Faculty Bibliography

The data described in this article refers to Chatterjee et al. (2015) "In vivo genome-wide analysis of multiple tissues identifies gene regulatory networks, novel functions and downstream regulatory genes for Bapx1 and its co-regulation with Sox9 in the mammalian vertebral column" (GEO GSE35649) [1]. Transcriptional profiling combined with genome wide binding data is a powerful tool to elucidate the molecular mechanism behind vertebrate organogenesis. It also helps to uncover multiple roles of a single gene in different organs. In the above mentioned report we reveal the function of the homeobox gene Bapx1 during the embryogenesis of five distinct organs (vertebral column, spleen, gut, forelimb and hindlimb) at a relevant developmental stage (E12.5), microarray analysis of isolated wildtype and mutant cells in is compared in conjunction with ChIP-Seq analysis. We also analyzed the development of the vertebral column by comparing microarray and ChIP-Seq data for Bapx1 with similarly generated data sets for Sox9 to generate a gene regulatory network controlling various facets of the organogenesis.

The risk of Hirschsprung disease (HSCR) is approximately 15/100 000 live births per newborn but has been reported to show significant inter-individual variation from the effects of seven common susceptibility alleles at the RET, SEMA3 and NRG1 loci. We show, by analyses of these variants in 997 samples from 376 HSCR families of European ancestry, that significant genetic risk can only be detected at RET (rs2435357 and rs2506030) and at SEMA3 (rs11766001), but not at NRG1. RET rs2435357 also showed significant frequency differences by gender, segment length of aganglionosis and familiality. Further, in combination, disease risk varied >30-fold between individuals with none and up to 6 susceptibility alleles. Thus, these polymorphisms can be used to stratify the newborn population into distinct phenotypic classes with defined risks to understand HSCR etiology.

BACKGROUND:Vertebrate organogenesis is a highly complex process involving sequential cascades of transcription factor activation or repression. Interestingly a single developmental control gene can occasionally be essential for the morphogenesis and differentiation of tissues and organs arising from vastly disparate embryological lineages. RESULTS:Here we elucidated the role of the mammalian homeobox gene Bapx1 during the embryogenesis of five distinct organs at E12.5 - vertebral column, spleen, gut, forelimb and hindlimb - using expression profiling of sorted wildtype and mutant cells combined with genome wide binding site analysis. Furthermore we analyzed the development of the vertebral column at the molecular level by combining transcriptional profiling and genome wide binding data for Bapx1 with similarly generated data sets for Sox9 to assemble a detailed gene regulatory network revealing genes previously not reported to be controlled by either of these two transcription factors. CONCLUSIONS:The gene regulatory network appears to control cell fate decisions and morphogenesis in the vertebral column along with the prevention of premature chondrocyte differentiation thus providing a detailed molecular view of vertebral column development.

BACKGROUND: Hirschsprung disease (HSCR) is a neurocristopathy characterized by absence of intramural ganglion cells along variable lengths of the gastrointestinal tract in neonates. Three polymorphisms, rs2435357, within a conserved transcriptional enhancer of RET, and, rs7835688 and rs16879552, within intron 1 of NRG1, have been shown to be associated with isolated forms of HSCR. We wished to replicate these findings, and study the interactions between these variants, in Indonesian HSCR patients. METHODS: Sixty isolated HSCR patients and 124 controls were ascertained for this study. The three genetic markers were examined using TaqMan Genotyping Assays in genomic DNA for association studies. RESULTS: RET rs2435357 showed the strongest association with HSCR both by case-control analysis (p=2.5 x 10(-8)) and transmission disequilibrium test (p=4.2 x 10(-6)). NRG1 rs7835688 was modestly associated with HSCR only by case-control analysis (p=4.3 x 10(-3)), whereas rs16879552 demonstrated no association (p>0.097). Two locus analyses of variants showed significant interactions with increased and decreased disease risks of HSCR at NRG1 but conditional on rs2435357 genotype. CONCLUSIONS: RET and NRG1 variants are common susceptibility factors for HSCR in Indonesia. These common variants demonstrate that development of HSCR requires joint effects of RET and NRG1 early in gut development.

