The Saccharomyces cerevisiae Histone Chaperone Rtt106 Mediates the Cell Cycle Recruitment of SWI/SNF and RSC to the HIR-Dependent Histone Genes

In Saccharomyces cerevisiae, three out of the four histone gene pairs (HTA1-HTB1, HHT1-HHF1, and HHT2-HHF2) are regulated by the HIR co-repressor complex. The histone chaperone Rtt106 has recently been shown to be present at these histone gene loci throughout the cell cycle in a HIR- and Asf1-dependent manner and involved in their transcriptional repression. The SWI/SNF and RSC chromatin remodeling complexes are both recruited to the HIR-dependent histone genes; SWI/SNF is required for their activation in S phase, whereas RSC is implicated in their repression outside of S phase. Even though their presence at the histone genes is dependent on the HIR complex, their specific recruitment has not been well characterized. In this study we focused on characterizing the role played by the histone chaperone Rtt106 in the cell cycle-dependent recruitment of SWI/SNF and RSC complexes to the histone genes.Using GST pull-down and co-immunoprecipitation assays, we showed that Rtt106 physically interacts with both the SWI/SNF and RSC complexes in vitro and in vivo. We then investigated the function of this interaction with respect to the recruitment of these complexes to HIR-dependent histone genes. Using chromatin immunoprecipitation assays (ChIP), we found that Rtt106 is important for the recruitment of both SWI/SNF and RSC complexes to the HIR-dependent histone genes. Furthermore, using synchronized cell cultures, we showed by ChIP assays that the Rtt106-dependent SWI/SNF recruitment to these histone gene loci is cell cycle regulated and restricted to late G1 phase just before the peak of histone gene expression in S phase.Overall, these data strongly suggest that the interaction between the histone chaperone Rtt106 and both the SWI/SNF and RSC chromatin remodeling complexes is important for the cell cycle regulated recruitment of these two complexes to the HIR-dependent histone genes

Prioritization of gene regulatory interactions from large-scale modules in yeast

<p>Abstract</p> <p>Background</p> <p>The identification of groups of co-regulated genes and their transcription factors, called transcriptional modules, has been a focus of many studies about biological systems. While methods have been developed to derive numerous modules from genome-wide data, individual links between regulatory proteins and target genes still need experimental verification. In this work, we aim to prioritize regulator-target links within transcriptional modules based on three types of large-scale data sources.</p> <p>Results</p> <p>Starting with putative transcriptional modules from ChIP-chip data, we first derive modules in which target genes show both expression and function coherence. The most reliable regulatory links between transcription factors and target genes are established by identifying intersection of target genes in coherent modules for each enriched functional category. Using a combination of genome-wide yeast data in normal growth conditions and two different reference datasets, we show that our method predicts regulatory interactions with significantly higher predictive power than ChIP-chip binding data alone. A comparison with results from other studies highlights that our approach provides a reliable and complementary set of regulatory interactions. Based on our results, we can also identify functionally interacting target genes, for instance, a group of co-regulated proteins related to cell wall synthesis. Furthermore, we report novel conserved binding sites of a glycoprotein-encoding gene, CIS3, regulated by Swi6-Swi4 and Ndd1-Fkh2-Mcm1 complexes.</p> <p>Conclusion</p> <p>We provide a simple method to prioritize individual TF-gene interactions from large-scale transcriptional modules. In comparison with other published works, we predict a complementary set of regulatory interactions which yields a similar or higher prediction accuracy at the expense of sensitivity. Therefore, our method can serve as an alternative approach to prioritization for further experimental studies.</p

Comprehensive reanalysis of transcription factor knockout expression data in Saccharomyces cerevisiae reveals many new targets

Transcription factor (TF) perturbation experiments give valuable insights into gene regulation. Genome-scale evidence from microarray measurements may be used to identify regulatory interactions between TFs and targets. Recently, Hu and colleagues published a comprehensive study covering 269 TF knockout mutants for the yeast Saccharomyces cerevisiae. However, the information that can be extracted from this valuable dataset is limited by the method employed to process the microarray data. Here, we present a reanalysis of the original data using improved statistical techniques freely available from the BioConductor project. We identify over 100 000 differentially expressed genes—nine times the total reported by Hu et al. We validate the biological significance of these genes by assessing their functions, the occurrence of upstream TF-binding sites, and the prevalence of protein–protein interactions. The reanalysed dataset outperforms the original across all measures, indicating that we have uncovered a vastly expanded list of relevant targets. In summary, this work presents a high-quality reanalysis that maximizes the information contained in the Hu et al. compendium. The dataset is available from ArrayExpress (accession: E-MTAB-109) and it will be invaluable to any scientist interested in the yeast transcriptional regulatory system

