Altered RNA Polymerase Research Articles

Base-excision repair pathway regulates transcription-replication conflicts in pancreatic ductal adenocarcinoma

Oncogenic mutations (such as in KRAS) can dysregulate transcription and replication, leading to transcription-replication conflicts (TRCs). Here, we demonstrate that TRCs are enriched in human pancreatic ductal adenocarcinoma (PDAC) compared to other common solid tumors or normal cells. Several orthogonal approaches demonstrated that TRCs are oncogene dependent. A small interfering RNA (siRNA) screen identified several factors in the base-excision repair (BER) pathway as main regulators of TRCs in PDAC cells. Inhibitors of BER pathway (methoxyamine and CRT) enhanced TRCs. Mechanistically, BER pathway inhibition severely altered RNA polymerase II (RNAPII) and R-loop dynamics at nascent DNA, causing RNAPII trapping and contributing to enhanced TRCs. The ensuing DNA damage activated the ATR-Chk1 pathway. Co-treatment with ATR inhibitor (VX970) and BER inhibitor (methoxyamine) at clinically relevant doses synergistically enhanced DNA damage and reduced cell proliferation in PDAC cells. The study provides mechanistic insights into the regulation of TRCs in PDAC by the BER pathway, which has biologic and therapeutic implications.

Dynamic control of gene expression by ISGF3 and IRF1 during IFNβ and IFNγ signaling

Type I interferons (IFN-I, including IFNβ) and IFNγ produce overlapping, yet clearly distinct immunological activities. Recent data show that the distinctness of global transcriptional responses to the two IFN types is not apparent when comparing their immediate effects. By analyzing nascent transcripts induced by IFN-I or IFNγ over a period of 48 h, we now show that the distinctiveness of the transcriptomes emerges over time and is based on differential employment of the ISGF3 complex as well as of the second-tier transcription factor IRF1. The distinct transcriptional properties of ISGF3 and IRF1 correspond with a largely diverse nuclear protein interactome. Mechanistically, we describe the specific input of ISGF3 and IRF1 into enhancer activation and the regulation of chromatin accessibility at interferon-stimulated genes (ISG). We further report differences between the IFN types in altering RNA polymerase II pausing at ISG 5’ ends. Our data provide insight how transcriptional regulators create immunological identities of IFN-I and IFNγ.

Abstract C065: Base excision repair pathway regulates transcription-replication conflicts in pancreatic ductal adenocarcinoma

Abstract Oncogenic mutations (such as in KRAS) can dysregulate transcription and replication leading to transcription conflicts (TRCs). Unresolved TRCs can cause lethal DNA damage. Here, we sought to investigate the causal role of oncogenic mutations in regulating TRCs in pancreatic ductal adenocarcinoma (PDAC). Further, we characterize the adaptive pathways that mitigate TRCs for PDAC survival. Human PDAC demonstrated 30-120-fold higher levels of TRC genomic signature compared to breast, colon and lung cancer (p<0.001). Gene expression analysis demonstrated high levels of known TRC resolution factors in human tumors compared to normal tissues. TRCs as measured by RNAPII-PCNA proximity ligation assay were significantly enriched in human PDAC cells (Panc-1, BxPC3, MiaPaca2) compared to immortalized normal HPNE cells (p<0.001). Similarly transcriptomic complexes were enriched in nascent DNA immunoprecipitates from Panc1 cells but not HPNE cells. Ectopic expression of oncogenic KRAS(G12D) in human pancreatic ductal epithelial (HPNE) cells enhanced TRCs, and TRC-related DNA:RNA hybrids (R-loops). Inhibition of KRAS or downstream effectors abrogated TRCs in Panc1 and MiaPaca2 cells. An siRNA screen identified several factors in the base-excision repair (BER) pathway as regulators of TRCs. Sub-lethal exposure to hydrogen peroxide enhanced TRCs, whereas N-acetyl cysteine abrogated TRCs. These findings were validated by a pharmacologic approach using inhibitors of APE1 endonuclease in BER pathway (Methoxyamine and CRT). Mechanistic studies revealed that BER pathway inhibition severely altered RNA polymerase II dynamics at nascent DNA; causing RNAPII trapping and contributing to enhanced TRCs. The ensuing DNA damage activated Chk2-ATR pathway but not Chk1-ATM pathway. Co-treatment with ATR inhibitor (VX970, 10-80nM) and BER inhibitor (methoxyamine, 6-12mM) at clinically relevant doses, synergistically enhanced DNA damage and reduced clonogenic survival in PDAC cells. The study uncovers a novel role of BER pathway defects and oxidative DNA damage in promoting TRCs. Our studies provide mechanistic insights into the regulation of TRCs in PDAC which has implications for genome instability and therapy in PDAC. Citation Format: Fan Meng, Anup K. Singh, Tiane Li, Marc Attiyeh, Fatemeh Kohram, Terence Williams, Yilun Liu, Mustafa Raoof. Base excision repair pathway regulates transcription-replication conflicts in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C065.

