Increased histone acetylation is the signature of repressed state on the genes transcribed by RNA polymerase III.

Several covalent modifications are found associated with the transcriptionally active chromatin regions constituted by the genes transcribed by RNA polymerase (pol) II. Pol III-transcribed genes code for the small, stable RNA species, which participate in many cellular processes, essential for survival. Pol III transcription is repressed under most of the stress conditions by its negative regulator Maf1. We found that most of the histone acetylations increase with starvation-induced repression on several genes transcribed by the yeast pol III. On one of these genes, SNR6 (coding for the U6snRNA), a strongly positioned nucleosome in the gene upstream region plays regulatory role under repression. On this nucleosome, the changes in H3K9 and H3K14 acetylations show different dynamics. During repression, acetylation levels on H3K9 show steady increase whereas H3K14 acetylation increases with a peak at 40 min after which levels reduce. Both the levels settle by 2 hr to a level higher than the active state, which revert to normal levels with nutrient repletion. The increase in H3 acetylations is seen in the mutants reported to show reduced SNR6 transcription but not in the maf1Δ cells. This increase on a regulatory nucleosome may be part of the signaling mechanisms, which prepare cells for the stress-related quick repression as well as reactivation. The contrasting association of the histone acetylations with pol II and pol III transcription may be an important consideration to make in research studies focused on drug developments targeting histone modifications.

Structural Features of the Nucleosomal DNA Modulate the Functional Binding of a Transcription Factor and Productive Transcription.

A small non-histone protein of budding yeast, Nhp6 has been reported to specifically influence the transcription of a yeast gene, SNR6. The gene is essential, transcribed by the enzyme RNA polymerase III, and codes for the U6snRNA required for mRNA splicing. A translationally positioned nucleosome on the gene body enables the assembly factor TFIIIC binding by juxtaposing its otherwise widely separated binding sites, boxes A and B. We found histone depletion results in the loss of U6 snRNA production. Changing the rotational phase of the boxes and the linear distance between them with deletions in 5 bp steps displayed a helical periodicity in transcription, which gradually reduced with incremental deletions up to 40 bp but increased on further deletions enclosing the pseudoA boxes. Nhp6 influences the transcription in a dose-dependent manner, which is modulated by its previously reported co-operator, an upstream stretch of seven T residues centered between the TATA box and transcription start site. Nhp6 occupancy on the gene in vivo goes up at least 2-fold under the repression conditions. Nhp6 absence, T7 disruption, or shorter A-B box distance all cause the downstream initiation of transcription. The right +1 site is selected with the correct placement of TFIIIC before the transcription initiation factor TFIIIB. Thus, the T7 sequence and Nhp6 help the assembly and placement of the transcription complex at the right position. Apart from the chromatin remodelers, the relative rotational orientation of the promoter elements in nucleosomal DNA, and Nhp6 regulate the transcription of the SNR6 gene with precision.

Regulatory networking of the three RNA polymerases helps the eukaryotic cells cope with environmental stress.

Environmental stress influences the cellular physiology in multiple ways. Transcription by all the three RNA polymerases (Pols I, II, or III) in eukaryotes is a highly regulated process. With latest advances in technology, which have made many extensive genome-wide studies possible, it is increasingly recognized that all the cellular processes may be interconnected. A comprehensive view of the current research observations brings forward an interesting possibility that Pol II-associated factors may be directly involved in the regulation of expression from the Pol III-transcribed genes and vice versa, thus enabling a cross-talk between the two polymerases. An equally important cross-talk between the Pol I and Pol II/III has also been documented. Collectively, these observations lead to a change in the current perception that looks at the transcription of a set of genes transcribed by the three Pols in isolation. Emergence of an inclusive perspective underscores that all stress signals may converge on common mechanisms of transcription regulation, requiring an extensive cross-talk between the regulatory partners. Of the three RNA polymerases, Pol III turns out as the hub of these cross-talks, an essential component of the cellular stress-response under which the majority of the cellular transcriptional activity is shut down or re-aligned.

Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes.

