES). We also observed larger contact domains containing two or more neighboring genes, which were dependent on accessible border regions. Binding of transcription factors to accessible TSS/TES regions formed these gene domains. We successfully simulated these Hi-C contact maps via computational modeling using chromatin accessibility as input. Our results demonstrate that gene domains establish basic 3D chromatin architecture units that likely contribute to higher-order 3D genome folding in plants.

Remodeling of the 3D chromatin architecture in the marine microalga Nannochloropsis oceanica during lipid accumulation

BackgroundGenomic three-dimensional (3D) spatial organization plays a key role in shaping gene expression and associated chromatin modification, and it is highly sensitive to environmental stress conditions. In microalgae, exposure to nitrogen stress can drive lipid accumulation, yet the associated functional alterations in the spatial organization of the microalgal genome have yet to be effectively characterized.ResultsAccordingly, the present study employed RNA-seq, Hi-C, and ChIP-seq approaches to explore the relationship between 3D chromosomal architecture and gene expression during lipid accumulation in the marine microalga Nannochloropsis oceanica in response to nitrogen deprivation (ND). These analyses revealed that ND resulted in various changes in chromosomal organization, including A/B compartment transitions, topologically associating domain (TAD) shifts, and the disruption of short-range interactions. Significantly higher levels of gene expression were evident in A compartments and TAD boundary regions relative to B compartments and TAD interior regions, consistent with observed histone modification enrichment in these areas. ND-induced differentially expressed genes (DEGs) were notably enriched in altered TAD-associated regions and regions exhibiting differential genomic contact. These DEGs were subjected to Gene Ontology (GO) term analyses that indicated they were enriched in the ‘fatty acid metabolism’, ‘response to stress’, ‘carbon fixation’ and ‘photosynthesis’ functional categories, in line with the ND treatment conditions used to conduct this study. These data indicate that Nannochloropsis cells exhibit a clear association between chromatin organization and transcriptional activity under nitrogen stress conditions. Pronounced and extensive histone modifications were evident in response to ND. Observed changes in chromatin architecture were linked to shifts in histone modifications and gene expression.ConclusionsOverall, the reprogramming of many lipid metabolism-associated genes was evident under nitrogen stress conditions with respect to both histone modifications and chromosomal organization. Together these results revealed that higher-order chromatin architecture represents a new layer that can guide efforts to understand the transcriptional regulation of lipid metabolism in nitrogen-deprived microalgae.

Abstract B017: SETD2-loss mediated H3K36me3 causes disruptions in topologically associated domains (TADs) to promote oncogenic expression in clear cell renal cell carcinoma

Abstract SETD2 is an RNA Polymerase II (Pol II)-associated histone methyltransferase that catalyzes the co-transcriptional methylation of H3K36me2 to create H3K36me3 marks in gene bodies of actively transcribed genes. SETD2 is one of the most frequently mutated chromatin-modifying genes across different cancer types, with the highest prevalence (13%) in clear cell renal cell carcinoma (ccRCC). ccRCC has been recognized as a VHL loss-driven disease in which VHL is mutated or silenced in up to 80-90% of tumors, and the heterozygous loss of chromosome 3p where VHL resides is identified in >90% of ccRCC. The other three top mutated genes in ccRCC are also located at chromosome 3p, including PBRM1 (~40%), SETD2 (8-13%), and BAP1 (6-10%). Notably, deletion of Vhl alone is insufficient to induce kidney cancer in mice, whereas combined deletion of Vhl and Pbrm1 results in multifocal, transplantable non-metastatic clear cell kidney cancer. Human ccRCC genomics and cancer evolution studies revealed that SETD2 mutations often co-occur with VHL and PBRM1 mutations and SETD2 mutations associate with tumor progression. Previously, using VHLMT PBRM1MT SETD2MT metastatic ccRCC patient-derived cell line and xenograft models we found that H3K36me3 restoration reduced distant metastases of ccRCC in mice, establishing SEDT2 as a metastatic tumor suppressor in kidney cancer. An integrated ATAC-seq, ChIP-seq, and transcriptome analysis uncovered a tumor suppressor model of SETD2 in which loss of SETD2-mediated H3K36me3 creates an epigenetic landscape consisting of widespread open chromatin that amplifies oncogenic transcriptional output through aberrant activation of enhancers. Here, we hypothesize that loss of SETD2-mediated H3K36me3 alters 3D chromosome architecture to increase chromatin accessibility and enhancer-promoter interactions, which in turn activates oncogenic transcription, including pro-metastatic genes such as MMP1, HGF, MYC, and ETV1. Here, we performed chromosome conformation capture (Hi-C) to interrogate 3D chromatin architecture in kidney cancer with defined mutations in VHL, PBRM1, or SETD2 for the first time. Our preliminary global analysis comparing SETD2 mutant and wildtype HiC samples showed disruptions in Topologically Associated Domains (TADs) and increased long-range interaction with loss of SETD2 function. Further understanding of how dysregulated epigenetics conferred by SETD2 loss promotes tumor initiation and metastasis will allow the discovery of novel therapeutic vulnerabilities associated with the loss of SETD2. Citation Format: Amrita M. Nargund, Rui Yang. SETD2-loss mediated H3K36me3 causes disruptions in topologically associated domains (TADs) to promote oncogenic expression in clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B017.

