Takehisa Watanabe

The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome

Significance The retrovirus human T-lymphotropic virus type 1 (HTLV-1) causes inflammatory and malignant diseases in humans. To maintain latency and avoid immune detection in vivo, HTLV-1 minimizes expression of genes on the plus-strand of the integrated provirus but allows constitutive expression of the minus-strand gene, which maintains clonal persistence. It is not understood how this gene expression is regulated. We show that CTCF, a master regulator of chromatin structure and gene expression, binds to HTLV-1, forms loops between the provirus and host genome, and alters expression of proviral and host genes. Because a typical HTLV-1–infected host carries >104 infected T-cell clones, each containing a provirus integrated in a different genomic site, CTCF binding gives HTLV-1 the potential to cause widespread abnormalities in the human genome. Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in ∼10% of infected people. A typical host has between 104 and 105 clones of HTLV-1–infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.

Higher-Order Chromatin Regulation and Differential Gene Expression in the Human Tumor Necrosis Factor/Lymphotoxin Locus in Hepatocellular Carcinoma Cells

ABSTRACT The three-dimensional context of endogenous chromosomal regions may contribute to the regulation of gene clusters by influencing interactions between transcriptional regulatory elements. In this study, we investigated the effects of tumor necrosis factor (TNF) signaling on spatiotemporal enhancer-promoter interactions in the human tumor necrosis factor (TNF)/lymphotoxin (LT) gene locus, mediated by CCCTC-binding factor (CTCF)-dependent chromatin insulators. The cytokine genes LTα, TNF, and LTβ are differentially regulated by NF-κB signaling in inflammatory and oncogenic responses. We identified at least four CTCF-enriched sites with enhancer-blocking activities and a TNF-responsive TE2 enhancer in the TNF/LT locus. One of the CTCF-enriched sites is located between the early-inducible LTα/TNF promoters and the late-inducible LTβ promoter. Depletion of CTCF reduced TNF expression and accelerated LTβ induction. After TNF stimulation, via intrachromosomal dynamics, these insulators mediated interactions between the enhancer and the LTα/TNF promoters, followed by interaction with the LTβ promoter. These results suggest that insulators mediate the spatiotemporal control of enhancer-promoter associations in the TNF/LT gene cluster.

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.

Evaluation of sorafenib treatment and hepatic arterial infusion chemotherapy for advanced hepatocellular carcinoma: a comparative study using the propensity score matching method

While sorafenib (SFN) is the established worldwide standard therapeutic agent for advanced hepatocellular carcinoma (HCC), hepatic arterial infusion chemotherapy (HAIC) is also considered a favorable treatment for some advanced HCCs. This study aimed to evaluate each treatment and provide an optimal therapeutic choice for advanced HCCs. We analyzed 72 patients treated with SFN and 128 patients receiving HAIC. Both treatment groups were analyzed for prognostic and disease progression factors, and matched pair analysis was performed using the propensity score matching method. The preferable status of intrahepatic lesions, that is, no lesions or only a single (<3 cm) intrahepetic lesion, was positively associated with good prognosis and negatively associated with disease progression in the SFN group. Maximum tumor size (>5 cm) and low albumin (≤3.4 g/dL) were poor prognostic and disease progression factors in the HAIC group. Analysis of 53 patients selected from each of the SFN and HAIC groups based on the propensity score matching method showed no significant differences in survival or disease progression between the two matched subgroups. On the other hand, progression‐free survival (PFS) in the HAIC‐matched subgroup was significantly longer than in the SFN‐matched subgroup, particularly in patients with portal vein invasion (PVI) and/or without extrahepatic spread (EHS). The treatment efficacy of HAIC is similar to that of SFN regarding survival and disease progression. Longer PFS might be expected for HAIC compared with SFN, particularly in patients with PVI and/or without EHS.

Pancreatic metastasis from mixed adenoneuroendocrine carcinoma of the uterine cervix: a case report.

