OCT2 pre-positioning facilitates cell fate transition and chromatin architecture changes in humoral immunity.

During the germinal center (GC) reaction, B cells undergo profound transcriptional, epigenetic and genomic architectural changes. How such changes are established remains unknown. Mapping chromatin accessibility during the humoral immune response, we show that OCT2 was the dominant transcription factor linked to differential accessibility of GC regulatory elements. Silent chromatin regions destined to become GC-specific super-enhancers (SEs) contained pre-positioned OCT2-binding sites in naive B cells (NBs). These preloaded SE 'seeds' featured spatial clustering of regulatory elements enriched in OCT2 DNA-binding motifs that became heavily loaded with OCT2 and its GC-specific coactivator OCAB in GC B cells (GCBs). SEs with high abundance of pre-positioned OCT2 binding preferentially formed long-range chromatin contacts in GCs, to support expression of GC-specifying factors. Gain in accessibility and architectural interactivity of these regions were dependent on recruitment of OCAB. Pre-positioning key regulators at SEs may represent a broadly used strategy for facilitating rapid cell fate transitions.

Smc3 dosage regulates B cell transit through germinal centers and restricts their malignant transformation.

During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and three-dimensional reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3, encoding the cohesin ATPase subunit, abrogated GC formation, while, in marked contrast, Smc3 haploinsufficiency resulted in GC hyperplasia, skewing of GC polarity and impaired plasma cell (PC) differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by the lymphoma epigenetic tumor suppressors Tet2 and Kmt2d and failure of Smc3-haploinsufficient GC B cells to switch from B cell- to PC-defining transcription factors. Smc3 haploinsufficiency preferentially impaired the connectivity of enhancer elements controlling various lymphoma tumor suppressor genes, and, accordingly, Smc3 haploinsufficiency accelerated lymphomagenesis in mice with constitutive Bcl6 expression. Collectively, our data indicate a dose-dependent function for cohesin in humoral immunity to facilitate the B cell to PC phenotypic switch while restricting malignant transformation.

Histone demethylase LSD1 is required for germinal center formation and BCL6-driven lymphomagenesis.

Germinal center (GC) B cells feature repression of many gene enhancers to establish their characteristic transcriptome. Here we show that conditional deletion of Lsd1 in GCs significantly impaired GC formation, associated with failure to repress immune synapse genes linked to GC exit, which are also direct targets of the transcriptional repressor BCL6. We found that BCL6 directly binds LSD1 and recruits it primarily to intergenic and intronic enhancers. Conditional deletion of Lsd1 suppressed GC hyperplasia caused by constitutive expression of BCL6 and significantly delayed BCL6-driven lymphomagenesis. Administration of catalytic inhibitors of LSD1 had little effect on GC formation or GC-derived lymphoma cells. Using a CRISPR-Cas9 domain screen, we found instead that the LSD1 Tower domain was critical for dependence on LSD1 in GC-derived B cells. These results indicate an essential role for LSD1 in the humoral immune response, where it modulates enhancer function by forming repression complexes with BCL6.

Unique Immune Cell Coactivators Specify Locus Control Region Function and Cell Stage.

Locus control region (LCR) functions define cellular identity and have critical roles in diseases such as cancer, although the hierarchy of structural components and associated factors that drive functionality are incompletely understood. Here we show that OCA-B, a B cell-specific coactivator essential for germinal center (GC) formation, forms a ternary complex with the lymphoid-enriched OCT2 and GC-specific MEF2B transcription factors and that this complex occupies and activates an LCR that regulates the BCL6 proto-oncogene and is uniquely required by normal and malignant GC B cells. Mechanistically, through OCA-B-MED1 interactions, this complex is required for Mediator association with the BCL6 promoter. Densely tiled CRISPRi screening indicates that only LCR segments heavily bound by this ternary complex are essential for its function. Our results demonstrate how an intimately linked complex of lineage- and stage-specific factors converges on specific and highly essential enhancer elements to drive the function of a cell-type-defining LCR.

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.

Childhood bullying victimization, emotion regulation, rumination, distress tolerance, and depressive symptoms: A cross-national examination among young adults in seven countries.