3D organization of regulatory elements for transcriptional regulation in Arabidopsis

BackgroundAlthough spatial organization of compartments and topologically associating domains at large scale is relatively well studied, the spatial organization of regulatory elements at fine scale is poorly understood in plants.ResultsHere we perform high-resolution chromatin interaction analysis using paired-end tag sequencing approach. We map chromatin interactions tethered with RNA polymerase II and associated with heterochromatic, transcriptionally active, and Polycomb-repressive histone modifications in Arabidopsis. Analysis of the regulatory repertoire shows that distal active cis-regulatory elements are linked to their target genes through long-range chromatin interactions with increased expression of the target genes, while poised cis-regulatory elements are linked to their target genes through long-range chromatin interactions with depressed expression of the target genes. Furthermore, we demonstrate that transcription factor MYC2 is critical for chromatin spatial organization, and propose that MYC2 occupancy and MYC2-mediated chromatin interactions coordinately facilitate transcription within the framework of 3D chromatin architecture. Analysis of functionally related gene-defined chromatin connectivity networks reveals that genes implicated in flowering-time control are functionally compartmentalized into separate subdomains via their spatial activity in the leaf or shoot apical meristem, linking active mark- or Polycomb-repressive mark-associated chromatin conformation to coordinated gene expression.ConclusionThe results reveal that the regulation of gene transcription in Arabidopsis is not only by linear juxtaposition, but also by long-range chromatin interactions. Our study uncovers the fine scale genome organization of Arabidopsis and the potential roles of such organization in orchestrating transcription and development.

G-quadruplex DNA contributes to RNA polymerase II-mediated 3D chromatin architecture.

High-order chromatin organization plays an important role in biological processes and disease development. Previous studies revealed a widespread occurrence of guanine quadruplex (G4) structures in the human genome, with enrichment in gene regulatory regions, especially in promoters. However, it remains unclear whether G4 structures contribute to RNA polymerase II (RNAPII)-mediated long-range DNA interactions and transcription activity. In this study, we conducted an intuitive overlapping analysis of previously published RNAPII ChIA-PET (chromatin interaction analysis with paired-end tag) and BG4 ChIP-seq (chromatin immunoprecipitation followed by sequencing using a G4 structure-specific antibody) data. We observed a strong positive correlation between RNAPII-linked DNA loops and G4 structures in chromatin. Additionally, our RNAPII HiChIP-seq (in situ Hi-C followed by ChIP-seq) results showed that treatment of HepG2 cells with pyridostatin (PDS), a small-molecule G4-binding ligand, could diminish RNAPII-linked long-range DNA contacts, with more pronounced diminutions being observed for those contacts involving G4 structure loci. RNA sequencing data revealed that PDS treatment modulates the expression of not only genes with G4 structures in their promoters, but also those with promoters being connected with distal G4sthrough RNAPII-linked long-range DNA interactions. Together, our data substantiate the function of DNA G4s in RNAPII-associated DNA looping and transcription regulation.