The FACT (FAcilitates Chromatin Transactions) complex influences transcription initiation and enables passage of RNA polymerase (pol) II through gene body nucleosomes during elongation. In the budding yeast, ~280 non-coding RNA genes highly transcribed in vivo by pol III are found in the nucleosome-free regions bordered by positioned nucleosomes. The downstream nucleosome dynamics was found to regulate transcription via controlling the gene terminator accessibility and hence, terminator-dependent pol III recycling. As opposed to the enrichment at the 5'-ends of pol II-transcribed genes, our genome-wide mapping found transcription-dependent enrichment of the FACT subunit Spt16 near the 3'-end of all pol III-transcribed genes. Spt16 physically associates with the pol III transcription complex and shows gene-specific occupancy levels on the individual genes. On the non-tRNA pol III-transcribed genes, Spt16 facilitates transcription by reducing the nucleosome occupany on the gene body. On the tRNA genes, it maintains the position of the nucleosome at the 3' gene-end and affects transcription in gene-specific manner. Under nutritional stress, Spt16 enrichment is abolished in the gene downstream region of all pol III-transcribed genes and reciprocally changed on the induced or repressed pol II-transcribed ESR genes. Under the heat and replicative stress, its occupancy on the pol III-transcribed genes increases significantly. Our results show that Spt16 elicits a differential, gene-specific and stress-responsive dynamics, which provides a novel stress-sensor mechanism of regulating transcription against external stress. By primarily influencing the nucleosomal organization, FACT links the downstream nucleosome dynamics to transcription and environmental stress on the pol III-transcribed genes.

Yeast PAF1 complex counters the pol III accumulation and replication stress on the tRNA genes.

The RNA polymerase (pol) III transcribes mostly short, house-keeping genes, which produce stable, non-coding RNAs. The tRNAs genes, highly transcribed by pol III in vivo are known replication fork barriers. One of the transcription factors, the PAF1C (RNA polymerase II associated factor 1 complex) is reported to associate with pol I and pol II and influence their transcription. We found low level PAF1C occupancy on the yeast pol III-transcribed genes, which is not correlated with nucleosome positions, pol III occupancy and transcription. PAF1C interacts with the pol III transcription complex and causes pol III loss from the genes under replication stress. Genotoxin exposure causes pol III but not Paf1 loss from the genes. In comparison, Paf1 deletion leads to increased occupancy of pol III, γ-H2A and DNA pol2 in gene-specific manner. Paf1 restricts the accumulation of pol III by influencing the pol III pause on the genes, which reduces the pol III barrier to the replication fork progression.

Interactome of the yeast RNA polymerase III transcription machinery constitutes several chromatin modifiers and regulators of the genes transcribed by RNA polymerase II.

Eukaryotic transcription is a highly regulated fundamental life process. A large number of regulatory proteins and complexes, many of them with sequence-specific DNA-binding activity are known to influence transcription by RNA polymerase (pol) II with a fine precision. In comparison, only a few regulatory proteins are known for pol III, which transcribes genes encoding small, stable, non-translated RNAs. The pol III transcription is precisely regulated under various stress conditions. We used pol III transcription complex (TC) components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits and mass spectrometry to identify their potential interactors in vivo. A large interactome constituting chromatin modifiers, regulators and factors of transcription by pol I and pol II supports the possibility of a crosstalk between the three transcription machineries. The association of proteins and complexes involved in various basic life processes like ribogenesis, RNA processing, protein folding and degradation, DNA damage response, replication and transcription underscores the possibility of the pol III TC serving as a signaling hub for communication between the transcription and other cellular physiological activities under normal growth conditions. We also found an equally large number of proteins and complexes interacting with the TC under nutrient starvation condition, of which at least 25% were non-identical under the two conditions. The data reveal the possibility of a large number of signaling cues for pol III transcription against adverse conditions, necessary for an efficient co-ordination of various cellular functions.

Regulation of tRNA gene transcription by the chromatin structure and nucleosome dynamics.

