Masafumi Nakamoto

Quantitative assessment of higher‐order chromatin structure of the INK4/ARF locus in human senescent cells

Somatic cells can be reset to oncogene‐induced senescent (OIS) cells or induced pluripotent stem (iPS) cells by expressing specified factors. The INK4/ARF locus encodes p15INK4b, ARF, and p16INK4a genes in human chromosome 9p21, the products of which are known as common key reprogramming regulators. Compared with growing fibroblasts, the CCCTC‐binding factor CTCF is remarkably up‐regulated in iPS cells with silencing of the three genes in the locus and is reversely down‐regulated in OIS cells with high expression of p15INK4b and p16INK4a genes. There are at least three CTCF‐enriched sites in the INK4/ARF locus, which possess chromatin loop‐forming activities. These CTCF‐enriched sites and the p16INK4a promoter associate to form compact chromatin loops in growing fibroblasts, while CTCF depletion disrupts the loop structure. Interestingly, the loose chromatin structure is found in OIS cells. In addition, the INK4/ARF locus has an intermediate type of chromatin compaction in iPS cells. These results suggest that senescent cells have distinct higher‐order chromatin signature in the INK4/ARF locus.

DNA methylation-independent removable insulator controls chromatin remodeling at the HOXA locus via retinoic acid signaling

Chromatin insulators partition the genome into functional units to control gene expression, particularly in complex chromosomal regions. The CCCTC-binding factor (CTCF) is an insulator-binding protein that functions in transcriptional regulation and higher-order chromatin formation. Variable CTCF-binding sites have been identified to be cell type-specific partly due to differential DNA methylation. Here, we show that DNA methylation-independent removable CTCF insulator is responsible for retinoic acid (RA)-mediated higher-order chromatin remodeling in the human HOXA gene locus. Detailed chromatin analysis characterized multiple CTCF-enriched sites and RA-responsive enhancers at this locus. These regulatory elements and transcriptionally silent HOXA genes are closely positioned under basal conditions. Notably, upon RA signaling, the RAR/RXR transcription factor induced loss of adjacent CTCF binding and changed the higher-order chromatin conformation of the overall locus. Targeted disruption of a CTCF site by genome editing with zinc finger nucleases and CRISPR/Cas9 system showed that the site is required for chromatin conformations that maintain the initial associations among insulators, enhancers and promoters. The results indicate that the initial chromatin conformation affects subsequent RA-induced HOXA gene activation. Our study uncovers that a removable insulator spatiotemporally switches higher-order chromatin and multiple gene activities via cooperation of CTCF and key transcription factors.

Predictive value of the combination of SMAD4 expression and lymphocyte infiltration in malignant transformation of oral leukoplakia

Oral leukoplakia (OL) is a common, potentially malignant disorder of the oral cavity. SMAD4 was initially identified as a tumor suppressor and central mediator of transforming growth factor (TGF)‐β signaling. In this study, we aimed to determine the expression patterns of SMAD4 in OL, its relationship with the degree of inflammation, and its clinical implications as a biomarker for OL malignant transformation. A total of 150 patients with OL were enrolled in this study. Paraffin‐embedded sections obtained from biopsy or resection specimens were subjected to immunohistochemical analysis. Associations among the status of epithelial SMAD4 expression, stromal lymphocyte infiltration, and malignant transformation of OL were examined. Malignant transformation was significantly associated with the status of SMAD4 expression (P = 0.0017) and lymphocyte infiltration status (P = 0.0054). Cox regression analysis, based on the event‐free survival (EFS), revealed that a low SMAD4 expression was a significant prognostic factor in OL patients (hazard ratio, 2.632; P = 0.043). In addition, a low SMAD4 expression was closely correlated with high lymphocyte infiltration (P = 0.00035), resulting in a significant correlation between the combination of low SMAD4 expression and high lymphocyte infiltration with malignant transformation of OL (P = 0.00027). The combination of the status of epithelial SMAD4 expression and stromal lymphocyte infiltration may be a useful biomarker for predicting malignant transformation in OL patients. These results suggest that not only epithelial SMAD4 loss, but also stromal features, may regulate the risk of malignant transformation of OL.

The glucocorticoid receptor regulates the ANGPTL4 gene in a CTCF-mediated chromatin context in human hepatic cells

Glucocorticoid signaling through the glucocorticoid receptor (GR) plays essential roles in the response to stress and in energy metabolism. This hormonal action is integrated to the transcriptional control of GR-target genes in a cell type-specific and condition-dependent manner. In the present study, we found that the GR regulates the angiopoietin-like 4 gene (ANGPTL4) in a CCCTC-binding factor (CTCF)-mediated chromatin context in the human hepatic HepG2 cells. There are at least four CTCF-enriched sites and two GR-binding sites within the ANGPTL4 locus. Among them, the major CTCF-enriched site is positioned near the ANGPTL4 enhancer that binds GR. We showed that CTCF is required for induction and subsequent silencing of ANGPTL4 expression in response to dexamethasone (Dex) and that transcription is diminished after long-term treatment with Dex. Although the ANGPTL4 locus maintains a stable higher-order chromatin conformation in the presence and absence of Dex, the Dex-bound GR activated transcription of ANGPTL4 but not that of the neighboring three genes through interactions among the ANGPTL4 enhancer, promoter, and CTCF sites. These results reveal that liganded GR spatiotemporally controls ANGPTL4 transcription in a chromosomal context.