Computational prediction and experimental validation of novel Hedgehog-responsive enhancers linked to genes of the Hedgehog pathway

Abstract Background The Hedgehog (Hh) signaling pathway, acting through three homologous transcription factors (GLI1, GLI2, GLI3) in vertebrates, plays multiple roles in embryonic organ development and adult tissue homeostasis. At the level of the genome, GLI factors bind to specific motifs in enhancers, some of which are hundreds of kilobases removed from the gene promoter. These enhancers integrate the Hh signal in a context-specific manner to control the spatiotemporal pattern of target gene expression. Importantly, a number of genes that encode Hh pathway molecules are themselves targets of Hh signaling, allowing pathway regulation by an intricate balance of feed-back activation and inhibition. However, surprisingly few of the critical enhancer elements that control these pathway target genes have been identified despite the fact that such elements are central determinants of Hh signaling activity. Recently, ChIP studies have been carried out in multiple tissue contexts using mouse models carrying FLAG-tagged GLI proteins (GLIFLAG). Using these datasets, we tested whether a meta-analysis of GLI binding sites, coupled with a machine learning approach, could reveal genomic features that could be used to empirically identify Hh-regulated enhancers linked to loci of the Hh signaling pathway. Results A meta-analysis of four existing GLIFLAG datasets revealed a library of GLI binding motifs that was substantially more restricted than the potential sites predicted by previous in vitro binding studies. A machine learning method (kmer-SVM) was then applied to these datasets and enriched k-mers were identified that, when applied to the mouse genome, predicted as many as 37,000 potential Hh enhancers. For functional analysis, we selected nine regions which were annotated to putative Hh pathway molecules and found that seven exhibited GLI-dependent activity, indicating that they are directly regulated by Hh signaling (78 % success rate). Conclusions The results suggest that Hh enhancer regions share common sequence features. The kmer-SVM machine learning approach identifies those features and can successfully predict functional Hh regulatory regions in genomic DNA surrounding Hh pathway molecules and likely, other Hh targets. Additionally, the library of enriched GLI binding motifs that we have identified may allow improved identification of functional GLI binding sites.http://deepblue.lib.umich.edu/bitstream/2027.42/134520/1/12861_2016_Article_106.pd

WNT5a in Tongue and Fungiform Papilla Development

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71558/1/j.1749-6632.2009.04369.x.pd

A YY1-dependent increase in aerobic metabolism is indispensable for intestinal organogenesis

During late gestation, villi extend into the intestinal lumen to dramatically increase the surface area of the intestinal epithelium, preparing the gut for the neonatal diet. Incomplete development of the intestine is the most common gastrointestinal complication in neonates, but the causes are unclear. We provide evidence in mice that Yin Yang 1 (Yy1) is crucial for intestinal villus development. YY1 loss in the developing endoderm had no apparent consequences until late gestation, after which the intestine differentiated poorly and exhibited severely stunted villi. Transcriptome analysis revealed that YY1 is required for mitochondrial gene expression, and ultrastructural analysis confirmed compromised mitochondrial integrity in the mutant intestine. We found increased oxidative phosphorylation gene expression at the onset of villus elongation, suggesting that aerobic respiration might function as a regulator of villus growth. Mitochondrial inhibitors blocked villus growth in a fashion similar to Yy1 loss, thus further linking oxidative phosphorylation with late-gestation intestinal development. Interestingly, we find that necrotizing enterocolitis patients also exhibit decreased expression of oxidative phosphorylation genes. Our study highlights the still unappreciated role of metabolic regulation during organogenesis, and suggests that it might contribute to neonatal gastrointestinal disorders

