Final Report Summary - HETCHROMPROJECT (Study of the Determinants of Heterochromatin Formation and Maintenance)
Publishable summary
Project objectives:
Heterochromatin is a complex and dynamic structure that has key roles in many critical cellular processes, including gene regulation, the maintenance of chromosomal structures and in the establishment and transmission of epigenetic information. As such, having a complete understanding of the mechanisms of heterochromatin formation is essential to better assess its roles in these key cellular processes. To study the mechanisms involved in heterochromatin formation, we proposed two independent approaches (see below). The experiments were performed in the genetically tractable model organism Schizosaccharomyces pombe which is an ideal system due to the fundamentally conserved nature of much of the biological apparatus.
Work performed:
Aim 1. To analyze the contribution of the fission yeast Centromere protein-B (CENP-B) homolog Abp1 (ARS-binding protein 1) to genomic expression and heterochromatin formation.
Aim 1.1. Study of small RNAs (sRNAs) accumulation in the absence of Abp1.
Preliminary unpublished results from my host laboratory indicated that Abp1 could be regulating sRNAs silencing, as deletion of the abp1 gene results in a strong accumulation of sRNAs. In collaboration with the Functional Genomics and Bioinformatics/Biostatistics Core Facilities of our host institute –the Institute for Research in Biomedicine or IRB- we identified by Solexa-sequencing the sRNAs. We validated the increase in sRNA coming from different types of genomic features by qPCR. We performed the ChIP-sequencing of Abp1 and the initiating form of the RNA Polymerase II DNA binding sites to correlate them with the sRNAs location over the yeast genome. We studied by qPCR after gel purification of the sRNAs fraction whether the sRNAs also accumulated in mutants for genes involved in chromatin-mediated gene silencing and/or RNA degradation pathways and their potential genetic interactions with the abp1 gene deletion. We studied by ChIP the binding of Abp1 in a mutant for a gene involved in nucleosome positioning (hip3Δ).
Aim 1.2. Determination of Abp1 interacting partners.
We generated a C-terminal TAP-tagged version of Abp1 and confirm that this protein behaves like its untagged counterparts. We used the tandem affinity purification (TAP)-tagging method to isolate proteins from this yeast strain under non-denaturing conditions and purified Abp1-TAP by a 2-step purification procedure. Protein species that specifically associated with Abp1-TAP were isolated from the silver-stained gel and their identities determined in collaboration with the Mass Spectrometry Core Facility of the IRB. We also realized an analysis of the purified proteins by mixture mass spectrometry.
Aim 1.3 Study of the contribution of Abp1 to the structural organization of heterochromatin.
We assessed the effect of the deletion of abp1 on the size of the nucleolus. We discovered an enlargement of the nucleolar structure in the absence of Abp1 that will lead to further investigation on the role of Abp1 in organizing the chromatin at the nucleolar levels.
Aim 2. To identify proteins involved in the regulation of heterochromatin formation.
We realized a genome-wide screen to identify novel regulators of heterochromatin. First, we selected for strains that are sensitive to the drug carbendazim. Because intact centromeric structures are necessary to connect chromosomes to the mitotic spindle through the assembly of the kinetochore, cells with compromised centromeric heterochromatin often display sensitivity to carbendazim. We replica plated a yeast haploid single gene deletion library (Bioneer Corporation) on media with and without drug and selected for the strains presenting a slower growth in presence of carbendazim. We then further selected for the genes directly involved in centromeric heterochromatin formation by looking at the de-repression of the dg/dh repeats, a hallmark of the disturbance of the pericentromeric heterochromatin structure. To do this, we prepared total RNA from the wild-type (wt) and the drug-sensitive strains, and looked by RT-qPCR at the de-repression of the forward and reverse strands of the dh and dg repeats. Eight positive results of the screen were validated by deleting the selected genes in a wt strain and in a strain containing a ura4 gene in the centromeric repeats.
