Elongation Factor For RNA Polymerase Research Articles

Structure of the transcribing RNA polymerase II–Elongin complex

Elongin is a heterotrimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. Here, we report three cryo-EM structures of human Elongin bound to transcribing Pol II. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB–ELOC subunit heterodimer. ELOA contains a ‘latch’ that binds between the end of the Pol II bridge helix and funnel helices, thereby inducing a conformational change near the polymerase active center. The latch is required for the elongation-stimulatory activity of Elongin, but not for Pol II binding, indicating that Elongin functions by allosterically regulating the conformational mobility of the polymerase active center. Elongin binding to Pol II is incompatible with association of the super elongation complex, PAF1 complex and RTF1, which also contain an elongation-stimulatory latch element.

Characterizing the Interaction between the HTLV-1 Transactivator Tax-1 with Transcription Elongation Factor ELL2 and Its Impact on Viral Transactivation.

The human T-cell leukemia virus type 1 (HTLV-1)-encoded transactivator and oncoprotein Tax-1 is essential for HTLV-1 replication. We recently found that Tax-1 interacts with transcription elongation factor for RNA polymerase II 2, ELL2, which enhances Tax-1-mediated transactivation of the HTLV-1 promotor. Here, we characterize the Tax-1:ELL2 interaction and its impact on viral transactivation by confocal imaging, co-immunoprecipitation, and luciferase assays. We found that Tax-1 and ELL2 not only co-precipitate, but also co-localize in dot-like structures in the nucleus. Tax-1:ELL2 complex formation occurred independently of Tax-1 point mutations, which are crucial for post translational modifications (PTMs) of Tax-1, suggesting that these PTMs are irrelevant for Tax-1:ELL2 interaction. In contrast, Tax-1 deletion mutants lacking either N-terminal (aa 1–37) or C-terminal regions (aa 150–353) of Tax-1 were impaired in interacting with ELL2. Contrary to Tax-1, the related, non-oncogenic Tax-2B from HTLV-2B did not interact with ELL2. Finally, we found that ELL2-R1 (aa 1–353), which carries an RNA polymerase II binding domain, and ELL2-R3 (aa 515–640) are sufficient to interact with Tax-1; however, only ELL2-truncations expressing R1 could enhance Tax-1-mediated transactivation of the HTLV-1 promoter. Together, this study identifies domains in Tax-1 and ELL2 being required for Tax-1:ELL2 complex formation and for viral transactivation.

Genetic Variants That Influence Fetal Hemoglobin Expression from Hydroxyurea Treatment

Introduction: Hydroxyurea is a potent therapeutic agent for sickle cell anemia (SCA), and treatment at maximum tolerated dose (MTD) is becoming the standard of care. Hydroxyurea exerts its disease-modifying effects primarily through induction of fetal hemoglobin (HbF), although the cellular and molecular mechanisms by which hydroxyurea increases HbF expression remain unclear. Children with SCA treated with hydroxyurea at MTD have substantial phenotypic variation, however, as some have higher HbF responses than others. We hypothesized that unknown quantitative trait loci modulate the pharmacological induction of HbF, so we performed a large genome wide association study (GWAS) of hydroxyurea-associated HbF responses for children with SCA treated prospectively with dose escalation to MTD. Methods: We analyzed genomic DNA from 831 children with SCA enrolled in pediatric research trials from the US (HUSTLE, SWiTCH, TWiTCH), the Caribbean (EXTEND, SACRED) and sub-Saharan Africa (REACH, NOHARM); all of these trials reported robust treatment responses with average HbF >20%. Study participants received hydroxyurea with dose escalation to MTD based on mild myelosuppression. Whole blood DNA was genotyped using the H3Africa SNP array (Illumina) with whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform. A transformed z-score for each study cohort gave a standardized measure of HbF induction relative to their steady-state level and their treatment HbF level at MTD. These standardized z-score HbF values were then used as a continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, using age, hydroxyurea dose at MTD, and sex as co-variates. We first performed an initial GWAS discovery using hydroxyurea response data from four distinct African populations (n=377). Single nucleotide variants (SNVs) with nominal significance (p<0.001) in the discovery step were then selected for replication using an additional African cohort (n=168). Variants that were significant in both the discovery and replication cohorts were then verified using a cohort of US (n=200) and Caribbean (n=86) children with SCA, identifying genomic loci with consistent associations for HbF induction across all cohorts. Results: In the discovery GWAS step, no variant passed genome wide significance (p<10-8) for the MTD HbF phenotype, including no significant associations with known genetic modifiers of endogenous HbF (BCL11A, HBS1L-MYB, HBG2). A total of 2057 low frequency and common SNVs had at least nominal association (p<0.001) with the hydroxyurea treatment responses, of which 44 were also significant (p<0.05) and with the same direction of association with HbF induction in the replication cohort. In the final verification step, these 44 significant variants were then tested in additional independent SCA cohorts with at least three demonstrating a strong effect (Table 1). The rs10978155 variant in the PTPRD gene and the rs55695413 variant in the RPH3AL gene were both consistently associated (p<0.05) with lower HbF treatment responses. Another variant (rs75442556) near the ELL2 gene approached statistical significance (p=0.08) in the verification cohort and was also associated with lower HbF expression. The allele frequencies for these PTPRD, RPH3AL, and ELL2 variants were 0.32, 0.017, and 0.25, respectively, and did not affect baseline HbF levels. Children with these PTPRD, RPH3AL, and ELL2 genetic variants still had substantial HbF induction, but achieved lower hydroxyurea MTD HbF levels on average by 2.9%, 9.8%, and 2.7%, respectively. Conclusions: This large GWAS using global cohorts of children with SCA and robust prospective HbF phenotype data has identified genetic predictors of HbF hydroxyurea treatment responses. Three novel genetic loci, PTPRD, RPH3AL, and ELL2 have SNVs associated with lower HbF responses. PTPRD is a protein tyrosine phosphatase receptor involved in cellular processes such as cell growth and differentiation, while RPH3AL, a rabphilin 3A like protein, is known to be involved in calcium-ion-dependent exocytosis. ELL2 is an elongation factor for RNA polymerase II and could modify RNA processing under the cytostatic effects of hydroxyurea. These genes and variants will be investigated to determine how they impact individual HbF responses to hydroxyurea treatment. Disclosures Aygun: National Heart, Lung, and Blood Institute: Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Research Funding; National Institute of Nursing Research: Research Funding; Patient-Centered Outsomes Research Institute: Research Funding.