The short, non-coding genes transcribed by the RNA polymerase (pol) III, necessary for survival of a cell, need to be repressed under the stress conditions in vivo. The pol III-transcribed genes have adopted several novel chromatin-based regulatory mechanisms to their advantage. In the budding yeast, the sub-nucleosomal size tRNA genes are found in the nucleosome-free regions, flanked by positioned nucleosomes at both the ends. With their chromosomes-wide distribution, all tRNA genes have a different chromatin context. A single nucleosome dynamics controls the accessibility of the genes for transcription. This dynamics operates under the influence of several chromatin modifiers in a gene-specific manner, giving the scope for differential regulation of even the isogenes within a tRNA gene family. The chromatin structure around the pol III-transcribed genes provides a context conducive for steady-state transcription as well as gene-specific transcriptional regulation upon signaling from the environmental cues. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.

Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III-transcribed genes in budding yeast.

The association of RNA polymerase III (Pol III)-transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III-transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements-centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III-transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III-transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III-dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III-transcribed genes required active transcription. We conclude that the association of Pol III-transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization.

Yeast Bud27 modulates the biogenesis of Rpc128 and Rpc160 subunits and the assembly of RNA polymerase III.

Yeast Bud27, an unconventional prefoldin is reported to affect the expression of nutrient-responsive genes, translation initiation and assembly of the multi-subunit eukaryotic RNA polymerases (pols), at a late step. We found that Bud27 associates with pol III in active as well as repressed states. Pol III transcription and occupancy at the target genes reduce with the deletion of BUD27. It promotes the interaction of pol III with the chromatin remodeler RSC found on most of the pol III targets, and with the heat shock protein Ssa4, which helps in nuclear import of the assembled pol III. Under nutrient-starvation, Ssa4-pol III interaction increases, while pol III remains inside the nucleus. Bud27 but not Ssa4 is required for RSC-pol III interaction, which reduces under nutrient-starvation. In the bud27Δ cells, total protein level of the largest pol III subunit Rpc160 but not of Rpc128, Rpc34 and Rpc53 subunits is reduced. This is accompanied by lower transcription of RPC128 gene and lower RPC160 translation due to reduced association of mRNA with the ribosomes. The resultant alteration in the normal cellular ratio of the two largest subunits of pol III core leads to reduced association of other pol III subunits and hampers the normal assembly of pol III at an early step in the cytoplasm. Our results show that Bud27 is required in multiple activities responsible for pol III biogenesis and activity.

Genome-wide mapping of yeast histone chaperone anti-silencing function 1 reveals its role in condensin binding with chromatin.

Genome-wide participation and importance of the histone chaperone Asf1 (Anti-Silencing Function 1) in diverse DNA transactions like replication, repair, heterochromatic silencing and transcription are well documented. Yet its genome-wide targets have not been reported. Using ChIP-seq method, we found that yeast Asf1 associates with 590 unique targets including centromeres, telomeres and condensin-binding sites. It is found selectively on highly transcribed regions, which include replication fork pause sites. Asf1 preferentially associates with the genes transcribed by RNA polymerase (pol) III where its presence affects RNA production and replication-independent histone exchange. On pol II-transcribed genes, a negative correlation is found between Asf1 and nucleosome occupancy. It is not enriched on most of the reported sites of histone exchange or on the genes, which are misregulated in the asf1Δ cells. Interestingly, chromosome-wide distributions of Asf1 and one of the condensin subunits, Brn1 show a nearly identical pattern. Moreover, Brn1 shows reduced occupancy at various condensin-binding sites in asf1Δ cells. These results along with high association of Asf1 with heterochromatic centromeres and telomeres ascribe novel roles to Asf1 in condensin loading and chromatin dynamics.

Systematic characterization of the conformation and dynamics of budding yeast chromosome XII.