Transcription Defects in SF3B1K700E Induce Targetable Alterations in the Chromatin Landscape

Introduction: Acquired mutations, most commonly in RNA splicing factors and epigenetic regulators are thought to be disease drivers of clonal myeloid disorders such as MDS and AML. Both MDS and AML are amenable to epigenetic therapies regardless of underlying mutational subtypes. This prompted our hypothesis that alterations to epigenetic and chromatin landscape is important in pathobiology of clonal myeloid disorders driven by splicing factor mutations. In this study, we demonstrate that SF3B1 mutations induce distinct changes to epigenetic landscape and chromatin organization by altering RNA Polymerase II (Pol2) transcription kinetics. Critically, such epigenetic changes are also targetable - inhibiting components in the H3K4me/Sin3/HDAC pathway reverses Pol2 transcription defects and downstream effects on chromatin organization and genomic integrity. Methods: We generated inducible isogenic K562 cell lines that express single mutant allele of SF3B1 K700Ecombining two genome editing approaches (AAV-intron trap with CRISPR/Cas9 (Boddu et al, Comms Biol, 2021)) (Fig1A). RNAPII transcription kinetics changes were assessed using ChIP-seq for Pol2 and nascent-transcriptome assays (including GRO-seq and transient transcriptome-time lapse sequencing (TT-TL-seq). Chromatin accessibility/nucleosome density and histone marks (H3K4me3, H3K27ac, H3K27me3) were assessed using ATAC-seq and low-input CUT&RUN, respectively. Key results were replicated in patient samples with SF3B1 mutations and in a Sf3b1K700E mouse model. Results: Combining genome-wide approaches (ChIP-seq (Fig1B), GRO-seq, and TT-TL seq (Fig1C)), we found that Pol2 in SF3B1 K700E redistributes into the gene body due to a primary gene-body elongation defect. This transcriptional dysregulation was reflected in downstream effects such as excess R-loops (S9.6 microscopy; Fig1D), transcription-replication conflicts (proximity ligation assay for PCNA-Pol2; Fig1E), and S-phase arrest (flow cytometry; Fig1F). Importantly, normalized Pol2 density at proximal-promoter (p-p) region was reduced in SF3B1 K700E (Fig1G). This correlated with a reduction in p-p H3K4me3 in SF3B1 K700E determined by Cut and Run (Fig1H). ATAC-seq showed a reduction in promoter accessibility (Fig1I) and increased nucleosome promoter density (Fig1J). ChIP-seq for CDK9, a component of p-TEFb pause release complex, showed higher occupancy at promoters in SF3B1 K700E (Fig1K), suggesting that premature promoter pause release of Pol2 is the major driver for the observed reduction in p-p Pol2 density. Consistent with the importance of p-p Pol2 in maintaining accessible chromatin states, our findings suggest that depletion of p-p Pol2 leads to nucleosome repositioning at promoters and loss of chromatin accessibility (Gilchrist et al, Cell, 2010). Given the extensive cross talk between chromatin and transcription, we speculated that modulating epigenetic regulators might improve Pol2 elongation defects and rescue cells from downstream effects. An unbiased shRNA rescue screen, targeting epigenetic regulators and chromatin modifier protein classes, enriched for proteins in the Sin3/HDAC pathway (ING2, HDAC2) (Fig1L). Conversely, knocking down (KD) for WDR5 (H3K4me3 writer complex) further worsened survival. HDAC2 and ING2 KD cells showed reduced R-loops and DNA damage, lower gene-body Pol2 ChIP densities (Fig1M), normalization of p-p H3K4me3 densities (Fig1N), and rescue from S-phase arrest. These effects of WDR5 and HDAC2/ING2 KD on survival in SF3B1-mutant isogenic cells was confirmed in both MDS patient samples (Fig1O)- and Sf3b1K700E murine models (Fig1P)- contexts, using in vitro colony assays. Conclusion: Our findings show that SF3B1 K700E-induced disruption of Pol2 elongation kinetics is reflected in reduced Pol2 density at proximal promoter sites and results in a corresponding reduction in chromatin accessibility and promoter H3K4me3. Through an unbiased shRNA screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, improved the transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes affect chromatin organization through their effects on Pol II transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy.