Pyrin Modulates the Intracellular Distribution of PSTPIP1

PSTPIP1 is a cytoskeleton-associated adaptor protein that links PEST-type phosphatases to their substrates. Mutations in PSTPIP1 cause PAPA syndrome (Pyogenic sterile Arthritis, Pyoderma gangrenosum, and Acne), an autoinflammatory disease. PSTPIP1 binds to pyrin and mutations in pyrin result in familial Mediterranean fever (FMF), a related autoinflammatory disorder. Since disease-associated mutations in PSTPIP1 enhance pyrin binding, PAPA syndrome and FMF are thought to share a common pathoetiology. The studies outlined here describe several new aspects of PSTPIP1 and pyrin biology. We document that PSTPIP1, which has homology to membrane-deforming BAR proteins, forms homodimers and generates membrane-associated filaments in native and transfected cells. An extended FCH (Fes-Cip4 homology) domain in PSTPIP1 is necessary and sufficient for its self-aggregation. We further show that the PSTPIP1 filament network is dependent upon an intact tubulin cytoskeleton and that the distribution of this network can be modulated by pyrin, indicating that this is a dynamic structure. Finally, we demonstrate that pyrin can recruit PSTPIP1 into aggregations (specks) of ASC, another pyrin binding protein. ASC specks are associated with inflammasome activity. PSTPIP1 molecules with PAPA-associated mutations are recruited by pyrin to ASC specks with particularly high efficiency, suggesting a unique mechanism underlying the robust inflammatory phenotype of PAPA syndrome

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis

Construction of an ~700-kb transcript map around the Familial Mediterranean Fever locus on human chromosome 16p13.3

We used a combination of cDNA selection, exon amplification, and computational prediction from genomic sequence to isolate transcribed sequences from genomic DNA surrounding the familial Mediterranean fever (FMF) locus. Eighty-seven kb of genomic DNA around D16S3370, a marker showing a high degree of linkage disequilibrium with FMF, was sequenced to completion, and the sequence annotated. A transcript map reflecting the minimal number of genes encoded within the ∼700 kb of genomic DNA surrounding the FMF locus was assembled. This map consists of 27 genes with discreet messages detectable on Northerns, in addition to three olfactory-receptor genes, a cluster of 18 tRNA genes, and two putative transcriptional units that have typical intron–exon splice junctions yet do not detect messages on Northerns. Four of the transcripts are identical to genes described previously, seven have been independently identified by the French FMF Consortium, and the others are novel. Six related zinc-finger genes, a cluster of tRNAs, and three olfactory receptors account for the majority of transcribed sequences isolated from a 315-kb FMF central region (betweenD16S468/D16S3070 and cosmid 377A12). Interspersed among them are several genes that may be important in inflammation. This transcript map not only has permitted the identification of the FMF gene (MEFV), but also has provided us an opportunity to probe the structural and functional features of this region of chromosome 16.Michael Centola, Xiaoguang Chen, Raman Sood, Zuoming Deng, Ivona Aksentijevich, Trevor Blake, Darrell O. Ricke, Xiang Chen, Geryl Wood, Nurit Zaks, Neil Richards, David Krizman, Elizabeth Mansfield, Sinoula Apostolou, Jingmei Liu, Neta Shafran, Anil Vedula, Melanie Hamon, Andrea Cercek, Tanaz Kahan, Deborah Gumucio, David F. Callen, Robert I. Richards, Robert K. Moyzis, Norman A. Doggett, Francis S. Collins, P. Paul Liu, Nathan Fischel-Ghodsian and Daniel L. Kastne

Analysis of Germline GLI1 Variation Implicates Hedgehog Signalling in the Regulation of Intestinal Inflammatory Pathways

Charlie Lees and colleagues identify a reduced-function variant of the hedgehog signaling pathway protein GLI1 that associates with inflammatory bowel disease, and investigate its role in a mouse model of colitis

Hedgehog Signaling in Intestinal Development, Homeostasis and Disease

The hedgehog (Hh) signaling pathway plays several diverse regulatory and patterning roles during organogenesis of the intestine and in the regulation of adult intestinal homeostasis. In the embryo, fetus, and adult, intestinal Hh signaling is paracrine: Hh ligands are expressed in the endodermally derived epithelium, while signal transduction is confined to the mesenchymal compartment, where at least a dozen distinct cell types are capable of responding to Hh signals. Epithelial Hh ligands not only regulate a variety of mesenchymal cell behaviors, but they also direct these mesenchymal cells to secrete additional soluble factors (e.g., Wnts, Bmps, inflammatory mediators) that feed back to regulate the epithelial cells themselves. Evolutionary conservation of the core Hh signaling pathway, as well as conservation of epithelial/mesenchymal cross talk in the intestine, has meant that work in many diverse model systems has contributed to our current understanding of the role of this pathway in intestinal organogenesis, which is reviewed here.http://deepblue.lib.umich.edu/bitstream/2027.42/175364/2/annurev-physiol-031620-094324.pdfPublished versionDescription of annurev-physiol-031620-094324.pdf : Published versio