ATAC-seq and ChIP-seq targeting PBX1 and PBX3 in the chick FEZ at stage 22

During craniofacial development, sonic hedgehog (SHH) signaling from the frontonasal ectodermal zone (FEZ) plays a critical role in the control of Frontonasal Process (FNP) morphogenesis. Along with SHH, pre-B-cell leukemia homeobox (PBX) transcription factors regulate midfacial prominence fusion partially through PBX-dependent epithelial apoptosis and epithelial-to-mesenchymal transition. Despite the critical functions of PBX transcription factors in the cephalic epithelium, their specific roles in regulating SHH expression in the FEZ have not been fully understood. Elucidating PBX-dependent mechanisms that mediate regulation of SHH expression during early embryonic development will provide novel insights into understanding the etiology of craniofacial birth defects and may improve clinical interventions and outcomes. We hypothesized that PBX1 and PBX3 regulate SHH expression in the FEZ by activating or repressing its transcription. The hypothesis was tested by manipulating PBX1/3 expression in chick embryos and profiling epigenomic landscapes at early developmental stages. PBX1/3 expression was perturbed in the chick FEZ beginning at stage 10 (HH10) using RCAS viruses, and the resulting SHH expression was assessed at HH22. Overexpression of PBX1 expanded SHH expression, while overexpression of PBX3 decreased SHH expression. Also, reducing PBX1 expression decreased SHH expression, but reducing PBX3 led to increased and ectopic SHH expression. To assess the extent to which the two PBX transcription factors directly targeted the SHH locus, we performed ATAC-seq and ChIP-seq for PBX1 and PBX3 on normal chick embryonic FEZ cells at HH22. The high-throughput data confirmed that all PBX1/3-bound sequences span accessible chromatin regions and uncovered a 400 bp PBX1-enriched element within intron 1 of the SHH gene (chr2:8,173,222-8,173,621). Enhancer activity of this element was demonstrated by electroporation of reporter constructs in ovo and luciferase reporter assays in vitro. When bound by PBX1, this element upregulates SHH transcription, while it downregulates transcription when bound by PBX3. The present study identifies a regulatory sequence that interacts with PBX1/3 to regulate SHH expression in the FEZ and establishes that PBX1 and PBX3 play complementary roles in SHH regulation.
This genome-wide sequencing data set displays open accessible chromatin regions and binding sites for PBX1 and PBX3 in the embryonic chick frontonasal ectodermal tissue at stage 22 (HH22) by ATAC-seq and ChIP-seq. SHH is a key regulator of the upper jaw morphogenesis, and its expression pattern in the frontonasal process defines a signaling center, the frontonasal ectodermal zone (FEZ), at Hamburger and Hamilton by stage 22 (HH22). Expression of SHH is regulated by homeodomain transcription factors, PBX1 and PBX3, in a complementary manner. To evaluate their direct interaction with chromatin in the chick FEZ at HH22, we generated these ATAC-seq data and ChIP-seq data targeting PBX1 and PBX3 (n=2). With this data set, we identified a 400 bp-long open chromatin region within Intron 1 of SHH where both PBX1 and PBX3 bind. This intronic element modulates transcription through complementary mechanisms by PBX transcription factors; transcription is activated when bound by PBX1 while transcription is repressed when bound by PBX3. We named this regulatory element SHH FEZ Enhancer 1 (SFE1).