A parallelized, automated platform enabling individual or sequential ChIP of histone marks and transcription factors

Daniel Alpern, Antonio Carlos Alves Meireles Filho, Riccardo Dainese, Bart Deplancke, Vincent Roland Julien Gardeux, Jia Yuan Jiang, Gerard Llimos Aubach
NATL ACAD SCIENCES, 2020
Article

Résumé

Despite its popularity, chromatin immunoprecipitation followed by sequencing (ChIP-seq) remains a tedious (>2 d), manually intensive, low-sensitivity and low-throughput approach. Here, we combine principles of microengineering, surface chemistry, and molecular biology to address the major limitations of standard ChIP-seq. The resulting technology, FloChIP, automates and miniaturizes ChIP in a beadless fashion while facilitating the downstream library preparation process through on-chip chromatin tagmentation. FloChIP is fast (

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Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

Elsevier2015

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Many organisms exhibit temporal rhythms in gene expression that propel diurnal cycles in physiology. In the liver of mammals, these rhythms are controlled by transcription-translation feedback loops of the core circadian clock and by feeding-fasting cycles. To better understand the regulatory interplay between the circadian clock and feeding rhythms, we mapped DNase I hypersensitive sites (DHSs) in the mouse liver during a diurnal cycle. The intensity of DNase I cleavages cycled at a substantial fraction of all DHSs, suggesting that DHSs harbor regulatory elements that control rhythmic transcription. Using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq), we found that hypersensitivity cycled in phase with RNA polymerase II (Pol II) loading and H3K27ac histone marks. We then combined the DHSs with temporal Pol II profiles in wild-type (WT) and Bmal1(-/-) livers to computationally identify transcription factors through which the core clock and feeding-fasting cycles control diurnal rhythms in transcription. While a similar number of mRNAs accumulated rhythmically in Bmal1(-/-) compared to WT livers, the amplitudes in Bmal1(-/-) were generally lower. The residual rhythms in Bmal1(-/-) reflected transcriptional regulators mediating feeding-fasting responses as well as responses to rhythmic systemic signals. Finally, the analysis of DNase I cuts at nucleotide resolution showed dynamically changing footprints consistent with dynamic binding of CLOCK: BMAL1 complexes. Structural modeling suggested that these footprints are driven by a transient heterotetramer binding configuration at peak activity. Together, our temporal DNase I mappings allowed us to decipher the global regulation of diurnal transcription rhythms in the mouse liver.

Public Library of Science2017

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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.

EPFL2019

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

Riccardo Dainese

EPFL2019