Genomic footprinting uncovers global transcription factor responses to amino acids in Escherichia coli.

Our knowledge of transcriptional responses to changes in nutrient availability comes primarily from few well-studied transcription factors (TFs), often lacking an unbiased genome-wide perspective. Leveraging recent advances allowing bacterial genomic footprinting, we comprehensively mapped the genome-wide regulatory responses of Escherichia coli to exogenous leucine, methionine, alanine, and lysine. The global TF Lrp was found to individually sense three amino acids and mount three different target gene responses. Overall, 531 genes had altered RNA polymerase occupancy, and 32 TFs responded directly or indirectly to the presence of amino acids, including regulators of membrane and osmotic pressure homeostasis. About 70% of the detected TF-DNA interactions had not been reported before. We thus identified 682 previously unknown TF-binding locations, for a subset of which the involved TFs were identified by affinity purification. This comprehensive map of amino acid regulation illustrates the incompleteness of the known transcriptional regulation network, even in E.coli.

The CDK7 Inhibitor THZ1 Alters RNA Polymerase Dynamics at the 5‘ and 3‘ Ends of Genes

The t(8;21) is one of the most frequent chromosomal translocations in acute myeloid leukemia (AML). This translocation encodes a fusion protein containing the DNA binding domain of RUNX1, a master regulator of hematopoiesis, linked to the majority of the myeloid translocation gene on chromosome 8 (MTG8, also known as eight-twenty-one or ETO; gene name RUNX1T1). This chimeric fusion protein binds to the RUNX1 DNA elements to control gene expression by recruiting transcriptional cofactors via the MTG8/ETO moiety. In addition, MTG/ETO family proteins co-purified in a complex that also contained “E proteins” and the cyclin dependent kinase CDK9, a component of the positive transcription elongation factor b (P-TEFb).P-TEFb contains Cyclin T1 and CDK9 and several potential CDK9 inhibitors have shown promise in the clinics for t(8;21) AML, such as flavopiridol (alvocidib). Transcription initiation, promoter-proximal pausing of RNA polymerase II (RNAPII), and transcription elongation are events that are highly regulated for genes involved in proliferation and differentiation such as MYC and BCL2 . CDK7 phosphorylates Ser5 of the C-terminal domain of RNAPII to trigger promoter-proximal pausing of RNA polymerase II, whereas CDK9 phosphorylates Ser2 of RNAPII and the negative elongation factors DSIF and NELF to release the polymerase into the body of the gene.In this study, we used inhibitors of CDK7 and CDK9 to probe for transcriptional vulnerabilities in t(8;21) AML. We used both broad spectrum (flavopiridol, dinacicilib) and a specific CDK9 inhibitor PHA767491 along with THZ1 (CDK7 inhibitor). Since CDK7 is also a CAK (CDK activating kinase) that activates cell-cycle CDKs and mutations in Cyclin D2 are common in t(8;21) AML, we also tested the highly selective CDK4/6 inhibitor palbociclib (PD-0332991). We observed that the t(8;21)-containing AML cell lines SKNO-1 and Kasumi-1, as well as primary AML patient cells, were extremely sensitive to all the CDK7/9 inhibitors, but not palbociclib. We used precision global run-on transcription sequencing (PROseq) to define the mechanism of action of these compounds. This technique creates high resolution genomic maps of the active RNA polymerases to define the effects on RNA polymerase dynamics. CDK9 inhibitors caused wide spread RNA Polymerase II promoter proximal pausing at nearly 80% of the expressed genes, as well as at