scholarly journals A flexible repertoire of transcription factor binding sites and diversity threshold determines enhancer activity in embryonic stem cells

Author(s):  
Gurdeep Singh ◽  
Shanelle Mullany ◽  
Sakthi D Moorthy ◽  
Richard Zhang ◽  
Tahmid Mehdi ◽  
...  

ABSTRACTTranscriptional enhancers are critical for development, phenotype evolution and often mutated in disease contexts; however, even in well-studied cell types, the sequence code conferring enhancer activity remains unknown. We found genomic regions with conserved binding of multiple transcription factors in mouse and human embryonic stem cells (ESCs) contain on average 12.6 conserved transcription factor binding sites (TFBS). These TFBS are a diverse repertoire of 70 different sequences representing the binding sites of both known and novel ESC regulators. Remarkably, using a diverse set of TFBS from this repertoire was sufficient to construct short synthetic enhancers with activity comparable to native enhancers. Site directed mutagenesis of conserved TFBS in endogenous enhancers or TFBS deletion from synthetic sequences revealed a requirement for more than ten different TFBS. Furthermore, specific TFBS, including the OCT4:SOX2 co-motif, are dispensable, despite co-binding the OCT4, SOX2 and NANOG master regulators of pluripotency. These findings reveal a TFBS diversity threshold overrides the need for optimized regulatory grammar and individual TFBS that bind specific master regulators.

2020 ◽  
Vol 118 (8) ◽  
pp. 2015-2026 ◽  
Author(s):  
Matan Goldshtein ◽  
Meir Mellul ◽  
Gai Deutch ◽  
Masahiko Imashimizu ◽  
Koh Takeuchi ◽  
...  

2018 ◽  
Author(s):  
Xiao-Yong Li ◽  
Michael B. Eisen

AbstractHere we explore how mutating different sequences in an enhancer that regulates patterned gene expression in Drosophila melanogaster embryos can affect its output. We used quantitative imaging to analyze the effects of a wide variety of mutations in the hunchback distal anterior enhancer. This enhancer has been shown to respond to the anterior morphogen Bicoid, but we found that mutations in only one of the five strong Bicoid sites in the enhancer has a significant effect on its binding. The pioneer factor Zelda, which binds to this enhancer and is the only other factor implicated in its activity besides Bicoid. However, we found that mutations of all its sites only has modest effect that is limited to reduction of its output in more posterior regions of the embryo, where Bicoid levels are low. In contrast to the modest effects of mutating known transcription factor binding sites, randomizing the sequences between Zelda and Bicoid sites significantly compromised enhancer activity. Finer mapping suggested that the sequences that determine activity are broadly distributed in the enhancer. Mutations in short sequences flanking Bicoid binding sites have stronger effects than mutations to Bicoid sites themselves, highlighting the complex and counterintuitive nature of the relationship between enhancer sequence and activity.


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