Temtamy preaxial brachydactyly syndrome is caused by loss-of-function mutations in chondroitin synthase 1, a potential target of BMP signaling.

Altered Bone Morphogenetic Protein (BMP) signaling leads to multiple developmental defects, including brachydactyly and deafness. Here we identify chondroitin synthase 1 (CHSY1) as a potential mediator of BMP effects. We show that loss of human CHSY1 function causes autosomal-recessive Temtamy preaxial brachydactyly syndrome (TPBS), mainly characterized by limb malformations, short stature, and hearing loss. After mapping the TPBS locus to chromosome 15q26-qterm, we identified causative mutations in five consanguineous TPBS families. In zebrafish, antisense-mediated chsy1 knockdown causes defects in multiple developmental processes, some of which are likely to also be causative in the etiology of TPBS. In the inner ears of zebrafish larvae, chsy1 is expressed similarly to the BMP inhibitor dan and in a complementary fashion to bmp2b. Furthermore, unrestricted Bmp2b signaling or loss of Dan activity leads to reduced chsy1 expression and, during epithelial morphogenesis, defects similar to those that occur upon Chsy1 inactivation, indicating that Bmp signaling affects inner-ear development by repressing chsy1. In addition, we obtained strikingly similar zebrafish phenotypes after chsy1 overexpression, which might explain why, in humans, brachydactyly can be caused by mutations leading either to loss or to gain of BMP signaling.

A specific mutation in the distant sonic hedgehog (SHH) cis-regulator (ZRS) causes Werner mesomelic syndrome (WMS) while complete ZRS duplications underlie Haas type polysyndactyly and preaxial polydactyly (PPD) with or without triphalangeal thumb.

Werner mesomelic syndrome (WMS) is an autosomal dominant disorder with unknown molecular etiology characterized by hypo- or aplasia of the tibiae in addition to the preaxial polydactyly (PPD) of the hands and feet and/or five-fingered hand with absence of thumbs. We show that point mutations of a specific nucleotide within the sonic hedgehog (SHH) regulatory region (ZRS) cause WMS. In a previously unpublished WMS family, we identified the causative G>A transition at position 404 of the ZRS, and in six affected family members of a second WMS family we found a 404G>C mutation of the ZRS. The 404G>A ZRS mutation is known as the "Cuban mutation" of PPD type II (PPD2). Interestingly, the index patient of that family had tibial hypoplasia as well. These data provide the first evidence that WMS is caused by a specific ZRS mutation, which leads to strong ectopic SHH expression. In contrast, we show that complete duplications of the ZRS region lead to type Haas polysyndactyly or triphalangeal thumb-polysyndactyly syndrome, but do not affect lower limb development. We suggest the term "ZRS-associated syndromes" and a clinical subclassification for the continuum of limb malformations caused by different molecular alterations of the ZRS.

Transcriptome profiling of estrogen-regulated genes in human primary osteoblasts reveals an osteoblast-specific regulation of the insulin-like growth factor binding protein 4 gene.

Estradiol (E2) is believed to modulate physiological functions relevant to osteoblast biology through the actions of estrogen receptors (ERs) that in turn regulate the expression of target genes. The molecular effects of estrogen action in bone remain to be fully elucidated. This study reports a genome-wide molecular and computational analysis of the interaction between ER and regulatory elements on the DNA of target genes in human primary osteoblasts. Of approximately 54,000 gene probes surveyed in this study, a total of 375 genes were up-regulated and 418 genes were down-regulated on exposure to E2, with only 46 of these being direct target genes after 24 h, as determined by concomitant cycloheximide treatment. Computational analysis discovered several pathways where E2 co-regulates multiple functionally linked components. Examination of the genomic sequence of IGF binding protein 4 located ER response elements within the first intron. Using by chromatin immunoprecipitation, we show a site- and cell-specific recruitment of transcription factors to this newly identified regulatory region. Transient transfection studies revealed that this intronic region acts as a functional promoter in human osteoblasts. Taken together, this analysis provides a comprehensive gene transcription profile and identifies several genes of potential physiological importance in controlling estrogen-mediated signaling in primary osteoblasts.

Mechanistic role of a disease-associated genetic variant within the ADAM33 asthma susceptibility gene.

BACKGROUND: ADAM33 has been identified as an asthma-associated gene in an out-bred population. Genetic studies suggested that the functional role of this metalloprotease was in airway remodeling. However, the mechanistic roles of the disease-associated SNPs have yet to be elucidated especially in the context of the pathophysiology of asthma. One disease-associated SNP, BC+1, which resides in intron BC toward the 5' end of ADAM33, is highly associated with the disease. METHODS: The region surrounding this genetic variant was cloned into a model system to determine if there is a regulatory element within this intron that influences transcription. RESULTS: The BC+1 protective allele did not impose any affect on the transcription of the reporter gene. However, the at-risk allele enforced such a repressive affect on the promoter that no protein product from the reporter gene was detected. These results indicated that there exists within intron BC a regulatory element that acts as a repressor for gene expression. Moreover, since SNP BC+1 is a common genetic variant, this region may interact with other undefined regulatory elements within ADAM33 to provide a rheostat effect, which modulates pre-mRNA processing. Thus, SNP BC+1 may have an important role in the modulation of ADAM33 gene expression. CONCLUSION: These data provide for the first time a functional role for a disease-associated SNP in ADAM33 and begin to shed light on the deregulation of this gene in the pathophysiology of asthma.

Androgen receptor binding sites identified by a GREF_GATA model.

Changes in transcriptional regulation can be permissive for tumor progression by allowing for selective growth advantage of tumor cells. Tumor suppressors can effectively inhibit this process. The PMEPA1 gene, a potent inhibitor of prostate cancer cell growth is an androgen-regulated gene. We addressed the question of whether or not androgen receptor can directly bind to specific PMEPA1 promoter upstream sequences. To test this hypothesis we extended in silico prediction of androgen receptor binding sites by a modeling approach and verified the actual binding by in vivo chromatin immunoprecipitation assay. Promoter upstream sequences of highly androgen-inducible genes were examined from microarray data of prostate cancer cells for transcription factor binding sites (TFBSs). Results were analyzed to formulate a model for the description of specific androgen receptor binding site context in these sequences. In silico analysis and subsequent experimental verification of the selected sequences suggested that a model that combined a GREF and a GATA TFBS was sufficient for predicting a class of functional androgen receptor binding sites. The GREF matrix family represents androgen receptor, glucocorticoid receptor and progesterone receptor binding sites and the GATA matrix family represents GATA binding protein 1-6 binding sites. We assessed the regulatory sequences of the PMEPA1 gene by comparing our model-based GREF_GATA predictions to weight matrix-based predictions. Androgen receptor binding to predicted promoter upstream sequences of the PMEPA1 gene was confirmed by chromatin immunoprecipitation assay. Our results suggested that androgen receptor binding to cognate elements was consistent with the GREF_GATA model. In contrast, using only single GREF weight matrices resulted in additional matches, apparently false positives. Our findings indicate that complex models based on datasets selected by biological function can be superior predictors as they recognize TFBSs in their functional context.