Identification of novel craniofacial regulatory domains located far upstream of SOX9 and disrupted in Pierre Robin sequence.

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.

Disruption of a long distance regulatory region upstream of SOX9 in isolated disorders of sex development.

BACKGROUND: The early gonad is bipotential and can differentiate into either a testis or an ovary. In XY embryos, the SRY gene triggers testicular differentiation and subsequent male development via its action on a single gene, SOX9. The supporting cell lineage of the bipotential gonad will differentiate as testicular Sertoli cells if SOX9 is expressed and conversely will differentiate as ovarian granulosa cells when SOX9 expression is switched off. RESULTS: Through copy number variation mapping this study identified duplications upstream of the SOX9 gene in three families with an isolated 46,XX disorder of sex development (DSD) and an overlapping deletion in one family with two probands with an isolated 46,XY DSD. The region of overlap between these genomic alterations, and previously reported deletions and duplications at the SOX9 locus associated with syndromic and isolated cases of 46,XX and 46,XY DSD, reveal a minimal non-coding 78 kb sex determining region located in a gene desert 517-595 kb upstream of the SOX9 promoter. CONCLUSIONS: These data indicate that a non-coding regulatory region critical for gonadal SOX9 expression and subsequent normal sex development is located far upstream of the SOX9 promoter. Its copy number variations are the genetic basis of isolated 46,XX and 46,XY DSDs of variable severity (ranging from mild to complete sex reversal). It is proposed that this region contains a gonad specific SOX9 transcriptional enhancer(s), the gain or loss of which results in genomic imbalance sufficient to activate or inactivate SOX9 gonadal expression in a tissue specific manner, switch sex determination, and result in isolated DSD.

Altered SOX9 genital tubercle enhancer region in hypospadias.

Human mutations in the SOX9 gene or its regulatory region can disrupt testicular development, leading to disorders of sex development (DSDs). Our previous work involving the genomic analysis of isolated DSD patients revealed a 78kb minimal sex determining region (RevSex) far upstream of SOX9 that was duplicated in 46,XX and deleted in 46,XY DSDs. It was postulated that RevSex contains a gonadal enhancer. However, the most highly conserved sub-region within RevSex, called SR4, was neither responsive to sex determining factors in vitro nor active in the gonads of transgenic mice, suggesting that SR4 may not be functioning as a testicular enhancer. Interestingly, SR4 transgenic mice showed reporter activity in the genital tubercle, the primordium of the penis and clitoris, a previously unreported domain of Sox9 expression. SOX9 protein was detected in the genital tubercle, notably in the urethral plate epithelium, preputial glands, ventral surface ectoderm and corpus cavernosa. SR4 may therefore function as a Sox9 genital tubercle enhancer, mutations of which could possibly lead to hypospadias, a birth defect seen in the DSD patients in the RevSex study. SR4 activity and the observed SOX9 expression pattern suggest that SR4 may function as a Sox9 genital tubercle enhancer. However, conditional ablation of Sox9 in the genital tubercle using Shh-Cre/+;Sox9flox/flox mice revealed no genital tubercle abnormalities, possibly due to compensation by similar Sox factors. To conclude, we have identified a novel regulatory enhancer driving Sox9 expression during external genitalia development.

Deposition of 5-Methylcytosine on Enhancer RNAs Enables the Coactivator Function of PGC-1α.

The Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a transcriptional co-activator that plays a central role in adapted metabolic responses. PGC-1α is dynamically methylated and unmethylated at the residue K779 by the methyltransferase SET7/9 and the Lysine Specific Demethylase 1A (LSD1), respectively. Interactions of methylated PGC-1α[K779me] with the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, the Mediator members MED1 and MED17, and the NOP2/Sun RNA methytransferase 7 (NSUN7) reinforce transcription, and are concomitant with the m(5)C mark on enhancer RNAs (eRNAs). Consistently, loss of Set7/9 and NSun7 in liver cell model systems resulted in depletion of the PGC-1α target genes Pfkl, Sirt5, Idh3b, and Hmox2, which was accompanied by a decrease in the eRNAs levels associated with these loci. Enrichment of m(5)C within eRNA species coincides with metabolic stress of fasting in vivo. Collectively, these findings illustrate the complex epigenetic circuitry imposed by PGC-1α at the eRNA level to fine-tune energy metabolism.

[Cis-ruptions of highly conserved non-coding genomic elements distant from the SOX9 gene in the Pierre Robin sequence]. / Cis-ruptions d'éléments génomiques hautement conservés non codants à distance du gène SOX9 dans la séquence de Pierre Robin.

Major developmental genes, exhibiting complex expression patterns, are often embedded within a genic desert particularly rich in regions, which though non-coding are highly conserved. The developmental expression of these genes in many areas requires coordinated regulation in time and space, which is orchestrated by some of these conserved non-coding regions, acting as transcriptional regulators. SOX9 is an essential gene for many developmental processes, such as chondrogenesis, migration and differentiation of neural crest cells and testis development. In agreement with these major expression areas, SOX9 haploinsufficiency, linked to alterations in coding sequence, leads to a polymorphic malformation syndrome - campomelic dysplasia - whose major symptoms are a bone anomaly, a Pierre Robin sequence, and a sexual differentiation anomaly (Disorder of Sex Development, DSD). SOX9 is located in a  ~2.5 Mb gene desert extremely rich in conserved sequences. We have used the SOX9 locus and campomelic dysplasia as a model to show that one or several endophenotypes within a complex syndrome may arise from a tissue-specific deregulation of a major developmental gene transcription. Our work has focused on one of these endophenotypes, SPR, characterized by the triad micro- and/or retrognathy, glossoptosis and cleft palate. Here we report in detail how we identified alterations (translocations, deletions, point mutations) in non-coding regions, located far away (more than 1.2 Mb) upstream and downstream of SOX9, in clustered or sporadic SPR cases.

Disruption of long-distance highly conserved noncoding elements in neurocristopathies.

One of the key discoveries of vertebrate genome sequencing projects has been the identification of highly conserved noncoding elements (CNEs). Some characteristics of CNEs include their high frequency in mammalian genomes, their potential regulatory role in gene expression, and their enrichment in gene deserts nearby master developmental genes. The abnormal development of neural crest cells (NCCs) leads to a broad spectrum of congenital malformation(s), termed neurocristopathies, and/or tumor predisposition. Here we review recent findings that disruptions of CNEs, within or at long distance from the coding sequences of key genes involved in NCC development, result in neurocristopathies via the alteration of tissue- or stage-specific long-distance regulation of gene expression. While most studies on human genetic disorders have focused on protein-coding sequences, these examples suggest that investigation of genomic alterations of CNEs will provide a broader understanding of the molecular etiology of both rare and common human congenital malformations.

Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence.

Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06-1.23 Mb upstream of SOX9, and microdeletions both approximately 1.5 Mb centromeric and approximately 1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.