Characterization of the enhancer and promoter landscape of inflammatory bowel disease from human colon biopsies.

Inflammatory bowel disease (IBD) is a chronic intestinal disorder, with two main types: Crohn's disease (CD) and ulcerative colitis (UC), whose molecular pathology is not well understood. The majority of IBD-associated SNPs are located in non-coding regions and are hard to characterize since regulatory regions in IBD are not known. Here we profile transcription start sites (TSSs) and enhancers in the descending colon of 94 IBD patients and controls. IBD-upregulated promoters and enhancers are highly enriched for IBD-associated SNPs and are bound by the same transcription factors. IBD-specific TSSs are associated to genes with roles in both inflammatory cascades and gut epithelia while TSSs distinguishing UC and CD are associated to gut epithelia functions. We find that as few as 35 TSSs can distinguish active CD, UC, and controls with 85% accuracy in an independent cohort. Our data constitute a foundation for understanding the molecular pathology, gene regulation, and genetics of IBD.

Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2.

The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52% are not annotated. A subset of these has the potential to confer change in protein function due to protein domain exclusion. Moreover, we locate 890 transcribed enhancer candidates, where ∼50% are changing in usage after TNF-α stimulation. These enhancers share motif enrichments with similarly responding gene promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers.

Genome-wide analysis of CDX2 binding in intestinal epithelial cells (Caco-2).

The CDX2 transcription factor is known to play a crucial role in inhibiting proliferation, promoting differentiation and the expression of intestinal specific genes in intestinal cells. The overall effect of CDX2 in intestinal cells has previously been investigated in conditional knock-out mice, revealing a critical role of CDX2 in the formation of the normal intestinal identity. The identification of direct targets of transcription factors is a key problem in the study of gene regulatory networks. The ChIP-seq technique combines chromatin immunoprecipitation (ChIP) with next generation sequencing resulting in a high throughput experimental method of identifying direct targets of specific transcription factors. The method was applied to CDX2, leading to the identification of the direct binding of CDX2 to several known and novel target genes in the intestinal cell. Examination of the transcript levels of selected genes verified the regulatory role of CDX2 binding. The results place CDX2 as a key node in a transcription factor network controlling the proliferation and differentiation of intestinal cells.

Control of intestinal promoter activity of the cellular migratory regulator gene ELMO3 by CDX2 and SP1.

An important aspect of the cellular differentiation in the intestine is the migration of epithelial cells from the crypt to the villus tip. As homeodomaine transcription factor CDX2 has been suggested to influence cell migration, we performed a genome-wide promoter analysis for CDX2 binding in the differentiated human intestinal cancer cell line Caco-2 in order to identify CDX2-regulated genes involved in cellular migration. The engulfment and cell motility 3 (ELMO3) gene was identified as a potential CDX2 target gene. ELMO3 is an essential upstream regulator of the GTP-binding protein RAC during cell migration. However, no information is available about the transcriptional regulation of the ELMO3 gene. The aim of this study was to investigate the potential role of CDX2 in the regulation of the ELMO3 promoter activity. Electrophoretic mobility shift assays showed that CDX2 bound to conserved CDX2 sequences and mutations of the CDX2-binding sites, significantly reduced the promoter activity. Reporter gene assays demonstrated that the region mediating ELMO3 basal transcriptional activity to be located between -270 and -31 bp. Sequence analysis revealed no typical TATA-box, but four GC-rich sequences. In vitro analyses (electrophoretic mobility shift assays and promoter analyses) demonstrate that the SP1-binding sites are likely to play an important role in regulating the ELMO3 promoter activity. Furthermore, we showed here that CDX2 and SP1 can activate the ELMO3 promoter. Taken together, the present study reports the first characterization of the ELMO3 promoter and suggests a significant role of CDX2 in the basal transcriptional regulation of the intestine-specific expression of ELMO3, possibly through interaction with SP1.