Main results:
Aim 1: Our main results for this aim is the finding that Abp1 is responsible for the genome wide transcriptional repression of sRNAs of a length spanning between 50 and 200 bp. The strongest silencing occurs on the Tf2 retrotransposons and the ribosomal DNA repeats (rDNA). At least some of these sRNAs are polyadenylated and we are showing genetic interactions between the abp1 deletion and the cid14 and dis3 mutants strain involved in targeting unwanted transcript for degradation by the exosome. In the absence of Abp1, initiating RNA Polymerase II complexes are enriched at the sites of sRNAs production, in particular on the Tf2 retrotransposon and the rDNA repeats. We found that the sRNAs are also de-repressed in strains deleted for the clr3 and clr6 histone deacetylase genes and the hip3 HIRA complex gene, genes whose products are involved in gene silencing through regulation of the chromatin structure. We propose that these sRNAs are akin to the CUTs (“cryptic unstable transcripts”) and are the product of unwanted or “cryptic” transcription that arise following a perturbation of a repressing chromatin structure in the absence of Abp1. The gain of access of the RNAPII to the rDNA repeats in the absence of Abp1 was unexpected and very interesting and prompted us to initiate the study of the role of Abp1 in the nucleolus structure.
Aim 2: The completion of a 2-steps screen using a fission yeast haploid single gene deletion library gave us 83 deletion strains to further analyze. We started the study of 8 of these deletion mutants and validated the phenotype of transcriptional de-repression of the centromeric repeats, they are therefore good candidates to present an alteration of the centromeric heterochromatic structure. Some of these genes can be related to pathways involved in heterochomatin regulation, others are showing the implication of new pathways and will open new angles of investigation in the field of heterochromatin regulation.
Expected final results.
We report a novel Abp1 function in preventing pervasive transcription. We show that loss of Abp1 results in the accumulation of CUTs, mostly from the Tf2 retrotransposons and the rDNA repeats. This study provides new insights into the role of Abp1 in controlling unwanted transcription by regulating the chromatin structure.
The two-step genome-wide screen based on the properties of pericentromeric heterochromatin provides new determinants of heterochromatin formation and maintenance. Their study will improve our knowledge on the regulation of pericentromeric heterochromatin.
Project objectives:
Heterochromatin is a complex and dynamic structure that has key roles in many critical cellular processes, including gene regulation, the maintenance of chromosomal structures and in the establishment and transmission of epigenetic information. As such, having a complete understanding of the mechanisms of heterochromatin formation is essential to better assess its roles in these key cellular processes. To study the mechanisms involved in heterochromatin formation, we proposed two independent approaches (see below). The experiments were performed in the genetically tractable model organism Schizosaccharomyces pombe which is an ideal system due to the fundamentally conserved nature of much of the biological apparatus.
Work performed:
Aim 1. To analyze the contribution of the fission yeast Centromere protein-B (CENP-B) homolog Abp1 (ARS-binding protein 1) to genomic expression and heterochromatin formation.
Aim 1.1. Study of small RNAs (sRNAs) accumulation in the absence of Abp1.
Preliminary unpublished results from my host laboratory indicated that Abp1 could be regulating sRNAs silencing, as deletion of the abp1 gene results in a strong accumulation of sRNAs. In collaboration with the Functional Genomics and Bioinformatics/Biostatistics Core Facilities of our host institute –the Institute for Research in Biomedicine or IRB- we identified by Solexa-sequencing the sRNAs. We validated the increase in sRNA coming from different types of genomic features by qPCR. We performed the ChIP-sequencing of Abp1 and the initiating form of the RNA Polymerase II DNA binding sites to correlate them with the sRNAs location over the yeast genome. We studied by qPCR after gel purification of the sRNAs fraction whether the sRNAs also accumulated in mutants for genes involved in chromatin-mediated gene silencing and/or RNA degradation pathways and their potential genetic interactions with the abp1 gene deletion. We studied by ChIP the binding of Abp1 in a mutant for a gene involved in nucleosome positioning (hip3Δ).
Aim 1.2. Determination of Abp1 interacting partners.
