RNA Polymerase II-transcribed Genes Research Articles

Potential role of N6-adenine DNA methylation in alternative splicing and endosymbiosis in Paramecium bursaria

N6-adenine DNA methylation (6mA), a rediscovered epigenetic mark in eukaryotic organisms, diversifies in abundance, distribution, and function across species, necessitating its study in more taxa. Paramecium bursaria is a typical model organism with endosymbiotic algae of the species Chlorella variabilis. This consortium therefore serves as a valuable system to investigate the functional role of 6mA in endosymbiosis, as well as the evolutionary importance of 6mA among eukaryotes. In this study, we report the first genome-wide, base pair-resolution map of 6mA in P.bursaria and identify its methyltransferase PbAMT1. Functionally, 6mA exhibits a bimodal distribution at the 5' end of RNA polymerase II-transcribed genes and possibly participates in transcription by facilitating alternative splicing. Evolutionarily, 6mA co-evolves with gene age and likely serves as a reverse mark of endosymbiosis-related genes. Our results offer new insights for the functional diversification of 6mA in eukaryotes as an important epigenetic mark.

RNA Polymerase I Activators Count and Adjust Ribosomal RNA Gene Copy Number

Ribosome is the most abundant RNA-protein complex in a cell, and many copies of the ribosomal RNA gene (rDNA) have to be maintained. However, arrays of tandemly repeated rDNA genes can lose the copies by intra-repeat recombination. Loss of the rDNA copies of Saccharomyces cerevisiae is counteracted by gene amplification whereby the number of rDNA repeats stabilizes around 150 copies, suggesting the presence of a monitoring mechanism that counts and adjusts the number. Here, we report that, in response to rDNA copy loss, the upstream activating factor (UAF) for RNA polymerase I that transcribes the rDNA is released and directly binds to a RNA polymerase II-transcribed gene, SIR2, whose gene products silence rDNA recombination, to repress. We show that the amount of UAF determines the rDNA copy number that is stably maintained. UAF ensures rDNA production not only by rDNA transcription activation but also by its copy-number maintenance.

Structure and Functions of the Mediator Complex.

Mediator is a key factor in the regulation of expression of RNA polymerase II-transcribed genes. Recent studies have shown that Mediator acts as a coordinator of transcription activation and participates in maintaining chromatin architecture in the cell nucleus. In this review, we present current concepts on the structure and functions of Mediator.

Control of RNA polymerase II-transcribed genes by direct binding of TOR kinase.

Under conditions of nutrient limitation and cellular stress, or by addition of rapamycin, the mechanistic target of rapamycin complex 1 (mTORC1) is inhibited. This results in downregulation of genes that encode rRNA and ribosomal proteins. While most of the mTORC1 functions that have been previously characterized at a mechanistic level take place in the cytoplasm, nuclear roles have also been reported, including direct association of TOR kinase with rRNA genes. This review highlights the recent observation that Saccharomyces cerevisiae Tor1p also binds directly to the RNA polymerase II-transcribed gene encoding Hmo1p, a protein that is involved in communicating mTORC1 activity to downstream targets. A reduction in HMO1 mRNA levels in response to DNA damage or addition of rapamycin requires Tor1p, suggesting a role for TOR kinase in control of gene activity by direct binding to target genes. Potential targets for chromatin-bound Tor1p are discussed and the possibility that Tor1p similarly contributes to control of other genes linked to ribosome biogenesis is considered.

DNA damage regulates direct association of TOR kinase with the RNA polymerase II-transcribed HMO1 gene.

The mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient sufficiency and cellular stress. When mTORC1 is inhibited, protein synthesis is reduced in an intricate process that includes a concerted down-regulation of genes encoding rRNA and ribosomal proteins. The Saccharomyces cerevisiae high-mobility group protein Hmo1p has been implicated in coordinating this response to mTORC1 inhibition. We show here that Tor1p binds directly to the HMO1 gene (but not to genes that are not linked to ribosome biogenesis) and that the presence of Tor1p is associated with activation of gene activity. Persistent induction of DNA double-strand breaks or mTORC1 inhibition by rapamycin results in reduced levels of HMO1 mRNA, but only in the presence of Tor1p. This down-regulation is accompanied by eviction of Ifh1p and recruitment of Crf1p, followed by concerted dissociation of Hmo1p and Tor1p. These findings uncover a novel role for TOR kinase in control of gene activity by direct association with an RNA polymerase II-transcribed gene.

