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Rajesh C Rao, Qiang Li, Fengbao Mao, Yali Dou; COMPASS acts as a temporal rheostat to control retinal neuroectoderm versus mesoderm fate choice.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):571.
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How ubiquitously expressed proteins, such as those required for survival, achieve time-sensitive & tissue-specific functions remains poorly understood. We tested the hypothesis that the ubiquitiously expressed mammalian COMPASS complex acts as a "hidden" regulator of retinal differentiation.
We electroporated a piggybac transposon encoding doxycycline (dox)-inducible COMPASS transgene (wildtype or pocket mutant) into Rx:GFP knockin pluripotent stem cells (PSCs). CRISPR/Cas9 was used to knockout endogenous COMPASS, such that COMPASS expression was dependent on dox alone (n=3 independently edited PSC lines), allowing loss-of-function (LOF) & rescue studies. We integrated RNA-seq, ATAC-seq, & ChIP-seq to identify COMPASS direct target genes, and changes in the transcription and chromatin accessibility due to COMPASS LOF.
When COMPASS is expressed during early PSC differentiation, Rx+ retinal organoids form; when deleted, PSCs die. After transient COMPASS deletion, cell proliferation increases and differentiation skews toward mesoderm, with formation of contractile cardiomyocyte-containing embryoid bodies (EBs) in serum-free conditions or hematopoietic progenitors in methylcellulose culture. RNA-seq, ATAC-seq, & ChIP-seq studies reveal that transient COMPASS loss triggers a transition from one chromatin accessibility state to another, such that Wdr5 re-entry into chromatin spurs transcription of mesoderm—rather than retinal neuroectoderm—genes. Introduction of COMPASS mutants that disrupt a critical binding pocket results in a loss of retinal differentiation, leading to cardiomyocyte differentiation.
We identify a novel master regulator of retinal differentiation, the ubiquitious epigenetic complex COMPASS. When uncoupled from cell survival functions, COMPASS strikes the proper balance between retinal neuroectoderm & mesoderm through regulation of chromatin accessibility in a time-dependent manner. We identify a targetable binding pocket that controls PSC fate choice, which raises the possibility that neuroectoderm & mesoderm fates from PSCs can easily be toggled by small-molecules. Our studies point to a "dark" layer of temporal cell-fate control mediated by ubiquitous proteins whose function in stem cell differentiation has traditionally been masked by their conventionally accepted roles as "housekeeping" genes required for cell viability and chromatin function.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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