enhancers where there was a general impairment of production of eRNAs and lncRNAs. Conversely, inhibition of CDK7, a critical component of TFIIH caused a loss of paused RNA polymerase II at promoters, which was associated with an increase of polymerases in the body of genes. While RUNX1 was reported to be regulated by CDK7 in T-ALL, there was no impact on the expression of RUNX1 or the fusion protein in t(8;21)-containing cells. Although enhancers were not affected, super enhancers were modestly affected by THZ1. Interestingly, within a subset of genes that showed increased RNA polymerase accumulation within the gene body, CDK7 inhibition altered 3' end transcription termination dynamics within 1 hr and this effect remained even at 2 hr. A time course of THZ1 treatment at 0, 15', 30', and 60' suggested that CDK7 inhibition altered RNA polymerase dynamics at 5'ends of the genes, and it's these effects that eventually affected transcription termination at the 3' ends of the genes. DisclosuresSavona:Amgen: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Takeda: Research Funding; Karyopharm: Consultancy, Equity Ownership; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Consultancy, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in MYC-driven medulloblastoma.

MYC-driven medulloblastoma is a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. MYC gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in MYC-amplified medulloblastoma, we employ a CRISPR-Cas9 essentiality screen targeting 1,140 genes. We identify CDK7 as a mediator of medulloblastoma tumorigenesis. Using chemical inhibitors and genetic depletion, we observe cessation of tumor growth in xenograft mouse models and increases in apoptosis. The results are attributed to repression of a core set of MYC-driven transcriptional programs mediating DNA repair. CDK7 inhibition alters RNA polymerase II (RNA Pol II) and MYC association at DNA repair genes. Blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage, extending survival and tumor latency in xenograft mouse models. Our studies establish the selective inhibition of MYC-driven medulloblastoma by CDK7 inhibition combined with radiation as a viable therapeutic strategy.

AlgU, a Conserved Sigma Factor Regulating Abiotic Stress Tolerance and Promoting Virulence in Pseudomonas syringae.

Pseudomonas syringae can rapidly deploy specialized functions to deal with abiotic and biotic stresses. Host niches pose specific sets of environmental challenges driven, in part, by immune defenses. Bacteria use a "just-in-time" strategy of gene regulation, meaning that they only produce the functions necessary for survival as needed. Extracytoplasmic function (ECF) sigma factors transduce a specific set of environmental signals and change gene expression patterns by altering RNA polymerase promoter specificity, to adjust bacterial physiology, structure, or behavior, singly or in combination, to improve chances of survival. The broadly conserved ECF sigma factor AlgU affects virulence in both animal and plant pathogens. Pseudomonas syringae AlgU controls expression of more than 800 genes, some of which contribute to suppression of plant immunity and bacterial fitness in plants. This review discusses AlgU activation mechanisms, functions controlled by AlgU, and how these functions contribute to P. syringae survival in plants.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.

E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation.

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

Experimental Evolution of Extreme Resistance to Ionizing Radiation in Escherichia coli after 50 Cycles of Selection.

