scholarly journals p53-Dependent p21 mRNA Elongation Is Impaired when DNA Replication Is Stalled

2006 ◽  
Vol 27 (4) ◽  
pp. 1309-1320 ◽  
Author(s):  
Melissa Mattia ◽  
Vanesa Gottifredi ◽  
Kristine McKinney ◽  
Carol Prives
Keyword(s):  
Dna Replication ◽  
Rna Polymerase Ii ◽  
Target Genes ◽  
Large Subunit ◽  
Transcriptional Elongation ◽  
Mrna Transcription ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Replication Checkpoint ◽  
Dna Replication Checkpoint

ABSTRACT We have previously reported that when DNA replication is blocked in some human cell lines, p53 is impaired in its ability to induce a subset of its key target genes, including p21 WAF1/CIP1 . Here, we investigated the reason for this impairment by comparing the effects of two agents, hydroxyurea (HU), which arrests cells in early S phase and impairs induction of p21, and daunorubicin, which causes a G2 block and leads to robust activation of p21 by p53. HU treatment was shown to inhibit p21 mRNA transcription rather than alter its mRNA stability. Nevertheless, chromatin immunoprecipitation assays revealed that HU impacts neither p53 binding nor acetylation of histones H3 and H4 within the p21 promoter. Furthermore, recruitment of the TFIID/TATA-binding protein complex and the large subunit of RNA polymerase II (RNA Pol II) are equivalent after HU and daunorubicin treatments. Relative to daunorubicin treatment, however, transcription elongation of the p21 gene is significantly impaired in cells treated with HU, as evidenced by reduced occupancy of RNA Pol II at regions downstream of the start site. Likewise, in the p21 downstream region after administration of HU, there is less of a specifically phosphorylated form of RNA Pol II (Pol II-C-terminal domain serine 2P) which occurs only when the polymerase is elongating RNA. We propose that while the DNA replication checkpoint is unlikely to regulate the assembly of a p21 promoter initiation complex, it signals to one or more factors involved in the process of transcriptional elongation.

scholarly journals HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Yongkang Yang ◽  
Haiquan Lu ◽  
Chelsey Chen ◽  
Yajing Lyu ◽  
Robert N. Cole ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Transcription ◽  
Target Genes ◽  
Large Subunit ◽  
Hypoxia Inducible Factor ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Pol Ii

AbstractHypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O2) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30–60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

scholarly journals HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription

2002 ◽  
Vol 364 (3) ◽  
pp. 649-657 ◽  
Author(s):  
Sergei NEKHAI ◽  
Meisheng ZHOU ◽  
Anne FERNANDEZ ◽  
William S. LANE ◽  
Ned J.C. LAMB ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Viral Gene ◽  
Transcriptional Elongation ◽  
Rna Pol Ii ◽  
Hela Nuclear Extract ◽  
Pol Ii ◽  
Terminal Domain ◽  
Hiv 1

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246–256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.

scholarly journals c-Myc Is Required for the ChREBP-Dependent Activation of Glucose-Responsive Genes

2010 ◽  
Vol 24 (6) ◽  
pp. 1274-1286 ◽  
Author(s):  
Pili Zhang ◽  
Mallikarjurna R. Metukuri ◽  
Sharell M. Bindom ◽  
Edward V. Prochownik ◽  
Robert M. O'Doherty ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Time Course ◽  
Target Genes ◽  
Process Time ◽  
Glucose Response ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Molecular Events ◽  
Regulated Gene Expression

Abstract Glucose regulates programs of gene expression that orchestrate changes in cellular phenotype in several metabolically active tissues. Carbohydrate response element-binding protein (ChREBP) and its binding partner, Mlx, mediate glucose-regulated gene expression by binding to carbohydrate response elements on target genes, such as the prototypical glucose-responsive gene, liver-type pyruvate kinase (Pklr). c-Myc is also required for the glucose response of the Pklr gene, although the relationship between c-Myc and ChREBP has not been defined. Here we describe the molecular events of the glucose-mediated activation of Pklr and determine the effects of decreasing the activity or abundance of c-Myc on this process. Time-course chromatin immunoprecipitation revealed a set of transcription factors [hepatocyte nuclear factor (HNF)1α, HNF4α, and RNA polymerase II (Pol II)] constitutively resident on the Pklr promoter, with a relative enrichment of acetylated histones 3 and 4 in the same region of the gene. Glucose did not affect HNF1α binding or the acetylation of histones H3 or H4. By contrast, glucose promoted the recruitment of ChREBP and c-Myc and increased the occupancy of HNF4α and RNA Pol II, which were coincident with the glucose-mediated increase in transcription as determined by a nuclear run-on assay. Depletion of c-Myc activity using a small molecule inhibitor (10058-F4/1RH) abolished the glucose-mediated recruitment of HNF4α, ChREBP, and RNA Pol II, without affecting basal gene expression, histone acetylation, and HNF1α or basal HNF4α occupancy. The activation and recruitment of ChREBP to several glucose-responsive genes were blocked by 1RH, indicating a general necessity for c-Myc in this process.

scholarly journals TFIIF Facilitates Dissociation of RNA Polymerase II from Noncoding RNAs That Lack a Repression Domain

2009 ◽  
Vol 30 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Stacey D. Wagner ◽  
Jennifer F. Kugel ◽  
James A. Goodrich
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Noncoding Rnas ◽  
Mrna Transcription ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
General Transcription Factors ◽  
B2 Rna ◽  
Repression Domain

