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Genome-scale discovery of developmental regulatory elements

Presented at the Neonatal Society 2012 Autumn Meeting.

Biddie SC1,2,3, Attanasio C2, Nord AS2, Blow MJ3, Visel A2,3, Pennacchio LA2,3

1 Faculty of Medicine and Dentistry, University of Bristol, Bristol, UK
2 Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
3 United States Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA

Background: Mutations in coding regions of the human genome are well known to cause disease, for example exonic mutations causing cystic fibrosis and MCADD. However, genome-wide association (GWA) studies have identified extensive genetic polymorphisms in non-coding elements that confer disease risk. The noncoding DNA sequences represent ~98% of the human genome, and harbour regulatory elements such as enhancers that act to regulate gene expression. Enhancers regulate genes by the binding of regulatory factors, co-factors and through the modification of histones, the DNA compacting protein complexes. Mutations in regulatory elements could therefore alter gene expression in disease. The identification of enhancers across the genome has previously been challenging as their activities differ between tissues and change during development. However, the recent emergence of whole-genome sequencing technologies has aided enhancer identification. Mapping of an enhancer-bound co-activator protein, p300, has been successfully used in unbiased discovery of tissue-specific enhancers active in the developing mouse embryo and human foetal heart (1,2). However, p300 is associated only with a subset of enhancers, leaving an unknown number of undiscovered enhancers. Regulatory elements can also act as repressors that silence gene expression, a class of genomic elements that remains poorly investigated. New methods and markers of enhancer and repressors would expand our understanding of non-coding elements that might be associated with disease.

Methods: Active regulatory elements were identified by chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq) of the enhancer-associated chromatin remodelling subunit Brg1 (SMARCA4) in E11.5 mouse heart, forebrain and limb bud. Due to limited availability of antibodies against Brg1, FLAGtagged Brg1 knock-in mice were generated, with anti-Flag antibodies used for immunoprecipitation. Putative enhancer elements were validated in vivo using a LacZ transgenic mouse assay.

Results: Brg1 binding sites were found to be highly tissue-specific, consistent with the possibility that they are involved in tissue-specific gene regulation. A subset of Brg1 binding sites were found to be associated with enhancer-associated chromatin marks including H3K27ac. A substantial proportion of predicted Brg1- associated enhancer elements showed reproducible tissue-specific enhancer activity in vivo in a transgenic mouse assay. Furthermore we observed a proportion of Brg1 peaks overlapping tissue-specific binding sites for repressor-associated chromatin signatures across the genome, suggesting that Brg1 is also commonly associated with repressors. Incorporating GWA data with Brg1 at human and mouse conserved DNA elements, we found elements harbouring single nucleotide polymorphisms associated with disease.

Conclusion: Whole-genome mapping of Brg1 occupancy in developing mouse tissues identified genome-wide sets of candidate regulatory sequences that are likely to be involved in tissue-specific transcriptional regulation in embryo development. Comparison with tissue-specific histone marks, as well as in vivo enhancer assays in transgenic mice, indicate that Brg1 is widely associated with both tissue-specific enhancers and repressor elements. Our results provide genome-scale evidence for a dual role of this key regulatory protein in both tissue-specific activation and repression of developmentally expressed genes. These elements identified through this study provide candidate sequences in the search for non-coding sequence polymorphisms that might underlie human congenital disease.

Corresponding author: simon.biddie@doctors.org.uk

1. Visel, A et al.,2009.ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature, 457(7231)
2. May, D et al.,2011.Large-scale discovery of enhancers from human heart tissue. Nature Genetics, 44(1)

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