Transcriptional enhancement by GATA1-occupied DNA segments is strongly associated with evolutionary constraint on the binding site motif

Abstract

Tissue development and function are exquisitely dependent on proper regulation of gene expression, but it remains controversial whether the genomic signals controlling this process are subject to strong selective constraint. While some studies show that highly constrained noncoding regions act to enhance transcription, other studies show that DNA segments with biochemical signatures of regulatory regions, such as occupancy by a transcription factor, are seemingly unconstrained across mammalian evolution. To test the possible correlation of selective constraint with enhancer activity, we used chromatin immunoprecipitation as an approach unbiased by either evolutionary constraint or prior knowledge of regulatory activity to identify DNA segments within a 66-Mb region of mouse chromosome 7 that are occupied by the erythroid transcription factor GATA1. DNA segments bound by GATA1 were identified by hybridization to high-density tiling arrays, validated by quantitative PCR, and tested for gene regulatory activity in erythroid cells. Whereas almost all of the occupied segments contain canonical WGATAR binding site motifs for GATA1, in only 45% of the cases is the motif deeply preserved (found at the orthologous position in placental mammals or more distant species). However, GATA1-bound segments with high enhancer activity tend to be the ones with an evolutionarily preserved WGATAR motif, and this relationship was confirmed by a loss-of-function assay. Thus, GATA1 binding sites that regulate gene expression during erythroid maturation are under strong selective constraint, while nonconstrained binding may have only a limited or indirect role in regulation.

Identification of Biologically Relevant Enhancers in Human Erythroid Cells

“…1). As in previous reports, erythroid transcription factor binding was very common in intron 1 of RefSeq genes (data not shown) (44,59). These data suggest that transcription factors mark enhancers more commonly than p300 in erythroid cells because bona fide enhancers are expected to act in the genomic vicinity of their cognate genes (35).…”

“…Recent studies have used PhastCons analyses and RP scores to predict whether or not a region of DNA contains a functional CRM (54,59,65). PhastCons uses a hidden Markov model method on aligned genomic sequences to estimate a probability that any nucleotide is conserved (66).…”

Dissecting the regulatory switches of development: lessons from enhancer evolution in Drosophila

“…For example, mammalian sequences with multiple binding sites for the GATA1 TF will continue to function as an enhancer if only a single binding site is intact. However, a very particular site must remain intact: eliminating two of the three GATA1 sites in an enhancer may not have any effect on its function, but the single loss of the third crucial site can completely abolish enhancer activity in mice (Cheng et al, 2008). This example stresses the functional importance of a subset of possible binding sites, although the issue of overall TFBS architecture at mammalian enhancers remains largely unexplored.…”

Erythroid GATA1 function revealed by genome-wide analysis of transcription factor occupancy, histone modifications, and mRNA expression

“…These data, along with the expression results, can be viewed and downloaded from a custom genome browser at http://main.genome-browser.bx.psu.edu/, on the mouse mm8 (Feb 2006) genome assembly. A summary view from a 66-Mb region of mouse chromosome 7 studied previously (Cheng et al 2008;Zhang et al 2009) shows substantial ChIP signal above the background and a striking congruence of the signals for GATA1 occupancy between the two genome-wide data sets (Fig. 1B).…”

“…2A). This set includes 56 out of the 63 validated GATA1 OSs determined previously (Cheng et al 2008), indicating a sensitivity of 90%, while none of the 70 false-positive regions (i.e., ChIP with microarray hybridization [ChIP-chip] peaks not validated by quantitative PCR) are included, indicating high specificity.…”