In previous work (D. R. Harris et al., J Bacteriol 191:5240-5252, 2009, https://doi.org/10.1128/JB.00502-09; B. T. Byrne et al., Elife 3:e01322, 2014, https://doi.org/10.7554/eLife.01322), we demonstrated that Escherichia coli could acquire substantial levels of resistance to ionizing radiation (IR) via directed evolution. Major phenotypic contributions involved adaptation of organic systems for DNA repair. We have now undertaken an extended effort to generate E. coli populations that are as resistant to IR as Deinococcus radiodurans After an initial 50 cycles of selection using high-energy electron beam IR, four replicate populations exhibit major increases in IR resistance but have not yet reached IR resistance equivalent to D. radiodurans Regular deep sequencing reveals complex evolutionary patterns with abundant clonal interference. Prominent IR resistance mechanisms involve novel adaptations to DNA repair systems and alterations in RNA polymerase. Adaptation is highly specialized to resist IR exposure, since isolates from the evolved populations exhibit highly variable patterns of resistance to other forms of DNA damage. Sequenced isolates from the populations possess between 184 and 280 mutations. IR resistance in one isolate, IR9-50-1, is derived largely from four novel mutations affecting DNA and RNA metabolism: RecD A90E, RecN K429Q, and RpoB S72N/RpoC K1172I. Additional mechanisms of IR resistance are evident.IMPORTANCE Some bacterial species exhibit astonishing resistance to ionizing radiation, with Deinococcus radiodurans being the archetype. As natural IR sources rarely exceed mGy levels, the capacity of Deinococcus to survive 5,000 Gy has been attributed to desiccation resistance. To understand the molecular basis of true extreme IR resistance, we are using experimental evolution to generate strains of Escherichia coli with IR resistance levels comparable to Deinococcus Experimental evolution has previously generated moderate radioresistance for multiple bacterial species. However, these efforts could not take advantage of modern genomic sequencing technologies. In this report, we examine four replicate bacterial populations after 50 selection cycles. Genomic sequencing allows us to follow the genesis of mutations in populations throughout selection. Novel mutations affecting genes encoding DNA repair proteins and RNA polymerase enhance radioresistance. However, more contributors are apparent.

The CDK7 inhibitor THZ1 alters RNA polymerase dynamics at the 5' and 3' ends of genes.

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). We found that t(8;21) AML were extremely sensitive to THZ1, which triggered apoptosis after only 4 h. We used precision nuclear run-on transcription sequencing (PROseq) to define the global effects of THZ1 and other CDK inhibitors on RNA polymerase II dynamics. Inhibition of CDK7 using THZ1 caused wide-spread loss of promoter-proximal paused RNA polymerase. This loss of 5′ pausing was associated with accumulation of polymerases in the body of a large number of genes. However, there were modest effects on genes regulated by ‘super-enhancers’. At the 3′ ends of genes, treatment with THZ1 suppressed RNA polymerase ‘read through’ at the end of the last exon, which resembled a phenotype associated with a mutant RNA polymerase with slower elongation rates. Consistent with this hypothesis, polyA site-sequencing (PolyA-seq) did not detect differences in poly A sites after THZ1 treatment. PROseq analysis after short treatments with THZ1 suggested that these 3′ effects were due to altered CDK7 activity at the 5′ end of long genes, and were likely to be due to slower rates of elongation.

Changes in long-range rDNA-genomic interactions associate with altered RNA polymerase II gene programs during malignant transformation

The three-dimensional organization of the genome contributes to its maintenance and regulation. While chromosomal regions associate with nucleolar ribosomal RNA genes (rDNA), the biological significance of rDNA-genome interactions and whether they are dynamically regulated during disease remain unclear. rDNA chromatin exists in multiple inactive and active states and their transition is regulated by the RNA polymerase I transcription factor UBTF. Here, using a MYC-driven lymphoma model, we demonstrate that during malignant progression the rDNA chromatin converts to the open state, which is required for tumor cell survival. Moreover, this rDNA transition co-occurs with a reorganization of rDNA-genome contacts which correlate with gene expression changes at associated loci, impacting gene ontologies including B-cell differentiation, cell growth and metabolism. We propose that UBTF-mediated conversion to open rDNA chromatin during malignant transformation contributes to the regulation of specific gene pathways that regulate growth and differentiation through reformed long-range physical interactions with the rDNA.