A five-gene signature to predict the overall survival time of patients with esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the six most commonly diagnosed tumor types in the Chinese population. Gene expression profiles help to predict the prognosis of patients with ESCC. Disease recurrence as the survival endpoint has been analyzed in the majority of previous studies; therefore, the aim of the present study was to construct a robust gene signature in order to determine the overall survival (OS) of patients with ESCC. The gene expression and clinical data of patients with ESCC were downloaded from The Cancer Genome Atlas (TCGA) database. Of the selected data (172 samples from surviving patients), 72 samples were randomly selected as modeling data, and verification was conducted using the entire dataset. Data from the Gene Expression Omnibus was analyzed simultaneously, and a venn diagram was constructed to determine the intersection between these two sets of results; a total of 97 genes were found to be associated with OS. Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that these genes were primarily associated with specific pathways (Homo sapiens), including DNA replication, protein processing in endoplasmic reticulum and influenza A. A five-gene signature was identified with a robust likelihood-based survival modeling approach. Using regression coefficient modeling, a prognostic model consisting of the C-X-C motif chemokine ligand 8, DNA damage inducible transcript 3, RAB27A, member RAS oncogene family, replication factor C subunit 2 and elongation factor for RNA polymerase II 2 genes was constructed and validated. Based on these results, patients were subdivided into high and low-risk groups. Compared with the high-risk group, the OS time of patients in the low-risk group was significantly increased. Furthermore, it was determined that the five genes were all differentially expressed in ESCC tissues compared with normal tissues, indicating the potential role of these genes in ESCC initiation and progression. In another independent cohort, this five-gene signature was further confirmed and was considered as an independent prognostic biomarker for OS prediction in patients with ESCC. In conclusion, the OS of patients with ESCC may be predicted using this five-gene signature, which may be useful in identifying patients with high-risk ESCC.

ELL2 Is Downregulated and Associated with Galactose-Deficient IgA1 in IgA Nephropathy.

Background Galactose-deficient IgA1 (Gd-IgA1) is an important causal factor in IgA nephropathy; however, the underlying mechanism for the production of Gd-IgA1 is unknown. The elongation factor for RNA polymerase II (ELL2), which encoded a key component of the superelongation complex (SEC), drives secretory-specific Ig mRNA production. Methods We enrolled 21 patients with IgAN, 18 healthy controls, and 20 patients with non-IgAN glomerulonephritis. The differential expression of ELL2 was compared using publically available data from Gene Expression Omnibus (GEO) datasets. The relationship between ELL2 expressions and galactose-deficient IgA1 (Gd-IgA1) levels in serum were also studied. At last, the results were validated by shELL2 treatment experiment. Results We found that the number of CD19+ B cells was increased in IgAN patients compared to healthy controls. The expression level of ELL2 in patients with IgAN was significantly lower than that of healthy control and disease control. Consistent with present results, the lower ELL2 expression in IgAN patients was observed in microarray expression profiles from GEO datasets. Pearson correlation analysis showed that ELL2 expression negatively correlated with Gd-IgA1 levels. Furthermore, in an in vitro experiment, we found that loss of ELL2 function in human B lymphoma DAKIKI cells, an IgA1-producing cell line, increased the levels of Gd-IgA1, which confirmed that ELL2 modulated the levels of Gd-IgA1. Conclusion Our findings implied that decreased ELL2 expression was negatively correlated with the numbers of B cells and aberrant glycosylation of IgA1 in IgAN.

Regulation of ELL2 stability and polyubiquitination by EAF2 in prostate cancer cells.

