Final Report Summary - CHROMOSOME STABILITY (Chromosomal addresses and mechanism of chromatin binding by the Scc2/Scc4 cohesin and condensin loading complex)
Accurate control of the mechanisms implicated in sister chromatids cohesion after DNA replication and chromosome condensation during mitosis are essential for faithful chromosome segregation during cell division. A fundamental part of these mechanisms are the Structural Maintenance of Chromosomes (SMC) protein complexes, which can be found from bacteria to humans. SMC proteins function as core components of the Cohesin and Condensin complexes in eukaryotes. These are key elements of the structural and functional organisation of chromosomes. Cohesin and Condensin depend in their chromosomal association on a loading factor, the Scc2/Scc4 complex. Little is known about how and where Scc2/Scc4 binds to chromosomes, and how it facilitates the chromosomal loading of Cohesin and Condensin.
The aim of this project was to determine the chromatin features underlying the loading of Scc2/Scc4 onto chromosomes and thus Cohesin and Condensin localisation along the genome.
Description of the work performed
In the first year of this project different techniques were performed to determine the binding sites of Scc2/Scc4 along the genome.
Chromatin immunoprecipitation of Scc2 or Scc4 followed by high-throughput sequencing showed the binding pattern of the Scc2/Scc4 complex genome wide. 25 % of the binding sites correspond to tRNA genes, the promoter region of Ribosomal Protein genes (RP genes thereafter) account for another 25 % of the binding sites and the remaining 50 % correspond to genes that are not tRNA or RP genes.
The preferential binding to tRNA and PR genes prompted us to analyse if any of the transcription factors involved in their transcription was involved in the Scc2/Scc4 complex loading to chromosomes. However, no interaction was observed between the Scc2/Scc4 complex and the tRNA transcription factor TFIIIC or the RP genes transcription factor Fhl1, and deletion of these transcription factors did not affect the binding of Scc2/Scc4.
We also compared our data set with reported chromatin features such are histone modifications and histone variants, transcription factors and structural proteins such as Hmo1. No correlation could be found.
We then compared our data set with the nucleosome occupancy along the genome reported in Lee et al, 2007. In this paper, the authors cluster the genes according to the nucleosome occupancy around the Transcription Start Site (TSS). They find 4 clusters depending on the size of the nucleosome free region (NFR) that spans between the-1 and the + 1 nucleosomes. We found a correlation between the Scc2/Scc4 complex binding pattern and the cluster of genes that show a NFR that spans for at least 400 bp, found at the promoter of highly transcribed genes such are tRNA and RP genes. The other clusters, in which only one nucleosome is missing right before the TSS or the nucleosome occupancy at the promoter is very high, show a negative correlation with the Scc2/Scc4 binding sites meaning that the Scc2/Scc4 complex is excluded from sites occupied by nucleosomes.
To test our hypothesis that the Scc2/Scc4 complex binds NFR, we analysed the nucleosome landscape under different conditions by paired-end deep sequencing. When yeast cells are subjected to a heat shock, a nucleosome is loaded at the promoter of RP genes, inducing their silencing. This response is perfect for our model as RP genes are one of the preferred binding sites of the Scc2/Scc4 complex. We tested whether the Scc2/Scc4 complex was released from RP genes upon nucleosome loading after a heat shock and indeed it was the case, ChIP followed by qPCR showed a decrease in the binding of the Scc2/Scc4 complex at the sites where a nucleosome has been loaded. However, transcription was also affected under this condition so we cannot exclude the possibility that a transcription factor mediating the Scc2/Scc4 complex loading is responsible for the release of Scc2/Scc4. To address this, we had to find a way to inhibit transcription without changing the nucleosome occupancy at the promoter, to do so, we deleted the TATA box of the promoter of a gene bound by Scc2/Scc4 in order to avoid the binding of the TBP and inhibit transcription without affecting the signals for nucleosome occupancy. Under these conditions, the Scc2/Scc4 complex remains bound to the NFR even when transcription is impaired by the lack of TBP binding to the TATA box.
We then decided to address if the Scc2/Scc4 complex is somehow involved in the maintenance of these NFR or if it is simply binding there. Using a thermo sensible (ts) allele we could inactivate Scc2 to then map the nucleosomes along the genome. Scc2 inactivation led to massive changes in the nucleosome occupancy at its binding sites. Prompted by this observation, we analysed the relationship between the Scc2/Scc4 complex and chromatin remodelling complexes. It's been previously reported the interaction between Cohesin and the ISWI chromatin remodelling complex in human cells (Hakimi et al, 2002) so we decided to check if there was any interaction between the Scc2/Scc4 complex and the chromatin remodelling complex of the ISWI family RSC in yeast. Co-immunoprecipitation analysis showed a physical interaction between Scc2 and the RSC subunit Sth1.
A description of the main results achieved so far
The Scc2/Scc4 complex binds to Nucleosome Free Regions mainly found at the promoters of highly transcribed genes. Loading of a nucleosome at the NFR leads to a decrease in the binding of Scc2/Scc4 to that very site. Transcription does not have an effect on the loading of the Scc2/Scc4 complex to the promoters.
We have also showed that the Scc2/Scc4 complex interacts with the RSC chromatin remodelling complex and its inactivation affects the nucleosome occupancy at its binding sites.
