Large-scale discovery and validation of functional elements in the human genome

A report on the genomics workshop 'Identification of Functional Elements in Mammalian Genomes', Cold Spring Harbor, New York, 11-13 November 2004

DNA-Protein Interactions in High Definition

An elegant, genome-wide approach to define the precise DNA sequences bound by transcription factors has been developed by Rhee and Pugh

Development and validation of a T7 based linear amplification for genomic DNA

BACKGROUND: Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure. These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation. To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified. Current protocols use PCR-based approaches to amplify in exponential fashion. However, exponential amplification protocols are highly susceptible to bias. Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis. These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail. RESULTS: We have developed a novel linear amplification protocol for genomic DNA. Terminal transferase is used to add polyT tails to the ends of DNA fragments. Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP. Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT). Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material. In contrast, we find that PCR amplification is biased towards species of greater size. Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach. CONCLUSION: We present a T7-based linear amplification protocol for genomic DNA. Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments

Global nucleosome occupancy in yeast

BACKGROUND: Although eukaryotic genomes are generally thought to be entirely chromatin-associated, the activated PHO5 promoter in yeast is largely devoid of nucleosomes. We systematically evaluated nucleosome occupancy in yeast promoters by immunoprecipitating nucleosomal DNA and quantifying enrichment by microarrays. RESULTS: Nucleosome depletion is observed in promoters that regulate active genes and/or contain multiple evolutionarily conserved motifs that recruit transcription factors. The Rap1 consensus was the only binding motif identified in a completely unbiased search of nucleosome-depleted promoters. Nucleosome depletion in the vicinity of Rap1 consensus sites in ribosomal protein gene promoters was also observed by real-time PCR and micrococcal nuclease digestion. Nucleosome occupancy in these regions was increased by the small molecule rapamycin or, in the case of the RPS11B promoter, by removing the Rap1 consensus sites. CONCLUSIONS: The presence of transcription factor-binding motifs is an important determinant of nucleosome depletion. Most motifs are associated with marked depletion only when they appear in combination, consistent with a model in which transcription factors act collaboratively to exclude nucleosomes and gain access to target sites in the DNA. In contrast, Rap1-binding sites cause marked depletion under steady-state conditions. We speculate that nucleosome depletion enables Rap1 to define chromatin domains and alter them in response to environmental cues

Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions

Mammalian gene regulation is dependent on tissue-specific enhancers that can act across large distances to influence transcriptional activity1-3. Mapping experiments have identified hundreds of thousands of putative enhancers whose functionality is supported by cell type–specific chromatin signatures and striking enrichments for disease-associated sequence variants4-11. However, these studies did not address the in vivo functions of the putative elements or their chromatin states and could not determine which genes, if any, a given enhancer regulates. Here we present a strategy to investigate endogenous regulatory elements by selectively altering their chromatin state using programmable reagents. Transcription activator–like (TAL) effector repeat domains fused to the LSD1 histone demethylase efficiently remove enhancer-associated chromatin modifications from target loci, without affecting control regions. We find that inactivation of enhancer chromatin by these fusion proteins frequently causes down-regulation of proximal genes, revealing enhancer target genes. Our study demonstrates the potential of ‘epigenome editing’ tools to characterize an important class of functional genomic elements

Genomic Distribution and Inter-Sample Variation of Non-CpG Methylation across Human Cell Types

DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set