KLF4 inhibits early neural differentiation of ESCs by coordinating specific 3D chromatin structure.

Neural differentiation of embryonic stem cells (ESCs) requires precisely orchestrated gene regulation, a process governed in part by changes in 3D chromatin structure. How these changes regulate gene expression in this context remains unclear. In this study, we observed enrichment of the transcription factor KLF4 at some poised or closed enhancers at TSS-linked regions of genes associated with neural differentiation. Combination analysis of ChIP, HiChIP and RNA-seq data indicated that KLF4 loss in ESCs induced changes in 3D chromatin structure, including increased chromatin interaction loops between neural differentiation-associated genes and active enhancers, leading to upregulated expression of neural differentiation-associated genes and therefore early neural differentiation. This study suggests KLF4 inhibits early neural differentiation by regulation of 3D chromatin structure, which is a new mechanism of early neural differentiation.

A HOTAIR regulatory element modulates glioma cell sensitivity to temozolomide through long-range regulation of multiple target genes.

Temozolomide (TMZ) is a frequently used chemotherapy for glioma; however, chemoresistance is a major problem limiting its effectiveness. Thus, knowledge of mechanisms underlying this outcome could improve patient prognosis. Here, we report that deletion of a regulatory element in the HOTAIR locus increases glioma cell sensitivity to TMZ and alters transcription of multiple genes. Analysis of a combination of RNA-seq, Capture Hi-C, and patient survival data suggests that CALCOCO1 and ZC3H10 are target genes repressed by the HOTAIR regulatory element and that both function in regulating glioma cell sensitivity to TMZ. Rescue experiments and 3C data confirmed this hypothesis. We propose a new regulatory mechanism governing glioma cell TMZ sensitivity.

Enhancer architecture-dependent multilayered transcriptional regulation orchestrates RA signaling-induced early lineage differentiation of ESCs.

Signaling pathway-driven target gene transcription is critical for fate determination of embryonic stem cells (ESCs), but enhancer-dependent transcriptional regulation in these processes remains poorly understood. Here, we report enhancer architecture-dependent multilayered transcriptional regulation at the Halr1-Hoxa1 locus that orchestrates retinoic acid (RA) signaling-induced early lineage differentiation of ESCs. We show that both homeobox A1 (Hoxa1) and Hoxa adjacent long non-coding RNA 1 (Halr1) are identified as direct downstream targets of RA signaling and regulated by RARA/RXRA via RA response elements (RAREs). Chromosome conformation capture-based screens indicate that RA signaling promotes enhancer interactions essential for Hoxa1 and Halr1 expression and mesendoderm differentiation of ESCs. Furthermore, the results also show that HOXA1 promotes expression of Halr1 through binding to enhancer; conversely, loss of Halr1 enhances interaction between Hoxa1 chromatin and four distal enhancers but weakens interaction with chromatin inside the HoxA cluster, leading to RA signaling-induced Hoxa1 overactivation and enhanced endoderm differentiation. These findings reveal complex transcriptional regulation involving synergistic regulation by enhancers, transcription factors and lncRNA. This work provides new insight into intrinsic molecular mechanisms underlying ESC fate determination during RA signaling-induced early differentiation.

Three-Dimensional Gene Regulation Network in Glioblastoma Ferroptosis.

Ferroptosis is an iron-dependent form of cell death, which is reported to be associated with glioma progression and drug sensitivity. Targeting ferroptosis is a potential therapeutic approach for glioma. However, the molecular mechanism of glioma cell ferroptosis is not clear. In this study, we profile the change of 3D chromatin structure in glioblastoma ferroptosis by using HiChIP and study the 3D gene regulation network in glioblastoma ferroptosis. A combination of an analysis of HiChIP and RNA-seq data suggests that change of chromatin loops mediated by 3D chromatin structure regulates gene expressions in glioblastoma ferroptosis. Genes that are regulated by 3D chromatin structures include genes that were reported to function in ferroptosis, like HDM2 and TXNRD1. We propose a new regulatory mechanism governing glioblastoma cell ferroptosis by 3D chromatin structure.

CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA.

Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer-enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.

Regulation Network of Colorectal-Cancer-Specific Enhancers in the Progression of Colorectal Cancer.

Enhancers regulate multiple genes via higher-order chromatin structures, and they further affect cancer progression. Epigenetic changes in cancer cells activate several cancer-specific enhancers that are silenced in normal cells. These cancer-specific enhancers are potential therapeutic targets of cancer. However, the functions and regulation networks of colorectal-cancer-specific enhancers are still unknown. In this study, we profile colorectal-cancer-specific enhancers and reveal their regulation network through the analysis of HiChIP data that were derived from a colorectal cancer cell line and Hi-C and RNA-seq data that were derived from tissue samples by in silico analysis and in vitro experiments. Enhancer-promoter loops in colorectal cancer cells containing colorectal-cancer-specific enhancers are involved in more than 50% of the topological associated domains (TADs) changed in colorectal cancer cells compared to normal colon cells. In addition, colorectal-cancer-specific enhancers interact with 152 genes that are significantly and highly expressed in colorectal cancer cells. These colorectal-cancer-specific enhancer target genes include ITGB4, RECQL4, MSLN, and GDF15. We propose that the regulation network of colorectal-cancer-specific enhancers plays an important role in the progression of colorectal cancer.

Bivalent activity of super-enhancer RNA LINC02454 controls 3D chromatin structure and regulates glioma sensitivity to temozolomide.

Glioma cell sensitivity to temozolomide (TMZ) is critical for effective treatment and correlates with patient survival, although mechanisms underlying this activity are unclear. Here, we reveal a new mechanism used by glioma cells to modulate TMZ sensitivity via regulation of SORBS2 and DDR1 genes by super-enhancer RNA LINC02454. We report that LINC02454 activity increases glioma cell TMZ sensitivity by maintaining long-range chromatin interactions between SORBS2 and the LINC02454 enhancer. By contrast, LINC02454 activity also decreased glioma cell TMZ sensitivity by promoting DDR1 expression. Our study suggests a bivalent function for super-enhancer RNA LINC02454 in regulating glioma cell sensitivity to TMZ.

Two Enhancers Regulate HoxB Genes Expression During Retinoic Acid-Induced Early Embryonic Stem Cells Differentiation Through Long-Range Chromatin Interactions.

Homeobox B cluster (HoxB) genes play important roles in retinoic acid (RA)-induced early embryonic stem cells (ESCs) differentiation. Knowledge of regulation network of HoxB is important to further unveil the mechanism of ESCs differentiation. In this study, we identified two enhancers that were activated by RA treatment and 4C data showed long-range interactions between HoxB genes and the two enhancers. CRISPR/Cas9-mediated individual or compound deletion of the two enhancers significantly inhibits HoxB gene expression, and transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in the enhancer KO cells. We propose new mechanism by which two enhancers regulate HoxB gene expression by different regulation modes during RA-induced early ESCs differentiation through long-range chromatin interactions.