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Chromatin dynamics during DNA replication

Periodic Reporting for period 4 - NChIP (Chromatin dynamics during DNA replication)

Reporting period: 2019-12-01 to 2020-05-31

Chromatin assembly is a fundamental cellular process necessary for the maintenance of genome integrity and transcriptional programs. Understanding the effect of DNA replication on histone protein dynamics is also a prerequisite for understanding the role of chromatin in epigenetic inheritance. Epigenetic phenomena are thought to influence cellular differentiation and cancer formation, as well as the impact of environmental factors on early development and later predispositions to disease. While epigenetic inheritance of chromatin components is, in theory, accepted as the driver of such phenomena, chromatin state inheritance per se has only been demonstrated for a few specific cases.
NChIP's goal was to develop high throughput systems for directly measuring movements of histones and chromatin regulators during genomic replication in S.cerevisiae to determine, how chromatin states survive the perturbations associated with replication.

Overall our results upend the commonly adopted linear thinking that chromatin state inheritance is a prerequisite for transcription state inheritance. Active chromatin states are inherited but their inheritance does not instruct the cell to reestablish the same transcription state at its underlying genes. Transcription states are instead transmitted together with chromatin states directly through recycling of the transcription machinery and rather than being a consequence of chromatin state inheritance transcription actually contributes to the reestablishment of chromatin states on both replicated gene copies.
The NChIP project consisted of four specific aims: 1.,Tracking maternal nucleosomes at targeted genomic loci, 2. Heterochromatin inheritance: tracking Sir complex redistribution after DNA replication, 3. Chromatin configuration re-establishment after DNA replication and 4. Global degradation of maternal histones and consequences for the maintenance of cellular phenotype.
We successfully completed aims 2 and 3 and developed the experimental technique for aim 1. Aim 4 had to be abandon because our cloning of the necessary yeast strains did not yield viable cells.
Aim 1: Tracking maternal nucleosomes at targeted genomic loci
The goal was to measure replication-mediated dispersion of maternal nucleosomes in specific chromatin domains. We have developed a technique for controlled in vivo labelling of histone H3 genome wide and in yeast heterochromatin which we call ChAP for Chromatin Avidine Pull-down. Our genome wide in vivo labelling of histone H3 provides an alternative method for measuring nucleosome turnover that doesn’t require the expression of exogenous tagged histone genes.
Aim 2: Heterochromatin inheritance: tracking Sir complex redistribution after DNA replication,
Our goal was to understand how yeast heterochromatin is maintained after genome replication and cell division. The budding yeast SIR complex (Silent Information Regulator) is the principal actor in heterochromatin formation, which causes epigenetically regulated gene silencing phenotypes. We measured genome wide turnover rates of the SIR subunit Sir3 before and after exit from stationary phase and show that old Sir3 subunits are completely replaced with newly synthesized Sir3 during the first cell cycle after release from stationary phase. Our analyses of genome-wide transcription dynamics show that Sir3 binding to subtelomeric regions is necessary for genome wide gene silencing during stationary phase and for the optimal activation of “growth” genes after release from stationary phase. We have uncovered a new role for the Sir complex that suggests that yeast heterochromatin does not just act to locally silence genes. Heterochromatin integrity is also key for global regulation of transcription programs during environmental challenges.
We have submitted our study to bioRxiv: Galic et al. (2019). bioRxiv, 603613., and a revised version with more functional RNA-seq experiments in Sir mutants will be submitted for publication before the end of 2020.
Aim 3:Chromatin configuration re-establishment after DNA replication
Our goal was to measure the dynamics of chromatin feature reestablishment after genome replication. To this end we have developed a method for genome wide parallel mapping of chromatin structure on both replicated daughter chromatids which we call NChAP (Nascent Chromatin Avidin Pulldown). We produced the following publications.
1. Vasseur et al. (2016). Cell Reports 16, 2651-2665.
This study describes NChAP. We measure a key aspect of chromatin structure dynamics during replication – how rapidly nucleosome positions are established on the newly-replicated daughter genomes. We find that nucleosomes rapidly adopt their mid-log positions at highly-transcribed genes, consistent with a role for transcription in positioning nucleosomes in vivo. We also characterized nucleosome positions on the leading and lagging strand genomes, uncovering differences in chromatin maturation dynamics at hundreds of genes that correlate with the direction of transcription relative to replication fork orientation.
2. Ziane et al. (2019). bioRxiv, 553669.
The revised version will be submitted for publication before the end of August 2020. In order to better understand what role chromatin features play in the transmission of gene expression states from one cell generation to the next we have ChIP with NChAP and used it to monitor the distribution of RNAPol2 and new and old H3 histones on newly-replicated daughter genomes in S. Cerevisiae. We uncovered inherently asymmetric distribution of RNAPol2, and histones on daughter chromatids after replication. We show that lagging and leading strand replication is not simultaneous at a majority of genes. Nucleosomes and RNApol2 preferentially bind to either the leading or the lagging strand gene copy depending on which one replicated first. RNApol2 shifts to the other copy later on. We propose a two-step model of chromatin assembly on nascent DNA . The model describes how chromatin and transcription states are first restored on one and then the other replicated gene copy, thus ensuring that after division each cell will have “inherited” a gene copy with identical gene expression and chromatin states.
"Our methods for genome wide mapping of chromatin features' dynamics on newly replicated DNA brought unprecedented focus to a poorly understood but fundamental cellular process. We show that the inheritance of chromatin features characteristic of actively transcribed chromatin is independent from the inheritance of the transcription machinery. The binding of both to newly replicated DNA is constrained by the mechanics and interactions of replication and transcription ahead of the replication fork which direct old nucleosomes and the transcription machinery to the strand that replicated first. The transcription machinery later switches to the second strand and directs the establishment of histone marks characteristic of active transcription onto new nucleosomes. So new histones don't acquire the ""active"" marks carried by old histones through a hypothetical direct copying mechanism . The marks are put on new nucleosomes indirectly as a consequence of transcription. So even though both replicated gene copies ultimately end up with the same chromatin features, the ""active"" histone marks are actually not truly epigenetic as they are a consequence and not a cause of transcriptional activity."
Diagram of the RNAPol2 ChIP-NChAP experiment.
The distribution of old and new nucleosomes after replication