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Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns. The boundaries of topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. Here the authors find that CTCF positions cohesin through its N-terminus but does not control its overall binding dynamics on chromatin, and show how the orientation of CTCF binding sites translates into genome folding patterns.

Multiple myeloma (MM) arises through oncogenic transformation of immunoglobulin-secreting plasma cells. MM often co-opts the central endoplasmic reticulum (ER)-stress mitigator, inositol-requiring enzyme 1 (IRE1), to sustain malignant growth. While certain MMs require enzymatic IRE1-dependent activation of the transcription factor XBP1s, others display a nonenzymatic IRE1 dependency that is not yet mechanistically understood. Here we identify interferon regulatory factor 4 (IRF4), which stimulates genes that promote immune-cell proliferation, as a key conduit for IRE1’s nonenzymatic control of cell-cycle progression in MM. IRE1 silencing increased inhibitory S114/S270 phosphorylation on IRF4, disrupting IRF4’s chromatin-binding and transcriptional activity. IRF4 knockdown recapitulated, whereas IRF4 repletion reversed, the anti-proliferative phenotype of IRE1 silencing. Furthermore, phospho-deficient, but not phospho-mimetic, IRF4 mutants rescued proliferation under IRE1 silencing. Functional studies revealed that IRF4 engages the E2F1 and CDC25A genes and promotes CDK2 activation to drive cell-cycle progression. Our results advance mechanistic understanding of IRE1 and IRF4 in MM.

Death of pancreatic β cells is a pathological hallmark of type 1 diabetes (T1D). However, the molecular mechanisms of β cell death and its regulation are poorly understood. Here we describe a unique regulatory pathway of β cell death that comprises microRNA-21, its target tumor suppressor PDCD4, and its upstream transcriptional activator nuclear factor-κB (NF-κB). In pancreatic β cells, c-Rel and p65 of the NF-κB family activated the mir21 gene promoter and increased miR-21 RNA levels; miR-21 in turn decreased the level of PDCD4, which is able to induce cell death through the Bax family of apoptotic proteins. Consequently, PDCD4 deficiency in pancreatic β cells renders them resistant to death, and PDCD4 deficiency in NOD or C57BL/6 mice conferred resistance to spontaneous diabetes and diabetes induced by autoimmune T cells or the β cell toxin streptozotocin (STZ). Thus, the NF-κB–microRNA-21–PDCD4 axis plays a crucial role in T1D and represents a unique therapeutic target for treating the disease.

TEAD (transcriptional enhanced associate domain) transcription factors (TEAD1-4) serve as the primary effectors of the Hippo signaling pathway in various cancers. Targeted therapy leads to the emergence of resistance and the underlying mechanism of resistance to TEAD inhibition in cancers is less characterized. We uncover that upregulation of the AP-1 (activator protein-1) transcription factors, along with restored YAP (yes-associated protein) and TEAD activity, drives resistance to GNE-7883, a pan-TEAD inhibitor. Acute GNE-7883 treatment abrogates YAP-TEAD binding and attenuates FOSL1 (FOS like 1) activity. TEAD inhibitor resistant cells restore YAP and TEAD chromatin occupancy, acquire additional FOSL1 binding and exhibit increased MAPK (mitogen-activated protein kinase) pathway activity. FOSL1 is required for the chromatin binding of YAP and TEAD. This study describes a clinically relevant interplay between the Hippo and MAPK pathway and highlights the key role of MAPK pathway inhibitors in mitigating resistance to TEAD inhibition in Hippo pathway dependent cancers. The underlying mechanism of acquired resistance to targeted therapy in cancer remains to be explored. Here, the authors show that upregulation of the FOSL1 transcription factor restores YAP/TEAD occupancy on chromatin to drive resistance to GNE-7883, an allosteric TEAD inhibitor.

Background DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statistical significance, but are also high cost if performed genome-wide. Here, we describe a simplified targeted bisulfite sequencing approach in which DNA sequencing libraries are prepared following sodium bisulfite conversion and two rounds of PCR for target enrichment and sample barcoding, termed BisPCR 2 . Results We have applied the BisPCR 2 technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR 2 compared to prior array-based approaches. Conclusion This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies.

The loss of insulin-secreting β cells is characteristic among type I and type II diabetes. Stimulating proliferation to expand sources of β cells for transplantation remains a challenge because adult β cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human β cells. Expression of p57 is regulated by the DNA methylation status of the imprinting control region 2 (ICR2), which is commonly hypomethylated in Beckwith-Wiedemann syndrome patients who exhibit massive β cell proliferation. We hypothesized that targeted demethylation of the ICR2 using a transcription activator-like effector protein fused to the catalytic domain of TET1 (ICR2-TET1) would repress p57 expression and promote cell proliferation. We report here that overexpression of ICR2-TET1 in human fibroblasts reduces p57 expression levels and increases proliferation. Furthermore, human islets overexpressing ICR2-TET1 exhibit repression of p57 with concomitant upregulation of Ki-67 while maintaining glucose-sensing functionality. When transplanted into diabetic, immunodeficient mice, the epigenetically edited islets show increased β cell replication compared with control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing β cell proliferation, which may one day alleviate the scarcity of transplantable β cells for the treatment of diabetes.

