The focal facial dermal dysplasias (FFDDs) are a group of inherited developmental disorders in which the characteristic diagnostic feature is bitemporal scar-like lesions that resemble forceps marks. To date, the genetic defects underlying these ectodermal dysplasias have not been determined. To identify the gene defect causing autosomal-recessive Setleis syndrome (type III FFDD), homozygosity mapping was performed with genomic DNAs from five affected individuals and 26 members of the consanguineous Puerto Rican (PR) family originally described by Setleis and colleagues. Microsatellites D2S1397 and D2S2968 were homozygous in all affected individuals, mapping the disease locus to 2q37.3. Haplotype analyses of additional markers in the PR family and a consanguineous Arab family further limited the disease locus to approximately 3 Mb between D2S2949 and D2S2253. Of the 29 candidate genes in this region, the bHLH transcription factor, TWIST2, was initially sequenced on the basis of its known involvement in murine facial development. Homozygous TWIST2 nonsense mutations, c.324C>T and c.486C>T, were identified in the affected members of the Arab and PR families, respectively. Characterization of the expressed mutant proteins, p.Q65X and p.Q119X, by electrophoretic mobility shift assays and immunoblot analyses indicated that they were truncated and unstable. Notably, Setleis syndrome patients and Twist2 knockout mice have similar facial features, indicating the gene's conserved role in mammalian development. Although human TWIST2 and TWIST1 encode highly homologous bHLH transcription factors, the finding that TWIST2 recessive mutations cause an FFDD and dominant TWIST1 mutations cause Saethre-Chotzen craniocynostosis suggests that they function independently in skin and bone development.
Cranial neural crest cells (crNCCs) migrate from the neural tube to the pharyngeal arches (PAs) of the developing embryo and, subsequently, differentiate into bone and connective tissue to form the mandible. Within the PAs, crNCCs respond to local signaling cues to partition into the proximo-distally oriented subdomains that convey positional information to these developing tissues. Here, we show that the distal-most of these subdomains, the distal cap, is marked by expression of the transcription factor Hand1 (H1) and gives rise to the ectomesenchymal derivatives of the lower incisors. We uncover a H1 enhancer sufficient to drive reporter gene expression within the crNCCs of the distal cap. We show that bone morphogenic protein (BMP) signaling and the transcription factor HAND2 (H2) synergistically regulate H1 distal cap expression. Furthermore, the homeodomain proteins distal-less homeobox 5 (DLX5) and DLX6 reciprocally inhibit BMP/H2-mediated H1 enhancer regulation. These findings provide insights into how multiple signaling pathways direct transcriptional outcomes that pattern the developing jaw.Bmp | DLX | HAND1 | cranial neural crest cells | development A fter migrating to specific locations within the developing embryo, neural crest cells (NCCs), a multipotent cell population originating from the dorsal lip of the neural tube, respond to local morphogenetic signaling cues to pattern and differentiate (1). Following migration to the pharyngeal arches (PAs), cranial NCCs (crNCCs) respond to endothelin 1 (EDN1) and bone morphogenic protein (BMP) signaling from surrounding pharyngeal epithelia to subdivide the PA ectomesenchyme into discrete proximo-distal domains (2). In the mandibular arch (MD1), these nested PA subdomains, characterized by the expression of DLX homeobox and/or HAND basic helix-loop-helix (bHLH) transcription factors, are integral to the development of specific jaw structures, including bone, tongue mesenchyme, and heterogeneous teeth (3). HAND factors regulate mandibular incisor development (4), whereas DLX proteins influence maxillary molar development (2). The mechanisms by which DLX-and HANDdependent transcriptional programs establish proximo-distal PA subdomains are poorly understood. Indeed, the dearth of identified cis-regulatory elements active in postmigratory NCCs has hampered the elucidation of the gene regulatory networks that establish regional identity within the developing mandible.BMP and EDN1 signaling initially overlaps within the distal murine MD1, but by embryonic day (E)10.5, these signaling pathways become spatially segregated. The distal-most tip of the PAs, known as the distal cap, is transiently devoid of active EDN1 signaling. BMP signaling is ostensibly restricted to this distal cap by the localized expression of Bmp antagonists (3). Here, we provide evidence that DLX5 and DLX6 act as transcriptional repressors of BMP signaling specifically within the cranial PAs. We show that, within the crNCCs, the BMP-dependent transcription factors Smads, H2, and GATA2/3 provi...