Chromosomes architecture is viewed as a key component of gene regulation, but principles of chromosomal folding remain elusive. Here we used high-throughput live cell microscopy to characterize the conformation and dynamics of the longest chromosome of Saccharomyces cerevisiae (XII). Chromosome XII carries the ribosomal DNA (rDNA) that defines the nucleolus, a major hallmark of nuclear organization. We determined intranuclear positions of 15 loci distributed every ~100 kb along the chromosome, and investigated their motion over broad time scales (0.2-400 s). Loci positions and motions, except for the rDNA, were consistent with a computational model of chromosomes based on tethered polymers and with the Rouse model from polymer physics, respectively. Furthermore, rapamycin-dependent transcriptional reprogramming of the genome only marginally affected the chromosome XII internal large-scale organization. Our comprehensive investigation of chromosome XII is thus in agreement with recent studies and models in which long-range architecture is largely determined by the physical principles of tethered polymers and volume exclusion.

A unique nucleosome arrangement, maintained actively by chromatin remodelers facilitates transcription of yeast tRNA genes.

BACKGROUND: RNA polymerase (pol) III transcribes a unique class of genes with intra-genic promoters and high transcriptional activity. The major contributors to the pol III transcriptome, tRNAs genes are found scattered on all chromosomes of yeast. A prototype tDNA of <150 bp length, is generally considered nucleosome-free while some pol III-transcribed genes have been shown to have nucleosome-positioning properties. RESULTS: Using high resolution ChIP-chip and ChIP-seq methods, we found several unique features associated with nucleosome profiles on all tRNA genes of budding yeast, not seen on nucleosome-dense counterparts in fission yeast and resting human CD4+ T cells. The nucleosome-free region (NFR) on all but three yeast tDNAs is found bordered by an upstream (US) nucleosome strongly positioned at -140 bp position and a downstream (DS) nucleosome at variable positions with respect to the gene terminator. Perturbation in this nucleosomal arrangement interferes with the tRNA production. Three different chromatin remodelers generate and maintain the NFR by targeting different gene regions. Isw1 localizes to the gene body and makes it nucleosome-depleted, Isw2 maintains periodicity in the upstream nucleosomal array, while RSC targets the downstream nucleosome. Direct communication of pol III with RSC serves as a stress-sensory mechanism for these genes. In its absence, the downstream nucleosome moves towards the gene terminator. Levels of tRNAs from different families are found to vary considerably as different pol III levels are seen even on isogenes within a family. Pol III levels show negative correlation with the nucleosome occupancies on different genes. CONCLUSIONS: Budding yeast tRNA genes maintain an open chromatin structure, which is not due to sequence-directed nucleosome positioning or high transcription activity of genes. Unlike 5' NFR on pol II-transcribed genes, the tDNA NFR, which facilitates tDNA transcription, results from action of chromatin remodeler Isw1, aided by Isw2 and RSC. The RSC-regulated nucleosome dynamics at the 3' gene-end serves as a novel regulatory mechanism for pol III transcription in vivo, probably by controlling terminator-dependent facilitated recycling of pol III. Salient features of yeast tDNA chromatin structure reported in this study can explain the basis of the novel non-transcriptional roles ascribed to tDNAs.

Epigenetic regulation of transcription by RNA polymerase III.

Chromatin participates actively in all DNA transactions and all phenomena directly under the influence of chromatin are explained by epigenetic mechanisms. The genes transcribed by RNA polymerase (pol) III are generally found in regions free of nucleosomes, the structural units of chromatin. Yet, histone modifications and positions of nucleosomes in the gene flanking regions have been reported to show direct correlation with activity status of these genes. Gene-specific as well as genome-wide studies have also revealed association of several epigenetic components with pol III-transcribed genes. This review presents a summary of the research in past many years, which have gathered enough evidence to conclude that pol III-transcribed genes are important components of an epigenome.

Yeast H2A.Z, FACT complex and RSC regulate transcription of tRNA gene through differential dynamics of flanking nucleosomes.