Vorinostat-induced acetylation of RUNX3 reshapes transcriptional profile through long-range enhancer-promoter interactions in natural killer cells

Natural killer (NK) cells are an essential part of the innate immune system that helps control infections and tumors. Recent studies have shown that Vorinostat, a histone deacetylase (HDAC) inhibitor, can cause significant changes in gene expression and signaling pathways in NK cells. Since gene expression in eukaryotic cells is closely linked to the complex three-dimensional (3D) chromatin architecture, an integrative analysis of the transcriptome, histone profiling, chromatin accessibility, and 3D genome organization is needed to gain a more comprehensive understanding of how Vorinostat impacts transcription regulation of NK cells from a chromatin-based perspective. The results demonstrate that Vorinostat treatment reprograms the enhancer landscapes of the human NK-92 NK cell line while overall 3D genome organization remains largely stable. Moreover, we identified that the Vorinostat-induced RUNX3 acetylation is linked to the increased enhancer activity, leading to elevated expression of immune response-related genes via long-range enhancerpromoter chromatin interactions. In summary, these findings have important implications in the development of new therapies for cancer and immune-related diseases by shedding light on the mechanisms underlying Vorinostat's impact on transcriptional regulation in NK cells within the context of 3D enhancer network.

Morphine Re-arranges Chromatin Spatial Architecture of Primate Cortical Neurons

The expression of linear DNA sequence is precisely regulated by the three-dimensional (3D) architecture of chromatin. Morphine-induced aberrant gene networks of neurons have been extensively investigated; however, how morphine impacts the 3D genomic architecture of neurons is still unknown. Here, we applied digestion-ligation-only high-throughput chromosome conformation capture (DLO Hi-C) technology to investigate the effects of morphine on the 3D chromatin architecture of primate cortical neurons. After receiving continuous morphine administration for 90 days on rhesus monkeys, we discovered that morphine re-arranged chromosome territories, with a total of 391 segmented compartments being switched. Morphine altered over half of the detected topologically associated domains (TADs), most of which exhibited a variety of shifts, followed by separating and fusing types. Analysis of the looping events at kilobase-scale resolution revealed that morphine increased not only the number but also the length of differential loops. Moreover, all identified differentially expressed genes from the RNA sequencing data were mapped to the specific TAD boundaries or differential loops, and were further validated for changed expression. Collectively, an altered 3D genomic architecture of cortical neurons may regulate the gene networks associated with morphine effects. Our finding provides critical hubs connecting chromosome spatial organization and gene networks associated with the morphine effects in humans.

Topologically associating domain boundaries are required for normal genome function

Topologically associating domain (TAD) boundaries partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotypes1–3, but the overall extent to which this occurs remains unknown. Here we demonstrate that targeted deletions of TAD boundaries cause a range of disruptions to normal in vivo genome function and organismal development. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11–80 kb in size) from the genome. All deletions examined resulted in detectable molecular or organismal phenotypes, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. We observed changes in local 3D chromatin architecture in 7 of 8 (88%) cases, including the merging of TADs and altered contact frequencies within TADs adjacent to the deleted boundary. For 5 of 8 (63%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and reinforce the critical need to carefully consider the potential pathogenicity of noncoding deletions affecting TAD boundaries in clinical genetics screening.

