👤 Masahide Sakabe

Alternative RNA splicing adds diverse variations to gene function, and its abnormalities are occasionally associated with the etiology of disease. We examined this possibility in pre-eclampsia. We performed transcriptome analysis of placentas from pre-eclamptic and normotensive pregnancies and screened for disease-specific aberrant splicing. We identified aberrant splicing at exon 14 in the ZC3H4 gene. This in-frame exon is generally skipped in placentas from normal pregnancies but often observed in those from pre-eclampsia patients. The level of exon inclusion did not correlate with disease severity, such as blood pressure or fetal weight, but showed an association with the decrease in placental weight. Significantly, placental blood flow resistance measured by Doppler ultrasound correlated with the level of ZC3H4 exon 14 inclusion, suggesting that this retention leads to the onset and/or symptoms of pre-eclampsia. ZC3H4 is known to act on transcriptional regulation via suppression of lncRNA expression. Moreover, the SOD1 gene, encoding superoxide dismutase that eliminates toxic free superoxide radicals, was identified in the downstream gene group for ZC3H4. Indeed, the expression of SOD1 was found in this current study to be decreased in the pre-eclamptic placenta in correlation with the levels of ZC3H4 exon 14 retention. Aberrant splicing of ZC3H4 gene may induce excessive oxidative stress in the placenta via the downregulation of downstream SOD1 expression thereby leading to the onset and development of pre-eclampsia. Show less

Endocardial cushion tissue is primordia of the valves and septa of the adult heart, and its malformation causes various congenital heart diseases (CHDs). Tricuspid atresia (TA) is defined as congenital absence or agenesis of the tricuspid valve caused by endocardial cushion defects. However, little is known about what type of endocardial cushion defect causes TA. Using three-dimensional volume rendering image analysis, we demonstrated morphological changes of endocardial cushion tissue in developing Hey2/Hrt2 KO mouse embryos that showed malformation of the tricuspid valve, which resembled human TA at neonatal period. In control embryos, atrioventricular (AV) endocardial cushion tissues showed rightward shift to form a tricuspid valve. However, the rightward shift of endocardial cushion tissue was disrupted in Hey2/Hrt2 KO embryos, leading to the misalignment of AV cushions. We also found that muscular tissue filled up the space between the right atrium and ventricle, resulting in the absence of the tricuspid valve. Moreover, analysis using tissue-specific conditional KO mice showed that HEY2/HRT2-expressing myocardium may physically regulate the AV shift. Disruption of rightward cushion movement is an initial cue of TA phenotype, and myocardial HEY2/HRT2 is necessary for the regulation of proper alignment of AV endocardial cushion tissue. Show less

Vascular homeostasis and pathophysiology are tightly regulated by mechanical forces generated by hemodynamics. Vascular disorders such as atherosclerotic diseases largely occur at curvatures and bifurcations where disturbed blood flow activates endothelial cells while unidirectional flow at the straight part of vessels promotes endothelial health. Integrated analysis of the endothelial transcriptome, the 3D epigenome, and human genetics systematically identified the SNP-enriched cistrome in vascular endothelium subjected to well-defined atherosclerosis-prone disturbed flow or atherosclerosis-protective unidirectional flow. Our results characterized the endothelial typical- and super-enhancers and underscored the critical regulatory role of flow-sensitive endothelial super-enhancers. CRISPR interference and activation validated the function of a previously unrecognized unidirectional flow-induced super-enhancer that upregulates antioxidant genes NQO1, CYB5B, and WWP2, and a disturbed flow-induced super-enhancer in endothelium which drives prothrombotic genes EDN1 and HIVEP in vascular endothelium. Our results employing multiomics identify the cis-regulatory architecture of the flow-sensitive endothelial epigenome related to atherosclerosis and highlight the regulatory role of super-enhancers in mechanotransduction mechanisms. Show less

Genome-wide association studies (GWAS) have identified many disease-associated variants, yet mechanisms underlying these associations remain unclear. To understand obesity-associated variants, we generate gene regulatory annotations in adipocytes and hypothalamic neurons across cellular differentiation stages. We then test variants in 97 obesity-associated loci using a massively parallel reporter assay and identify putatively causal variants that display cell type specific or cross-tissue enhancer-modulating properties. Integrating these variants with gene regulatory information suggests genes that underlie obesity GWAS associations. We also investigate a complex genomic interval on 16p11.2 where two independent loci exhibit megabase-range, cross-locus chromatin interactions. We demonstrate that variants within these two loci regulate a shared gene set. Together, our data support a model where GWAS loci contain variants that alter enhancer activity across tissues, potentially with temporally restricted effects, to impact the expression of multiple genes. This complex model has broad implications for ongoing efforts to understand GWAS. Show less

The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1(ko) /H2(ko) ) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1(ko) /H2(eko) ) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1(ko) /H2(ko) phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1(ko) /H2(eko) embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency. Show less

The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. Interferon-alpha (IFN-α)/5-fluorouracil (5-FU) therapy is reportedly effective in some HCC patients. In the present study, to improve HCC prognosis, we identified the genes that are sensitizing to these agents. The screening strategy was dependent on the concentration of ribozymes that rendered HepG2 cells resistant to 5-FU by the repeated transfection of ribozymes into the cells. After 10 cycles of transfection, which was initiated by 5,902,875 sequences of a ribozyme library, three genes including protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (PRKAG2); transforming growth factor-beta receptor II (TGFBR2); and exostosin 1 (EXT1) were identified as 5-FU-sensitizing genes. Adenovirus-mediated transfer of TGFBR2 and EXT1 enhanced IFN-α/5-FU-induced cytotoxicity as well as 5-FU, although the overexpression of these genes in the absence of IFN-α/5-FU did not induce cell death. This effect was also observed in a tumor xenograft model. The mechanisms of TGFBR2 and EXT1 include activation of the TGF-β signal and induction of endoplasmic reticulum stress, resulting in apoptosis. In HCC patients treated with IFN-α/5-FU therapy, the PRKAG2 mRNA level in HCC tissues was positively correlated with survival period, suggesting that PRKAG2 enhances the effect of IFN-α/5-FU and serves as a prognostic marker for IFN-α/5-FU therapy. In conclusion, we identified three genes that chemosensitize the effects of 5-FU and IFN-α/5-FU on HCC cells and demonstrated that PRKAG2 mRNA can serve as a prognostic marker for IFN-α/5-FU therapy. Show less