Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility.

Chromatin accessibility is a hallmark of active regulatory regions and is functionally linked to transcriptional networks and cell identity. However, the molecular mechanisms and networks that govern chromatin accessibility have not been thoroughly studied. Here we conducted a genome-wide CRISPR screening combined with an optimized ATAC-see protocol to identify genes that modulate global chromatin accessibility. In addition to known chromatin regulators like CREBBP and EP400, we discovered a number of previously unrecognized proteins that modulate chromatin accessibility, including TFDP1, HNRNPU, EIF3D and THAP11 belonging to diverse biological pathways. ATAC-seq analysis upon their knockouts revealed their distinct and specific effects on chromatin accessibility. Remarkably, we found that TFDP1, a transcription factor, modulates global chromatin accessibility through transcriptional regulation of canonical histones. In addition, our findings highlight the manipulation of chromatin accessibility as an approach to enhance various cell engineering applications, including genome editing and induced pluripotent stem cell reprogramming.

Borealin-Derived Peptides as Survivin-Targeting Cancer Imaging and Therapeutic Agents.

Survivin is overexpressed in most cancer cells but is rarely expressed in normal adult tissues. It is associated with poor prognosis and resistance to radiation therapy and chemotherapy. In this study, we designed and synthesized borealin-derived small peptides (Bor peptides) to function as survivin-targeting agents for the diagnosis and treatment of cancers. These peptides exhibited binding affinities for recombinant human survivin (Kd = 49.6-193 nM), with Bor65-75 showing the highest affinity (Kd = 49.6 nM). Fluorescence images of fluorescein isothiocyanate-labeled Bor65-75 showed its co-localization with survivin expression in the human pancreatic cancer cell line, MIA PaCa-2. In the WST-1 assay, cell penetrable nona-d-arginine-conjugated Bor65-75 (r9-Bor65-75) inhibited the growth of MIA PaCa-2 cells and MDA-MB-231 cells (89 and 88% inhibition at 10 µM, respectively), whereas it had almost no effect on the human mammary epithelial cell line, MCF-10A, that inherently does not have high survivin expression. Flow cytometry with annexin V and propidium iodide staining revealed that r9-Bor65-75 induced apoptosis in MIA PaCa-2 cells in a dose-dependent manner. An increase in cleaved poly ADP-ribose polymerase protein expression was observed in MIA PaCa-2 cells exposed to r9-Bor65-75 by western blotting, suggesting that r9-Bor65-75 inhibits cell proliferation by inducing apoptosis. In vivo, r9-Bor65-75 significantly suppressed tumor growth in MIA PaCa-2 xenograft mice, without any marked weight loss. Hence, Bor peptides are promising candidates for the development of cancer imaging and anticancer agents targeting survivin.

Design, synthesis and biological activity of selective and orally available TF/FVIIa complex inhibitors containing non-amidine P1 ligands.

We found the novel selective and orally available non-amidine TF/FVIIa complex inhibitor 21e, 4-({[(1S)-(aminocarbonyl)-3-methylbutyl]amino}carbonyl)-2'-({[4- (aminomethyl)phenyl]amino}carbonyl)-4'-(methylamino)biphenyl-2- carboxylic acid. The derivatives were synthesized by conversions of the isobutyl moiety and the introduction of alkylamino groups to 4'-position of the central phenyl ring of compounds 2a and 2b reported previously. Some compounds show increased in vitro anti-TF/FVIIa and PT prolongation activities. Among them, compound 21e reached and sustained micromolar plasma concentration levels of up to 2h after oral administration in mice. Moreover, compound 21e did not prolong the bleeding time even at the highest dose level in cynomolgus monkeys, while PT was prolonged 3.7-fold increases at this dose.

Modulation of retinoid signaling by a cytoplasmic viral protein via sequestration of Sp110b, a potent transcriptional corepressor of retinoic acid receptor, from the nucleus.

Hepatitis C virus (HCV) core protein (core) plays a significant role in the development of chronic liver diseases caused by HCV infection. We have discovered that the core sensitized all-trans-retinoic acid (ATRA)-induced cell death in MCF-7 cells. Activation of retinoic acid receptor alpha (RARalpha)-mediated transcription by the core was also seen in all the cell lines tested. By use of a yeast two-hybrid system, we identified Sp110b as a candidate for a core-interacting cellular factor. Although the function of Sp110b has remained unknown, we observed that Sp110b interacts with RARalpha and suppresses RARalpha-mediated transcription. These data suggest that Sp110b is a transcriptional cofactor negatively regulating RARalpha-mediated transcription. RNA interference-mediated reduction of endogenous Sp110b levels depressed the ability of the core to activate RARalpha-mediated transcription, suggesting an essential role for Sp110b in this pathway. The normal nuclear subcellular localization of Sp110b was altered by molecular interaction with the core to the cytoplasmic surface of the endoplasmic reticulum. This evidence suggests a model in which the core sequesters Sp110b from the nucleus and inactivates its corepressor function to activate RARalpha-mediated transcription. These findings likely describe a novel system in which a cytoplasmic viral protein regulates host cell transcription.