A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination

Interactions between transcription and cohesin-mediated loop extrusion can influence 3D chromatin architecture. However, their relevance in biology is unclear. Here, we report a direct role for such interactions in the mechanism of antibody class switch recombination (CSR) at the murine immunoglobulin heavy chain locus (Igh). Using Tri-C to measure higher-order multiway interactions on single alleles, we find that the juxtaposition (synapsis) of transcriptionally active donor and acceptor Igh switch (S)sequences, an essential step in CSR, occurs via the interaction of loop extrusion complexes with a de novo topologically associating domain (TAD) boundary formed via transcriptional activity across S regions. Surprisingly, synapsis occurs predominantly in proximity to the 3' CTCF-binding element (3'CBE) rather than the Igh super-enhancer, suggesting a two-step mechanism whereby transcription of S regions is not topologically coupled to synapsis, as has been previously proposed. Altogether, these insights advance our understanding of how 3D chromatin architecture regulates CSR.

Reorganization of three-dimensional chromatin architecture in Medicago truncatula under phosphorus deficiency.

Emerging evidence reveals that the three-dimensional (3D) chromatin architecture plays a key regulatory role in various biological processes of plants. However, information on the 3D chromatin architecture of the legume model plant Medicago truncatula and its potential roles in the regulation of response to mineral nutrient deficiency are very limited. Using high-resolution chromosome conformation capture sequencing, we identified the 3D genome structure of M. truncatula in terms of A/B compartments, topologically associated domains (TADs) and chromatin loops. The gene density, expressional level, and active histone modification were higher in A compartments than in B compartments. Moreover, we analysed the 3D chromatin architecture reorganization in response to phosphorus (P) deficiency. The intra-chromosomal cis-interaction proportion was increased by P deficiency, and a total of 748 A/B compartment switch regions were detected. In these regions, density changes in H3K4me3 and H3K27ac modifications were associated with expression of P deficiency-responsive genes involved in root system architecture and hormonal responses. Furthermore, these genes enhanced P uptake and mobilization by increasing root surface area and strengthening signal transduction under P deficiency. These findings advance our understanding of the potential roles of 3D chromatin architecture in responses of plants in general, and in particular in M. truncatula, to P deficiency.

K27M in canonical and noncanonical H3 variants occurs in distinct oligodendroglial cell lineages in brain midline gliomas.

Canonical (H3.1/H3.2) and noncanonical (H3.3) histone 3 K27M-mutant gliomas have unique spatiotemporal distributions, partner alterations and molecular profiles. The contribution of the cell of origin to these differences has been challenging to uncouple from the oncogenic reprogramming induced by the mutation. Here, we perform an integrated analysis of 116 tumors, including single-cell transcriptome and chromatin accessibility, 3D chromatin architecture and epigenomic profiles, and show that K27M-mutant gliomas faithfully maintain chromatin configuration at developmental genes consistent with anatomically distinct oligodendrocyte precursor cells (OPCs). H3.3K27M thalamic gliomas map to prosomere 2-derived lineages. In turn, H3.1K27M ACVR1-mutant pontine gliomas uniformly mirror early ventral NKX6-1+/SHH-dependent brainstem OPCs, whereas H3.3K27M gliomas frequently resemble dorsal PAX3+/BMP-dependent progenitors. Our data suggest a context-specific vulnerability in H3.1K27M-mutant SHH-dependent ventral OPCs, which rely on acquisition of ACVR1 mutations to drive aberrant BMP signaling required for oncogenesis. The unifying action of K27M mutations is to restrict H3K27me3 at PRC2 landing sites, whereas other epigenetic changes are mainly contingent on the cell of origin chromatin state and cycling rate.

Chromatin architectural alterations due to null mutation of a major CG methylase in rice.

Associations between 3D chromatin architectures and epigenetic modifications have been characterized in animals. However, any impact of DNA methylation on chromatin architecture in plants is understudied, which is confined to Arabidopsis thaliana. Because plant species differ in genome size, composition, and overall chromatin packing, it is unclear to what extent findings from A. thaliana hold in other species. Moreover, the incomplete chromatin architectural profiles and the low-resolution high-throughput chromosome conformation capture (Hi-C) data from A. thaliana have hampered characterizing its subtle chromatin structures and their associations with DNA methylation. We constructed a high-resolution Hi-C interaction map for the null OsMET1-2 (the major CG methyltransferase in rice) mutant (osmet1-2) and isogenic wild-type rice (WT). Chromatin structural changes occurred in osmet1-2, including intra-/inter-chromosomal interactions, compartment transition, and topologically associated domains (TAD) variations. Our findings provide novel insights into the potential function of DNA methylation in TAD formation in rice and confirmed DNA methylation plays similar essential roles in chromatin packing in A. thaliana and rice.

