IBD Genetics: Focus on (Dys) Regulation in Immune Cells and the Epithelium

Abstract

See “Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium,” by Mokry M, Middendorp S, Wiegerinck CL, et al, on page 1040. Genome-wide association studies (GWAS) in inflammatory bowel disease (IBD) have been a real bonanza. The latest round of international collaborative studies has increased the number of genetic loci that are associated with risk for IBD to 163.1Jostins L. Ripke S. Weersma R.K. et al.Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.Nature. 2012; 491: 119-124Crossref PubMed Scopus (3181) Google Scholar Except for very few loci, such as those harboring NOD22Hugot J.P. Chamaillard M. Zouali H. et al.Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.Nature. 2001; 411: 599-603Crossref PubMed Scopus (4647) Google Scholar and ATG16L1,3Hampe J. Franke A. Rosenstiel P. et al.A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1.Nat Genet. 2007; 39: 207-211Crossref PubMed Scopus (1526) Google Scholar neither the genes nor the causal alleles at the risk loci are yet known with a reasonable level of confidence.1Jostins L. Ripke S. Weersma R.K. et al.Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.Nature. 2012; 491: 119-124Crossref PubMed Scopus (3181) Google Scholar Although linkage disequilibrium (LD), the correlation among alleles due to short stretches of shared ancestry, has enabled GWAS to efficiently probe the entire genome, such correlations make the task of identifying causal alleles at known risk loci extremely challenging (it is very difficult to tell apart alleles in near perfect LD only using statistical association). Moreover, LD often spans hundreds to tens of thousands of base pairs, making the search space of plausible causal alleles extremely large. Also, the vast majority of risk loci for IBD localize in non–protein-coding regions of the genome (Figure 1). So, the analytic gap between genetics and genes remains profound for understanding the role of genetics in risk for IBD. In the current issue of Gastroenterology, Mokry et al4Mokry M. Middendorp S. Wiegerinck C.L. et al.Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar report that genetic risk loci for IBD are enriched within regulatory elements in cell types directly relevant to human IBD, the intestinal epithelium and immune cells of the myeloid and lymphoid lineages. This underscores the role of regulation in the genetic risk for IBD thus opening new avenues for IBD research. The vast majority of GWAS-identified risk variants (within the field of IBD, NOD2, and ATG16L1 are again exceptions in this regard) are located in non–protein-coding regions of the genome,1Jostins L. Ripke S. Weersma R.K. et al.Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.Nature. 2012; 491: 119-124Crossref PubMed Scopus (3181) Google Scholar making their functional interpretation challenging. Messenger RNA transcript expression levels in primary cells or their derivatives (eg, Epstein Barr virus–transformed, immortalized B lymphoblastoid cells lines) have been correlated with single nucleotide polymorphisms (SNP) variants, establishing expression quantitative trait loci and highlighting that genetic variants can affect the level of transcription of specific target genes in (closer or wider) proximity.5Dendrou C.A. Plagnol V. Fung E. et al.Cell-specific protein phenotypes for the autoimmune locus IL2RA using a genotype-selectable human bioresource.Nat Genet. 2009; 41: 1011-1015Crossref PubMed Scopus (221) Google Scholar, 6Fairfax B.P. Makino S. Radhakrishnan J. et al.Genetics of gene expression in primary immune cells identifies cell type-specific master regulators and roles of HLA alleles.Nat Genet. 2012; 44: 502-510Crossref PubMed Scopus (330) Google Scholar In fact, expression quantitative trait loci analysis has contributed to candidate gene prioritization at multigene risk loci, although this is still mostly based on a computer algorithm called GRAIL. This uses proximity analysis of GWAS SNPs and co-occurrence of gene names in PubMed abstracts as the basis for inferring relationships and consequently naming candidate genes.7Raychaudhuri S. Plenge R.M. Rossin E.J. et al.Identifying relationships among genomic disease regions: predicting genes at pathogenic SNP associations and rare deletions.PLoS Genet. 