CTCF-mediated chromatin looping provides a topological framework for the formation of phase-separated transcriptional condensates.

CTCF is crucial to the organization of mammalian genomes into loop structures. According to recent studies, the transcription apparatus is compartmentalized and concentrated at super-enhancers to form phase-separated condensates and drive the expression of cell-identity genes. However, it remains unclear whether and how transcriptional condensates are coupled to higher-order chromatin organization. Here, we show that CTCF is essential for RNA polymerase II (Pol II)-mediated chromatin interactions, which occur as hyperconnected spatial clusters at super-enhancers. We also demonstrate that CTCF clustering, unlike Pol II clustering, is independent of liquid-liquid phase-separation and resistant to perturbation of transcription. Interestingly, clusters of Pol II, BRD4, and MED1 were found to dissolve upon CTCF depletion, but were reinstated upon restoration of CTCF, suggesting a potent instructive function for CTCF in the formation of transcriptional condensates. Overall, we provide evidence suggesting that CTCF-mediated chromatin looping acts as an architectural prerequisite for the assembly of phase-separated transcriptional condensates.

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. [BMB Reports 2023; 56(7): 398-403].

CTCF controls three-dimensional enhancer network underlying the inflammatory response of bone marrow-derived dendritic cells.

Dendritic cells are antigen-presenting cells orchestrating innate and adaptive immunity. The crucial role of transcription factors and histone modifications in the transcriptional regulation of dendritic cells has been extensively studied. However, it is not been well understood whether and how three-dimensional chromatin folding controls gene expression in dendritic cells. Here we demonstrate that activation of bone marrow-derived dendritic cells induces extensive reprogramming of chromatin looping as well as enhancer activity, both of which are implicated in the dynamic changes in gene expression. Interestingly, depletion of CTCF attenuates GM-CSF-mediated JAK2/STAT5 signaling, resulting in defective NF-κB activation. Moreover, CTCF is necessary for establishing NF-κB-dependent chromatin interactions and maximal expression of pro-inflammatory cytokines, which prime Th1 and Th17 cell differentiation. Collectively, our study provides mechanistic insights into how three-dimensional enhancer networks control gene expression during bone marrow-derived dendritic cells activation, and offers an integrative view of the complex activities of CTCF in the inflammatory response of bone marrow-derived dendritic cells.

p53 expression confers sensitivity to 5-fluorouracil via distinct chromatin accessibility dynamics in human colorectal cancer.

One of the most commonly used drugs in chemotherapy, 5-fluorouracil (5-FU) has been shown to be effective in only 10-15% of patients with colon cancer. Thus, studies of the mechanisms affecting 5-FU sensitivity in these patients are necessary. The tumor suppressor protein p53 is a transcription factor that serves important roles in cell apoptosis by regulating the cell cycle. It has also been characterized as a key factor influencing drug sensitivity. Furthermore, accessible chromatin is a hallmark of active DNA regulatory elements and functions as a crucial epigenetic factor regulating cancer mechanisms. The present study assessed the genetic regulatory landscape in colon cancer by performing RNA sequencing and Assay for Transposase-Accessible Chromatin sequencing, and investigated the effects of 5-FU on chromatin accessibility and gene expression. Notably, while treatment with 5-FU mediated global increases in chromatin accessibility, chromatin organization in several genomic regions differed depending on the expression status of p53. Since the occupancy of p53 does not overlap with accessible chromatin regions, the 5-FU-mediated changes in chromatin accessibility were not regulated by direct binding of p53. In the p53-expressing condition, the 5-FU-mediated accessible chromatin region was primarily associated with genes encoding cell death pathways. Additionally, 5-FU was revealed to induce open chromatin conformation at regions containing binding motifs for AP-1 family transcription factors, which may drive expression of apoptosis pathway genes. In conclusion, expression of p53 may confer 5-FU sensitivity by regulating chromatin accessibility of distinct genes associated with cell apoptosis in a transcription-independent manner.

Liver-Specific Deletion of Mouse CTCF Leads to Hepatic Steatosis via Augmented PPARγ Signaling.

