Bullen, M. J. | Search bioRxiv

bioRxiv2020

Histone demethylome map reveals combinatorial gene regulatory functions in embryonic stem cells

Epigenetic regulators and transcription factors establish distinct regulatory networks for gene regulation to maintain the embryonic stem cell (ESC) state. Although much has been learned regarding individual epigenetic regulators, their combinatorial functions remain elusive. Here, we report previously unknown combinatorial functions of histone demethylases (HDMs) in gene regulation of mouse ESCs. Generation of a histone demethylome (HDMome) map of 20 well-characterized HDMs based on their genome-wide binding revealed co-occupancy of HDMs in different combinations: KDM1A-KDM4B-KDM6A and JARID2-KDM2B-KDM4A-KDM4C-KDM5B largely co-occupy at enhancers and promoters, respectively. Mechanistic studies uncover that KDM1A-KDM6A combinatorially modulates P300/H3K27ac, H3K4me2 deposition and OCT4 recruitment that directs the OCT4/CORE regulatory network for target gene expression; while co-operative actions of JARID2-KDM2B-KDM4A-KDM4C-KDM5B control H2AK119ub1 and bivalent marks of polycomb-repressive complexes that facilitate the PRC regulatory network for target gene repression. Thus, combinatorial functions of HDMs differentially impact gene expression programs in mESCs.

genomics↗

bioRxiv2019

A saturating mutagenesis CRISPR-Cas9 mediated functional genomic screen identifies cis- and trans-regulatory elements of Oct4 in embryonic stem cells

Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the non-coding genome and control gene expression programs either in -cis or in - trans. Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the [~]12kb cis-region (Cis-REs; CREs) of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs, as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling Oct4 gene expression. A subset of active CREs and TREs physically interact with the Oct4 promoter to varying degrees; specifically, a greater number of active CREs compared to active TREs, physically interact with the Oct4 promoter. Moreover, comparative genomics analysis reveals that more number of active CREs than active TREs are evolutionary conserved between mouse and primates, including human. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs, and investigate their roles in the regulation of transcriptional output of a target gene (in this case Oct4) in their native context.

genomics↗