The association of the RSC remodeler complex with chromatin is influenced by the prefoldin-like Bud27 and determines nucleosome positioning and polyadenylation sites usage in Saccharomyces cerevisiae.

The tripartite interaction between the chromatin remodeler complex RSC, RNA polymerase subunit Rpb5 and prefoldin-like Bud27 is necessary for proper RNA pol II elongation. Indeed lack of Bud27 alters this association and affects transcription elongation. This work investigates the consequences of lack of Bud27 on the chromatin association of RSC and RNA pol II, and on nucleosome positioning. Our results demonstrate that RSC binds chromatin in gene bodies and lack of Bud27 alters this association, mainly around polyA sites. This alteration impacts chromatin organization and leads to the accumulation of RNA pol II molecules around polyA sites, likely due to pausing or arrest. Our data suggest that RSC is necessary to maintain chromatin organization around those sites, and any alteration of this organization results in the widespread use of alternative polyA sites. Finally, we also find a similar molecular phenotype that occurs upon TOR inhibition with rapamycin, which suggests that alternative polyadenylation observed upon TOR inhibition is likely Bud27-dependent.

Genome-wide regulation of Pol II, FACT, and Spt6 occupancies by RSC in Saccharomyces cerevisiae.

RSC (remodels the structure of chromatin) is an essential ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae. RSC utilizes its ATPase subunit, Sth1, to slide or remove nucleosomes. RSC has been shown to regulate the width of the nucleosome-depleted regions (NDRs) by sliding the flanking nucleosomes away from NDRs. As such, when RSC is depleted, nucleosomes encroach NDRs, leading to transcription initiation defects. In this study, we examined the effects of the catalytic-dead Sth1 on transcription and compared them to those observed during acute and rapid Sth1 depletion by auxin-induced degron strategy. We found that rapid depletion of Sth1 reduces recruitment of TBP and Pol II in highly transcribed genes, as would be expected considering its role in regulating chromatin structure at promoters. In contrast, cells harboring the catalytic-dead Sth1 (sth1-K501R) exhibited a severe reduction in TBP binding, but, surprisingly, also displayed a substantial accumulation in Pol II occupancies within coding regions. The Pol II occupancies further increased upon depleting endogenous Sth1 in the catalytic-dead mutant, suggesting that the inactive Sth1 contributes to Pol II accumulation in coding regions. Notwithstanding the Pol II increase, the ORF occupancies of histone chaperones, FACT and Spt6 were significantly reduced in the mutant. These results suggest a potential role for RSC in recruiting/retaining these chaperones in coding regions. Pol II accumulation despite substantial reductions in TBP, FACT, and Spt6 occupancies in the catalytic-dead mutant could indicate severe transcription elongation and termination defects. Such defects would be consistent with studies showing that RSC is recruited to coding regions in a transcription-dependent manner. Thus, these findings imply a role for RSC in transcription elongation and termination processes, in addition to its established role in transcription initiation.

Measuring occupancies of the nucleosome and nucleosome-interacting factors in vivo in Saccharomyces cerevisiae genome-wide.

Nucleosomes are the repeating units of chromatin. The presence of nucleosomes poses a major impediment to all DNA-dependent processes. As a result, access to DNA in chromatin is dynamically regulated by many factors, including ATP-dependent chromatin remodeling complexes. Digestion of chromatin by micrococcal nuclease (MNase) followed by chromatin immunoprecipitation (ChIP) and sequencing can be leveraged to determine nucleosome occupancy, positioning, and the ability of chromatin interacting factors to alter chromatin accessibility. Here we describe the procedure for performing MNase and MNase ChIP-seq in detail.

Genome-Wide Regulations of the Preinitiation Complex Formation and Elongating RNA Polymerase II by an E3 Ubiquitin Ligase, San1.