ABSTRACT Noncoding RNAs (ncRNAs) have recently been found to regulate multiple steps in mammalian mRNA transcription. Mouse B2 RNA and human Alu RNA bind RNA polymerase II (Pol II) and repress mRNA transcription, using regions of the ncRNAs referred to as repression domains. Two other ncRNAs, mouse B1 RNA and human small cytoplasmic Alu (scAlu) RNA, bind Pol II with high affinity but lack repression domains and hence do not inhibit transcription. To better understand the interplay between ncRNAs that bind Pol II and their functions in transcription, we studied how Pol II binding and transcriptional repression are controlled by general transcription factors. We found that TFIIF associates with B1 RNA/Pol II and scAlu RNA/Pol II complexes and decreases their kinetic stability. Both subunits of TFIIF are required for this activity. Importantly, fusing a repression domain to B1 RNA stabilizes its interaction with Pol II in the presence of TFIIF. These results suggest a new role for TFIIF in regulating the interaction of ncRNAs with Pol II; specifically, it destabilizes interactions with ncRNAs that are not transcriptional repressors. These studies also identify a new function for ncRNA repression domains: they stabilize interactions of ncRNAs with Pol II in the presence of TFIIF.

scholarly journals DOT1L complex regulates transcriptional initiation in human erythroleukemic cells

2021 ◽  
Vol 118 (27) ◽  
pp. e2106148118
Author(s):  
Aiwei Wu ◽  
Junhong Zhi ◽  
Tian Tian ◽  
Ali Cihan ◽  
Murat A. Cevher ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Target Genes ◽  
Initiation Factor ◽  
Transcriptional Elongation ◽  
Saga Complex ◽  
Transcriptional Initiation ◽  
Pol Ii ◽  
Transcription Initiation Factor ◽  
Erythroleukemic Cells

DOT1L, the only H3K79 methyltransferase in human cells and a homolog of the yeast Dot1, normally forms a complex with AF10, AF17, and ENL or AF9, is dysregulated in most cases of mixed-lineage leukemia (MLLr), and has been believed to regulate transcriptional elongation on the basis of its colocalization with RNA polymerase II (Pol II), the sharing of subunits (AF9 and ENL) between the DOT1L and super elongation complexes, and the distribution of H3K79 methylation on both promoters and transcribed regions of active genes. Here we show that DOT1L depletion in erythroleukemic cells reduces its global occupancy without affecting the traveling ratio or the elongation rate (assessed by 4sUDRB-seq) of Pol II, suggesting that DOT1L does not play a major role in elongation in these cells. In contrast, analyses of transcription initiation factor binding reveal that DOT1L and ENL depletions each result in reduced TATA binding protein (TBP) occupancies on thousands of genes. More importantly, DOT1L and ENL depletions concomitantly reduce TBP and Pol II occupancies on a significant fraction of direct (DOT1L-bound) target genes, indicating a role for the DOT1L complex in transcription initiation. Mechanistically, proteomic and biochemical studies suggest that the DOT1L complex may regulate transcriptional initiation by facilitating the recruitment or stabilization of transcription factor IID, likely in a monoubiquitinated H2B (H2Bub1)-enhanced manner. Additional studies show that DOT1L enhances H2Bub1 levels by limiting recruitment of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex. These results advance our understanding of roles of the DOT1L complex in transcriptional regulation and have important implications for MLLr leukemias.

scholarly journals DIPG-03. THERAPEUTIC TARGETING OF TRANSCRIPTIONAL ELONGATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii287-iii287
Author(s):  
Hiroaki Katagi ◽  
Nozomu Takata ◽  
Yuki Aoi ◽  
Yongzhan Zhang ◽  
Emily J Rendleman ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Transcriptional Elongation ◽  
Pontine Glioma ◽  
Elongation Complex ◽  
Pol Ii ◽  
Repair Genes ◽  
Novel Therapeutic

Abstract Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

Overlapping Roles of the Spindle Assembly and DNA Damage Checkpoints in the Cell-Cycle Response to Altered Chromosomes in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 521-534
Author(s):  
Peter M Garber ◽  
Jasper Rine
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Spindle Checkpoint ◽  
Dna Lesions ◽  
Replication Proteins ◽  
Replication Checkpoint ◽  
Dna Damage Checkpoints ◽  
Dna Replication Checkpoint ◽  
Mcm Proteins ◽  
Stalled Replication Forks

Abstract The MAD2-dependent spindle checkpoint blocks anaphase until all chromosomes have achieved successful bipolar attachment to the mitotic spindle. The DNA damage and DNA replication checkpoints block anaphase in response to DNA lesions that may include single-stranded DNA and stalled replication forks. Many of the same conditions that activate the DNA damage and DNA replication checkpoints also activated the spindle checkpoint. The mad2Δ mutation partially relieved the arrest responses of cells to mutations affecting the replication proteins Mcm3p and Pol1p. Thus a previously unrecognized aspect of spindle checkpoint function may be to protect cells from defects in DNA replication. Furthermore, in cells lacking either the DNA damage or the DNA replication checkpoints, the spindle checkpoint contributed to the arrest responses of cells to the DNA-damaging agent methyl methanesulfonate, the replication inhibitor hydroxyurea, and mutations affecting Mcm2p and Orc2p. Thus the spindle checkpoint was sensitive to a wider range of chromosomal perturbations than previously recognized. Finally, the DNA replication checkpoint did not contribute to the arrests of cells in response to mutations affecting ORC, Mcm proteins, or DNA polymerase δ. Thus the specificity of this checkpoint may be more limited than previously recognized.

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