Ancient Human Endogenous Retroviruses Contribute to Genetic Evolution and Regulate Cancer Cell Type–Specific Gene Expression

Genome-wide analyses show that human endogenous retroviruses mediate cancer cell type-specific gene expression, epigenetic modification, and 3D chromatin architecture, elucidating the relationship between HERVs and diverse cancers.

Prolonged FOS activity disrupts a global myogenic transcriptional program by altering 3D chromatin architecture in primary muscle progenitor cells

BackgroundThe AP-1 transcription factor, FBJ osteosarcoma oncogene (FOS), is induced in adult muscle satellite cells (SCs) within hours following muscle damage and is required for effective stem cell activation and muscle repair. However, why FOS is rapidly downregulated before SCs enter cell cycle as progenitor cells (i.e., transiently expressed) remains unclear. Further, whether boosting FOS levels in the proliferating progeny of SCs can enhance their myogenic properties needs further evaluation.MethodsWe established an inducible, FOS expression system to evaluate the impact of persistent FOS activity in muscle progenitor cells ex vivo. We performed various assays to measure cellular proliferation and differentiation, as well as uncover changes in RNA levels and three-dimensional (3D) chromatin interactions.ResultsPersistent FOS activity in primary muscle progenitor cells severely antagonizes their ability to differentiate and form myotubes within the first 2 weeks in culture. RNA-seq analysis revealed that ectopic FOS activity in muscle progenitor cells suppressed a global pro-myogenic transcriptional program, while activating a stress-induced, mitogen-activated protein kinase (MAPK) transcriptional signature. Additionally, we observed various FOS-dependent, chromosomal re-organization events in A/B compartments, topologically associated domains (TADs), and genomic loops near FOS-regulated genes.ConclusionsOur results suggest that elevated FOS activity in recently activated muscle progenitor cells perturbs cellular differentiation by altering the 3D chromosome organization near critical pro-myogenic genes. This work highlights the crucial importance of tightly controlling FOS expression in the muscle lineage and suggests that in states of chronic stress or disease, persistent FOS activity in muscle precursor cells may disrupt the muscle-forming process.

Rearrangement of T Cell genome architecture regulates GVHD

SummaryWAPL, cohesin’s DNA release factor, regulates three-dimensional (3D) chromatin architecture. The 3D chromatin structure and its relevance to mature T cell functions is not well understood. We show that in vivo lymphopenic expansion, and alloantigen-driven proliferation, alters the 3D structure and function of the genome in mature T cells. Conditional deletion of WAPL, cohesin’s DNA release factor, in T cells reduced long-range genomic interactions and altered chromatin A/B compartments and interactions within topologically associating domains (TADs) of the chromatin in T cells at baseline. WAPL deficiency in T cells reduced loop extensions, changed expression of cell cycling genes and reduced proliferation following in vitro and in vivo stimulation, and reduced severity of graft-versus-host disease (GVHD) following experimental allogeneic hematopoietic stem cell transplantation. These data collectively characterize 3D genomic architecture of T cells in vivo and demonstrate biological and clinical implications for its disruption by cohesin release factor WAPL.

Reorganization of 3D chromatin architecture in doxorubicin-resistant breast cancer cells.