Setleis Syndrome (OMIM ID: 227260) is a rare autosomal recessive disease characterized by abnormal facial development. Recently, we have reported that two nonsense mutations (c.486C>T [Q119X] and c.324C>T [Q65X]) of the basic helix-loop-helix (bHLH) transcription factor TWIST2 cause Setleis Syndrome. Here we show that periostin, a cell adhesion protein involved in connective tissue development and maintenance, is down-regulated in Setleis Syndrome patient fibroblast cells and that periostin positively responds to manipulations in TWIST2 levels, suggesting that TWIST2 is a transactivator of periostin. Functional analysis of the TWIST2 mutant form (Q119X) revealed that it maintains the ability to localize to the nucleus, forms homo and heterodimers with the ubiquitous bHLH protein E12, and binds to dsDNA. Reporter gene assays using deletion constructs of the human periostin promoter also reveal that TWIST2 can activate this gene more specifically than Twist1, while the Q119X mutant results in no significant transactivation. Chromatin immunoprecipitation assays show that both wild-type TWIST2 and the Q119X mutant bind the periostin promoter, however only wild-type TWIST2 is associated with higher levels of histone acetylation across the 5′-regulatory region of periostin. Taken together, these data suggest that the C-terminal domain of TWIST2, which is missing in the Q119X mutant form of TWIST2, is responsible for proper transactivation of the periostin gene. Improper regulation of periostin by the mutant form of TWIST2 could help explain some of the soft tissue abnormalities seen in these patients therefore providing a genotype-phenotype relationship for Setleis Syndrome.
Periostin (POSTN), a cell adhesion protein involved in connective tissue development and maintenance, is regulated positively by the basic Helix‐Loop‐Helix (bHLH) transcription factor TWIST2. Two naturally occurring nonsense mutations of TWIST2 (Q119X and Q65X) were found in Puerto Rican and Omani Setleis Syndrome (SS) patients. The Yeast‐Two‐Hybrid assay was used to assay the dimerization properties of TWIST2 mutants. Wildtype and Q119X forms of TWIST2 form homodimers as well as heterodimers with E12 (a ubiquitous bHLH factor), while the Q65X TWIST2 mutant lacks ability to dimerize with itself and with E12. Microarray analysis of human skin fibroblasts suggests that TWIST2 seems to act as a positive regulator more frequently than as a repressor in human skin fibroblasts. POSTN is down‐regulated in SS patient fibroblasts when compared to normal controls, as confirmed by real time RT‐PCR. RNAi and TWIST2 over‐expression techniques provide evidence that POSTN is positively regulated by TWIST2. Knockdown of TWIST2 shows a decrease of POSTN expression over a 72hr time course in fibroblastic cells. Similarly, TWIST2 over‐expressed in these same cells results in increased POSTN expression over a similar time course. Overexpression of Twist2 in SS patient skin fibroblasts caused an increase in POSTN gene expression. This research is supported by MBRS RISE Grant R25GM061838, and RCMI Grant G12RR03051.
Setleis Syndrome (SS, MIM #227260) is a rare autosomal recessive disorder characterized by abnormal facial and skin development. Recently, we have shown that nonsense mutations in the basic helix‐loop‐helix (bHLH) transcription factor TWIST2 cause Setleis Syndrome. Microarray analysis suggests that periostin (POSTN), a cell adhesion protein involved in connective tissue development and maintenance, is down regulated in Setleis Syndrome patient fibroblasts. Overexpression and knockdown of TWIST2 resulted in increased and decreased expression of POSTN mRNA, respectively. Functional analysis of the Q119X TWIST2 mutant form revealed that it maintains the ability to localize to the nucleus, form homo and heterodimers with the ubiquitous bHLH protein E12 and shows binding to dsDNA. Chromatin immunoprecipitation assays suggest that both wild‐type TWIST2 and its mutant form can bind the POSTN regulatory region, however only wild‐type TWIST2 is associated with higher levels of acetylated histone H3 of the POSTN promoter. Reporter gene assays using deletion constructs of the POSTN regulatory region also reveal that wild‐type TWIST2 can activate this gene, but not the Q119X TWIST2 mutant. This data suggests that the C‐terminal domain of TWIST2, which is missing in Q119X TWIST2, is responsible for proper transactivation of POSTN. This research is supported by MBRS RISE Grant R25GM061838 and RCMI Grant G12RR03051.
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