2009; 5: e1000534Crossref PubMed Scopus (335) Google Scholar The massive decline in sequencing cost, owing to the advent and ever-accelerating refinement of next-generation sequencing technology, has made it feasible to survey activity along the DNA double helix by directly analyzing chromatin. One such technique is chromatin immunoprecipitation (ChIP) for transcription factors or specifically modified histones followed by sequencing the DNA contained within these complexes (ChIP-Seq). Covalent modification of histones contains critical information regarding the functional state of the underlying DNA sequence. Amongst those important histone modifications is acetylation of histone 3 at lysine (K) 27 (abbreviated H3K27ac), which marks active regulatory elements.8Bernstein B.E. Birney E. Dunham I. et al.An integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (10847) Google Scholar, 9Li B. Carey M. Workman J.L. The role of chromatin during transcription.Cell. 2007; 128: 707-719Abstract Full Text Full Text PDF PubMed Scopus (2658) Google Scholar Data from ChIP-Seq can be combined with those from other techniques that, for example, detect open chromatin (DNase I hypersensitivity mapping through DNase-Seq), a prerequisite for transcription factors gaining access to promoters and other regulatory elements, or techniques that measure detailed transcriptional output. This is the basic toolbox of methodologic approaches utilized by the massive Encyclopedia of DNA Elements (ENCODE) project that aims to systematically map active regulatory loci, transcription factor association, chromatin structure, and histone modification. ENCODE has already assigned biochemical functions to 80% of the genome, hence ascribing deep functionality to non–protein-coding regions of the genome.8Bernstein B.E. Birney E. Dunham I. et al.An integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (10847) Google Scholar It has vastly advanced our understanding of induction and regulation of genes, ranging from transcription factor binding and cooperation to the role of specific histone modifications. One of the most exciting findings from ENCODE has been the fact that for a variety of different diseases and traits up to 71% of GWAS risk SNPs (along with SNPs in LD with them) overlie a DNase I hypersensitivity site, and 31% overlap with transcription factor binding sites,8Bernstein B.E. Birney E. Dunham I. et al.An integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (10847) Google Scholar, 10Schaub M.A. Boyle A.P. Kundaje A. et al.Linking disease associations with regulatory information in the human genome.Genome Res. 2012; 22: 1748-1759Crossref PubMed Scopus (510) Google Scholar, 11Maurano M.T. Humbert R. Rynes E. et al.Systematic localization of common disease-associated variation in regulatory DNA.Science. 2012; 337: 1190-1195Crossref PubMed Scopus (2156) Google Scholar indicating that the majority of (common) risk SNPs typically surveyed in GWAS may indeed affect transcriptional regulation. In the current issue of Gastroenterology, Mokry et al4Mokry M. Middendorp S. Wiegerinck C.L. et al.Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar report the first genome-wide map of regulatory regions identified by H3K27ac immunoprecipitation in a cell type directly relevant to human IBD, namely the intestinal epithelium.4Mokry M. Middendorp S. Wiegerinck C.L. et al.Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar The authors used this newly generated data set along with already publicly available H3K27ac data from bona fide immune cells (monocytes, macrophages, CD4 and CD8 naïve and memory lymphocytes) to ask whether genetic risk loci of IBD overlap with active regulatory regions in the genomes of these cells. Indeed, similarly to other traits,11Maurano M.T. Humbert R. Rynes E. et al.Systematic localization of common disease-associated variation in regulatory DNA.Science. 2012; 337: 1190-1195Crossref PubMed Scopus (2156) Google Scholar IBD risk SNPs identified through GWAS and their LD partners do overlap.4Mokry M. Middendorp S. Wiegerinck C.L. et al.Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Reassuringly, such overlap was not found upon examining risk SNPs from nonimmunologic diseases. Finally, the authors use in vitro and an in vivo zebrafish model to demonstrate that at least some of the regulatory regions can be modeled in cell lines and seem to be functionally conserved across species.4Mokry M. Middendorp S. Wiegerinck C.L. et al.Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Altogether, this is an important advance and allows us to (epi-)genetically home in on cell types likely relevant to IBD. In that regard, it is important to bear in mind that ENCODE data mostly focus on cell lines, such as the erythroleukemia cell line K562, the B-lymphoblastoid cell line GM12878, the cervical carcinoma cell line HeLa-S3, and others, to deduce general principles of gene regulation, whereas it does not necessarily address regulation in specific tissues and cell types that might be relevant for actual diseases.12Trynka G. Sandor C. Han B. et al.Chromatin marks identify critical cell types for fine mapping complex trait variants.Nat Genet. 2013; 45: 124-130Crossref PubMed Scopus (380) Google Scholar Experimentally probing how genetic association mechanistically predisposes for disease has ≥2 important prerequisites: First, knowledge of the causal variant(s) and gene(s) at each of these loci and how the causal variant(s) impacts on its function; and second, identification of the cell types in which individual risk genes and variants exert their function.12Trynka G. Sandor C. Han B. et al.Chromatin marks identify critical cell types for fine mapping complex trait variants.Nat Genet. 2013; 45: 124-130Crossref PubMed Scopus (380) Google Scholar In fact, these 2 facets are critically intertwined: SNPs located in regulatory regions seem to affect gene transcription in a remarkably cell type-dependent manner.5Dendrou C.A. Plagnol V. Fung E. et al.Cell-specific protein phenotypes for the autoimmune locus IL2RA using a genotype-selectable human bioresource.Nat Genet. 2009; 41: 1011-1015Crossref PubMed Scopus (221) Google Scholar Even opposite directions of regulation in different cell types has been reported.6Fairfax B.P. Makino S. Radhakrishnan J. et al.Genetics of gene expression in primary immune cells identifies cell type-specific master regulators and roles of HLA alleles.Nat Genet. 2012; 44: 502-510Crossref PubMed Scopus (330) Google Scholar Only when this critical information is available may the newly gained genetic landscape be translated into novel biological understanding. Overlaying GWAS association data onto chromatin activity is indeed a powerful means to predict cell types that play a critical role in relaying individual loci’s genetic risk to disease pathophysiology,12Trynka G. Sandor C. Han B. et al.Chromatin marks identify critical cell types for fine mapping complex trait variants.Nat Genet. 2013; 45: 124-130Crossref PubMed Scopus (380) Google Scholar as shown in the paper by Mokry et al.4Mokry M. Middendorp S. Wiegerinck C.L. et al.Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar The transcriptional programs arising from open and transcriptionally active chromatin are inherent to and characteristic for individual cell types and indeed specify the cell type’s identity and biological function. In this context, the Mokry et al paper is an important first step in analyzing cell types local and characteristic to IBD.4Mokry M. Middendorp S. Wiegerinck C.L. et al.Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar The authors sequenced H3K27ac-bound DNA in chromatin obtained from primary intestinal epithelial cells, specifically small and large intestinal crypts, and from intestinal organoids cultured from intestinal stem cells, obtained from healthy individuals and patients with Crohn’s disease; overall, 7 specimens from 4 patients were studied. The heterogeneity of this sample set could potentially be considered a weakness of the paper, but could similarly be viewed as a feature in that it captures a variety of different conditions, anatomic locales, and isolation techniques as a template for the mapping of GWAS loci. Needless to say, the current study is an important step in the right direction. The “bona fide” immune cells that Mokry et al included in their analysis provide interesting insight from the mapping of IBD GWAS risk SNPs, but also point to a hurdle that needs to be overcome in future studies: These cells have been isolated or derived from peripheral blood cells,13Pham T.H. Benner C. Lichtinger M. et al.Dynamic epigenetic enhancer signatures reveal key transcription factors associated with monocytic differentiation states.Blood. 