Diversity and Complexity in Chromatin Recognition by TFII-I Transcription Factors in Pluripotent Embryonic Stem Cells and Embryonic Tissues

<div><p><em>GTF2I</em> and <em>GTF2IRD1</em> 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 <em>cis</em>-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.</p> </div

Chromatin isolation and ChIP-chip analysis.

<p>(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.</p

Target recognition by TFII-I factors. (

<p>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 <i>Ezh2</i> and <i>Nsd1</i>. 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.</p

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

<p>(A) TFII-I and BEN bind to the promoters of <i>Ednra, Edn1, Sox2, Sox3, Hoxa1</i> and <i>Gata3</i> implicated in neural crest and craniofacial development. (B) TFII-I occupies the promoters of <i>Emx1, Emx2, Zic1</i> and <i>Neurod4</i> involved in brain development. The notation and labeling are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044443#pone-0044443-g002" target="_blank">Figure 2B</a>. (C) TFII-I and BEN occupy the promoter regions of the <i>HoxA</i> cluster (<i>Hoxa1, Hoxa7</i> and <i>Hoxa13</i>) in mouse ESCs and ETs. (D) TFII-I and BEN recognize the same <i>cis</i>-regulatory element in the promoters of <i>Dicer, Cdx2</i> and <i>Olfr480</i> in stem cells and embryonic tissues. (E) TFII-I binds to the promoters of <i>Twist1, Snail2, Ezh2 and Nsd1</i> (red lines) in ESCs, although BEN does not bind to these promoters (blue lines). siRNA-mediated knockdown of TFII-I down-regulates expression of <i>Twist</i>, <i>Snail1, Ezh2</i> and <i>Nsd1</i> in embryonic neural crest cells (JoMa1.3 line). Error bars represent the standard deviation calculated from three independent knockdown experiments.</p

Promoter recognition by the TFII-I family.

<p>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).</p

Colocalization of bivalent chromatin with TFII-I bound sites.

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

In-Depth Characterization of microRNA Transcriptome in Melanoma

<div><p>The full repertoire of human microRNAs (miRNAs) that could distinguish common (benign) nevi from cutaneous (malignant) melanomas remains to be established. In an effort to gain further insight into the role of miRNAs in melanoma, we applied Illumina next-generation sequencing (NGS) platform to carry out an in-depth analysis of miRNA transcriptome in biopsies of nevi, thick primary (>4.0 mm) and metastatic melanomas with matched normal skin in parallel to melanocytes and melanoma cell lines (both primary and metastatic) (n = 28). From this data representing 698 known miRNAs, we defined a set of top-40 list, which properly classified normal from cancer; also confirming 23 (58%) previously discovered miRNAs while introducing an additional 17 (42%) known and top-15 putative novel candidate miRNAs deregulated during melanoma progression. Surprisingly, the miRNA signature distinguishing specimens of melanoma from nevus was significantly different than that of melanoma cell lines from melanocytes. Among the top list, miR-203, miR-204-5p, miR-205-5p, miR-211-5p, miR-23b-3p, miR-26a-5p and miR-26b-5p were decreased in melanomas vs. nevi. In a validation cohort (n = 101), we verified the NGS results by qRT-PCR and showed that receiver-operating characteristic curves for miR-211-5p expression accurately discriminated invasive melanoma (AUC = 0.933), melanoma in situ (AUC = 0.933) and dysplastic (atypical) nevi (AUC = 0.951) from common nevi. Target prediction analysis of co-transcribed miRNAs showed a cooperative regulation of key elements in the MAPK signaling pathway. Furthermore, we found extensive sequence variations (isomiRs) and other non-coding small RNAs revealing a complex melanoma transcriptome. Deep-sequencing small RNAs directly from clinically defined specimens provides a robust strategy to improve melanoma diagnostics.</p></div

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

<p>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 <i>Hoxa1</i> and H3K27me3 at <i>Hoxa13, Hdac4</i> and <i>Nsd1</i>. (D) ChIP revealed that TFII-I depletion affects H3K27me3 at the promoters of <i>Hoxa13, Hdac3</i> and <i>Nsd1</i> and H3K4me3 at the <i>Hoxa1</i> promoter. H3K27m3 is in red; H3K4me3 is in green.</p

Clinicopathologic characteristics for discovery cohort.

*, #, ˆ<p>denote samples matched with normal control tissues. The abbreviations are: PCM, primary cutaneous melanoma (invasive); ALM, acrolentiginous melanoma; NM, nodular melanoma; SSM, superficial spreading melanoma; NS, normal skin; CN, common nevus; PCM, primary cutaneous melanoma; MMLN, metastatic melanoma to lymph node; MMS, metastatic melanoma to skin; SLN, sentinel lymph node; NA, not available.</p

Bioinformatics pipeline, heat map-clustering analysis on top-40 miRNAs differentially expressed in melanoma specimens and cell lines.

<p>The flowchart describes the programs and steps used to process the raw small RNA-sequence data to miRNA signature and target gene prediction (A). Clustering analysis of the top-40 miRNAs identified by NGS appropriately segregated primary cutaneous melanoma (PCM), normal skin (NS), common nevus (CN), metastatic melanoma to lymph node (MMLN) and metastatic melanoma to skin (MMS); and cultured primary melanoma (CPM), cultured metastatic melanoma (CMM) and cultured melanocytes (CMEL) (B). The miRNA signature for melanoma specimens is dramatically dissimilar to cell lines. A clustering analysis using 698 distinct mature known miRNAs appropriately segregated PCM from MMLN from MMS (C) and correctly segregated two melanoma patients with biopsy-proven microscopic metastasis to sentinel lymph node (SLN) from two melanoma patients without metastatic disease (D).</p