Existing research suggests a robust association between childhood bullying victimization and depressive symptoms in adulthood, but less is known about potential mediators of this link. Furthermore, there is limited cross-national research evaluating similarities and differences in bullying victimization and its associations with mental health. The current study addressed gaps in the literature by evaluating cognitive and affective responses to stress (i.e., emotion regulation, rumination, and distress tolerance) as potential mediators of the link between recalled bullying victimization and current depressive symptoms among 5909 (70.6% female) college students from seven countries. Results revealed specific indirect associations of bullying victimization through distress tolerance and three out of four facets of rumination, as well as a persistent direct association of childhood bullying on adulthood depression. Emotion regulation strategies were not significantly associated with bullying victimization and did not mediate its association with depressive symptoms. Constrained multigroup models indicated that results were invariant across country and gender. Findings provide evidence of statistical mediation in a cross-sectional sample and await replication in prospective studies. Rumination and distress tolerance may be promising targets for resilience-promoting interventions among children experiencing peer victimization. Ongoing research is needed to clarify cross-national patterns in childhood bullying, identify additional mediators accounting for the remaining direct association, and evaluate emotion regulation as a potential moderator of associations between bullying victimization and adult mental health.

The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication.

SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here, we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single-cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factor p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro. These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication. IMPORTANCE The COVID-19 pandemic has caused significant mortality and morbidity around the world. Although effective vaccines have been developed, large parts of the world remain unvaccinated while new SARS-CoV-2 variants keep emerging. Furthermore, despite extensive efforts and large-scale drug screenings, no fully effective antiviral treatment options have been discovered yet. Therefore, it is of the utmost importance to gain a better understanding of essential factors driving SARS-CoV-2 replication to be able to develop novel approaches to target SARS-CoV-2 biology.

Reducing cyberbullying: A theory of reasoned action-based video prevention program for college students.

Few studies have evaluated the effectiveness of cyberbullying prevention/intervention programs. The goals of the present study were to develop a Theory of Reasoned Action (TRA)-based video program to increase cyberbullying knowledge (1) and empathy toward cyberbullying victims (2), reduce favorable attitudes toward cyberbullying (3), decrease positive injunctive (4) and descriptive norms about cyberbullying (5), and reduce cyberbullying intentions (6) and cyberbullying behavior (7). One hundred sixty-seven college students were randomly assigned to an online video cyberbullying prevention program or an assessment-only control group. Immediately following the program, attitudes and injunctive norms for all four types of cyberbullying behavior (i.e., unwanted contact, malice, deception, and public humiliation), descriptive norms for malice and public humiliation, empathy toward victims of malice and deception, and cyberbullying knowledge significantly improved in the experimental group. At one-month follow-up, malice and public humiliation behavior, favorable attitudes toward unwanted contact, deception, and public humiliation, and injunctive norms for public humiliation were significantly lower in the experimental than the control group. Cyberbullying knowledge was significantly higher in the experimental than the control group. These findings demonstrate a brief cyberbullying video is capable of improving, at one-month follow-up, cyberbullying knowledge, cyberbullying perpetration behavior, and TRA constructs known to predict cyberbullying perpetration. Considering the low cost and ease with which a video-based prevention/intervention program can be delivered, this type of approach should be considered to reduce cyberbullying.

BTG1 mutation yields supercompetitive B cells primed for malignant transformation.

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Alterations in transcriptional networks in cancer: the role of noncoding somatic driver mutations.

Aberrant gene expression is a cancer hallmark and it is known that almost every tumor acquires somatic mutations in transcription factors, chromatin regulators, or the DNA regulatory elements that are critical for transcriptional control and cell phenotype. While the role of transcription factors and chromatin regulators has been widely studied, relatively few noncoding driver mutations have been identified and functionally characterized to date. Here, we review the current understanding of somatic variants in noncoding regions of the cancer genome and their impact on chromatin architecture and transcriptional networks. We also discuss approaches and ongoing challenges for noncoding driver discovery, and highlight insights gained from recent studies exploring the nature and impact of noncoding drivers on tumor formation.

Increased tumor glycolysis is associated with decreased immune infiltration across human solid tumors.

Response to immunotherapy across multiple cancer types is approximately 25%, with some tumor types showing increased response rates compared to others (i.e. response rates in melanoma and non-small cell lung cancer (NSCLC) are typically 30-60%). Patients whose tumors are resistant to immunotherapy often lack high levels of pre-existing inflammation in the tumor microenvironment. Increased tumor glycolysis, acting through glucose deprivation and lactic acid accumulation, has been shown to have pleiotropic immune suppressive effects using in-vitro and in-vivo models of disease. To determine whether the immune suppressive effect of tumor glycolysis is observed across human solid tumors, we analyzed glycolytic and immune gene expression patterns in multiple solid malignancies. We found that increased expression of a glycolytic signature was associated with decreased immune infiltration and a more aggressive disease across multiple tumor types. Radiologic and pathologic analysis of untreated estrogen receptor (ER)-negative breast cancers corroborated these observations, and demonstrated that protein expression of glycolytic enzymes correlates positively with glucose uptake and negatively with infiltration of CD3+ and CD8+ lymphocytes. This study reveals an inverse relationship between tumor glycolysis and immune infiltration in a large cohort of multiple solid tumor types.

