Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis

Abstract

Alx1 is a conserved regulator of skeletogenesis in echinoderms and evolutionary changes in Alx1 sequence and expression have played a pivotal role in modifying programs of skeletogenesis within the phylum. Alx1 regulates a large suite of effector genes that control the morphogenetic behaviors and biomineral-forming activities of skeletogenic cells. To better understand the gene regulatory control of skeletogenesis by Alx1, we used genome-wide ChIP-seq to identify Alx1-binding sites and direct gene targets. Our analysis revealed that many terminal differentiation genes receive direct transcriptional inputs from Alx1. In addition, we found that intermediate transcription factors previously shown to be downstream of Alx1 all receive direct inputs from Alx1. Thus, Alx1 appears to regulate effector genes by indirect, as well as direct, mechanisms. We tested 23 high-confidence ChIP-seq peaks using GFP reporters and identified 18 active cisregulatory modules (CRMs); this represents a high success rate for CRM discovery. Detailed analysis of a representative CRM confirmed that a conserved, palindromic Alx1-binding site was essential for expression. Our work significantly advances our understanding of the gene regulatory circuitry that controls skeletogenesis in sea urchins and provides a framework for evolutionary studies.

ALX 1‐ related frontonasal dysplasia results from defective neural crest cell development and migration

“…Evidence of gene compensation has previously been reported in animal studies (Beverdam et al , ; Dee et al , ). In studies of sea urchins, Alx4 was shown to be directly regulated by Alx1 (Rafiq et al , ; Khor et al , ). The question remains how the different ALX family members regulate craniofacial development, through transcriptional activation or repression of shared and unique target genes.…”

The evolution of a new cell type was associated with competition for a signaling ligand

“…Alx1 has positive inputs into at least half of the >400 effector genes expressed selectively by PMCs and an even larger proportion of such genes that are expressed at high levels [24]. Recent chromatin immunoprecipitation sequencing (ChIP-seq) studies have shown that many of these inputs are direct [64]. Alx1 also functions in PMCs to suppress alternative mesodermal regulatory states [24,29], and misexpression of alx1 in nonskeletogenic mesoderm cells is sufficient to convert them to a PMC-like fate [17].…”

Transcription Factors of the Alx Family: Evolutionarily Conserved Regulators of Deuterostome Skeletogenesis

“…Alx1 provides positive inputs into almost half of the ~420 genes that are differentially expressed by PMCs, highlighting the pivotal role of Alx1 in establishing skeletogenic cell identity (Rafiq et al, 2014). A recent chromatin immunoprecipitation sequencing (ChIP-seq) study determined that many of these genes, including both regulatory (i.e., transcription factorencoding) and effector (i.e., differentiation) genes, are direct targets of alx1 (Khor et al, 2019). A second transcription factor, Ets1, collaborates with Alx1 in the co-regulation of a large fraction of genes differentially expressed by PMCs (Rafiq et al, 2014), in many cases through a feed-forward mechanism (i.e., Ets1 > Alx1, Ets + Alx1 > effector gene; Yamasu and Wilt, 1999;Amore and Davidson, 2006;Oliveri et al, 2008;Yajima et al, 2010;Shashikant et al, 2018;Khor et al, 2019).…”

“…A recent chromatin immunoprecipitation sequencing (ChIP-seq) study determined that many of these genes, including both regulatory (i.e., transcription factorencoding) and effector (i.e., differentiation) genes, are direct targets of alx1 (Khor et al, 2019). A second transcription factor, Ets1, collaborates with Alx1 in the co-regulation of a large fraction of genes differentially expressed by PMCs (Rafiq et al, 2014), in many cases through a feed-forward mechanism (i.e., Ets1 > Alx1, Ets + Alx1 > effector gene; Yamasu and Wilt, 1999;Amore and Davidson, 2006;Oliveri et al, 2008;Yajima et al, 2010;Shashikant et al, 2018;Khor et al, 2019). Downstream effector genes that are regulated by Alx1 include those that directly mediate biomineralization (e.g., those that encode secreted spicule matrix proteins that are incorporated into the biomineral) and those that mediate skeletogenesis through signaling pathways and morphogenetic cell behaviors (Figure 2).…”

“…Positive regulatory inputs by Ets1 and Alx1 into msp130, sm50, and vegf-Ig-10 are described in (Oliveri et al, 2008). Direct targets of the sea urchin Alx1 (Khor et al, 2019) define a genetic subcircuit that impinges on almost all aspect of PMC morphogenesis, including directional cell migration, extracellular matrix (ECM) remodeling, cell-cell fusion, and biomineralization. Dashed arrows indicate interactions that may be indirect.…”