FACT, the Bur Kinase Pathway, and the Histone Co-Repressor HirC Have Overlapping Nucleosome-Related Roles in Yeast Transcription Elongation

Gene transcription is constrained by the nucleosomal nature of chromosomal DNA. This nucleosomal barrier is modulated by FACT, a conserved histone-binding heterodimer. FACT mediates transcription-linked nucleosome disassembly and also nucleosome reassembly in the wake of the RNA polymerase II transcription complex, and in this way maintains the repression of ‘cryptic’ promoters found within some genes. Here we focus on a novel mutant version of the yeast FACT subunit Spt16 that supplies essential Spt16 activities but impairs transcription-linked nucleosome reassembly in dominant fashion. This Spt16 mutant protein also has genetic effects that are recessive, which we used to show that certain Spt16 activities collaborate with histone acetylation and the activities of a Bur-kinase/Spt4–Spt5/Paf1C pathway that facilitate transcription elongation. These collaborating activities were opposed by the actions of Rpd3S, a histone deacetylase that restores a repressive chromatin environment in a transcription-linked manner. Spt16 activity paralleling that of HirC, a co-repressor of histone gene expression, was also found to be opposed by Rpd3S. Our findings suggest that Spt16, the Bur/Spt4–Spt5/Paf1C pathway, and normal histone abundance and/or stoichiometry, in mutually cooperative fashion, facilitate nucleosome disassembly during transcription elongation. The recessive nature of these effects of the mutant Spt16 protein on transcription-linked nucleosome disassembly, contrasted to its dominant negative effect on transcription-linked nucleosome reassembly, indicate that mutant FACT harbouring the mutant Spt16 protein competes poorly with normal FACT at the stage of transcription-linked nucleosome disassembly, but effectively with normal FACT for transcription-linked nucleosome reassembly. This functional difference is consistent with the idea that FACT association with the transcription elongation complex depends on nucleosome disassembly, and that the same FACT molecule that associates with an elongation complex through nucleosome disassembly is retained for reassembly of the same nucleosome

Silencing Mediated by the Schizosaccharomyces pombe HIRA Complex Is Dependent upon the Hpc2-Like Protein, Hip4

HIRA (or Hir) proteins are conserved histone chaperones that function in multi-subunit complexes to mediate replication-independent nucleosome assembly. We have previously demonstrated that the Schizosaccharomyces pombe HIRA proteins, Hip1 and Slm9, form a complex with a TPR repeat protein called Hip3. Here we have identified a new subunit of this complex.To identify proteins that interact with the HIRA complex, rapid affinity purifications of Slm9 were performed. Multiple components of the chaperonin containing TCP-1 complex (CCT) and the 19S subunit of the proteasome reproducibly co-purified with Slm9, suggesting that HIRA interacts with these complexes. Slm9 was also found to interact with a previously uncharacterised protein (SPBC947.08c), that we called Hip4. Hip4 contains a HRD domain which is a characteristic of the budding yeast and human HIRA/Hir-binding proteins, Hpc2 and UBN1. Co-precipitation experiments revealed that Hip4 is stably associated with all of the other components of the HIRA complex and deletion of hip4(+) resulted in the characteristic phenotypes of cells lacking HIRA function, such as temperature sensitivity, an elongated cell morphology and hypersensitivity to the spindle poison, thiabendazole. Moreover, loss of Hip4 function alleviated the heterochromatic silencing of reporter genes located in the mating type locus and centromeres and was associated with increased levels of non-coding transcripts derived from centromeric repeat sequences. Hip4 was also found to be required for the distinct form of silencing that controls the expression of Tf2 LTR retrotransposons.Overall, these results indicate that Hip4 is an integral component of the HIRA complex that is required for transcriptional silencing at multiple loci

Rtt106 is essential for recruitment of RSC to the HIR-dependent histone genes.