BACKGROUND & AIMS: The liver is the major organ for metabolizing lipids, and malfunction of the liver leads to various diseases. Nonalcoholic fatty liver disease is rapidly becoming a major health concern worldwide and is characterized by abnormal retention of excess lipids in the liver. CCCTC-binding factor (CTCF) is a highly conserved zinc finger protein that regulates higher-order chromatin organization and is involved in various gene regulation processes. Here, we sought to determine the physiological role of CTCF in hepatic lipid metabolism. METHODS: We generated liver-specific, CTCF-ablated and/or CD36 whole-body knockout mice. Overexpression or knockdown of peroxisome proliferator-activated receptor (PPAR)γ in the liver was achieved using adenovirus. Mice were examined for development of hepatic steatosis and inflammation. RNA sequencing was performed to identify genes affected by CTCF depletion. Genome-wide occupancy of H3K27 acetylation, PPARγ, and CTCF were analyzed by chromatin immunoprecipitation sequencing. Genome-wide chromatin interactions were analyzed by in situ Hi-C. RESULTS: Liver-specific, CTCF-deficient mice developed hepatic steatosis and inflammation when fed a standard chow diet. Global analysis of the transcriptome and enhancer landscape revealed that CTCF-depleted liver showed enhanced accumulation of PPARγ in the nucleus, which leads to increased expression of its downstream target genes, including fat storage-related gene CD36, which is involved in the lipid metabolic process. Hepatic steatosis developed in liver-specific, CTCF-deficient mice was ameliorated by repression of PPARγ via pharmacologic blockade or adenovirus-mediated knockdown, but hardly rescued by additional knockout of CD36. CONCLUSIONS: Our data indicate that liver-specific deletion of CTCF leads to hepatosteatosis through augmented PPARγ DNA-binding activity, which up-regulates its downstream target genes associated with the lipid metabolic process.

Prion protein-deficient mice exhibit decreased CD4 T and LTi cell numbers and impaired spleen structure.

The cellular prion protein is expressed in almost all tissues, including the central nervous system and lymphoid tissues. To investigate the effects of the prion protein in lymphoid cells and spleen structure formation, we used prion protein-deficient (Prnp(0/0)) Zürich I mice generated by inactivation of the Prnp gene. Prnp(0/0) mice had decreased lymphocytes, in particular, CD4 T cells and lymphoid tissue inducer (LTi) cells. Decreased CD4 T cells resulted from impaired expression of CCL19 and CCL21 in the spleen rather than altered chemokine receptor CCR7 expression. Importantly, some of the white pulp regions in spleens from Prnp(0/0) mice displayed impaired T zone structure as a result of decreased LTi cell numbers and altered expression of the lymphoid tissue-organizing genes lymphotoxin-α and CXCR5, although expression of the lymphatic marker podoplanin and CXCL13 by stromal cells was not affected. In addition, CD3(-)CD4(+)IL-7Rα(+) LTi cells were rarely detected in impaired white pulp in spleens of these mice. These data suggest that the prion protein is required to form the splenic white pulp structure and for development of normal levels of CD4 T and LTi cells.

Immunomodulatory Effects of ZYM-201 on LPS-stimulated B Cells.

ZYM-201 is a methyl ester of triterpenoid glycoside from Sanguisorba officinalis which has been used for treatment of inflammatory and metabolic diseases. In this study, immunomodulatory effects of ZYM-201 on B cells were examined in vitro and in vivo. When splenocytes were activated with lipopolysaccharide (LPS), the major population which had shown an increase in cell numbers was B cells. However, when the B cells were treated with ZYM-201 after LPS activation, their cell numbers and the expression of major costimulatory molecules, CD80 and CD86, were decreased. Furthermore, the effect of LPS, which induces activation of NF-κB, was abolished by ZYM-201: LPS-stimulated B cells showed decrease of phosphorylation after treatment of ZYM-201. The same results were shown in vivo experiments. These results suggest that ZYM-201 may play a role in the modulation of inflammatory responses through inhibiting NF-κB activation and downregulating the expression of costimulatory molecules on B cells.

Increased Lymphocyte Infiltration in Rheumatoid Arthritis Is Correlated with an Increase in LTi-like Cells in Synovial Fluid.

In this study, we compared the immune cell populations in rheumatoid arthritis (RA) synovial fluid, which shows lymphoid tissue-like structure, with those in tonsils, which are normal secondary lymphoid tissues. Firstly, we found that CD4(-)CD11b(+) macrophages were the major population in RA synovial fluid and that B cells were the major population in tonsils. In addition, synovial fluid from patients with osteoarthritis, which is a degenerative joint disease, contained CD4(+)CD11b(+) monocytes as the major immune cell population. Secondly, we categorized three groups based on the proportion of macrophages found in RA synovial fluid: (1) the macrophage-high group, which contained more than 80% macrophages; (2) the macrophage-intermediate group, which contained between 40% and 80% macrophages; and (3) the macrophage-low group, which contained less than 40% macrophages. In the macrophage-low group, more lymphoid tissue inducer (LTi)-like cells were detected, and the expression of OX40L and TRANCE in these cells was higher than that in the other groups. In addition, in this group, the suppressive function of regulatory T cells was downregulated. Finally, CXCL13 expression was higher in RA synovial fluid than in tonsils, but CCL21 expression was comparable in synovial fluid from all groups and in tonsils. These data demonstrate that increased lymphocyte infiltration in RA synovial fluid is correlated with an increase in LTi-like cells and the elevation of the chemokine expression.