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in the genome-wide association of TATA box binding protein (TBP; which nucleates preinitiation complex [PIC] formation for transcription initiation) and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences and, hence, PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1 but not the incorporation of centromeric histone, Cse4, into the active genes in the Δsan1 strain. Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms.

The RSC complex remodels nucleosomes in transcribed coding sequences and promotes transcription in Saccharomyces cerevisiae.

RSC (Remodels the Structure of Chromatin) is a conserved ATP-dependent chromatin remodeling complex that regulates many biological processes, including transcription by RNA polymerase II (Pol II). We report that RSC contributes in generating accessible nucleosomes in transcribed coding sequences (CDSs). RSC MNase ChIP-seq data revealed that RSC-bound nucleosome fragments were very heterogenous (∼80 bp to 180 bp) compared to a sharper profile displayed by the MNase inputs (140 bp to 160 bp), supporting the idea that RSC promotes accessibility of nucleosomal DNA. Notably, RSC binding to +1 nucleosomes and CDSs, but not with -1 nucleosomes, strongly correlated with Pol II occupancies, suggesting that RSC enrichment in CDSs is linked to transcription. We also observed that Pol II associates with nucleosomes throughout transcribed CDSs, and similar to RSC, Pol II-protected fragments were highly heterogenous, consistent with the idea that Pol II interacts with remodeled nucleosomes in CDSs. This idea is supported by the observation that the genes harboring high-levels of Pol II in their CDSs were the most strongly affected by ablating RSC function. Additionally, rapid nuclear depletion of Sth1 decreases nucleosome accessibility and results in accumulation of Pol II in highly transcribed CDSs. This is consistent with a slower clearance of elongating Pol II in cells with reduced RSC function, and is distinct from the effect of RSC depletion on PIC assembly. Altogether, our data provide evidence in support of the role of RSC in promoting Pol II elongation, in addition to its role in regulating transcription initiation.

Transcriptomic Analysis of Naïve Human Embryonic Stem Cells Cultured in Three-Dimensional PEG Scaffolds.

Naïve human embryonic stem cells (ESCs) are characterized by improved viability, proliferation, and differentiation capacity in comparison to traditionally derived primed human ESCs. However, currently used two-dimensional (2-D) cell culture techniques fail to mimic the three-dimensional (3-D) in vivo microenvironment, altering morphological and molecular characteristics of ESCs. Here, we describe the use of 3-D self-assembling scaffolds that support growth and maintenance of the naïve state characteristics of ESC line, Elf1. Scaffolds were formed via a Michael addition reaction upon the combination of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups. 3-D scaffold environment maintained the naïve state and supported the long-term growth of ESCs. RNA-sequencing demonstrated significant changes in gene expression profiles between 2-D and 3-D grown cells. Gene ontology analysis revealed upregulation of biological processes involved in the regulation of transcription and translation, extracellular matrix organization, and chromatin remodeling in 3-D grown cells. 3-D culture conditions also induced upregulation of genes associated with Wnt and focal adhesion signaling, while p53 signaling pathway associated genes were downregulated. Our findings, for the first time, provide insight into the possible mechanisms of self-renewal of naïve ESCs stimulated by the transduction of mechanical signals from the 3-D microenvironment.

Chromatin remodeler Ino80C acts independently of H2A.Z to evict promoter nucleosomes and stimulate transcription of highly expressed genes in yeast.

The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.

Mesenchymal stem cells: Cell therapy and regeneration potential.

