Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - NChIP (Chromatin dynamics during DNA replication)

Teaser

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...

Summary

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. Not much is known about histone “inheritance” beyond the facts that bulk maternal histones distribute equally among the daughter strands and are diluted two-fold after replication with newly synthesized “unmarked” histones, and that the majority of H3/H4 tetramers do not split before reassembly. We have shown previously that maternal nucleosomes stay on average within 400bp of their original binding site, implying that any potentially heritable chromatin encoded information, has to be inherited in ~1kb blocs, as smaller nucleosome domains would rapidly be diluted by new nucleosomes.
I propose 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. This will allow us to assess locus specific differences in the spread of maternal nucleosomes after replication, the effects of leading and lagging strand replication on nucleosome positioning and maternal nucleosome distribution, the renewal dynamics of posttranslational histone marks and chromatin binding proteins, and the kinetics of chromatin footprint re-establishment and gene (re)activation.

Work performed

Specific Aim 1: Tracking maternal nucleosomes at targeted genomic loci
This aim is to measure replication-mediated dispersion of maternal nucleosomes in specific chromatin domains. This will allow us to extend our prior inferences based on indirect measurements of bulk nucleosome distributions, and to determine whether histone spreading differs at different regions of the genome. This information will help us to better understand the epigenetic potential of nucleosomes and which chromatin features, if any, could be inherited.
Results:
We have created strains with the degron BirA constructs stably integrated in the genome, and optimized the BirA induction and degradation conditions. They have also shown that the avitag-H3 is fully functional and can completely replace wt H3, and that biotinH3, i.e. the old ancestral histones, remain in cells for at least 4 generations after the initial BirA pulse.

Specific Aim 2: Heterochromatin inheritance: tracking Sir complex redistribution after DNA replication.
The budding yeast Sir (Silent Information Regulator) complex (composed of three subunits Sir2-4) is the principal factor for heterochromatin formation, which causes epigenetically regulated gene silencing phenotypes. The maternal Sir complex has to be disassembled during replication and if heterochromatin is to be restored on both daughter strands, the Sir complex has to be reformed on both strands to pre-replication levels. How heterochromatic Sir complexes exchange their components during the cell cycle and how they are distributed to daughter chromatids after replication has important implications for how heterochromatic states may be inherited. I therefore propose to use a tag switch system and pulse chase labeling of Sir components in order to measure their genome wide turnover rates and distribution after replication, thus directly testing whether the Sir proteins are maintained locally after replication as might be expected.
Results:
Our genome wide mapping of Sir3 turnover rates after exit from starvation revealed a complete removal of the bound Sir3 from telomeric regions and silent mating type loci around the time of the first cell division after release from starvation. Sir3 is then gradually restored to its preferred genomic loci over several cell generations. Concomitantly, new Sir3 binds with high affinity to all tDNA loci, probably as a consequence of high tRNA gene expression after release from starvation. Our results are a striking example of the direct influence of environmental cue on changes in chromatin structure.

Specific Aim 3: Chromatin configuration re-establishment after DNA replication

The goal is to create comprehensive maps of chromatin assembly dynamics on newly replicated DNA, in order to understand how chromatin states are restored after perturbations caused by DNA replication. We will also explore the links between chromatin maturation and (re)activation of transcriptional activity. To achieve this goal we are developing a method for isolating chromatin assembled on newly replicated DNA, i.e. Nascent Chromatin Avidin Pulldown or NChAP, which will be used alone or in combination with ChIP-seq, i.e. ChIP-NChAP and experimental tools from Aims 1 and 2.
Results:
To study nucleosome positioning reestablishment after replication, we have combined EdU labeling 4, 5 of newly synthesized DNA with MNase digestion of chromatin to map nucleosome positioning following replication. Our methode- NChAP- is now fully optimized for use in S.Cerevisiae. We found that nucleosomes rapidly adopt their mid-log positions at highly transcribed genes, consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in Hir1Δ mutants reveal a role for HIR in nucleosome spacing. We also characterized nucleosome positions on the leading and lagging strand genomes, uncovering differences in chromatin maturation dynamics at hundreds of genes. Our results suggest that the gene copy rep

Final results

We will further explore the mechanism responsible for asymmetric distribution of new histones and RNApol2 on daughter chromatids using a selection of deletion mutants. We expect to have measured the kinetics of histone methylation mark reestablishment after replication on the two daughter chromatids using ChIP-NChAP, as well as maternal histone distribution on newly replicated DNA in growing and ageing cells using in vivo biotinylation of Histone H3 in combination with NChAP. We will also continue to explore the mechanism of yeast heterochromatin reestablishment following replication after release from quiescence and in exponentially growing cells. We also expect to complete the objectives from Aim 1, by measuring the replication dependent dispersal of heterochromatic maternal nucleosomes after replication, which should help us understand the epigenetic mechanism responsible for heterochromatin maintenance from one cell generation to the next.