Histone 3 Methyltransferases Alter Melanoma Initiation and Progression Through Discrete Mechanisms.

Perturbations to the epigenome are known drivers of tumorigenesis. In melanoma, alterations in histone methyltransferases that catalyze methylation at histone 3 lysine 9 and histone 3 lysine 27-two sites of critical post-translational modification-have been reported. To study the function of these methyltransferases in melanoma, we engineered melanocytes to express histone 3 lysine-to-methionine mutations at lysine 9 and lysine 27, which are known to inhibit the activity of histone methyltransferases, in a zebrafish melanoma model. Using this system, we found that loss of histone 3 lysine 9 methylation dramatically suppressed melanoma formation and that inhibition of histone 3 lysine 9 methyltransferases in human melanoma cells increased innate immune response signatures. In contrast, loss of histone 3 lysine 27 methylation significantly accelerated melanoma formation. We identified FOXD1 as a top target of PRC2 that is silenced in melanocytes and found that aberrant overexpression of FOXD1 accelerated melanoma onset. Collectively, these data demonstrate how histone 3 lysine-to-methionine mutations can be used to uncover critical roles for methyltransferases.

A nuclear cAMP microdomain suppresses tumor growth by Hippo pathway inactivation.

Cyclic AMP (cAMP) signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. Here, we present a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncover a nuclear cAMP microdomain that activates a tumor-suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397 and export of YAP from the nucleus with no change in YAP protein stability. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer; thus, drugs directed at the nuclear cAMP microdomain may provide avenues for the treatment of cancer.

Extracellular Matrix in Synthetic Hydrogel-Based Prostate Cancer Organoids Regulate Therapeutic Response to EZH2 and DRD2 Inhibitors.

Following treatment with androgen receptor (AR) pathway inhibitors, ≈20% of prostate cancer patients progress by shedding their AR-dependence. These tumors undergo epigenetic reprogramming turning castration-resistant prostate cancer adenocarcinoma (CRPC-Adeno) into neuroendocrine prostate cancer (CRPC-NEPC). No targeted therapies are available for CRPC-NEPCs, and there are minimal organoid models to discover new therapeutic targets against these aggressive tumors. Here, using a combination of patient tumor proteomics, RNA sequencing, spatial-omics, and a synthetic hydrogel-based organoid, putative extracellular matrix (ECM) cues that regulate the phenotypic, transcriptomic, and epigenetic underpinnings of CRPC-NEPCs are defined. Short-term culture in tumor-expressed ECM differentially regulated DNA methylation and mobilized genes in CRPC-NEPCs. The ECM type distinctly regulates the response to small-molecule inhibitors of epigenetic targets and Dopamine Receptor D2 (DRD2), the latter being an understudied target in neuroendocrine tumors. In vivo patient-derived xenograft in immunocompromised mice showed strong anti-tumor response when treated with a DRD2 inhibitor. Finally, we demonstrate that therapeutic response in CRPC-NEPCs under drug-resistant ECM conditions can be overcome by first cellular reprogramming with epigenetic inhibitors, followed by DRD2 treatment. The synthetic organoids suggest the regulatory role of ECM in therapeutic response to targeted therapies in CRPC-NEPCs and enable the discovery of therapies to overcome resistance.

The Case for Examining and Treating the Combined Effects of Parental Drug Use and Interparental Violence on Children in their Homes.

This review examines what have been, to this point, generally two divergent lines of research: (a) effects of parental drug abuse on children, and (b) effects of children's exposure to interparental violence. A small, but growing body of literature has documented the robust relationship between drug use and intimate partner violence. Despite awareness of the interrelationship, little attention has been paid to the combined effect of these deleterious parent behaviors on children in these homes. Thus, we argue for the need to examine the developmental impact of these behaviors (both individually and combined) on children in these homes and for treatment development to reflect how each of these parent behaviors may affect children of substance abusers.