QT interval variation is assumed to arise from variation in repolarization as evidenced from rare Na- and K-channel mutations in Mendelian QT prolongation syndromes. However, in the general population, common noncoding variants at a chromosome 1q locus are the most common genetic regulators of QT interval variation. In this study, we use multiple human genetic, molecular genetic, and cellular assays to identify a functional variant underlying trait association: a noncoding polymorphism (rs7539120) that maps within an enhancer of NOS1AP and affects cardiac function by increasing NOS1AP transcript expression. We further localized NOS1AP to cardiomyocyte intercalated discs (IDs) and demonstrate that overexpression of NOS1AP in cardiomyocytes leads to altered cellular electrophysiology. We advance the hypothesis that NOS1AP affects cardiac electrical conductance and coupling and thereby regulates the QT interval through propagation defects. As further evidence of an important role for propagation variation affecting QT interval in humans, we show that common polymorphisms mapping near a specific set of 170 genes encoding ID proteins are significantly enriched for association with the QT interval, as compared to genome-wide markers. These results suggest that focused studies of proteins within the cardiomyocyte ID are likely to provide insights into QT prolongation and its associated disorders.

Traditionally, conditional knockout studies in mouse have utilized the Cre or Flpe technology to activate the expression of reporter genes such as lacZ or PLAP. Employing these reporter genes, however, requires tissue fixation. To make way for downstream in vivo or in vitro applications, we have inserted enhanced green fluorescent protein (EGFP) into the endogenous Sox9 locus and generated a novel conditional Sox9 null allele, by flanking the entire Sox9 coding region with loxP sites and inserting an EGFP reporter gene into the 3'-UTR allowing for EGFP to be expressed upon Sox9 loss of function yet under the control of the endogenous Sox9 promoter. Mating this new allele to any Cre-expressing line, the fate of Sox9 null cells can be traced in the cell type of interest in vivo or in vitro after fluorescence-activated cell sorting.

Skeletogenesis is a complex multi-step process, which involves many genes and pathways. The tightly regulated interplay between these genes in these pathways ensures a correct and timely organogenesis and it is imperative that we have a fair understanding of the major genes and gene families involved in the process. This review aims to give a deeper insight into the roles of 3 major transcription factor families involved in skeleton formation: Sox, Runx and Pax and to look at the human skeleotogenic phenotypes associated with mutations in these genes.

The sequencing of many vertebrate species over the last decade has opened up the possibility of using comparative genomics as a powerful tool to elucidate regulatory elements in the vertebrate genome. The zebrafish has played a pivotal role in this process. Its genome has been used in large-scale genome comparisons to locate vertebrate specific regulatory elements and also it has been an excellent model system to test out the predicted DNA sequences for their ability to drive reporter gene expression in vivo. In spite of all the successes there have still been some issues in using the zebrafish as a model system for these kinds of assays. This review will shed some light on the successes and failures of the zebrafish in pushing forward regulatory genomics.

BACKGROUND:Identifying DNA sequences (enhancers) that direct the precise spatial and temporal expression of developmental control genes remains a significant challenge in the annotation of vertebrate genomes. Locating these sequences, which in many cases lie at a great distance from the transcription start site, has been a major obstacle in deciphering gene regulation. Coupling of comparative genomics with functional validation to locate such regulatory elements has been a successful method in locating many such regulatory elements. But most of these studies looked either at a single gene only or the whole genome without focusing on any particular process. The pressing need is to integrate the tools of comparative genomics with knowledge of developmental biology to validate enhancers for developmental transcription factors in greater detail RESULTS:Our results show that near four different genes (nkx3.2, pax9, otx1b and foxa2) in zebrafish, only 20-30% of highly conserved DNA sequences can act as developmental enhancers irrespective of the tissue the gene expresses in. We find that some genes also have multiple conserved enhancers expressing in the same tissue at the same or different time points in development. We also located non-conserved enhancers for two of the genes (pax9 and otx1b). Our modified Bacterial artificial chromosome (BACs) studies for these 4 genes revealed that many of these enhancers work in a synergistic fashion, which cannot be captured by individual DNA constructs and are not conserved at the sequence level. Our detailed biochemical and transgenic analysis revealed Foxa1 binds to the otx1b non-conserved enhancer to direct its activity in forebrain and otic vesicle of zebrafish at 24 hpf. CONCLUSION/CONCLUSIONS:Our results clearly indicate that high level of functional conservation of genes is not necessarily associated with sequence conservation of its regulatory elements. Moreover certain non conserved DNA elements might have role in gene regulation. The need is to bring together multiple approaches to bear upon individual genes to decipher all its regulatory elements.

Transgenics using bacterial artificial chromosomes (BACs) offers a great opportunity to look at gene regulation in a developing embryo. The modified BAC containing a reporter inserted just before the translational start site of the gene of interest allows for the visualization of spatio-temporal gene expression. Though this method has been used in the mouse model extensively, its utility in zebrafish studies is relatively new. This review aims to look at the utility of making BAC transgenics in zebrafish and its applications in functional genomics. We look at the various methods to modify the BAC, some limitations and what the future holds.