Contrasting roles of the RSC and ISW1/CHD1 chromatin remodelers in RNA polymerase II elongation and termination.

Most yeast genes have a nucleosome-depleted region (NDR) at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. We have examined the interplay between RSC (a conserved essential SWI/SNF-type complex that determines NDR size) and the ISW1, CHD1, and ISW2 nucleosome spacing enzymes in chromatin organization and transcription, using isogenic strains lacking all combinations of these enzymes. The contributions of these remodelers to chromatin organization are largely combinatorial, distinct, and nonredundant, supporting a model in which the +1 nucleosome is positioned by RSC and then used as a reference nucleosome by the spacing enzymes. Defective chromatin organization correlates with altered RNA polymerase II (Pol II) distribution. RSC-depleted cells exhibit low levels of elongating Pol II and high levels of terminating Pol II, consistent with defects in both termination and initiation, suggesting that RSC facilitates both. Cells lacking both ISW1 and CHD1 show the opposite Pol II distribution, suggesting elongation and termination defects. These cells have extremely disrupted chromatin, with high levels of closely packed dinucleosomes involving the second (+2) nucleosome. We propose that ISW1 and CHD1 facilitate Pol II elongation by separating closely packed nucleosomes.

Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation

Cells and organisms respond to nutrient deprivation by decreasing global rates of transcription, translation and DNA replication. To what extent such changes can be reversed is largely unknown. We examined the effect of maternal dietary restriction on RNA synthesis in the offspring. Low protein diet fed either throughout gestation or for the preimplantation period alone reduced cellular RNA content across fetal somatic tissues during challenge and increased it beyond controls in fetal and adult tissues after challenge release. Changes in transcription of ribosomal RNA, the major component of cellular RNA, were responsible for this phenotype as evidenced by matching alterations in RNA polymerase I density and DNA methylation at ribosomal DNA loci. Cellular levels of the ribosomal transcription factor Rrn3 mirrored the rRNA expression pattern. In cell culture experiments, Rrn3 overexpression reduced rDNA methylation and increased rRNA expression; the converse occurred after inhibition of Rrn3 activity. These observations define novel mechanism where poor nutrition before implantation irreversibly alters basal rates of rRNA transcription thereafter in a process mediated by rDNA methylation and Rrn3 factor.

Histone H3K36 Methylation is Required for Efficient Pre‐mRNA Splicing in Saccharomyces cerevisiae

Proper gene expression involves multiple steps, including RNA splicing where non‐protein coding regions of RNA are removed from the RNA transcript. RNA splicing is carried out by the large and dynamic spliceosome, which is comprised of small nucleoprotein complexes (snRNPs) that assemble on an RNA molecule in a precise order. Assembly of the spliceosome occurs co‐transcriptionally and RNA splicing is tightly coordinated with transcription to ensure precise and efficient gene expression. However, the mechanisms that underlie this coordination are poorly understood. In order to identify proteins that function to coordinate RNA splicing with transcription we carried out a genetic interaction study using Saccharomyces cerevisiae. We identified negative genetic interactions between genes encoding RNA splicing factors and the SET2 gene, a histone methyltransferase that methylates lysine36 on histone H3 (H3K36) to regulate transcription. Furthermore, we show that mutations that block H3K36 methylation also have negative genetic interactions with splicing factor genes, suggesting that H3K36 methylation is important for RNA splicing. Indeed, we have shown that deletion of SET2 or point mutation of H3K36 inhibits RNA splicing and exacerbates splicing defects in yeast strains harboring deletions of splicing factor genes. Using chromatin immunoprecipitation, we demonstrate that deletion of SET2 reduces the association of snRNPs with chromatin. Furthermore, we tested for reciprocal/reverse coordination between RNA splicing and transcription. We found that mutation of Prp28, a helicase protein that normally promotes RNA splicing, alters RNA polymerase II association with DNA. We are currently testing whether perturbation of RNA splicing alters H3K36 methylation. Thus, we provide evidence that H3K36 methylation is required for appropriate RNA splicing in yeast and suggest a model in which Set2 or H3K36 methylation help to recruit splicing factors to RNA during transcription.Support or Funding InformationCottrell College Science Award from the Research Corporation for Science Advancement (#20186) to T.L.KAmerican Cancer Society (RSG‐05‐137‐01‐GMC) to S.W.S National Institutes of Health (GM084246 to S.W.S and GM110058 to B.D.S) Cancer Prevention & Research Institute of Texas (RP101501) to M.R.SGateway to Graduate School funded by The National Science Foundation (NSF‐URM) to D.M.F