The Histone Acetyltransferase Mst2 Protects Active Chromatin from Epigenetic Silencing by Acetylating the Ubiquitin Ligase Brl1

SummaryFaithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report a positive feedback loop that preserves the transcription-competent state of RNA polymerase II-transcribed genes. We found that Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3-marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription and prevents ectopic heterochromatin assembly. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

Regulation of expression of human RNA polymerase II-transcribed snRNA genes.

In addition to protein-coding genes, RNA polymerase II (pol II) transcribes numerous genes for non-coding RNAs, including the small-nuclear (sn)RNA genes. snRNAs are an important class of non-coding RNAs, several of which are involved in pre-mRNA splicing. The molecular mechanisms underlying expression of human pol II-transcribed snRNA genes are less well characterized than for protein-coding genes and there are important differences in expression of these two gene types. Here, we review the DNA features and proteins required for efficient transcription of snRNA genes and co-transcriptional 3′ end formation of the transcripts.

Plant nucleolar DNA: Green light shed on the role of Nucleolin in genome organization

ABSTRACTThe nucleolus forms as a consequence of ribosome biogenesis, but it is also implicated in other cell functions. The identification of nucleolus-associated chromatin domains (NADs) in animal and plant cells revealed the presence of DNA sequences other than rRNA genes in and around the nucleolus. NADs display repressive chromatin signatures and harbour repetitive DNA, but also tRNA genes and RNA polymerase II-transcribed genes. Furthermore, the identification of NADs revealed a specific function of the nucleolus and the protein Nucleolin 1 (NUC1) in telomere biology. Here, we discuss the significance of these data with regard to nucleolar structure and to the role of the nucleolus and NUC1 in global genome organization and stability.

CDK9 inhibitors define elongation checkpoints at both ends of RNA polymerase II-transcribed genes.

Transcription through early-elongation checkpoints requires phosphorylation of negative transcription elongation factors (NTEFs) by the cyclin-dependent kinase (CDK)9. Using CDK9 inhibitors and global run-on sequencing (GRO-seq), we have mapped CDK9 inhibitor-sensitive checkpoints genome-wide in human (Homo sapiens) cells. Our data indicate that early-elongation checkpoints are a general feature of RNA polymerase (pol) II-transcribed human genes and occur independently of polymerase stalling. Pol II that has negotiated the early-elongation checkpoint can elongate in the presence of inhibitors but, remarkably, terminates transcription prematurely close to the terminal polyadenylation (poly(A)) site. Our analysis has revealed a hitherto-unsuspected poly(A)-associated elongation checkpoint, which has major implications for the regulation of gene expression. Interestingly, the pattern of modification of the carboxyl-terminal domain (CTD) of pol II terminated at this novel checkpoint largely mirrors the pattern normally found downstream of the poly(A) site, suggesting common mechanisms of termination.

Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.

Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

The Pattern and Evolution of Looped Gene Bendability

Gene looping, defined as the physical interaction between the promoter and terminator regions of a RNA polymerase II-transcribed gene, is widespread in yeast and mammalian cells. Gene looping has been shown to play important roles in transcription. Gene-loop formation is dependent on regulatory proteins localized at the 5' and 3' ends of genes, such as TFIIB. However, whether other factors contribute to gene looping remains to be elucidated. Here, we investigated the contribution of intrinsic DNA and chromatin structures to gene looping. We found that Saccharomyces cerevisiae looped genes show high DNA bendability around middle and 3/4 regions in open reading frames (ORFs). This bendability pattern is conserved between yeast species, whereas the position of bendability peak varies substantially among species. Looped genes in human cells also show high DNA bendability. Nucleosome positioning around looped ORF middle regions is unstable. We also present evidence indicating that this unstable nucleosome positioning is involved in gene looping. These results suggest a mechanism by which DNA bendability and unstable nucleosome positioning could assist in the formation of gene loops.