Metastatic cancers of the pancreas are rare, accounting for approximately 2-4% of all pancreatic malignancies. Renal cell carcinoma is the most common solid tumor that metastasizes to the pancreas. Here, we present a case of uterine cervical carcinoma metastasizing to the pancreas and review the literature regarding this rare event. A 44-year-old woman with a uterine cervical tumor had undergone radical hysterectomy and had been diagnosed pathologically with stage Ib mixed adenoneuroendocrine carcinoma in 2004. She underwent concurrent radiotherapy and chemotherapy postoperatively. Pulmonary metastases subsequently appeared in 2008 and 2011, and she underwent complete resection of the lung tumors by video-assisted thoracic surgery. Although she was followed up without any treatment and with no other recurrences, positron emission tomography revealed an area of abnormal uptake within the pancreatic body in 2012. Enhanced computed tomography demonstrated a 20-mm lesion in the pancreatic body and upstream pancreatic duct dilatation. Endoscopic ultrasonography-guided fine needle aspiration was performed and pathological examination suggested neuroendocrine carcinoma (NEC). On the basis of these results and the patients oncological background, lesions in the pancreatic body were diagnosed as secondary metastasis from the cervical carcinoma that had been treated 8 years earlier. No other distant metastases were visualized, and the patient subsequently underwent middle pancreatectomy. Pathological examination showed NEC consistent with pancreatic metastasis from the uterine cervical carcinoma. The patient has survived 7 months since the middle pancreatectomy without any signs of local recurrence or other metastatic lesions.

Outcomes of treatment with daclatasvir and asunaprevir for recurrent hepatitis C after liver transplantation

The development of direct‐acting oral agents has dramatically changed the treatment strategy of hepatitis C virus (HCV) infection. Here we aimed to reveal the efficacy and safety of daclatasvir (DCV) and asunaprevir (ASV) for recurrent HCV genotype 1 infection after liver transplantation (LT).

Modeling of the Weight Status and Risk of Nonalcoholic Fatty Liver Disease in Elderly Individuals: The Potential Impact of the Disulfide Bond‐Forming Oxidoreductase A‐Like Protein (DsbA‐L) Polymorphism on the Weight Status

Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity. Disulfide bond‐forming oxidoreductase A‐like protein (DsbA‐L) is known to be a key molecule in protection against obesity and obesity‐induced inflammation. In the present study, we used a modeling and simulation approach in an attempt to develop body mass index (BMI) and BMI‐based NAFLD prediction models incorporating the DsbA‐L polymorphism to predict the BMI and NAFLD in 341 elderly subjects. A nonlinear mixed‐effect model best represented the sigmoidal relationship between the BMI and the logit function of the probability of NAFLD prevalence. The final models for BMI and NAFLD showed that DsbA‐L rs1917760 polymorphism, age, and gender were associated with the BMI, whereas gender, patatin‐like phospholipase 3 rs738409 polymorphism, HbA1c, and high‐density and low‐density lipoprotein cholesterol levels were associated with the risk of NAFLD. This information may aid in the genetic‐based prevention of obesity and NAFLD in the general elderly population.

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.

Ledipasvir and sofosbuvir for recurrent hepatitis C after liver transplantation.

Management of recurrent hepatitis C following liver transplantation still remains a challenge. Here, we report five patients who achieved viral responses following combined treatment with ledipasvir and sofosbuvir. All the patients received tacrolimus for immunosuppression. No dose adjustment was made before the ledipasvir and sofosbuvir therapy. All completed the intended 12-week treatment course with the full dose of ledipasvir and sofosbuvir. There were no significant adverse events greater than grade 2. During the study period, no acute rejection episodes were detected. The trough levels of tacrolimus were maintained stably. Hepatitis C virus RNA was not detected at week 12 in any of the patients. Based on the findings from this pilot study, combined ledipasvir and sofosbuvir therapy for 12 weeks is effective and safe for living - donor liver transplantation recipients with recurrence of hepatitis C virus.

HTLV-1 inserts an ectopic CTCF-binding site into the human genome.

HTLV-1 genes are encoded on both strands of the provirus, such as tax in the plus and HBZ in the minus strand. The HBZ gene is constitutively expressed from the negative strand of the integrated provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is expressed only intermittently in vivo, perhaps to escape from host immune surveillance. However, it remains unknown what regulates this pattern of proviral transcription in vivo. We have found that CTCF binds to the HTLV-1 provirus. CTCF is a DNA-binding protein that plays a fundamental role in controlling higher-order chromatin structure and gene expression in vertebrates. We identified several candidate regions for CTCF binding in the HTLV-1 genome. Chromatin immunoprecipitation assays showed that CTCF bound selectively to the pX region of HTLV-1. Furthermore, electromobility shift assays revealed that CTCF bound directly to the pX DNA sequence. Consistent with the CTCF binding, there was a sharp border of histone modification patterns at the pX region, consistent with CTCFs role as a chromatin insulator. Finally, the CTCF-binding region (1bp) showed enhancer-blocking activity. The CTCF binding and epigenetic border were detectable not only in HTLV-1 cell lines and ATL cell lines but also in fresh PBMCs of ATL patients. These observations suggest that CTCF plays a central role in the regulation of HTLV-1 transcription. #Poster award winnder - 1st place