89 - PBX1-binding Element with Enhancer Activity at the SHH Locus in the Chick Frontonasal Ectodermal Zone

Poster Board Number: 89
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Abstract Body : Sonic hedgehog (SHH) signaling from the frontonasal ectodermal zone (FEZ) is one of the key regulatory signals that is required to control morphogenesis of the Frontonasal Process (FNP). In the chicken FEZ by stage 22, spatial expression of SHH is upregulated by pre-B-cell leukemia homeobox transcription factor 1 (PBX1) and downregulated by PBX3. Therefore, elucidating genomic regions bound by PBX1/3 and chromatin landscapes within the SHH locus during early embryonic development will provide novel insights into understanding the causes of craniofacial congenital defects and may lead to better clinical interventions and outcomes. We hypothesized that PBX1 and PBX3 will bind to accessible regulatory sequences within the SHH locus to regulate SHH expression. To test this hypothesis, PBX1 and PBX3 ChIP-seq and ATAC-seq samples were prepared from the FEZ of normal chick embryos (n=2) at stage 22. Single-end read (50 nt) ChIP-seq data and paired-end read (2×150 nt) ATAC-seq data were generated (HiSeq 4000, Illumina). After mapping to the reference genome (galGal6), peaks of reads were called by MACS2, and PBX1- and PBX3-enriched peaks were analyzed by DiffBind. Analyzing these high throughput sequencing data, we confirmed that all PBX1 and PBX3 binding sequences spanned accessible chromatin regions. We identified a 400 bp long PBX1-bound element located in SHH intron 2 (chr2:8,173,222-8,173,621) by comparing PBX1 and PBX3 binding profiles. To confirm enhancer activity of this 400 bp long element, a 1.8 kb region spanning this peak was cloned into the TKP-TK vector and electroporated into the chick FNP ectoderm at stage 20. After 24 hours, this fragment exhibited transcriptional activity confirming its cis-regulatory function. Subsequently, this 1.8 kb fragment was divided into three 600 bp segments to identify the minimal region with enhancer activity. Each of these segments were transfected into DF1 cells along with a PBX1 expression vector and a luciferase reporter construct. This luciferase assay demonstrated that the reporter activity was restricted to the middle 600 bp fragment, which has 3 consensus binding sites for PBX transcription factors. The present study established that PBX1 and PBX3 bind to open chromatin regions within the SHH locus in the chicken FEZ and identified an enhancer sequence that drives PBX1 expression at the FEZ. Further investigation on relationships between histone mark binding and PBX binding at the SHH locus will provide better understanding of PBX/SHH-dependent molecular regulatory mechanisms driving craniofacial morphogenesis.