Viral Nucleases Induce an mRNA Degradation-Transcription Feedback Loop in Mammalian Cells.

Gamma-herpesviruses encode a cytoplasmic mRNA-targeting endonuclease, SOX, that cleaves most cellular mRNAs. Cleaved fragments are subsequently degraded by the cellular 5′-3′ mRNA exonuclease Xrn1, thereby suppressing cellular gene expression and facilitating viral evasion of host defenses. We reveal that mammalian cells respond to this widespread cytoplasmic mRNA decay by altering RNA Polymerase II (RNAPII) transcription in the nucleus. Measuring RNAPII recruitment to promoters and nascent mRNA synthesis revealed that the majority of affected genes are transcriptionally repressed in SOX-expressing cells. The transcriptional feedback does not occur in response to the initial viral endonuclease-induced cleavage, but instead to degradation of the cleaved fragments by cellular exonucleases. In particular, Xrn1 catalytic activity is required for transcriptional repression. Notably, viral mRNA transcription escapes decay-induced repression, and this escape requires Xrn1. Collectively, these results indicate that mRNA decay rates impact transcription and that gamma-herpesviruses use this feedback mechanism to facilitate viral gene expression.

BRF1 mutations alter RNA polymerase III-dependent transcription and cause neurodevelopmental anomalies.

RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III–related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development.

Fitness costs of rifampicin resistance in Mycobacterium tuberculosis are amplified under conditions of nutrient starvation and compensated by mutation in the β′ subunit of RNA polymerase

Rifampicin resistance, a defining attribute of multidrug-resistant tuberculosis, is conferred by mutations in the β subunit of RNA polymerase. Sequencing of rifampicin-resistant (RIF-R) clinical isolates of Mycobacterium tuberculosis revealed, in addition to RIF-R mutations, enrichment of potential compensatory mutations around the double-psi β-barrel domain of the β' subunit comprising the catalytic site and the exit tunnel for newly synthesized RNA. Sequential introduction of the resistance allele followed by the compensatory allele in isogenic Mycobacterium smegmatis showed that these mutations respectively caused and compensated a starvation enhanced growth defect by altering RNA polymerase activity. While specific combinations of resistance and compensatory alleles converged in divergent lineages, other combinations recurred among related isolates suggesting transmission of compensated RIF-R strains. These findings suggest nutrient poor growth conditions impose larger selective pressure on RIF-R organisms that results in the selection of compensatory mutations in a domain involved in catalysis and starvation control of RNA polymerase transcription.