Background: Doxorubicin resistance remains a major therapeutic challenge leading to poor survival prognosis and treatment failure in breast cancer. Although doxorubicin induces massive changes in the transcriptional landscape are well known, potential diagnostic or therapeutic targets associated with the reorganization of three-dimensional (3D) chromatin architecture have not yet been systematically investigated. Methods: Here we performed in situ high-throughput chromosome conformation capture (Hi-C) on parental and doxorubicin-resistant MCF7 (MCF7-DR) human breast cancer cells, followed by integrative analysis of HiC, ATAC-seq, RNA-seq and TCGA data. Results: It revealed that A/B compartment switching was positively correlated to genome-wide differential gene expression. The genome of MCF7-DR cells was spatially reorganized into smaller topologically associating domains (TADs) and chromatin loops. We also revealed the contribution of increased chromatin accessibility and potential transcription factor families, including CTCF, AP-1 and bHLH, to gained TADs or loops. Intriguingly, we observed two condensed genomic regions (∼20 kb) with decreased chromatin accessibility flanking TAD boundaries, which might play a critical role in the formation or maintenance of TADs. Finally, combining data from TCGA, we identified a number of gained and lost enhancer-promoter interactions and their corresponding differentially expressed genes involved in chromatin organization and breast cancer signaling pathways, including FA2H, FOXA1 and JRKL, which might serve as potential treatment targets for breast cancer. Conclusion: These data uncovered a close connection between 3D genome reorganization, chromatin accessibility as well as gene transcription and provide novel insights into the epigenomic mechanisms involving doxorubicin resistance in breast cancer.

DNA methylation in transposable elements buffers the connection between three-dimensional chromatin organization and gene transcription upon rice genome duplication

IntroductionPolyploidy is a major force in plant evolution and the domestication of cultivated crops. ObjectivesThe study aimed to explore the relationship and underlying mechanism between three-dimensional (3D) chromatin organization and gene transcription upon rice genome duplication. MethodsThe 3D chromatin structures between diploid (2C) and autotetraploid (4C) rice were compared using high-throughput chromosome conformation capture (Hi-C) analysis. The study combined genetics, transcriptomics, whole-genome bisulfite sequencing (WGBS-seq) and 3D genomics approaches to uncover the mechanism for DNA methylation in modulating gene transcription through 3D chromatin architectures upon rice genome duplication. ResultsWe found that 4C rice presents weakened intra-chromosomal interactions compared to its 2C progenitor in some chromosomes. In addition, we found that changes of 3D chromatin organizations including chromatin compartments, topologically associating domains (TADs), and loops, are uncorrelated with gene transcription. Moreover, DNA methylations in the regulatory sequences of genes in compartment A/B switched regions and TAD boundaries are unrelated to their expression. Importantly, although there was no significant difference in the methylation levels in transposable elements (TEs) in differentially expressed gene (DEG) and non-DEG promoters between 2C and 4C rice, we found that the hypermethylated TEs across genes in compartment A/B switched regions and TAD boundaries may suppress the expression of these genes. ConclusionThe study proposed that the rice genome doubling might modulate TE methylation to buffer the effects of chromatin architecture on gene transcription in compartment A/B switched regions and TAD boundaries, resulting in the disconnection between 3D chromatin structure alteration and gene transcription upon rice genome duplication.