2012; 119: e161-e171Crossref PubMed Scopus (106) Google Scholar, 14Bernstein B.E. Stamatoyannopoulos J.A. Costello J.F. et al.The NIH Roadmap Epigenomics Mapping Consortium.Nat Biotechnol. 2010; 28: 1045-1048Crossref PubMed Scopus (1224) Google Scholar but how much do they reflect the chromatin, hence transcriptional, and in consequence functional profile of lamina propria monocytes/macrophages, CD4 and CD8 lamina propria, or intraepithelial T cells? There is ample functional evidence for each of these cell types that the characteristics of their blood counterparts is profoundly different from their intestinal “siblings.”15Zigmond E. Jung S. Intestinal macrophages: well educated exceptions from the rule.Trends Immunol. 2013; 34: 162-168Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 16Cauley L.S. Lefrancois L. Guarding the perimeter: protection of the mucosa by tissue-resident memory T cells.Mucosal Immunol. 2013; 6: 14-23Crossref PubMed Scopus (71) Google Scholar Genetic risk may impact on many other, not-yet-studied intestinal cell types that are known to be involved in regulating intestinal homeostasis and inflammation, such as innate lymphoid cells,17Buonocore S. Ahern P.P. Uhlig H.H. et al.Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology.Nature. 2010; 464: 1371-1375Crossref PubMed Scopus (843) Google Scholar stromal cells, endothelial cells, and others,18Maloy K.J. Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease.Nature. 2011; 474: 298-306Crossref PubMed Scopus (1254) Google Scholar and their chromatin may need to be analyzed in a similar manner. Further, the intestinal epithelium is not a homogenous cell type either. Goblet, enteroendocrine and Paneth cells, the latter exhibiting secretory phenotypes in specific genetic susceptibility contexts of Crohn’s disease19Vandussen K.L. Liu T.C. Li D. et al.Genetic variants synthesize to produce Paneth cell phenotypes that define subtypes of Crohn's disease.Gastroenterology. 2014; 146: 200-209Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar and recently demonstrated as a site of origin of small intestinal Crohn’s disease-like inflammation in a murine model,20Adolph T.E. Tomczak M.F. Niederreiter L. et al.Paneth cells as a site of origin for intestinal inflammation.Nature. 2013; 503: 272-276Crossref PubMed Scopus (491) Google Scholar constitute minor fractions within the intestinal epithelium and their chromatin might be watered down beyond recognition within an unselected intestinal epithelial cell population. It is important to identify through which mechanisms (eg, regulation of transcription, as proposed by Mokry et al4Mokry M. Middendorp S. Wiegerinck C.L. et al.Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and through which specific cell types genetic variation contributes to risk for disease. As alluded to, this insight is the prerequisite for designing experiments aimed at unraveling the consequential biological mechanisms at a “3D” organismal level. However, with all our excitement about the genetics discoveries, we may not forget that genetic risk is only one, although clearly important, contributor to disease risk. The “environment,” which may account for as much as 85% (ulcerative colitis) and 50% (Crohn’s disease) of disease variance according to twin studies,18Maloy K.J. Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease.Nature. 2011; 474: 298-306Crossref PubMed Scopus (1254) Google Scholar, 21Halfvarson J. Bodin L. Tysk C. et al.Inflammatory bowel disease in a Swedish twin cohort: a long-term follow-up of concordance and clinical characteristics.Gastroenterology. 2003; 124: 1767-1773Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar remains the big unknown—and important to bear in mind in our hopeful pursuit to decipher the fundamental biology of IBD. Many Inflammatory Bowel Disease Risk Loci Include Regions That Regulate Gene Expression in Immune Cells and the Intestinal EpitheliumGastroenterologyVol. 146Issue 4PreviewThe contribution of genetic factors to the pathogenesis of inflammatory bowel disease (IBD) has been established by twin, targeted-sequencing, and genome-wide association studies. These studies identified many risk loci, and research is underway to identify causal variants. These studies have focused mainly on protein-coding genes. We investigated other functional elements in the human genome, such as regulatory regions. Full-Text PDF