Genome-wide analysis of functional sirtuin chromatin targets in yeast

BackgroundThe sirtuins are a conserved family of NAD+-dependent histone/protein deacetylases that regulate numerous cellular processes, including heterochromatin formation and transcription. Multiple sirtuins are encoded by each eukaryotic genome, raising the possibility of cooperativity or functional overlap. The scope and variety of chromatin binding sites of the sirtuins in any specific organism remain unclear.ResultsHere we utilize the ChIP-seq technique to identify and functionally characterize the genome-wide targets of the sirtuins, Sir2, Hst1 to Hst4, and the DNA binding partner of Hst1, Sum 1, in Saccharomyces cerevisiae. Unexpectedly, Sir2, Hst1 and Sum1, but not the other sirtuins, exhibit co-enrichment at several classes of chromatin targets. These include telomeric repeat clusters, tRNA genes, and surprisingly, the open reading frames (ORFs) of multiple highly expressed RNA polymerase II-transcribed genes that function in processes such as fermentation, glycolysis, and translation. Repression of these target genes during the diauxic shift is specifically dependent on Sir2/Hst1/Sum1 binding to the ORF and sufficiently high intracellular NAD+ concentrations. Sir2 recruitment to the ORFs is independent of the canonical SIR complex and surprisingly requires Sum1. The shared Sir2/Hst1/Sum1 targets also significantly overlap with condensin and cohesin binding sites, where Sir2, Hst1, and Sum1 were found to be important for condensin and cohesin deposition, suggesting a possible mechanistic link between metabolism and chromatin architecture during the diauxic shift.ConclusionsThis study demonstrates the existence of overlap in sirtuin function, and advances our understanding of conserved sirtuin-regulated functions, including the regulation of glycolytic gene expression and condensin loading.

Promoter-proximal polyadenylation sites reduce transcription activity

Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ~500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels.

Changing the Diet to Make More Mitochondria and Protect the Heart

### Lethal Mitochondrial Cardiomyopathy in a Hypomorphic Med30 Mouse Mutant is Ameliorated by Ketogenic Diet Krebs et al Proc Natl Acad Sci U S A. 2011;108:19678–19682. The complexity of mitochondrial diseases has made their treatment problematic. However, in a recent study from PNAS in 2011, researchers show how diet may be the key to better understanding and possibly fighting the expression of these diseases. In their study, Krebs and colleagues report that treatment with a ketogenic diet reduces lethality in a mouse model of mitochondrial cardiomyopathy.1 The cardiomyopathy is caused by a homozygous nonsynonymous A-to-T transversion in Med30 , a gene encoding one of the ≈30 subunits of the Mediator complex A. The homozygous mutation, which was induced on treatment with N-ethyl-N-nitrosurea, impairs oxidative phosphorylation and mitochondrial integrity leading to premature lethality within 2 to 3 weeks after weaning. Activity of the Mediator complex is required to express RNA-polymerase II-transcribed genes in the presence of gene-specific activators by promoting preinitiation complex assembly.2 As a result of the Med30 mutation, the authors observed a progressive decline in the transcription of genes specifically involved in oxidative phosphorylation and mitochondrial integrity. The transcriptional profile of the left ventricle of the 5 weeks old homozygous mutant mice displayed a considerable reduction in Ppargc1α and Esrra transcripts, which code for PGC1α (peroxisome proliferator-activated receptor-γ coactivator-1α) and ERRα (estrogen-related receptor-α), respectively. PGC1α and ERRα are known transcriptional factors for oxidative metabolism enzymes, including the respiratory chain, mitochondrial antioxidant defenses, and mitochondrial biogenesis (Figure).3 Accordingly, transcripts and activities of a number of PGC1α-ERRα–dependent mitochondrial enzymes were also decreased. The homozygous mutant mice, presumably as a result of …

A novel TBP-TAF complex on RNA Polymerase II-transcribed snRNA genes

Initiation of transcription of most human genes transcribed by RNA polymerase II (RNAP II) requires the formation of a preinitiation complex comprising TFIIA, B, D, E, F, H and RNAP II. The general transcription factor TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors. During transcription of snRNA genes, RNAP II does not appear to make the transition to long-range productive elongation, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3′ box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. For example, differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. As precedent, it already has been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5 has been shown to be associated with RNAP II-transcribed snRNA genes, the full complement of TAFs associated with these genes has remained unclear. Here we show, using a ChIP and siRNA-mediated approach, that the TBP/TAF complex on snRNA genes differs from that found on protein-coding genes. Interestingly, the largest TAF, TAF1, and the core TAFs, TAF10 and TAF4, are not detected on snRNA genes. We propose that this snRNA gene-specific TAF subset plays a key role in gene type-specific control of expression.