S101: GENETIC AND EPIGENETIC FACTORS DRIVING PRIMARY MEDIASTINAL B-CELL LYMPHOMA PATHOGENESIS AND OUTCOME

Background: Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive lymphoma affecting predominantly young female patients. Previous studies in this rare entity have focused on single genes or were limited in cohort size. Aims: To unravel the underlying genetic pathogenesis and its impact on outcome, we embarked on a comprehensive large-scale genetic investigation. Methods: Specimens of 486 previously untreated PMBCL patients were analyzed by paired tumor/normal whole-genome (WGS, n=14), whole-exome (WES, n=78) and targeted sequencing (TS, n=486). To understand the consequences of highly recurrent mutations in the chromatin-modifying gene ZNF217, we conducted functional and multi-omics analyses in CRISPR/Cas9 engineered cell lines. Results: WGS/WES revealed a complex genomic landscape in PMBCL with a median of 85 structural variants, a mutational burden of 5 mutations/Mb, 12 mutated coding candidate driver genes (CDG) and 4 focal somatic copy-number aberrations per sample (Fig.1a). Besides known targets, significant breakpoints were identified in genes previously not implicated in B-lymphomagenesis such as TOX and TP73 (36% and 21%). In addition, non-coding mutations clustered within the PAX5 enhancer region. With the identification of 50 recurrently mutated CDGs, we significantly expand the repertoire of known PMBCL drivers. The 10 most frequently mutated CDGs were SOCS1 (86%), B2M (67%), ITPKB (64%), ACTB (58%), STAT6 (58%), IGLL5 (56%), TNFAIP3 (53%), NFKBIE (49%), GNA13 (47%), and ZNF217 (36%), respectively. The operative mutational processes were attributed to aging, AID/APOBEC activity, defective MMR, and an unexpected infidelity of DNA-Polymerase ε. Next, we performed TS in 486 samples using a PMBCL-specific 106-gene panel. Recurrent lesions in 25 epigenetic modifiers were found in >90%, with ZNF217 being among the most frequently mutated genes (Fig.1b). After knockdown of ZNF217 in Karpas1106P and L428 cells, we demonstrated altered proliferation, migration, and apoptosis. Using mass spectrometry, we showed that ZNF217 is acting in a LSD1, CoREST and HDAC containing histone modifier complex. Accordingly, knockout of ZNF217 led to global changes in chromatin accessibility with an enrichment of differentially accessible motifs for crucial lymphoma-associated transcription factors, especially of the NF-κB, BATF/AP1, and IRF family, but also of CTCF, a major regulator of global 3D chromatin architecture. Resulting gene expression was characterized by changes in interferon-responsive genes and inflammation-associated transcription (Fig 1c). Clinical data were available for 329 cases, including 84 cases from clinical trials. Multivariate analysis using an IPI-corrected Cox regression model was performed. The estimated 5-year PFS and OS were 77% and 86%. Among the genetic lesions with the strongest association for poor outcome, we identified patients with mutatedCD58 having a significantly shorter survival (PFS: HR 2.96; p<.001; OS: HR 2.55; p=.006). In contrast, mutated DUSP2 indicated longer survival (PFS: HR 0.28; p=.002; OS: HR 0.15; p=.011) (Fig1d). Notably, DUSP2 mutated patients (25%) showed a similar outcome for CR rate, PFS and OS when comparing CHOP-like and intensified treatment regimens, suggesting no further benefit from treatment intensification in this very-low risk patient population. Image:Summary/Conclusion: Here, we present the genetic landscape of PMBCL highlighting a previously underappreciated role of chromatin modifying genes, identify novel treatment targets and provide a solid basis for guiding precision medicine approaches.

3D Genome Organization as an Epigenetic Determinant of Transcription Regulation in T Cells.

In the heart of innate and adaptive immunity lies the proper spatiotemporal development of several immune cell lineages. Multiple studies have highlighted the necessity of epigenetic and transcriptional regulation in cell lineage specification. This mode of regulation is mediated by transcription factors and chromatin remodelers, controlling developmentally essential gene sets. The core of transcription and epigenetic regulation is formulated by different epigenetic modifications determining gene expression. Apart from “classic” epigenetic modifications, 3D chromatin architecture is also purported to exert fundamental roles in gene regulation. Chromatin conformation both facilitates cell-specific factor binding at specified regions and is in turn modified as such, acting synergistically. The interplay between global and tissue-specific protein factors dictates the epigenetic landscape of T and innate lymphoid cell (ILC) lineages. The expression of global genome organizers such as CTCF, YY1, and the cohesin complexes, closely cooperate with tissue-specific factors to exert cell type-specific gene regulation. Special AT-rich binding protein 1 (SATB1) is an important tissue-specific genome organizer and regulator controlling both long- and short-range chromatin interactions. Recent indications point to SATB1’s cooperation with the aforementioned factors, linking global to tissue-specific gene regulation. Changes in 3D genome organization are of vital importance for proper cell development and function, while disruption of this mechanism can lead to severe immuno-developmental defects. Newly emerging data have inextricably linked chromatin architecture deregulation to tissue-specific pathophysiological phenotypes. The combination of these findings may shed light on the mechanisms behind pathological conditions.