Diversity and complexity in chromatin recognition by TFII-I transcription factors in pluripotent embryonic stem cells and embryonic tissues

Abstract

GTF2I and GTF2IRD1 encode a family of closely related transcription factors TFII-I and BEN critical in embryonic development. Both genes are deleted in Williams-Beuren syndrome, a complex genetic disorder associated with neurocognitive, craniofacial, dental and skeletal abnormalities. Although genome-wide promoter analysis has revealed the existence of multiple TFII-I binding sites in embryonic stem cells (ESCs), there was no correlation between TFII-I occupancy and gene expression. Surprisingly, TFII-I recognizes the promoter sequences enriched for H3K4me3/K27me3 bivalent domain, an epigenetic signature of developmentally important genes. Moreover, we discovered significant differences in the association between TFII-I and BEN with the cis-regulatory elements in ESCs and embryonic craniofacial tissues. Our data indicate that in embryonic tissues BEN, but not the highly homologous TFII-I, is primarily recruited to target gene promoters. We propose a "feed-forward model" of gene regulation to explain the specificity of promoter recognition by TFII-I factors in eukaryotic cells.

Figure 1. Chromatin isolation and ChIP-chip analysis.

(A) The ChIP-chip strategy. Chromatin was isolated from mouse ESCs and the craniofacial region (CF) of E10.5 mouse embryos. Chromatin immunoprecipitation (ChIP) was performed with TFII-I and BEN-specific antibodies. CF region is marked in red with dashed lines. (B) The correlation between the genome-wide promoter binding using goat anti-BEN and mouse anti-HA antibodies to BEN (left) and rabbit and goat polyclonal antibodies to TFII-I (right). Spearman’s rank correlation coefficient (r) is calculated for each antibody pair. (C) The overall statistics of TFII-I and BEN target genes in ESCs and embryonic craniofacial tissues. (D) Distribution of the TFII-I and BEN-bound genomic sites with respect to the gene structure. The 2 kb region upstream of the transcription start site (TSS) is arbitrarily divided into two 1 kb segments (‘enhancer region’ and ‘proximal promoter’). 0.5 kb region downstream from TSS is split into exon and intron sequences. Bars represent the standard deviation calculated from four (TFII-I) or six (BEN) chip hybridizations, *p<0.1, **p<0.05, ***p<0.01.

Figure 2. Genome-wide promoter recognition by TFII-I transcription factors in mouse ESCs. (

A) TFII-I and BEN bind to the distal element of the Gsc promoter. The conservation plot is downloaded from the UCSC genome browser (http://genome.ucsc.edu/). (B) TFII-I binds to the promoters of Cfl1, Tnnt2 and Tbx1 while BEN occupies the promoter of Fgf3. TFII-I and BEN recognize the same sequence in the Tbx4 promoter although they bind to different cis-regulatory motifs within the promoter of Olfr1045. Red – TFII-I binding, blue – BEN binding.

Figure 3. TFII-I factors occupy the promoters of key developmental regulators in ESCs and embryonic craniofacial tissues (ETs).

(A) TFII-I and BEN bind to the promoters of Ednra, Edn1, Sox2, Sox3, Hoxa1 and Gata3 implicated in neural crest and craniofacial development. (B) TFII-I occupies the promoters of Emx1, Emx2, Zic1 and Neurod4 involved in brain development. The notation and labeling are as in Figure 2B. (C) TFII-I and BEN occupy the promoter regions of the HoxA cluster (Hoxa1, Hoxa7 and Hoxa13) in mouse ESCs and ETs. (D) TFII-I and BEN recognize the same cis-regulatory element in the promoters of Dicer, Cdx2 and Olfr480 in stem cells and embryonic tissues. (E) TFII-I binds to the promoters of Twist1, Snail2, Ezh2 and Nsd1 (red lines) in ESCs, although BEN does not bind to these promoters (blue lines). siRNA-mediated knockdown of TFII-I down-regulates expression of Twist, Snail1, Ezh2 and Nsd1 in embryonic neural crest cells (JoMa1.3 line). Error bars represent the standard deviation calculated from three independent knockdown experiments.

Figure 4. Promoter recognition by the TFII-I family.

TFII-I (in red) and BEN (in blue) possess distinct promoter recognition properties in ESCs and embryonic craniofacial tissues (ETs). First, the majority of ESC promoters occupied by TFII-I become vacant in ETs (a); second, a large number of ESC promoters recognized by BEN recruit both transcription factors to the same site in ETs (b); third, the ESC promoters occupied by TFII-I and BEN are still recognized by both transcription factors in ETs, predominantly in the same sequence, although some sites lost their binding completely (d); and fourth, the promoters active in ETs recruit more BEN than TFII-I (e). The black numbers on the right indicate percentage expected from the random distribution of TFII-I or BEN binding. The green numbers indicate the observed distribution significantly deviated from the random distribution (chi-squared test).

Figure 5. Colocalization of bivalent chromatin with TFII-I bound sites.

TFII-I associates with the promoter regions of key developmental genes enriched for H3K4me3 and H3K27me3 marks.

Figure 6. TFII-I binding overlaps with bivalent domain in ESCs. (

A) The promoter occupancy by TFII-I and BEN correlates with genome-wide distribution of H3K27me3 and H3K4me3 bivalent marks (green bars). (B) Heat map indicates the co-localization frequency of TFII-I and BEN with bivalent domain. TF2I-BP, TFII-I bound promoters; TF2I-NBP, promoters free of TFII-I; BEN-BP, BEN bound promoters; BEN-NBP, promoters free of BEN. (C) Depletion of TFII-I by siRNA knockdown reduces H3K4me3 at Hoxa1 and H3K27me3 at Hoxa13, Hdac4 and Nsd1. (D) ChIP revealed that TFII-I depletion affects H3K27me3 at the promoters of Hoxa13, Hdac3 and Nsd1 and H3K4me3 at the Hoxa1 promoter. H3K27m3 is in red; H3K4me3 is in green.

Figure 7. Target recognition by TFII-I factors. (

A) Chromatin recognition by TFII-I transcription factors in ESCs and embryonic craniofacial tissues (ETs). (1), the majority of TFII-I-bound ESC promoters tend to lose TFII-I binding in ETs. (2), the majority of BEN-bound ESC promoters recruit TFII-I de novo in ETs. (3), most of TFII-I and BEN-bound ESC promoters continue to retain these factors in ETs. (4), the promoters active in ETs recruit more BEN than TFII-I. (B) The feed-forward model explains the lack of correlation between the promoter binding by TFII-I and expression of the corresponding genes (stage 1, ESCs). Transcription factors (TFs) activated by TFII-I at stage 2 (ETs) can also recognize the TFII-I target sites and together they could initiate expression of stage-specific genes and additionally activate chromatin-modifying genes Ezh2 and Nsd1. These epigenetic factors mark novel target promoters (genes F and G) for repression or activation (e.g. repressive mark H3K27me3 and active mark H3K36me3). Stage 1, embryonic stem cells; stage 2, embryonic tissues.