FACT complex is involved in elongation and ensures fidelity in the initiation step of transcription by RNA polymerase (pol) II. Histone variant H2A.Z is found in nucleosomes at the 5'-end of many genes. We report here H2A.Z-chaperone activity of the yeast FACT complex on the short, nucleosome-free, non-coding, pol III-transcribed yeast tRNA genes. On a prototype gene, yeast SUP4, chromatin remodeler RSC and FACT regulate its transcription through novel mechanisms, wherein the two gene-flanking nucleosomes containing H2A.Z, play different roles. Nhp6, which ensures transcription fidelity and helps load yFACT onto the gene flanking nucleosomes, has inhibitory role. RSC maintains a nucleosome abutting the gene terminator downstream, which results in reduced transcription rate in active state while H2A.Z probably helps RSC in keeping the gene nucleosome-free and serves as stress-sensor. All these factors maintain an epigenetic state which allows the gene to return quickly from repressed to active state and tones down the expression from the active SUP4 gene, required probably to maintain the balance in cellular tRNA pool.

Proteome profile of whole cerebellum of the mature rat.

Cerebellum is an important brain region involved in motor, cognition, learning and memory functions. Proteome mapping of the 21 days old rat cerebellum identified total 285 proteins, out of which 76 proteins were not reported earlier from rat brain. This includes 49 neuronal activity-specific proteins, 7 of which are reported for the first time from the cerebellum in this study. The protein sequence data for 31 proteins reported here have been integrated in the UniProt Knowledgebase.

Epigenetics to proteomics: from yeast to brain.

Brain is the most complex and least understood organ of the body. Recent research suggests that epigenetics of the brain may be behind the complex functions of this master organ. Yeast, the simplest eukaryote, had been the model for studying the complex physiology of higher eukaryotes, including humans. Current depth in understanding of mechanisms of gene regulation has been possible mainly because of the knowledge acquired by epigenetic studies on yeast while the research on the biochemistry and physiology of the brain has been tremendously benefitted by proteomic studies. The independent advances of research in both these fields are now converging. As the current understanding of epigenetics can be applied to understand the mysteries of normal brain function as well as various diseases, modern proteomic approaches can help find new therapeutic targets.

Proteome profile of the mature rat olfactory bulb.

Olfactory bulbs (OBs) are one of the few brain areas, which show active neurogenesis and neuronal migration processes in adult rats. We constructed a proteome map of the 21 days old rat OBs and identified total 196 proteins, out of which 76 proteins were not reported earlier from rat brain. This includes 24 neuronal activity-specific proteins present at high levels, 7 of which are reported for the first time from OBs.

Multiple sequence-directed possibilities provide a pool of nucleosome position choices in different states of activity of a gene.

BACKGROUND: Genome-wide mappings of nucleosome occupancy in different species have shown presence of well-positioned nucleosomes. While the DNA sequences may help decide their locations, the observed positions in vivo are end-results of chromatin remodeling, the state of gene activity and binding of the sequence-specific factors to the DNA, all of which influence nucleosome positions. Thus, the observed nucleosome locations in vivo do not reflect the true contribution of DNA sequence to the mapped position. Moreover, the naturally occurring nucleosome-positioning sequences are known to guide multiple translational positionings. RESULTS: We show that yeast SNR6, a gene transcribed by RNA polymerase III, constitutes nucleosome-positioning sequence. In the absence of a chromatin remodeler or any factor binding, the gene sequence confers a unique rotational phase to nucleosomes in the gene region, and directs assembly of several translationally positioned nucleosomes on approximately 1.2 kb DNA from the gene locus, including the short approximately 250 bp gene region. Mapping of all these gene sequence-directed nucleosome positions revealed that the array of nucleosomes in the gene upstream region occupy the same positions as those observed in vivo but the nucleosomes on the gene region can be arranged in three distinct registers. Two of these arrangements differ from each other in the position of only one nucleosome, and match with the nucleosome positions on the gene in repressed and active states in vivo, where the gene-specific factor is known to occupy the gene in both the states. The two positions are interchanged by an ATP-dependent chromatin remodeler in vivo. The third register represents the positions which block the access of the factor to the gene promoter elements. CONCLUSION: On a gene locus, multiple nucleosome positions are directed by a gene sequence to provide a pool of possibilities, out of which the preferred ones are selected by the chromatin remodeler and transcription factor of the gene under different states of activity of the gene.