Rapid advances in the isolation of multipotent progenitor cells, routinely called mesenchymal stromal/stem cells (MSCs), from various human tissues and organs have provided impetus to the field of cell therapy and regenerative medicine. The most widely studied sources of MSCs include bone marrow, adipose, muscle, peripheral blood, umbilical cord, placenta, fetal tissue, and amniotic fluid. According to the standard definition of MSCs, these clonal cells adhere to plastic, express cluster of differentiation (CD) markers such as CD73, CD90, and CD105 markers, and can differentiate into adipogenic, chondrogenic, and osteogenic lineages in vitro. However, isolated MSCs have been reported to vary in their potency and self-renewal potential. As a result, the MSCs used for clinical applications often lead to variable or even conflicting results. The lack of uniform characterization methods both in vitro and in vivo also contributes to this confusion. Therefore, the name "MSCs" itself has been increasingly questioned lately. As the use of MSCs is expanding rapidly, there is an increasing need to understand the potential sources and specific potencies of MSCs. This review discusses and compares the characteristics of MSCs and suggests that the variations in their distinctive features are dependent on the source and method of isolation as well as epigenetic changes during maintenance and growth. We also discuss the potential opportunities and challenges of MSC research with the hope to stimulate their use for therapeutic and regenerative medicine.

SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast.

The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of nucleosomes-during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.

Acetylation-Dependent Recruitment of the FACT Complex and Its Role in Regulating Pol II Occupancy Genome-Wide in Saccharomyces cerevisiae.

Histone chaperones, chromatin remodelers, and histone modifying complexes play a critical role in alleviating the nucleosomal barrier for DNA-dependent processes. Here, we have examined the role of two highly conserved yeast (Saccharomyces cerevisiae) histone chaperones, facilitates chromatin transcription (FACT) and Spt6, in regulating transcription. We show that the H3 tail contributes to the recruitment of FACT to coding sequences in a manner dependent on acetylation. We found that deleting a H3 histone acetyltransferase Gcn5 or mutating lysines on the H3 tail impairs FACT recruitment at ADH1 and ARG1 genes. However, deleting the H4 tail or mutating the H4 lysines failed to dampen FACT occupancy in coding regions. Additionally, we show that FACT depletion reduces RNA polymerase II (Pol II) occupancy genome-wide. Spt6 depletion leads to a reduction in Pol II occupancy toward the 3'-end, in a manner dependent on the gene length. Severe transcription and histone-eviction defects were also observed in a strain that was impaired for Spt6 recruitment (spt6Δ202) and depleted of FACT. Importantly, the severity of the defect strongly correlated with wild-type Pol II occupancies at these genes, indicating critical roles for Spt6 and Spt16 in promoting high-level transcription. Collectively, our results show that both FACT and Spt6 are important for transcription globally and may participate during different stages of transcription.

Recruitment of Saccharomyces cerevisiae Cmr1/Ydl156w to Coding Regions Promotes Transcription Genome Wide.

Cmr1 (changed mutation rate 1) is a largely uncharacterized nuclear protein that has recently emerged in several global genetic interaction and protein localization studies. It clusters with proteins involved in DNA damage and replication stress response, suggesting a role in maintaining genome integrity. Under conditions of proteasome inhibition or replication stress, this protein localizes to distinct sub-nuclear foci termed as intranuclear quality control (INQ) compartments, which sequester proteins for their subsequent degradation. Interestingly, it also interacts with histones, chromatin remodelers and modifiers, as well as with proteins involved in transcription including subunits of RNA Pol I and Pol III, but not with those of Pol II. It is not known whether Cmr1 plays a role in regulating transcription of Pol II target genes. Here, we show that Cmr1 is recruited to the coding regions of transcribed genes of S. cerevisiae. Cmr1 occupancy correlates with the Pol II occupancy genome-wide, indicating that it is recruited to coding sequences in a transcription-dependent manner. Cmr1-enriched genes include Gcn4 targets and ribosomal protein genes. Furthermore, our results show that Cmr1 recruitment to coding sequences is stimulated by Pol II CTD kinase, Kin28, and the histone deacetylases, Rpd3 and Hos2. Finally, our genome-wide analyses implicate Cmr1 in regulating Pol II occupancy at transcribed coding sequences. However, it is dispensable for maintaining co-transcriptional histone occupancy and histone modification (acetylation and methylation). Collectively, our results show that Cmr1 facilitates transcription by directly engaging with transcribed coding regions.

