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About Center for Spatial and Functional Genomics
The Center of Spatial and Functional Genomics has positioned over two decades of experience in locus discovery and Genome-wide association study (GWAS) in both academic and biotech settings (Struan Grant) and regulation of immunity and inflammatory gene expression (Andrew Wells) to make significant advances in the genetics of common diseases through the application of 3D genomics-based techniques. This work is already establishing the transcriptional architecture of loci implicated in the genetic susceptibility to disease and we are now applying these approaches through our federally funded projects at the whole-genome level in multiple human primary cells and tissues.
Techniques Utilized in Our Work
ChIP-seq represents a combination of two techniques, namely chromatin immuno-precipitation and high-throughput sequencing. This method for the identification and mapping of human DNA sequences that are bound by specific protein factor in vivo allows for a global location analysis for binding sites of a given histone mark (such as the enhancer mark H3k27ac), transcription factor, or another DNA-binding factor.
Genome-wide association study (GWAS) efforts only report genomic signals and not necessarily the precise localization of culprit genes. Given the need to improve upon the relatively low resolution of typical Hi-C approaches, we have expanded on a massively parallel, high-resolution Capture-C based method to characterize the genome-wide interactions of all human promoters in various cell settings. We have designed a custom Agilent SureSelect RNA library targeting DpnII restriction fragments overlapping promoters of protein-coding, noncoding, antisense, snRNA, miRNA, snoRNA and lincRNA genes.
We are now applying this method of genome-wide promoter-focused Capture C to multiple disease-relevant cell types. We also generate ATAC-seq open chromatin maps from the same samples to identify the most likely functional proxy SNPs for each GWAS locus for a given trait. By intersecting our sub-1kb genome-wide promoter-focused Capture C data with the ATAC-seq data, we observe consistent contacts between "open" promoters and proxy SNPs for various loci. Only by establishing which specific genes at such loci are regulated in the correct cellular context can one truly translate GWAS findings into more efficacious treatments for common diseases.
The greater than 3 billion nucleotide human genome is wrapped around histone octamers called nucleosomes, which are folded into higher-ordered chromatin fibers to allow packaging into the nucleus of the cell. For this reason, most of the genome is not accessible. Because all processes that regulate gene expression occur in regions of open chromatin, a map of the open chromatin 'landscape' represents a powerful resource that can focus research to regions with functional significance.
We generate genome-wide, cellular open chromatin maps using ATAC-seq (Assay for Transposase-Accessible Chromatin sequencing, Nat. Methods 10:1213, 2013). The key component of this process is a mutant hyperactive Tn5 transposable element that carries a sequence tag identifier and 'jumps' into accessible regions of nuclear DNA. These mutant elements lack the normal ability to repair double-stranded DNA cuts, resulting in excision.
We use RNA sequencing (RNA-Seq) as a highly sensitive and accurate approach for measuring coding and non-coding RNA transcription and gene expression across the genome. RNA-Seq offers several advantages over microarray approaches, including a broader dynamic range, higher sensitivity and accuracy, integration into existing NGS platforms and pipelines, and transcript analysis that is not limited by prior knowledge.
We use CRISPR/CAS9 to induce site-specific mutations in genes, putative regulatory elements, and SNPs in the human and mouse genomes. CRISPR stands for "clustered regularly interspaced short palindromic repeats" which, in combination with the prokaryotic RNA-directed DNA nuclease CAS9, acts as an adaptive bacterial immune system against DNA bacteriophages. In bacteria, complementary CRISPR RNAs (crRNA and tracrRNA) are transcribed and form an RNA duplex. This duplex is cleaved and subsequently acts to guide CAS9 to viral DNA sequences that are complementary to the crRNA.
Recently, CRISPR/CAS9 has been re-engineered into eukaryotic transient and stable expression systems as a powerful genome editing tool for eukaryotic cells. Importantly, we are applying this powerful approach to both human cells in vitro, and for generating transgenic mice that lack evolutionarily conserved regulatory elements to create new models to understand genetic disease susceptibility at an organismal level.
Our bioinformatics team combines in-house scripts with externally available software to establish pipelines and standard operating procedures for the different kinds of high-throughput data generated in our labs (e.g. ATAC-seq, Capture-C, ChIP-seq, RNA-seq, microarray, and DNA methylation). We also develop ad hoc analyses for specific questions of interest to the center and its collaborators, integrating results from such pipelines with publicly available functional genomics data, with the goal of discovering new insights into complex genetic diseases. In addition, our team has developed and maintains an internal relational database which tracks both bench and in silico work from our center and provides a user-friendly interface to upload and query our data.