Long-range upstream and downstream enhancers control distinct subsets of the complex spatiotemporal Sox9 expression pattern

Abstract

SOX9 is an evolutionary conserved transcription factor that is expressed in a variety of tissues, with essential functions in cartilage, testis, heart, glial cell, inner ear and neural crest development. By comparing human and pufferfish genomic sequences, we previously identified eight highly conserved sequence elements between 290 kb 5' and 450 kb 3' to human SOX9. In this study, we assayed the regulatory potential of elements E1 to E7 in transgenic mice using a lacZ reporter gene driven by a 529 bp minimal mouse Sox9 promoter. We found that three of these elements and the Sox9 promoter control distinct subsets of the tissue-specific expression pattern of Sox9. E3, located 251 kb 5' to SOX9, directs lacZ expression to cranial neural crest cells and to the inner ear. E1 is located 28 kb 5' to SOX9 and controls expression in the node, notochord, gut, bronchial epithelium and pancreas. Transgene expression in the neuroectoderm is mediated by E7, located 95 kb 3' to SOX9, which regulates expression in the telencephalon and midbrain, and by the Sox9 minimal promoter which controls expression in the ventral spinal cord and hindbrain. We show that E3-directed reporter gene expression in neural crest cells of the first but not of the second and third pharyngeal arch is dependent on beta-catenin, revealing a complex regulation of Sox9 in cranial neural crest cells. Moreover, we identify and discuss highly conserved transcription factor binding sites within enhancer E3 that are in good agreement with current models for neural crest and inner ear development. Finally, we identify enhancer E1 as a cis-regulatory element conserved between vertebrates and invertebrates, indicating that some cis-regulatory sequences that control developmental genes in vertebrates might be phylogenetically ancient.

NFκB-Independent Signaling to the Cyclin D1 Gene by Rac

“…Therefore, there must be at least one essential Rac-regulated transcriptional element far upstream or downstream of the transcription start site. In fact, the collagen promoter is known to have distant enhancers located 20 kb upstream of the transcription start site, 45,46 while genes like Bmp2 47 and Sox9 48 have enhancer elements hundreds of kb downstream of their promoters. Although the identification of the critical Rac-regulated factor(s) requires further study, we can now suggest a model of cyclin D1 gene regulation in MEFs: ERK activity induces mid-G 1 cyclin D1 gene expression independently of NFκB activity while Rac and NFκB signal in cooperative, but parallel, pathways to stimulate cyclin D1 gene expression.…”

miR-449a Regulates the Chondrogenesis of Human Mesenchymal Stem Cells Through Direct Targeting of Lymphoid Enhancer-Binding Factor-1

“…In addition, a previous report has indicated that the Sox 9 promoter region contained a binding site for LEF-1 [31], while another study showed direct regulation of Sox 9 expression by LEF-1 [32]. Based on such findings, we then sought to determine whether inhibiting LEF-1 via miR-449a would in turn translate into the proportional repression of Sox 9, and whether the opposite effect would be observed when a miR-449a antagomir was used.…”

Diversity in the molecular and cellular strategies of epithelium-to-mesenchyme transitions: Insights from the neural crest

“…Thus, Xenopus Snail-2 and mouse Sox-9 promoters were found to contain binding sites for the Lef/Tcf transcription factors, indicating direct regulation of their expression by the canonical Wnt signaling pathway. 202,203 In addition, both mouse Sox-9 and chick Sox-10 exhibit in their regulatory sequence enhancers that drive specifically precocious expression in cranial NC cells, thereby revealing the presence of unique gene regulatory networks in this particular NC population. 194,202 Interestingly, these enhancers contain binding sites for Ets-1, which is restricted to delaminating NC cells in the head.…”