Expected final results
We want to further analyse the functional relationship between the Scc2/Scc4 complex and the RSC chromatin remodelling complex. We aim to know if the Scc2/Scc4 complex is responsible for the recruitment of the RSC complex or if the binding of the Scc2/Scc4 complex follows the chromatin remodelling activity of the RSC complex. The evidence suggests that the Scc2/Scc4 complex is needed for the recruitment of the RSC complex as its inactivation leads to the loading of nucleosomes to former NFR.
Therefore, our findings could be extremely important to establish a link between local chromatin structure and higher order chromatin structure achieved by the loading of Condensin by the Scc2/Scc4 complex.
The aim of this project was to determine the chromatin features underlying the loading of Scc2/Scc4 onto chromosomes and thus Cohesin and Condensin localisation along the genome.
Description of the work performed
In the first year of this project different techniques were performed to determine the binding sites of Scc2/Scc4 along the genome.
Chromatin immunoprecipitation of Scc2 or Scc4 followed by high-throughput sequencing showed the binding pattern of the Scc2/Scc4 complex genome wide. 25 % of the binding sites correspond to tRNA genes, the promoter region of Ribosomal Protein genes (RP genes thereafter) account for another 25 % of the binding sites and the remaining 50 % correspond to genes that are not tRNA or RP genes.
The preferential binding to tRNA and PR genes prompted us to analyse if any of the transcription factors involved in their transcription was involved in the Scc2/Scc4 complex loading to chromosomes. However, no interaction was observed between the Scc2/Scc4 complex and the tRNA transcription factor TFIIIC or the RP genes transcription factor Fhl1, and deletion of these transcription factors did not affect the binding of Scc2/Scc4.
We also compared our data set with reported chromatin features such are histone modifications and histone variants, transcription factors and structural proteins such as Hmo1. No correlation could be found.
We then compared our data set with the nucleosome occupancy along the genome reported in Lee et al, 2007. In this paper, the authors cluster the genes according to the nucleosome occupancy around the Transcription Start Site (TSS). They find 4 clusters depending on the size of the nucleosome free region (NFR) that spans between the-1 and the + 1 nucleosomes. We found a correlation between the Scc2/Scc4 complex binding pattern and the cluster of genes that show a NFR that spans for at least 400 bp, found at the promoter of highly transcribed genes such are tRNA and RP genes. The other clusters, in which only one nucleosome is missing right before the TSS or the nucleosome occupancy at the promoter is very high, show a negative correlation with the Scc2/Scc4 binding sites meaning that the Scc2/Scc4 complex is excluded from sites occupied by nucleosomes.
To test our hypothesis that the Scc2/Scc4 complex binds NFR, we analysed the nucleosome landscape under different conditions by paired-end deep sequencing. When yeast cells are subjected to a heat shock, a nucleosome is loaded at the promoter of RP genes, inducing their silencing. This response is perfect for our model as RP genes are one of the preferred binding sites of the Scc2/Scc4 complex. We tested whether the Scc2/Scc4 complex was released from RP genes upon nucleosome loading after a heat shock and indeed it was the case, ChIP followed by qPCR showed a decrease in the binding of the Scc2/Scc4 complex at the sites where a nucleosome has been loaded. However, transcription was also affected under this condition so we cannot exclude the possibility that a transcription factor mediating the Scc2/Scc4 complex loading is responsible for the release of Scc2/Scc4. To address this, we had to find a way to inhibit transcription without changing the nucleosome occupancy at the promoter, to do so, we deleted the TATA box of the promoter of a gene bound by Scc2/Scc4 in order to avoid the binding of the TBP and inhibit transcription without affecting the signals for nucleosome occupancy. Under these conditions, the Scc2/Scc4 complex remains bound to the NFR even when transcription is impaired by the lack of TBP binding to the TATA box.
We then decided to address if the Scc2/Scc4 complex is somehow involved in the maintenance of these NFR or if it is simply binding there. Using a thermo sensible (ts) allele we could inactivate Scc2 to then map the nucleosomes along the genome. Scc2 inactivation led to massive changes in the nucleosome occupancy at its binding sites. Prompted by this observation, we analysed the relationship between the Scc2/Scc4 complex and chromatin remodelling complexes. It's been previously reported the interaction between Cohesin and the ISWI chromatin remodelling complex in human cells (Hakimi et al, 2002) so we decided to check if there was any interaction between the Scc2/Scc4 complex and the chromatin remodelling complex of the ISWI family RSC in yeast. Co-immunoprecipitation analysis showed a physical interaction between Scc2 and the RSC subunit Sth1.
A description of the main results achieved so far
The Scc2/Scc4 complex binds to Nucleosome Free Regions mainly found at the promoters of highly transcribed genes. Loading of a nucleosome at the NFR leads to a decrease in the binding of Scc2/Scc4 to that very site. Transcription does not have an effect on the loading of the Scc2/Scc4 complex to the promoters.
We have also showed that the Scc2/Scc4 complex interacts with the RSC chromatin remodelling complex and its inactivation affects the nucleosome occupancy at its binding sites.
Expected final results
We want to further analyse the functional relationship between the Scc2/Scc4 complex and the RSC chromatin remodelling complex. We aim to know if the Scc2/Scc4 complex is responsible for the recruitment of the RSC complex or if the binding of the Scc2/Scc4 complex follows the chromatin remodelling activity of the RSC complex. The evidence suggests that the Scc2/Scc4 complex is needed for the recruitment of the RSC complex as its inactivation leads to the loading of nucleosomes to former NFR.
Therefore, our findings could be extremely important to establish a link between local chromatin structure and higher order chromatin structure achieved by the loading of Condensin by the Scc2/Scc4 complex.