Coordinated Effects of Sequence Variation on DNA Binding, Chromatin Structure, and Transcription

DNA sequence variation has been associated with quantitative changes in molecular phenotypes such as gene expression, but its impact on chromatin states is poorly characterized. To understand the interplay between chromatin and genetic control of gene regulation, we quantified allelic variability in transcription factor binding, histone modifications, and gene expression within humans. We found abundant allelic specificity in chromatin and extensive local, short-range, and long-range allelic coordination among the studied molecular phenotypes. We observed genetic influence on most of these phenotypes, with histone modifications exhibiting strong context-dependent behavior. Our results implicate transcription factors as primary mediators of sequence-specific regulation of gene expression programs, with histone modifications frequently reflecting the primary regulatory event.

Nuclear Factor I genomic binding associates with chromatin boundaries

Giovanna Ambrosini, Philipp Bucher, Giovanni Dietler, Armelle Gaussin, Jan Kerschgens, Nicolas Mermod, Petar Pjanic

Background: The Nuclear Factor I (NFI) family of DNA binding proteins (also called CCAAT box transcription factors or CTF) is involved in both DNA replication and gene expression regulation. Using chromatin immuno-precipitation and high throughput sequencing (ChIP-Seq), we performed a genome-wide mapping of NFI DNA binding sites in primary mouse embryonic fibroblasts. Results: We found that in vivo and in vitro NFI DNA binding specificities are indistinguishable, as in vivo ChIP-Seq NFI binding sites matched predictions based on previously established position weight matrix models of its in vitro binding specificity. Combining ChIP-Seq with mRNA profiling data, we found that NFI preferentially associates with highly expressed genes that it up-regulates, while binding sites were under-represented at expressed but unregulated genes. Genomic binding also correlated with markers of transcribed genes such as histone modifications H3K4me3 and H3K36me3, even outside of annotated transcribed loci, implying NFI in the control of the deposition of these modifications. Positional correlation between + and - strand ChIP-Seq tags revealed that, in contrast to other transcription factors, NFI associates with a nucleosomal length of cleavage-resistant DNA, suggesting an interaction with positioned nucleosomes. In addition, NFI binding prominently occurred at boundaries displaying discontinuities in histone modifications specific of expressed and silent chromatin, such as loci submitted to parental allele-specific imprinted expression. Conclusions: Our data thus suggest that NFI nucleosomal interaction may contribute to the partitioning of distinct chromatin domains and to epigenetic gene expression regulation. NFI ChIP-Seq and input control DNA data were deposited at Gene Expression Omnibus (GEO) repository under accession number GSE15844. Gene expression microarray data for mouse embryonic fibroblasts are on GEO accession number GSE15871.

BioMed Central2013

What shapes eukaryotic transcriptional bursting?

Felix Naef, Damien Lionel Nicolas, Nicholas Edward Phillips

Isogenic cells in a common environment present a large degree of heterogeneity in gene expression. Part of this variability is attributed to transcriptional bursting: the stochastic activation and inactivation of promoters that leads to the discontinuous production of mRNA. The diversity in bursting patterns displayed by different genes suggests the existence of a connection between bursting and gene regulation. Experimental strategies such as single-molecule RNA FISH, MS2-GFP or short-lived protein reporters allow the quantification of transcriptional bursting and the comparison of bursting kinetics between conditions, allowing therefore the identification of molecular mechanisms modulating transcriptional bursting. In this review we recapitulate the impact on transcriptional bursting of different molecular aspects of transcription such as the chromatin environment, nucleosome occupancy, histone modifications, the number and affinity of regulatory elements, DNA looping and transcription factor availability. More specifically, we examine their role in tuning the burst size or the burst frequency. While some molecular mechanisms involved in transcription such as histone marks can affect every aspect of bursting, others predominantly influence the burst size (e.g. the number and affinity of cis-regulatory elements) or frequency (e.g. transcription factor availability).

Royal Society of Chemistry2017

Integrated microfluidic tools for the characterization of protein/DNA interactions in vitro and in vivo

Riccardo Dainese

The specific interaction between DNA and proteins constitutes one of the crucial elements in the regulation of gene expression. This thesis focuses on the development and optimization of two microfluidic-based technologies called SMiLE-seq and FloChIP that enable the sensitive and high-throughput analysis of two important aspects of protein/DNA interactions: respectively, 1) the transcription factor DNA-binding specificities and 2) the genome-wide distribution of chromatin-associated proteins. In Chapter 1, we describe the core motivation, operating principles, optimization and results obtained with SMiLE-seq. As opposed to existing solutions, SMiLE-seq offers the possibility to screen a large library of randomized DNA for sequence-specific protein ligands in a miniaturized context. SMiLE-seq integrates in a single microfluidic chip the advantages of both HT-SELEX and MITOMI. We first demonstrate as proof-of-principle that SMiLE-seq successfully recapitulates with robustness and reproducibility the binding specificities of known factors belonging to different species and TF families. Subsequently, we target the numerous although largely uncharacterized family of TFs called KRAB-ZFPs. We initially set out to redesign the microfluidic and protocol architecture in order to attain maximal throughput and sensitivity. Next, we add the ability of probing the sensitivity of these factors to methylation by synthesizing randomized methylated DNA libraries. Finally, we proceed to test 101 KRAB-ZFPs with both methylated and non-methylated DNA. We obtain high confidence motifs for 43 factors, of which 22 we not sensitive to methylation, 10 yielded motifs only with methylated DNA and 10 only with non-methylated DNA. By integrating our SMiLE-seq data with published ChIP-exo data and in silico predictions, we develop a framework for systematically identifying which zinc fingers directly contribute to DNA binding for a given KRAB-ZFPs. In Chapter 2, we describe the development and results obtained with FloChIP, a microfluidic implementation of the widespread ChIP-seq and sequential ChIP-seq protocols. FloChIP encompasses three main technological advances: 1) the multi-layered serface chemistry that allows any off-the-shelf antibody to be immobilized on-chip and and 2) a micropillar architecture that provides high surface-to-volume ratio and efficient removal of non-specific DNA. 3) The direct on-chip tagmentation of immunoprecipitated DNA. We validate our approach by first performing H3k27ac ChIP-seq on different number of sample inputs, i.e. from 500 to 10^6 cells. Next, we proceed to prove the flexibility of our approach by performing ChIP-seq on different histone marks â namely H3k27ac, H3k27me3, H3k4me3, H3k4me1 and H3k9me3 â and comparing them to existing ENCODE data. Both region-specific profiles and enrichment scores show high similarity with publicly available data. Subsequently, we set out to demonstrate the feasibility of on-chip sequential-ChIP-seq by performing consecutive H3k4me3 and H3k27me3 IP on chromatin derived from mouse embryonic stem cells. Finally, we illustrate the high-throughput feature of our technology by performing MEF2A-ChIP-seq on chromatin derived from 32 different lymphoblastoid cell lines.