The RSC complex localizes to coding sequences to regulate Pol II and histone occupancy.

ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions.

Histone deacetylases and phosphorylated polymerase II C-terminal domain recruit Spt6 for cotranscriptional histone reassembly.

Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C terminus that recognizes Pol II C-terminal domain (CTD) peptides phosphorylated on Ser2, Ser5, or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3' ends of genes, where phosphorylated Ser2 reaches its maximum level. In addition, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3' ends of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation, and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo.

Measuring dynamic changes in histone modifications and nucleosome density during activated transcription in budding yeast.

Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.

A role for phosphorylated Pol II CTD in modulating transcription coupled histone dynamics.

Histone acetylation modulates histone occupancy both at promoters and in coding sequences. Based on our recent observation that HDACs in the budding yeast, Saccharomyces cerevisiae, are co-transcriptionally recruited to coding regions by elongating polymerases, we propose a model in which Pol II facilitates recruitment of chromatin remodeling complexes as well as other factors required for productive elongation.

Phosphorylated Pol II CTD recruits multiple HDACs, including Rpd3C(S), for methylation-dependent deacetylation of ORF nucleosomes.

Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are cotranscriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their cotranscriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing cotranscriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is important for nucleosome eviction.

NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5.

NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

Sus1 is recruited to coding regions and functions during transcription elongation in association with SAGA and TREX2.

Gene transcription, RNA biogenesis, and mRNA transport constitute a complicated process essential for all eukaryotic cells. The transcription/export factor Sus1 plays a key role in coupling transcription activation with mRNA export, and it resides in both the SAGA and TREX2 complexes. Moreover, Sus1 is responsible for GAL1 gene gating at the nuclear periphery, which is important for its transcriptional status. Here, we show that Sus1 is required during transcription elongation and is associated with the elongating form of RNA Polymerase II (RNAP II) phosphorylated on Ser5 and Ser2 of the C-terminal domain (CTD). In addition, Sus1 copurifies with the essential mRNA export factors Yra1 and Mex67, which bind to the mRNA cotranscriptionally. Consistently, ChIP analysis reveals that Sus1 is present at coding regions dependent on transcription in a manner stimulated by Kin28-dependent CTD phosphorylation. Strikingly, eliminating the TREX2 component Sac3 or the SAGA subunit Ubp8 partially impairs Sus1 targeting to coding sequences and upstream activating sequences (UAS). We found, unexpectedly, that Sgf73 is necessary for association of Sus1 with both SAGA and TREX2, and that its absence dramatically reduces Sus1 occupancy of UAS and ORF sequences. Our results reveal that Sus1 plays a key role in coordinating gene transcription and mRNA export by working at the interface between the SAGA and TREX2 complexes during transcription elongation.

Gcn5 promotes acetylation, eviction, and methylation of nucleosomes in transcribed coding regions.

We report that coactivator SAGA, containing the HAT Gcn5p, occupies the GAL1 and ARG1 coding sequences during transcriptional induction, dependent on PIC assembly and Ser5 phosphorylation of the Pol II CTD. Induction of GAL1 increases H3 acetylation per nucleosome in the ORF, dependent on SAGA integrity but not the alternative Gcn5p-HAT complex ADA. Unexpectedly, H3 acetylation in ARG1 coding sequences does not increase during induction due to the opposing activities of multiple HDAs associated with the ORF. Remarkably, inactivation of Gcn5p decreases nucleosome eviction from both GAL1 and a long ( approximately 8 kb) ORF transcribed from the GAL1 promoter. This is associated with reduced Pol II occupancy at the 3' end and decreased mRNA production, selectively, for the long ORF. Gcn5p also enhances H3-K4 trimethylation in the ARG1 ORF and bulk histones. Thus, Gcn5p, most likely in SAGA, stimulates modification and eviction of nucleosomes in transcribed coding sequences and promotes Pol II elongation.