Discovery of Active Cis-Regulatory Elements and Transcription Factor Footprints in Nematodes Using Functional Genomics Approaches

Abstract

High throughput DNA sequencing has emerged as a versatile and inexpensive readout of functional activity in biological organisms. In this study I describe the implementation of DNaseI hypersensitivity assays using deep sequencing (DNase-seq) to systematically identify Caenorhabditis elegans cis -regulatory modules (CRMs) in embryonic and L1 arrest larval life stages in an unbiased and de novo manner. We validated our data by comparison to many known enhancers of lin-39/ceh-13 Hox complex and of hlh-1 , myo-2 , myo-3 , lin-26 , and other important developmental genes and are also able to predict novel cis -regulatory modules. We predict novel regulatory motifs from our DNase-seq data and predict potential regulatory functions using gene ontology and anatomy enrichment analysis. In addition, our data are high-resolution enough to identify binding sites of transcription factors in the genome. Our data provide support for many distal CRMs in C. elegans and for a significant portion of genes possessing multiple CRMs. DNase-seq data can also be used to refine prediction of tissue-specific genes such as those regulated by C. elegans pan-neuronal N1 and intestinal ELT-2 DNA motifs. Overall, we identify 24,128 putative CRMS containing over 55,000 footprints. In L1 arrest, we identify 15,841 putative CRMs in the L1 arrest larvae containing 32,000 TF footprints. From comparison of these datasets, we identify an additional 1,854 noncoding DHS that appear to be specific to the L1 arrest larvae condition. These genes include downstream targets of signaling pathways known to be regulated during L1 arrest such as insulin-like signaling via DAF-16/FOXO and Forkhead box transcription factor PHA-4/FOXA that impacts starvation survival in the L1 arrest condition. Having established the first proof-of-principle DNase-seq in nematodes using C. elegans , I am applying DNase-seq to a distantly related entomopathogenic nematode, Steinernema carpocapsae , with a recently sequenced genome and transcriptome. Finally, I am using a massively parallel reporter assay to test the functional activity of the CRMs we have discovered from DNase-seq using two reporter designs based on MPRA and STARR-seq and by performing DNA and RNA sequencing on transgenic C. elegans .