Long non-coding RNAs discriminate the stages and gene regulatory states of human humoral immune response.

lncRNAs make up a majority of the human transcriptome and have key regulatory functions. Here we perform unbiased de novo annotation of transcripts expressed during the human humoral immune response to find 30% of the human genome transcribed during this process, yet 58% of these transcripts manifest striking differential expression, indicating an lncRNA phylogenetic relationship among cell types that is more robust than that of coding genes. We provide an atlas of lncRNAs in naive and GC B-cells that indicates their partition into ten functionally categories based on chromatin features, DNase hypersensitivity and transcription factor localization, defining lncRNAs classes such as enhancer-RNAs (eRNA), bivalent-lncRNAs, and CTCF-associated, among others. Specifically, eRNAs are transcribed in 8.6% of regular enhancers and 36.5% of super enhancers, and are associated with coding genes that participate in critical immune regulatory pathways, while plasma cells have uniquely high levels of circular-RNAs accounted for by and reflecting the combinatorial clonal state of the Immunoglobulin loci.

CHD1 Loss Alters AR Binding at Lineage-Specific Enhancers and Modulates Distinct Transcriptional Programs to Drive Prostate Tumorigenesis.

Deletion of the gene encoding the chromatin remodeler CHD1 is among the most common alterations in prostate cancer (PCa); however, the tumor-suppressive functions of CHD1 and reasons for its tissue-specific loss remain undefined. We demonstrated that CHD1 occupied prostate-specific enhancers enriched for the androgen receptor (AR) and lineage-specific cofactors. Upon CHD1 loss, the AR cistrome was redistributed in patterns consistent with the oncogenic AR cistrome in PCa samples and drove tumor formation in the murine prostate. Notably, this cistrome shift was associated with a unique AR transcriptional signature enriched for pro-oncogenic pathways unique to this tumor subclass. Collectively, these data credential CHD1 as a tumor suppressor in the prostate that constrains AR binding/function to limit tumor progression.

TET2 Deficiency Causes Germinal Center Hyperplasia, Impairs Plasma Cell Differentiation, and Promotes B-cell Lymphomagenesis.

TET2 somatic mutations occur in ∼10% of diffuse large B-cell lymphomas (DLBCL) but are of unknown significance. Herein, we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation, and a preneoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit, such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP-mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP-mutant gene-expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence, TET2 plays a critical role in the GC reaction, and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals. SIGNIFICANCE: We show that TET2 is required for exit of the GC, B-cell differentiation, and is a tumor suppressor for mature B cells. Loss of TET2 phenocopies CREBBP somatic mutation. These results advocate for sequencing TET2 in patients with lymphoma and for the testing of epigenetic therapies to treat these tumors.See related commentary by Shingleton and Dave, p. 1515.This article is highlighted in the In This Issue feature, p. 1494.

Regulatory elements in molecular networks.

Regulatory elements determine the connectivity of molecular networks and mediate a variety of regulatory processes ranging from DNA looping to transcriptional, posttranscriptional, and posttranslational regulation. This review highlights our current understanding of the different types of regulatory elements found in molecular networks with a focus on DNA regulatory elements. We highlight technical advances and current challenges for the mapping of regulatory elements at the genome-wide scale, and describe new computational methods to uncover these elements via reconstructing regulatory networks from large genomic datasets. WIREs Syst Biol Med 2017, 9:e1374. doi: 10.1002/wsbm.1374 For further resources related to this article, please visit the WIREs website.

Widespread Mitotic Bookmarking by Histone Marks and Transcription Factors in Pluripotent Stem Cells.

During mitosis, transcription is halted and many chromatin features are lost, posing a challenge for the continuity of cell identity, particularly in fast cycling stem cells, which constantly balance self-renewal with differentiation. Here we show that, in pluripotent stem cells, certain histone marks and stem cell regulators remain associated with specific genomic regions of mitotic chromatin, a phenomenon known as mitotic bookmarking. Enhancers of stem cell-related genes are bookmarked by both H3K27ac and the master regulators OCT4, SOX2, and KLF4, while promoters of housekeeping genes retain high levels of mitotic H3K27ac in a cell-type invariant manner. Temporal degradation of OCT4 during mitotic exit compromises its ability both to maintain and induce pluripotency, suggesting that its regulatory function partly depends on its bookmarking activity. Together, our data document a widespread yet specific bookmarking by histone modifications and transcription factors promoting faithful and efficient propagation of stemness after cell division.