Re-evaluating the role of nucleosomal bivalency in early development

Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome1; posttranslational modifications of these histone proteins influence the activity of the associated genomic regions to regulate cell identity2–4. Traditionally, trimethylation of histone 3-lysine 4 (H3K4me3) is associated with transcriptional initiation5–10, whereas trimethylation of H3K27 (H3K27me3) is considered transcriptionally repressive11–15. The apparent juxtaposition of these opposing marks, termed “bivalent domains”16–18, was proposed to specifically demarcate of small set transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status19–22. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure H3K4me3/H3K27me3 bivalency genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model16,18: from naïve mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread, does not resolve with differentiation, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against interpreting bivalent domains as specific markers of developmentally poised genes.

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

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 (<2 h), has a wide dynamic range (from 106to 500 cells), is scalable and parallelized, and supports antibody- or sample-multiplexed ChIP on both histone marks and transcription factors. In addition, FloChIP’s interconnected design allows for straightforward chromatin reimmunoprecipitation, which allows this technology to also act as a microfluidic sequential ChIP-seq system. Finally, we ran FloChIP for the transcription factor MEF2A in 32 distinct human lymphoblastoid cell lines, providing insights into the main factors driving collaborative DNA binding of MEF2A and into its role in B cell-specific gene regulation. Together, our results validate FloChIP as a flexible and reproducible automated solution for individual or sequential ChIP-seq.

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

Despite its popularity, chromatin immunoprecipitation followed by sequencing (ChIP-seq) remains a tedious (>2d), 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 approach, FloChIP, automates and miniaturizes ChIP in a beadless fashion while facilitating the downstream library preparation process through on-chip chromatin tagmentation. FloChIP is fast (<2h), has a wide dynamic range (from 106 to 500 cells), is high-throughput (up to 64 parallel, antibody- or sample-multiplexed experiments) and is compatible with both histone mark and transcription factor ChIP. In addition, FloChIP’s interconnected design allows for straightforward chromatin re-immunoprecipitation, thus constituting the first example of a microfluidic sequential ChIP-seq system. Finally, we demonstrate FloChIP’s high-throughput capacity by performing ChIP-seq of the transcription factor MEF2A in 32 distinct human lymphoblastoid cell lines, providing novel insights into the main factors driving collaborative DNA binding of MEF2A and into its role in B-cell-specific gene regulation. Together, our results validate FloChIP as a flexible and reproducible automated solution for individual or sequential ChIP-seq.

Redundant and specific roles of cohesin STAG subunits in chromatin looping and transcription control

Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A and RAD21, cohesin contains in somatic cells one of two orthologous STAG subunits, SA1 or SA2. How these variable subunits affect the function of the cohesin complex is still unclear. SA1- and SA2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of SA1 and SA2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either SA1 or SA2. Following rapid depletion of either subunit, we perform high resolution Hi-C, RNA-sequencing and sequential ChIP studies to show that SA1 and SA2 do not co-occupy individual binding sites and have distinct ways how they affect looping and gene expression. These findings are supported at the single cell level by single-molecule localizations via dSTORM super-resolution imaging. Since somatic and congenital mutations of the SA subunits are associated with cancer (SA2) and intellectual disability syndromes with congenital abnormalities (SA1 and SA2), we verified SA1-/SA2-dependencies using human neural stem cells, hence highlighting their importance for understanding particular disease contexts.

Atrophin controls developmental signaling pathways via interactions with Trithorax-like

Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-pallidoluysian atrophy, a neurodegenerative disease. Drosophila Atrophin (Atro) mutants display many phenotypes, including neurodegeneration, segmentation, patterning and planar polarity defects. Despite Atro’s critical role in development and disease, relatively little is known about Atro’s binding partners and downstream targets. We present the first genomic analysis of Atro using ChIP-seq against endogenous Atro. ChIP-seq identified 1300 potential direct targets of Atro including engrailed, and components of the Dpp and Notch signaling pathways. We show that Atro regulates Dpp and Notch signaling in larval imaginal discs, at least partially via regulation of thickveins and fringe. In addition, bioinformatics analyses, sequential ChIP and coimmunoprecipitation experiments reveal that Atro interacts with the Drosophila GAGA Factor, Trithorax-like (Trl), and they bind to the same loci simultaneously. Phenotypic analyses of Trl and Atro clones suggest that Atro is required to modulate the transcription activation by Trl in larval imaginal discs. Taken together, these data indicate that Atro is a major Trl cofactor that functions to moderate developmental gene transcription.