Transcriptome Analysis of Developing Intestine [ChIP-Seq]

Background: The muscularis externa (ME) of the adult intestine consists of two layers of visceral smooth muscle (VISM), the inner circular muscle (ICM) and outer longitudinal muscle (OLM), that form sequentially beginning at embryonic day (E) 13 and E15 in the developing mouse. Coordinated contraction of these two layers facilitates the movement of food down the digestive tract. Though abnormal ME function or development has been linked to pseudoobstruction and irritable bowel syndrome, little is known about the molecular character of the smooth muscle that comprises this tissue. We performed transcriptome analysis to identify genes that are enriched in intestinal mesenchyme tissue at E14.5, when the inner circular muscle (ICM) is well established. Results: Expression patterns of enriched mesenchyme genes were examined in publically available in situ databases, revealing over one hundred genes that are expressed in the ICM. Examination of the promoter regions for these genes revealed enrichment for cJUN transcription factor binding sites and cJUN itself was also enriched in ICM. A cJUN ChIP-seq at E14.5 showed that cJUN regulatory regions contained characteristics of muscle enhancers. Overall design: E14.5 mouse intestines were harvested from one litter (8 embryos) and grown for 24 hours in a transwell culture. Intestines were dissociate into single cells, cross-linked and sonicated to an average DNA fragment size of 500 bp prior to incubation with cJUN antibody. Both cJUN immunoprecipitated and input DNA were submitted for sequencing.