Periodic Reporting for period 4 - RAPID (Chromatin dynamics resolved by rapid protein labeling and bioorthogonal capture)
Reporting period: 2021-07-01 to 2022-06-30
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. 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. 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. We have developed a method termed RAPID for rapid protein pulsing aimed at tracking a defined protein population over time. The method takes advantage of the highly efficient amber suppression technology in mammalian cells which I have developed and continuously improved. Combined with our quantitative MINUTE-ChIP, we can capture genome-wide chromatin dynamics in a quantitative manner and resolved over a period of time ranging from minutes to days. RAPID introduces a flexible time dimension in the form of pulse or pulse-chase experiments for studying genome-wide dynamic occupancy of a protein of interest by next-gen sequencing. It can also be coupled to other readouts such as mass spectrometry or microscopy.
RAPID is uniquely suited for studying cell cycle-linked processes, by defining when and where stable ‘marks’ are set in chromatin. RAPID will define fundamental rules for inheritance of histone and other chromatin-associated proteins. In a first application, we have profiled the dynamics of histone variant H3.3 elucidating its intriguing role in dynamic heterochromatin maintenance in mouse embryonic stem cells. Nucleosome turnover concomitant with incorporation of the replication-independent histone variant H3.3 is a hallmark of regulatory regions in the animal genome. In our current understanding, nucleosome turnover is universally linked to DNA accessibility and histone acetylation. However, we have shown that H3.3 contributes to a unique heterochromatin state at endogenous retroviral elements in mouse embryonic stem cells (mESC). We recently further demonstrated that fast histone turnover and H3.3 incorporation is compatible with stereotypical hallmarks of heterochromatin. We find that Smarcad1 ATPase triggers nucleosome eviction and histone H3.3 is required to maintain minimal DNA accessibility in this surprisingly dynamic heterochromatin state. Loss of H3.3 in mouse embryonic stem cells elicits a highly specific opening of interstitial heterochromatin with minimal effects on other silent or active regions of the genome. Following up on these results, we used RAPID to perform time-lapse interactomics using mass spectrometry and quantitative ChIP to understand the mechanism of H3.3 deposition and turnover (Katsori et. al., manuscript in preparation). We were able to assign distinct specific remodeling activities to different functional outcomes, e.g. heterochromatin maintenance versus the opening of enhancers.
We also apply our technology to understanding the function of Polycomb group (PcG) proteins, evolutionary conserved master regulators of cell identity and differentiation that have long been a paradigm for epigenetic gene control. Two major PcG complexes, PRC1 and PRC2, cooperate in the repression of target genes catalyzing histone H2A K119 monoubiquitination (H2Aub) and H3K27 trimethylation (H3K27me3), respectively. Applying our quantitative MINUTE-ChIP in mESCs lead us to a number of interesting findings: 1) Catalytic activity of both Polycomb complexes is not confined to so-called Polycomb targets (e.g. bivalent domains) but establishes a considerable ‘background’ across most of the genome both in mouse and human pluripotent cells. We did not find a dependence of PRC1 activity on presence of PRC2 or H3K27me3 as was also described by others. Instead, loss of H3K27me3 lead to a global increase in H2Aub (unpublished data), potentially consistent with a previously suggested competitive/compensatory interplay of PRC1 and PRC2. This and much emerging evidence in the field suggests that our understanding of PRC1 and PRC2 function needs to be fundamentally revisited. For example, PRC1 has recently been suggested to mediate promoter-enhancer contacts. In one implementation of RAPID, we developed a synthetic strategy to quickly deplete H2Aub, without disturbing PRC1 and PRC2 complexes itself. Surprisingly, we found that neither PRC1 nor PRC2 dissociate from the H2Aub-depleted chromatin immediately. A high degree of multivalent binding, or, as already documented in principle for PRC1, a phase separation phenomenon would explain such ‘resilient’ binding of PRC1.
We also apply our technology to understanding the function of Polycomb group (PcG) proteins, evolutionary conserved master regulators of cell identity and differentiation that have long been a paradigm for epigenetic gene control. Two major PcG complexes, PRC1 and PRC2, cooperate in the repression of target genes catalyzing histone H2A K119 monoubiquitination (H2Aub) and H3K27 trimethylation (H3K27me3), respectively. Applying our quantitative MINUTE-ChIP in mESCs lead us to a number of interesting findings: 1) Catalytic activity of both Polycomb complexes is not confined to so-called Polycomb targets (e.g. bivalent domains) but establishes a considerable ‘background’ across most of the genome both in mouse and human pluripotent cells. We did not find a dependence of PRC1 activity on presence of PRC2 or H3K27me3 as was also described by others. Instead, loss of H3K27me3 lead to a global increase in H2Aub (unpublished data), potentially consistent with a previously suggested competitive/compensatory interplay of PRC1 and PRC2. This and much emerging evidence in the field suggests that our understanding of PRC1 and PRC2 function needs to be fundamentally revisited. For example, PRC1 has recently been suggested to mediate promoter-enhancer contacts. In one implementation of RAPID, we developed a synthetic strategy to quickly deplete H2Aub, without disturbing PRC1 and PRC2 complexes itself. Surprisingly, we found that neither PRC1 nor PRC2 dissociate from the H2Aub-depleted chromatin immediately. A high degree of multivalent binding, or, as already documented in principle for PRC1, a phase separation phenomenon would explain such ‘resilient’ binding of PRC1.
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. The method takes advantage of the highly efficient amber suppression technology in mammalian cells which I have developed and continuously improved. Combined with our quantitative MINUTE-ChIP, we can capture genome-wide chromatin dynamics in a quantitative manner and resolved over a period of time ranging from minutes to days. RAPID introduces a flexible time dimension in the form of pulse or pulse-chase experiments for studying genome-wide dynamic occupancy of a protein of interest by next-gen sequencing. It can also be coupled to other readouts such as mass spectrometry or microscopy.
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. The method takes advantage of the highly efficient amber suppression technology in mammalian cells which I have developed and continuously improved. Combined with our quantitative MINUTE-ChIP, we can capture genome-wide chromatin dynamics in a quantitative manner and resolved over a period of time ranging from minutes to days. RAPID introduces a flexible time dimension in the form of pulse or pulse-chase experiments for studying genome-wide dynamic occupancy of a protein of interest by next-gen sequencing. It can also be coupled to other readouts such as mass spectrometry or microscopy.