Differential contribution of cis -regulatory elements to higher order chromatin structure and expression of the CFTR locus

Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure

A Genome-Wide Analysis of Open Chromatin in Human Epididymis Epithelial Cells Reveals Candidate Regulatory Elements for Genes Coordinating Epididymal Function1

The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33 542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium

A genome-wide analysis of open chromatin in human tracheal epithelial cells reveals novel candidate regulatory elements for lung function

Distal cell-type-specific regulatory elements may be located at very large distances from the genes that they control and are often hidden within intergenic regions or in introns of other genes. The development of methods that enable mapping of regions of open chromatin genome wide has greatly advanced the identification and characterisation of these elements

Mutation spectrum of NOD2 reveals recessive inheritance as a main driver of Early Onset Crohn’s Disease

Inflammatory bowel disease (IBD), clinically defined as Crohn’s disease (CD), ulcerative colitis (UC), or IBD-unclassified, results in chronic inflammation of the gastrointestinal tract in genetically susceptible hosts. Pediatric onset IBD represents ≥ 25% of all IBD diagnoses and often presents with intestinal stricturing, perianal disease, and failed response to conventional treatments. NOD2 was the first and is the most replicated locus associated with adult IBD, to date. However, its role in pediatric onset IBD is not well understood. We performed whole-exome sequencing on a cohort of 1,183 patients with pediatric onset IBD (ages 0–18.5 years). We identified 92 probands with biallelic rare and low frequency NOD2 variants accounting for approximately 8% of our cohort, suggesting a Mendelian inheritance pattern of disease. Additionally, we investigated the contribution of recessive inheritance of NOD2 alleles in adult IBD patients from a large clinical population cohort. We found that recessive inheritance of NOD2 variants explains ~ 7% of cases in this adult IBD cohort, including ~ 10% of CD cases, confirming the observations from our pediatric IBD cohort. Exploration of EHR data showed that several of these adult IBD patients obtained their initial IBD diagnosis before 18 years of age, consistent with early onset disease. While it has been previously reported that carriers of more than one NOD2 risk alleles have increased susceptibility to Crohn’s Disease (CD), our data formally demonstrate that recessive inheritance of NOD2 alleles is a mechanistic driver of early onset IBD, specifically CD, likely due to loss of NOD2 protein function. Collectively, our findings show that recessive inheritance of rare and low frequency deleterious NOD2 variants account for 7–10% of CD cases and implicate NOD2 as a Mendelian disease gene for early onset Crohn’s Disease

MAPPIN: Method for Annotating, Predicting Pathogenicity, and mode of Inheritance for Nonsynonymous variants

This directory includes software, readme files for installation and usage, associated data files for running MAPPIN, and prediction scores for all nsSNVs. The annotate.mappin.tar.gz tarball includes all the scripts and associated data files needed to annotate missense variants. The predict.mappin.tar.gz includes all the scripts and data files need to run predictions on the annotated missense variants file. The mappin_training_data_cross-validation tarball has the training datasets and other files, which can be run for training and cross-validation using the associated R script. The hg19.mappin.prediction.scores.tar.gz file has MAPPIN prediction scores (dominant, recessive, benign) for all possible nsSNVs in the genome. The hg38.mappin.prediction.scores.tar.gz file has MAPPIN prediction scores lifted over to hg38.<div><br></div><div><b>Release update 10/24/17: minor bug fixed; users only need to re-download predict_mappin.tar.gz.</b><br><div><br></div><div>Note: Although the license states CC BY, MAPPIN software can be used and modified freely for <b>non-commercial</b> purposes only. This work is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc/4.0/ or send a letter to Creative Commons, PO Box 1866, Mountain View, CA 94042, USA.</div></div

Chromatin Dynamics in the Regulation of CFTR Expression

The contribution of chromatin dynamics to the regulation of human disease-associated loci such as the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been the focus of intensive experimentation for many years. Recent technological advances in the analysis of transcriptional mechanisms across the entire human genome have greatly facilitated these studies. In this review we describe the complex machinery of tissue-specific regulation of CFTR expression, and put earlier observations in context by incorporating them into datasets generated by the most recent genomics methods. Though the gene promoter is required for CFTR expression, cell-type specific regulatory elements are located elsewhere in the gene and in flanking intergenic regions. Probably within its own topological domain established by the architectural proteins CTCF and cohesin, the CFTR locus utilizes chromatin dynamics to remodel nucleosomes, recruit cell-selective transcription factors, and activate intronic enhancers. These cis-acting elements are then brought to the gene promoter by chromatin looping mechanisms, which establish long-range interactions across the locus. Despite its complexity, the CFTR locus provides a paradigm for elucidating the critical role of chromatin dynamics in the transcription of individual human genes

Apple a Day Chicago (Semester Unknown) IPRO 359: Apple A Day Chicago IPRO 359 Abstract F08

Developing a website that will be capable of carrying out customer orders.  Conducting market research to help determine the ideal customer for the service that our business provides.  Designing a complete business. This involves mapping out the sourcing and distribution of products, determining an efficient supply chain, promoting and marketing the business so that people are aware that Apple-A-Day Chicago exists, determining the staffing requirements of the business, as well as analyzing what the projected costs and profits of the company will be.  Providing proof of concept of the operation of the business so that there is concrete evidence that Apple-A-Day Chicago is a viable solution to providing individuals with a convenient and healthy snack.Deliverable

Apple a Day Chicago (Semester Unknown) IPRO 359: Apple A Day Chicago IPRO 359 Poster2 F08

Developing a website that will be capable of carrying out customer orders.  Conducting market research to help determine the ideal customer for the service that our business provides.  Designing a complete business. This involves mapping out the sourcing and distribution of products, determining an efficient supply chain, promoting and marketing the business so that people are aware that Apple-A-Day Chicago exists, determining the staffing requirements of the business, as well as analyzing what the projected costs and profits of the company will be.  Providing proof of concept of the operation of the business so that there is concrete evidence that Apple-A-Day Chicago is a viable solution to providing individuals with a convenient and healthy snack.Deliverable

Apple a Day Chicago (Semester Unknown) IPRO 359

Developing a website that will be capable of carrying out customer orders.  Conducting market research to help determine the ideal customer for the service that our business provides.  Designing a complete business. This involves mapping out the sourcing and distribution of products, determining an efficient supply chain, promoting and marketing the business so that people are aware that Apple-A-Day Chicago exists, determining the staffing requirements of the business, as well as analyzing what the projected costs and profits of the company will be.  Providing proof of concept of the operation of the business so that there is concrete evidence that Apple-A-Day Chicago is a viable solution to providing individuals with a convenient and healthy snack.Deliverable