We generated a C-terminal TAP-tagged version of Abp1 and confirm that this protein behaves like its untagged counterparts. We used the tandem affinity purification (TAP)-tagging method to isolate proteins from this yeast strain under non-denaturing conditions and purified Abp1-TAP by a 2-step purification procedure. Protein species that specifically associated with Abp1-TAP were isolated from the silver-stained gel and their identities determined in collaboration with the Mass Spectrometry Core Facility of the IRB. We also realized an analysis of the purified proteins by mixture mass spectrometry.
Aim 1.3 Study of the contribution of Abp1 to the structural organization of heterochromatin.
We assessed the effect of the deletion of abp1 on the size of the nucleolus. We discovered an enlargement of the nucleolar structure in the absence of Abp1 that will lead to further investigation on the role of Abp1 in organizing the chromatin at the nucleolar levels.
Aim 2. To identify proteins involved in the regulation of heterochromatin formation.
We realized a genome-wide screen to identify novel regulators of heterochromatin. First, we selected for strains that are sensitive to the drug carbendazim. Because intact centromeric structures are necessary to connect chromosomes to the mitotic spindle through the assembly of the kinetochore, cells with compromised centromeric heterochromatin often display sensitivity to carbendazim. We replica plated a yeast haploid single gene deletion library (Bioneer Corporation) on media with and without drug and selected for the strains presenting a slower growth in presence of carbendazim. We then further selected for the genes directly involved in centromeric heterochromatin formation by looking at the de-repression of the dg/dh repeats, a hallmark of the disturbance of the pericentromeric heterochromatin structure. To do this, we prepared total RNA from the wild-type (wt) and the drug-sensitive strains, and looked by RT-qPCR at the de-repression of the forward and reverse strands of the dh and dg repeats. Eight positive results of the screen were validated by deleting the selected genes in a wt strain and in a strain containing a ura4 gene in the centromeric repeats.
Main results:
Aim 1: Our main results for this aim is the finding that Abp1 is responsible for the genome wide transcriptional repression of sRNAs of a length spanning between 50 and 200 bp. The strongest silencing occurs on the Tf2 retrotransposons and the ribosomal DNA repeats (rDNA). At least some of these sRNAs are polyadenylated and we are showing genetic interactions between the abp1 deletion and the cid14 and dis3 mutants strain involved in targeting unwanted transcript for degradation by the exosome. In the absence of Abp1, initiating RNA Polymerase II complexes are enriched at the sites of sRNAs production, in particular on the Tf2 retrotransposon and the rDNA repeats. We found that the sRNAs are also de-repressed in strains deleted for the clr3 and clr6 histone deacetylase genes and the hip3 HIRA complex gene, genes whose products are involved in gene silencing through regulation of the chromatin structure. We propose that these sRNAs are akin to the CUTs (“cryptic unstable transcripts”) and are the product of unwanted or “cryptic” transcription that arise following a perturbation of a repressing chromatin structure in the absence of Abp1. The gain of access of the RNAPII to the rDNA repeats in the absence of Abp1 was unexpected and very interesting and prompted us to initiate the study of the role of Abp1 in the nucleolus structure.
Aim 2: The completion of a 2-steps screen using a fission yeast haploid single gene deletion library gave us 83 deletion strains to further analyze. We started the study of 8 of these deletion mutants and validated the phenotype of transcriptional de-repression of the centromeric repeats, they are therefore good candidates to present an alteration of the centromeric heterochromatic structure. Some of these genes can be related to pathways involved in heterochomatin regulation, others are showing the implication of new pathways and will open new angles of investigation in the field of heterochromatin regulation.
Expected final results.
We report a novel Abp1 function in preventing pervasive transcription. We show that loss of Abp1 results in the accumulation of CUTs, mostly from the Tf2 retrotransposons and the rDNA repeats. This study provides new insights into the role of Abp1 in controlling unwanted transcription by regulating the chromatin structure.
The two-step genome-wide screen based on the properties of pericentromeric heterochromatin provides new determinants of heterochromatin formation and maintenance. Their study will improve our knowledge on the regulation of pericentromeric heterochromatin.