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Chromatin dynamics resolved by rapid protein labeling and bioorthogonal capture

Periodic Reporting for period 2 - RAPID (Chromatin dynamics resolved by rapid protein labeling and bioorthogonal capture)

Reporting period: 2018-07-01 to 2019-12-31

Histone proteins provide a dynamic packaging system for the eukaryotic genome. Chromatin integrates a multitude of signals to control gene expression, only some of which have the propensity to be maintained through replication and cell division. For our understanding of cellular memory and epigenetic inheritance we need to know what features characterize a stable, heritable chromatin state throughout the cell cycle. To unravel the molecular circuits that underlie epigenetic gene regulation and inheritance, my laboratory aims to increase the spatiotemporal resolution and biochemical precision of methods for studying chromatin in and from living cells.

Histone modifications have been studied in many aspects of human disease –– pre and postnatal development, behaviour, memory and neurodegeneration, genome stability and cancer, diabetes and cardiovascular diseases, AIDS, stem cells and reprogramming. However, it has been noted that our current, descriptive methodologies do not rule out the possibility that histones are mere bystanders rather than culprits in many pathologies. In the absence of unequivocal proof of locus-specific histone inheritance, the epigenetic nature of their marks remains debatable. It is therefore a major challenge of our field to devise experiments that can directly and conclusively test this hypothesis.
The first deliverable of our project is a multiplexed, quantitative ChIP-Seq method (Kumar and Elsässer, 2019). Quantitative measurements are essential for our aim to derive mechanistic insights from following the dynamic histone modifications and histone variants over time. Mint-ChIP, developed by the Bernstein lab (van Galen et al., 2016), provided a formidable starting point for our method with a streamlined one-pot chromatin barcoding and a post-ChIP linear amplification that requires only one adaptor per chromatin fragment. We introduce unique molecule (UMI) counting and paired-end mapping of the chromatin fragments to this method, which we then termed MINUTE-ChIP for multiplexed indexed unique molecule T7 amplification end-to-end sequencing. MINUTE-ChIP has a large linear dynamic range for accurately quantifying relative differences in genome-wide histone modification patterns across multiple pooled samples.

Second deliverable is a first quantitative characterization of Polycomb-mediated histone modifications in mouse embryonic stem cells, in naïve (2i) and Serum-primed growth conditions. We find compelling evidence for a broad H3K27me3 hypermethylation of the genome, while bivalent promoters stably retain high H3K27me3 levels in 2i. We show that DNA hypomethylation, as observed in 2i, is a contributor to genome-wide gain of H3K27me3, while active demethylation by JMJD3/UTX counteracts further accumulation of H3K27me3. In parallel, we find hypomethylation of H3K4me3, particularly at bivalent promoters, to be a characteristic of the 2i ground state. Serum stimulates H3K4me3 independent of GSK-3b and ERK signaling, suggesting that low H3K4me3 and high H3K27me3 levels at bivalent promoters are a product of two independent mechanisms that safeguard naïve pluripotency.
The need for quantitative ChIP-Seq has been widely appreciated and a number of quantitative methods have been proposed. Barcoding-first ChIP techniques have gained popularity because of the benefit of multiplexing (Arrigoni et al., 2018; Chabbert et al., 2018; van Galen et al., 2016; Lara-Astiaso et al., 2014). Pooling barcoded samples greatly increases throughput without complicated automation, while effectively removing technical variability between samples. Challenges remain for fragmentation and ligation of crude chromatin in a manner that maximizes barcoding efficiency while avoiding any technical variability or sample loss that could confound the subsequent quantitative measurement.

We further finished method development for the remaining components of the RAPID procedure and started acquiring preliminary data for the remaining deliverables, namely the dynamic incorporation and inheritance of histone variants associated with Polycomb domains, interstitial heterochromatin, during pluripotency and differentiation