Biology subjects

Rosen, B. P.

Publications and source records attributed to Rosen, B. P..

4 recordsLinked to original sources

bioRxiv2024

Discovery of NANOG enhancers and their essential roles in self-renewal and differentiation in human embryonic stem cells

Human embryonic stem cells (hESCs) are notable for their ability to self-renew and to differentiate into all tissue types in the body. NANOG is a core regulator of hESC identity, and dynamic control of its expression is crucial to maintain the balance between self-renewal and differentiation. Transcriptional regulation depends on enhancers, but NANOG enhancers in hESCs are not well characterized. Here we report two NANOG enhancers discovered from a CRISPR interference screen in hESCs. Deletion of a single copy of either enhancer significantly reduced NANOG expression, compromising self-renewal and increasing differentiation propensity. Interestingly, these two NANOG enhancers are involved in a tandem duplication event found in certain primates including humans but not in mice. However, the duplicated counterparts do not regulate NANOG expression. This work expands our knowledge of functional enhancers in hESCs, and highlights the sensitivity of the hESC state to the dosage of core regulators and their enhancers.

developmental biology↗

bioRxiv2023

Parallel genome-scale CRISPR screens distinguish pluripotency and self-renewal

Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.

developmental biology↗

bioRxiv2023

Dynamic network-guided CRISPRi screen reveals CTCF loop-constrained nonlinear enhancer-gene regulatory activity in cell state transitions

Comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Our network modeling revealed that nonlinear enhancer-gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Utilizing hESC definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core endoderm lineage-specifying transcription factors, and many enhancers had strong effects mid-transition but weak effects post-transition. Through integrating enhancer activity measurements and three-dimensional enhancer-promoter interaction information, we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.

genetics↗

bioRxiv2021

Identification of a MarR subfamily that regulates arsenic resistance genes

Members of the family of Multiple Antibiotic Resistance Regulators (MarR) often regulate genes that encode antibiotic resistance in bacteria. In this study, comprehensive analyses were performed to determine the function of an atypical MarR homolog in Achromobacter sp. As-55. Genomic analyses showed that this marR is located in an arsenic gene island in Achromobacter sp. As-55 adjacent to an arsV gene. ArsV is a flavin-dependent monooxygenase that confers resistance to the antibiotic methylarsenite (MAs(III)), the organoarsenic compound roxarsone(III) (Rox(III)), and the inorganic antimonite (Sb(III)). Similar marR genes are widely distributed in arsenic-resistant bacteria. Phylogenetic analyses showed that these MarRs are found in operons predicted to be involved in resistance to inorganic and organic arsenic species, so the subfamily was named MarRars. MarRars orthologs have three conserved cysteine residues, which are Cys36, Cys37 and Cys157 in Achromobacter sp. As-55, mutation of which compromises the response to MAs(III)/Sb(III). GFP-fluorescent biosensor assays show that AdMarRars (MarR protein of Achromobacter deleyi As-55) responds to trivalent As(III) and Sb(III) but not to pentavalent As(V) or Sb(V). The results of RT-qPCR assays show that arsV is expressed constitutively in a marR deletion mutant, indicating that marR represses transcription of arsV. Moreover, electrophoretic mobility shift assays (EMSA) demonstrate that AdMarRars binds to the promoters of both marR and arsV in the absence of ligands and that DNA binding is relieved upon binding of As(III) and Sb(III). Our results demonstrate that AdMarRars is a novel As(III)/Sb(III)-responsive transcriptional repressor that controls expression of arsV, which confers resistance to MAs(III), Roxarsone(III) and Sb(III). AdMarRars and its orthologs form a subfamily of MarR proteins that regulate genes conferring resistance to arsenic-containing antibiotics. IMPORTANCEIn this study, a MarR family member, AdMarRars was shown to regulate the arsV gene, which confers resistance to arsenic-containing antibiotics. It is a founding member of a distinct subfamily that we refer to as MarRars, regulating genes conferring resistance to arsenic and antimony antibiotic compounds. AdMarRars was shown to be a repressor containing conserved cysteine residues that are required to bind As(III) and Sb(III), leading to a conformational change and subsequent derepression. Here we show that members of the MarR family are involved in regulating arsenic-containing compounds.

microbiology↗