Next-generation selective estrogen receptor-α (ERα) antagonist/degraders (SERDs) are being developed for ER-positive breast cancer (ER + BC), with intentions of improving outcomes for patients. In recent clinical trials of metastatic ER + BC, next-generation SERDs demonstrated clinical activity, and elacestrant received an approval for advanced ESR1 -mutant disease. However, responses to these drugs were highly heterogeneous: across trials and independent of ESR1 status, 30–50% of patients progressed by their first follow-up scan while other patients sustained benefit for 2 years or more. Here, we interrogate the basis for heterogeneous responses by comparing biopsies from non-responding patients (NR; progression-free survival <2 months) and responding patients (Resp; PFS ≥ 2 months) who received the next-generation SERD giredestrant. While Resp tumors maintain high dependency on ERα signaling, NR tumors exhibit loss of luminal lineage identity and by extension, ERα dependence. NR tumors instead up-regulate multiple ERα-independent proliferative pathways, such as EGFR/MAPK and Hippo/TEAD, which may represent targetable dependencies in NR disease. Modeling resistance and lineage plasticity in vitro, we find that giredestrant-resistant ER + BC cell lines exhibit profound shifts in chromatin accessibility, with the transcription factors, FOXA1 and FOXM1, implicated in gene expression of NR-upregulated proliferative pathways.

Differentiation proceeds along a continuum of increasingly fate-restricted intermediates, referred to as canalization 1 , 2 . Canalization is essential for stabilizing cell fate, but the mechanisms that underlie robust canalization are unclear. Here we show that the BRG1/BRM-associated factor (BAF) chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells. Despite the establishment of a well-differentiated precardiac mesoderm, Brm −/− cells predominantly became neural precursors, violating germ layer assignment. Trajectory inference showed a sudden acquisition of a non-mesodermal identity in Brm −/− cells. Mechanistically, the loss of Brm prevented de novo accessibility of primed cardiac enhancers while increasing the expression of neurogenic factor POU3F1, preventing the binding of the neural suppressor REST and shifting the composition of BRG1 complexes. The identity switch caused by the Brm mutation was overcome by increasing BMP4 levels during mesoderm induction. Mathematical modelling supports these observations and demonstrates that Brm deletion affects cell fate trajectory by modifying saddle–node bifurcations 2 . In the mouse embryo, Brm deletion exacerbated mesoderm-deleted Brg1 -mutant phenotypes, severely compromising cardiogenesis, and reveals an in vivo role for Brm . Our results show that Brm is a compensable safeguard of the fidelity of mesoderm chromatin states, and support a model in which developmental canalization is not a rigid irreversible path, but a highly plastic trajectory. The BAF chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells.

Background/Aims: Epigenetic regulation is considered the main molecular mechanism underlying the developmental origin of health and disease’s (DOHAD) hypothesis. Previous studies that have investigated the role of paternal exercise on the metabolic health of the offspring did not control for the amount and intensity of the training or possible effects of adaptation to exercise and produced conflicting results regarding the benefits of parental exercise to the next generation. We employed a precisely regulated exercise regimen to study the transgenerational inheritance of improved metabolic health. Methods: We subjected male mice to a well-controlled exercise -training program to investigate the effects of paternal exercise on glucose tolerance and insulin sensitivity in their adult progeny. To investigate the molecular mechanisms of epigenetic inheritance, we determined chromatin markers in the skeletal muscle of the offspring and the paternal sperm. Results: Offspring of trained male mice exhibited improved glucose homeostasis and insulin sensitivity. Paternal exercise modulated the DNA methylation profile of PI3Kca and the imprinted H19/Igf2 locus at specific differentially methylated regions (DMRs) in the skeletal muscle of the offspring, which affected their gene expression. Remarkably, a similar DNA methylation profile at the PI3Kca, H19, and Igf2 genes was present in the progenitor sperm indicating that exercise-induced epigenetic changes that occurred during germ cell development contributed to transgenerational transmission. Conclusion: Paternal exercise might be considered as a strategy that could promote metabolic health in the offspring as the benefits can be inherited transgenerationally.

The recognition of β cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain β cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic β cells. Inducible ablation of LDB1 in mature β cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted β cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of β cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative β cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2) - factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of β cells and is a component of active enhancers in both murine and human islets.