An ENU-induced point mutation in the mouse Btaf1 gene causes post-gastrulation embryonic lethality and protein instability

The mouse Btaf1 gene, an ortholog of yeast MOT1, encodes a highly conserved general transcription factor. The function of this SNF2-like ATPase has been studied mainly in yeast and human cells, which has revealed that it binds directly to TBP, forming the B-TFIID complex. This complex binds to core promoters of RNA polymerase II-transcribed genes and, of crucial importance, BTAF1–TBP interactions have been shown to affect the kinetics of TBP–promoter interactions. Here we report the isolation of a mouse line carrying a Btaf1 allele containing an ENU-induced point mutation that causes a substitution mutation in the BTAF1 ATPase domain. Embryos homozygous for this loss-of-function mutation appear to be morphologically normal until early somite stages, but die between embryonic days 9 and 10.5 displaying growth arrest and edema. Analyses in vitro suggest that the altered protein is less stable and, independent from this, functionally impaired in releasing of TBP from chromatin, but not in binding to TBP.

Rapamycin increases rDNA stability by enhancing association of Sir2 with rDNA in Saccharomyces cerevisiae

The target of rapamycin (TOR) kinase is an evolutionarily conserved key regulator of eukaryotic cell growth and proliferation. Recently, it has been reported that inhibition of TOR signaling pathway can delay aging and extend lifespan in several eukaryotic organisms, but how lifespan extension is mediated by inhibition of TOR signaling is poorly understood. Here we report that rapamycin treatment and nitrogen starvation, both of which cause inactivation of TOR complex 1 (TORC1), lead to enhanced association of Sir2 with ribosomal DNA (rDNA) in Saccharomyces cerevisiae. TORC1 inhibition increases transcriptional silencing of RNA polymerase II-transcribed gene integrated at the rDNA locus and reduces homologous recombination between rDNA repeats that causes formation of toxic extrachromosomal rDNA circles. In addition, TORC1 inhibition induces deacetylation of histones at rDNA. We also found that Pnc1 and Net1 are required for enhancement of association of Sir2 with rDNA under TORC1 inhibition. Taken together, our findings suggest that inhibition of TORC1 signaling stabilizes the rDNA locus by enhancing association of Sir2 with rDNA, thereby leading to extension of replicative lifespan in S. cerevisiae.

ATAC and Mediator coactivators form a stable complex and regulate a set of non‐coding RNA genes

The Ada-Two-A-containing (ATAC) histone acetyltransferase and Mediator coactivator complexes regulate independent and distinct steps during transcription initiation and elongation. Here, we report the identification of a new stable molecular assembly formed between the ATAC and Mediator complexes in mouse embryonic stem cells. Moreover, we identify leucine zipper motif-containing protein 1 as a subunit of this meta-coactivator complex (MECO). Finally, we demonstrate that the MECO regulates a subset of RNA polymerase II-transcribed non-coding RNA genes. Our findings establish that transcription coactivator complexes can form stable subcomplexes to facilitate their combined actions on specific target genes.

A physiological role for gene loops in yeast

DNA loops that juxtapose the promoter and terminator regions of RNA polymerase II-transcribed genes have been identified in yeast and mammalian cells. Loop formation is transcription-dependent and requires components of the pre-mRNA 3'-end processing machinery. Here we report that looping at the yeast GAL10 gene persists following a cycle of transcriptional activation and repression. Moreover, GAL10 and a GAL1p-SEN1 reporter undergo rapid reactivation kinetics following a cycle of activation and repression-a phenomenon defined as "transcriptional memory"-and this effect correlates with the persistence of looping. We propose that gene loops facilitate transcriptional memory in yeast.

Gene Looping Is Conferred by Activator-dependent Interaction of Transcription Initiation and Termination Machineries

Gene looping juxtaposes the promoter and terminator regions of RNA polymerase II-transcribed genes in yeast and mammalian cells. Here we report an activator-dependent interaction of transcription initiation and termination factors during gene looping in budding yeast. Chromatin analysis revealed that MET16, INO1, and GAL1p-BUD3 are in a stable looped configuration during activated transcription. Looping was nearly abolished in the absence of transcription activators Met28, Ino2, and Gal4 of MET16, INO1, and GAL1p-BUD3 genes, respectively. The activator-independent increase in transcription was not accompanied by loop formation, thereby suggesting an essential role for activators in gene looping. The activators did not facilitate loop formation directly because they did not exhibit an interaction with the 3' end of the genes. Instead, activators physically interacted with the general transcription factor TFIIB when the genes were activated and in a looped configuration. TFIIB cross-linked to both the promoter and the terminator regions during the transcriptionally activated state of a gene. The presence of TFIIB on the terminator was dependent on the Rna15 component of CF1 3' end processing complex. Coimmunoprecipitation revealed a physical interaction of Rna15 with TFIIB. We propose that the activators facilitate gene looping through their interaction with TFIIB during transcriptional activation of genes.