<p>(A) Chromatin immunoprecipitation of the RSC complex were carried out as described in Material and Methods. An antibody directed against the TAP tag of the Rsc8-TAP subunit in wild-type and <i>rtt106Δ</i> strains was used. Presence of the indicated histone gene loci promoter sequence was monitored by qPCR. A non-transcribed region of chromosome V (InterV) was used as an internal background control. Error bars show the range between biological duplicates. (B) 25 µg of whole cell extract prepared from the <i>RSC8-TAP</i> (wild-type background) and <i>RSC8-TAP rtt106Δ</i> strains were loaded per lane. Protein levels of two representative RSC subunits, Rsc8-TAP and Sth1, were compared between the <i>RSC8-TAP</i> (lane 1) and <i>RSC8-TAP rtt106Δ</i> strain (lane 2) by western blots using an antibody targeting the TAP-tag in Rsc8-TAP, or an antibody against endogenous Sth1, which is the catalytic subunit of the RSC complex. Actin was used as a loading control. (C) ChIP assays were performed as in A to monitor the presence of the Rsc8-TAP tagged subunit at the <i>HTA1-HTB1</i> promoter in wild-type (WT) strain and strains deleted of <i>RTT106 (rtt106Δ)</i>, <i>ASF1 (asf1Δ)</i>, or <i>HPC2 (hpc2Δ)</i>. Enrichment of <i>HTA1-HTB1</i> promoter sequence in immunoprecipitated material was monitored by qPCR and normalized to the control region, InterV. The graph shows the average of duplicate IP reactions, and the error bars show the range between individual IPs.</p

Rtt106 is required for SWI/SNF localization to <i>HTA1-HTB1</i> loci.

<p>(A) Chromatin immunoprecipitation was carried out as described in Material and Methods. Antibodies specific for the N-terminus or C-terminus of the endogenous Swi2/Snf2 subunit were used for immunoprecipitation of the SWI/SNF complex in cross-linked chromatin extracts from the wild-type strain and strains deleted of <i>RTT106, ASF1</i>, or <i>HPC2</i>. The amount of <i>HTA1-HTB1</i> promoter (black bars) precipitated was monitored by qPCR and inter V region (grey bars) was used as an internal background control from a non-transcribed region on chromosome V. Data is expressed as percentage of ChIP/Input and error bars show the range of repeated IPs. (B) 25 µg of whole cell extracts prepared from the untagged wild-type and <i>rtt106Δ</i> strains were loaded per lane. Protein levels of four SWI/SNF subunits, Swi2/Snf2, Swi1, Snf5 and Swi3, were compared between the wild-type strain (lane 1) and the <i>rtt106Δ</i> strain (lane 2) by western blot analysis using specific antibodies against each individual protein. Actin was included as a loading control.</p

SWI/SNF recruitment to the <i>HTA1-HTB1</i> promoter is cell cycle regulated and dependent on Rtt106.

<p>(A) SWI/SNF binding to the <i>HTA1-HTB1</i> promoter in synchronized cultures of wild-type (blue line) and <i>rtt106Δ</i> (red line) strains was studied by chromatin immunoprecipitation (ChIP) assay on samples taken at the indicated time-points after release from G1 arrest induced by α-factor treatment. Samples for ChIP were cross-linked for 1h and SWI/SNF in chromatin extracts was immunoprecipitated using a specific antibody targeting the N-terminal end of Swi2/Snf2 subunit. Enrichment of <i>HTA1-HTB1</i> promoter relative to a control region (InterV) was quantified by real-time PCR. (B) Expression levels of mRNA from cell cycle regulated genes <i>CLN2</i> (solid line) and <i>CLB2</i> (dashed line) were monitored by RT-qPCR in <i>rtt106Δ</i> (red lines) and wild-type strain (blue lines) as a control for cell cycle progression and were normalized to expression of <i>ACT1</i>. Total RNA was purified from samples taken at the indicated time-points from the ChIP culture used in (A). (C) <i>HTA1</i> mRNA levels were monitored by RT-qPCR in wild-type (blue line) and <i>rtt106Δ</i> (red line) strains using the same samples used in (B) and were normalized to <i>ACT1</i> expression.</p