Heterogeneous Nuclear Ribonucleoprotein (HnRNP) K Genome-wide Binding Survey Reveals Its Role in Regulating 3′-End RNA Processing and Transcription Termination at the Early Growth Response 1 (EGR1) Gene through XRN2 Exonuclease*

The heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein that acts as a docking platform integrating signal transduction pathways to nucleic acid-related processes. Given that hnRNPK could be involved in other steps that compose gene expression the definition of its genome-wide occupancy is important to better understand its role in transcription and co-transcriptional processes. Here, we used chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) to analyze the genome-wide hnRNPK-DNA interaction in colon cancer cell line HCT116. 9.1/3.6 and 7.0/3.4 million tags were sequenced/mapped, then 1809 and 642 hnRNPK binding sites were detected in quiescent and 30-min serum-stimulated cells, respectively. The inspection of sequencing tracks revealed inducible hnRNPK recruitment along a number of immediate early gene loci, including EGR1 and ZFP36, with the highest densities present at the transcription termination sites. Strikingly, hnRNPK knockdown with siRNA resulted in increased pre-RNA levels transcribed downstream of the EGR1 polyadenylation (A) site suggesting altered 3'-end pre-RNA degradation. Further ChIP survey of hnRNPK knockdown uncovered decreased recruitment of the 5'-3' exonuclease XRN2 along EGR1 and downstream of the poly(A) signal without altering RNA polymerase II density at these sites. Immunoprecipitation of hnRNPK and XRN2 from intact and RNase A-treated nuclear extracts followed by shotgun mass spectrometry revealed the presence of hnRNPK and XRN2 in the same complexes along with other spliceosome-related proteins. Our data suggest that hnRNPK may play a role in recruitment of XRN2 to gene loci thus regulating coupling 3'-end pre-mRNA processing to transcription termination.

Abstract 3001: The expression of HER2 in human breast cancer cells leads to massive alteration of RNA polymerase II binding and gene activation

Abstract Background. Herceptin, which targets the protein HER2, has poor efficacy as a monotherapy. To better understand the role of HER2, we compared isogenic HER2 expressing and non-expressing cells for the distribution for RNA polymerase II (POL II) on promoters and RNA expression. Methods. Chromatin immunoprecipitation was performed with anti-RNA polymerase II (POL II) in high HER2-expressing and non-expressing MCF7 cells as well as two breast cancer cell lines with naturally amplified HER2; MDA453 and BT474. Binding of POL II was assessed on Agilent promoter arrays. Gene activation in the same cells was assessed by expression analysis using U133plus2 arrays. Results. De novo expression of HER2 in MCF7 cells causes POLII to interact strongly with 606 genes not bound in empty vector control cells (p < 0.05). 30% of these genes exhibited a significant increase in transcription (p < 0.05; Fold-change > 1.4). POL II also interacts 1079 genes that are common between HER2-expressing and control cells. Conversely, 678 genes were bound more tightly by POL II in the control (p < 0.05). 20% of these genes exhibited a significant decrease in transcription. An additional 825 genes, bind POLII “loosely” in MCF7HER2 cells but not in MCF7 cells. 30% of these genes were significantly differentially regulated relative to control. The transcription of 68 loosely bound genes was tested by qPCR for all three cell lines. When compared with the array data, the correlation coefficients were 0.74 - 0.90;(p < 0.01). In both HER2 and non-expressing cells, loosely bound POL II sites are largely associated with 3′ positions of the Agilent probes within the transcribed region, whereas strong binding sites were largely localized to + 250 bp of the transcription start sites (TSSs). The transcriptome of MCF7 was compared to MCF7HER2 and the two HER2-expressing cancer cell lines. 633 genes were differentially expressed between the control and the three lines. Conclusions. The expression of HER2 is associated with a marked alteration in the number of genes that interact with POL II, involving hundreds of genes. The resulting gene activity depends upon the binding strength of POL II. Strong localized binding in one part of the promoter is often associated with less transcription, and diffused looser binding is often associated with higher transcription, whereas a lack of POL II binding is usually associated with no transcription. This is consistent with a mass action model in which transcription is maximal for POL II sites with an optimal koff rate and declines with either an increase (stalled) or decrease (not interacting) in binding. 42%, of the HER2-dependent gene interactions and transcription changes occur in both of the human cancer cell lines with naturally amplified HER2. These observations reveal a much expanded role of HER2 in gene regulation and indicate a greatly extended role in gene activation in HER2-positive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3001. doi:1538-7445.AM2012-3001