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.