Elongation factor for RNA polymerase 2 (ELL2) and ELL associated factor 2 (EAF2) have been reported to have tumor suppressive properties in prostate epithelial cells. We investigated ELL2 expression in human prostate cancer specimens, and ELL2 protein stability and ubiquitination in prostate cancer cells. Immunostaining analysis of human prostate cancer specimens was used to determine ELL2 expression in tumor and normal tissues. ELL2 knockdown in prostate cancer cell lines LNCaP and C4-2 was used to compare proliferation and motility. Deletion and site-directed mutagenesis was used to identify amino acid residues in ELL2 that were important for degradation. ELL2 protein was downregulated in prostate cancer specimens and was up-regulated by androgens in prostate cancer cell lines LNCaP and C4-2. ELL2 knockdown enhanced prostate cancer cell proliferation and motility. ELL2 protein has a short half-life and was stabilized by proteasome inhibitor MG132. Amino acid residues K584 and K599 in ELL2 were important for ELL2 degradation. EAF2 could stabilize ELL2 and inhibited its polyubiquitination. Our findings provide further evidence that ELL2 is a potential tumor suppressor frequently down-regulated in clinical prostate cancer specimens and provides new insights into regulation of ELL2 protein level by polyubiquitination and EAF2 binding.

The multifunctional Ccr4–Not complex directly promotes transcription elongation

The Ccr4-Not complex has been implicated in the control of multiple steps of mRNA metabolism; however, its functions in transcription remain ambiguous. The discovery that Ccr4/Pop2 is the major cytoplasmic mRNA deadenylase and the detection of Not proteins within mRNA processing bodies have raised questions about the roles of the Ccr4-Not complex in transcription. Here we firmly establish Ccr4-Not as a positive elongation factor for RNA polymerase II (RNAPII). The Ccr4-Not complex is targeted to the coding region of genes in a transcription-dependent manner similar to RNAPII and promotes elongation in vivo. Furthermore, Ccr4-Not interacts directly with elongating RNAPII complexes and stimulates transcription elongation of arrested polymerase in vitro. Ccr4-Not can reactivate backtracked RNAPII using a mechanism different from that of the well-characterized elongation factor TFIIS. While not essential for its interaction with elongation complexes, Ccr4-Not interacts with the emerging transcript and promotes elongation in a manner dependent on transcript length, although this interaction is not required for it to bind RNAPII. Our comprehensive analysis shows that Ccr4-Not directly regulates transcription, and suggests it does so by promoting the resumption of elongation of arrested RNAPII when it encounters transcriptional blocks in vivo.

Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4–Spt5 in Transcription

Spt4-Spt5, a general transcription elongation factor for RNA polymerase II, also has roles in chromatin regulation. However, the relationships between these functions are not clear. Previously, we isolated suppressors of a Saccharomyces cerevisiae spt5 mutation in genes encoding members of the Paf1 complex, which regulates several cotranscriptional histone modifications, and Chd1, a chromatin remodeling enzyme. Here, we show that this suppression of spt5 can result from loss of histone H3 lysines 4 or 36 methylation, or reduced recruitment of Chd1 or the Rpd3S complex. These spt5 suppressors also rescue the synthetic growth defects observed in spt5 mutants that also lack elongation factor TFIIS. Using a FLO8 reporter gene, we found that a chd1 mutation caused cryptic initiation of transcription. We further observed enhancement of cryptic initiation in chd1 isw1 mutants and increased histone acetylation in a chd1 mutant. We suggest that, as previously proposed for H3 lysine 36 methylation and the Rpd3S complex, H3 lysine 4 methylation and Chd1 function to maintain normal chromatin structures over transcribed genes, and that one function of Spt4-Spt5 is to help RNA polymerase II overcome the repressive effects of these histone modifications and chromatin regulators on transcription.

RNA polymerase II complexes in the very early phase of transcription are not susceptible to TFIIS-induced exonucleolytic cleavage.

TFIIS is a transcription elongation factor for RNA polymerase II (pol II), which can suppress ribonucleotide misincorporation. We reconstituted transcription complexes in a highly purified pol II system on adenovirus Major-Late promoter constructs. We noted that these complexes have a high propensity for read-through upon GTP omission. Read-through occurred during the early stages at all registers analyzed. Addition of TFIIS reversed read-through of productive elongation complexes, which indicated that it was due to misincorporation. However, before register 13 transcription complexes were insensitive to TFIIS. These findings are discussed with respect to the structural models for pol II and we propose that TFIIS action is linked to the RNA:DNA hybrid.

Characterization of a HeLa cDNA clone encoding the human SII protein, an elongation factor for RNA polymerase II

We present the cloning and sequence characterization of a HeLa cDNA encoding the SII transcription elongation factor. This cDNA clone is distinct from those previously isolated from a human kidney cDNA library [Yoo et al., Nucleic Acids Res. 19 (1991) 1073–1079]. Southern analysis suggests that more than one gene may exist for SII in the human genome. A comparison of deduced amino acid sequences for SII-related proteins from a variety of eukaryotes demonstrates very high similarity, especially within the C-terminal domain.