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. 2013 Feb 1;374(1):234-44.
doi: 10.1016/j.ydbio.2012.11.017. Epub 2012 Nov 27.

"VSports注册入口" Pitx1 broadly associates with limb enhancers and is enriched on hindlimb cis-regulatory elements

Carlos R Infante  1 , Sungdae Park, Alexandra G Mihala, David M Kingsley, Douglas B Menke
Affiliations

Pitx1 broadly associates with limb enhancers and is enriched on hindlimb cis-regulatory elements (V体育ios版)

Carlos R Infante (V体育官网入口) et al. Dev Biol. .

"VSports最新版本" Abstract

Extensive functional analyses have demonstrated that the pituitary homeodomain transcription factor Pitx1 plays a critical role in specifying hindlimb morphology in vertebrates. However, much less is known regarding the target genes and cis-regulatory elements through which Pitx1 acts. Earlier studies suggested that the hindlimb transcription factors Tbx4, HoxC10, and HoxC11 might be transcriptional targets of Pitx1, but definitive evidence for direct regulatory interactions has been lacking. Using ChIP-Seq on embryonic mouse hindlimbs, we have pinpointed the genome-wide location of Pitx1 binding sites during mouse hindlimb development and identified potential gene targets for Pitx1. We determined that Pitx1 binding is significantly enriched near genes involved in limb morphogenesis, including Tbx4, HoxC10, and HoxC11. Notably, Pitx1 is bound to the previously identified HLEA and HLEB hindlimb enhancers of the Tbx4 gene and to a newly identified Tbx2 hindlimb enhancer. Moreover, Pitx1 binding is significantly enriched on hindlimb relative to forelimb-specific cis-regulatory features that are differentially marked by H3K27ac. However, our analysis revealed that Pitx1 also strongly associates with many functionally verified limb enhancers that exhibit similar levels of activity in the embryonic mesenchyme of forelimbs and hindlimbs. We speculate that Pitx1 influences hindlimb morphology both through the activation of hindlimb-specific enhancers as well as through the hindlimb-specific modulation of enhancers that are active in both sets of limbs. VSports手机版.

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Figures

Figure 1
Figure 1
Pitx1 binding is enriched near promoter regions and is associated with the canonical TAATCC bicoid-type binding motif. (A) Distribution of Pitx1 ChIP-seq peaks relative to transcription start sites compared to 1,000 replicates of randomized genomic regions. (B) Percentage of Pitx1 peaks found in promoter regions, regions downstream of genes, or within the exons/introns of genes. (C) Comparison of the in vitro determined Pitx1 binding motif (Pitx1 2312.1) to the top de novo motif enriched within ± 50 bp of Pitx1 ChIP-seq peak summits. (D) Distribution of de novo motif among all Pitx1 ChIP-seq peak summits.
Figure 2
Figure 2
Pitx1 associates with the HLEA and HLEB hindlimb enhancers of the Tbx4 gene. Whole-mount LacZ stained E11.5 mouse embryos carrying HLEA-hsp68LacZ (A) or HLEB-hsp68LacZ (B) transgenes. (C) Quantitative PCR analysis reveals the relative enrichment of HLEA and HLEB after Pitx1 ChIP. Values presented are the mean and standard error for eight (HLEA) or three (HLEB) independent ChIP experiments.
Figure 3
Figure 3
Pitx1 binding highlights functionally important subdomains of HLEA. (A) Conservation plot comparing the sequence identity between mouse and human HLEA reveals three regions of conservation. Aligned regions of at least 10bp and ≥70% sequence identity are shaded in pink (B) Pitx1 ChIP-seq signal across HLEA. The y-axis indicates the number of sequence tags per ten million reads. The position and orientation of sequences that match Pitx1 binding motifs are denoted by blue and red arrowheads. (C) Schematic of HLEA subregions tested for enhancer activity via Hsp68LacZ transgenes. (D–K) Representative whole-mount LacZ stained mouse embryos carrying different HLEA transgenes. Forelimbs and hindlimbs are outlined in white.
Figure 4
Figure 4
Pitx1 associates with limb cis-regulatory domains of the Tbx2 and Tbx4 genes. (A) Schematic of the genomic region surrounding Tbx2 and Tbx4. Regions of conservation ≥100 bp with ≥70% identity between mouse-human, mouse-chicken, and mouse-zebrafish are highlighted by colored peaks (blue=exons, pink=conserved non-coding regions). The Tbx4 HLEA and HLEB elements are highlighted in orange, and the putative Tbx2 limb enhancer region is shaded green. (B) Pitx1 ChIP-Seq and input control signal. An asterisk denotes the location of a Pitx1 peak just downstream of the Tbx4 open reading frame that was tested for enhancer activity. The y-axes of Pitx1 ChIP and input control plots are scaled to indicate the number of sequence tags per ten million reads. (C) Genomic regions covered by mouse Tbx2 BAC and Hsp68LacZ transgenes. Bars indicate the location of sequences contained within each transgene. Blue boxes within BAC transgenes indicate the insertion site of an IRES-LacZ cassette. Yellow bar indicates the position of the transgene containing a putative Tbx2 hindlimb enhancer, Red bar indicates the transgene containing a putative Tbx2 forelimb/hindlimb enhancer. (D-I) Representative whole-mount LacZ-stained mouse embryos carrying Tbx2 BAC transgenes (D, E, and F) and Hsp68LacZ transgenes (G, H, and I). Forelimbs and hindlimbs are outlined in white.
Figure 5
Figure 5
A Pitx1 ChIP-Seq peak is associated with a HoxC10/11 hindlimb regulatory domain. (A) Conservation plot comparing the mouse and human HoxC gene clusters. Regions of conservation ≥100 bp with ≥70% identity between mouse-human and mouse-chicken are highlighted by colored peaks (blue=exons, pink=conserved non-coding regions). (B) Pitx1 ChIP-Seq and input control signal. The y-axes of Pitx1 ChIP and input control plots are scaled to indicate the number of sequence tags per ten million reads. A Pitx1 peak located in the intergenic region between HoxC10 and HoxC11 falls within a 10 kb region (orange shading) that has been previously demonstrated to drive hindlimb-specific expression (Papenbrock et al., 2000).
Figure 6
Figure 6
Pitx1 binding is enriched on hindlimb cis-regulatory elements. (A) Comparison of overlap between Pitx1 ChIP-Seq peaks from E11.5 hindlimbs and H3K27ac ChIP-Seq peaks from E11.5 forelimbs and E11.5 hindlimbs (Cotney et al., 2012). (B) Comparison of overlap between embryonic hindlimb Pitx1 ChIP-Seq peaks and forelimb-biased and hindlimb-biased H3K27ac marks.
Figure 7
Figure 7
Pitx1 broadly associates with limb-specific enhancers and genes involved in limb and skeletal development. (A) Percent of known limb, heart, forebrain, midbrain, and hindbrain specific enhancers from the VISTA Enhancer Browser database (http://enhancer.lbl.gov/) that overlap with Pitx1 ChIP-Seq peaks. Also shown are the percentages of P300 ChIP-Seq peaks from E11.5 mouse limb, forebrain, and midbrain (Visel et al., 2009) that overlap with Pitx1 peaks. (B and C) Results from GREAT analysis. (B) Top 10 enriched terms from the GO Biological Processes database associated with Pitx1 ChIP-Seq peaks. (C) Top 10 enriched terms from the Mouse Phenotype database associated with Pitx1 ChIP-Seq peaks.
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