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.

scholarly journals Therapeutic targeting of transcriptional elongation in diffuse intrinsic pontine glioma

2021 ◽  
Author(s):  
Hiroaki Katagi ◽  
Nozomu Takata ◽  
Yuki Aoi ◽  
Yongzhan Zhang ◽  
Emily J Rendleman ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rna Polymerase Ii ◽  
Therapeutic Strategy ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Transcriptional Elongation ◽  
Pontine Glioma ◽  
Elongation Complex ◽  
Pol Ii ◽  
Chip Sequencing ◽  
Animal Survival

Abstract Background DIPG is associated with transcriptional dysregulation driven by H3K27 mutation. The super elongation complex (SEC) is required for transcriptional elongation through release of RNA polymerase II (Pol II). Inhibition of transcription elongation by SEC disruption can be an effective therapeutic strategy of H3K27M-mutant diffuse intrinsic pontine glioma (DIPG). Here, we tested the effect of pharmacological disruption of the SEC in H3K27M-mutant DIPG to advance understanding of the molecular mechanism and as a new therapeutic strategy for DIPG. Methods Short hairpin RNAs (shRNAs) were used to suppress the expression of AF4/FMR2 4 (AFF4), a central SEC component, in H3K27M-mutant DIPG cells. A peptidomimetic lead compound KL-1 was used to disrupt a functional component of SEC. Cell viability assay, colony formation assay, and apoptosis assay were utilized to analyze the effects of KL-1 treatment. RNA- and ChIP-sequencing were used to determine the effects of KL-1 on gene expression and chromatin occupancy. We treated mice bearing human H3K27M-mutant DIPG xenografts with KL-1. Intracranial tumor growth was monitored by bioluminescence image and therapeutic response was evaluated by animal survival. Results Depletion of AFF4 significantly reduced the cell growth of H3K27M-mutant DIPG. KL-1 increased genome-wide Pol II occupancy and suppressed transcription involving multiple cellular processes that promote cell proliferation and differentiation of DIPG. KL-1 treatment suppressed DIPG cell growth, increased apoptosis, and prolonged animal survival with human H3K27M-mutant DIPG xenografts. Conclusions SEC disruption by KL-1 increased therapeutic benefit in vitro and in vivo, supporting a potential therapeutic activity of KL-1 in H3K27M-mutant DIPG.

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

scholarly journals A machine learning-based framework for modeling transcription elongation

2021 ◽  
Vol 118 (6) ◽  
pp. e2007450118
Author(s):  
Peiyuan Feng ◽  
An Xiao ◽  
Meng Fang ◽  
Fangping Wan ◽  
Shuya Li ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
High Throughput Sequencing ◽  
Gene Expression Regulation ◽  
Transcription Elongation ◽  
Transcriptional Elongation ◽  
Sequencing Data ◽  
Pol Ii ◽  
High Throughput Sequencing Data ◽  
Pol Ii Pausing ◽  
Elongation Process

RNA polymerase II (Pol II) generally pauses at certain positions along gene bodies, thereby interrupting the transcription elongation process, which is often coupled with various important biological functions, such as precursor mRNA splicing and gene expression regulation. Characterizing the transcriptional elongation dynamics can thus help us understand many essential biological processes in eukaryotic cells. However, experimentally measuring Pol II elongation rates is generally time and resource consuming. We developed PEPMAN (polymerase II elongation pausing modeling through attention-based deep neural network), a deep learning-based model that accurately predicts Pol II pausing sites based on the native elongating transcript sequencing (NET-seq) data. Through fully taking advantage of the attention mechanism, PEPMAN is able to decipher important sequence features underlying Pol II pausing. More importantly, we demonstrated that the analyses of the PEPMAN-predicted results around various types of alternative splicing sites can provide useful clues into understanding the cotranscriptional splicing events. In addition, associating the PEPMAN prediction results with different epigenetic features can help reveal important factors related to the transcription elongation process. All these results demonstrated that PEPMAN can provide a useful and effective tool for modeling transcription elongation and understanding the related biological factors from available high-throughput sequencing data.

scholarly journals Histone H2B Ubiquitylation Is Associated with Elongating RNA Polymerase II

2005 ◽  
Vol 25 (2) ◽  
pp. 637-651 ◽  
Author(s):  
Tiaojiang Xiao ◽  
Cheng-Fu Kao ◽  
Nevan J. Krogan ◽  
Zu-Wen Sun ◽  
Jack F. Greenblatt ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transcription Elongation ◽  
Lysine Methylation ◽  
Coding Region ◽  
Elongation Complex ◽  
Histone H2b ◽  
Pol Ii ◽  
Paf1 Complex

ABSTRACT Rad6-mediated ubiquitylation of histone H2B at lysine 123 has been linked to transcriptional activation and the regulation of lysine methylation on histone H3. However, how Rad6 and H2B ubiquitylation contribute to the transcription and histone methylation processes is poorly understood. Here, we show that the Paf1 transcription elongation complex and the E3 ligase for Rad6, Bre1, mediate an association of Rad6 with the hyperphosphorylated (elongating) form of RNA polymerase II (Pol II). This association appears to be necessary for the transcriptional activities of Rad6, as deletion of various Paf1 complex members or Bre1 abolishes H2B ubiquitylation (ubH2B) and reduces the recruitment of Rad6 to the promoters and transcribed regions of active genes. Using the inducible GAL1 gene as a model, we find that the recruitment of Rad6 upon activation occurs rapidly and transiently across the gene and coincides precisely with the appearance of Pol II. Significantly, during GAL1 activation in an rtf1 deletion mutant, Rad6 accumulates at the promoter but is absent from the transcribed region. This fact suggests that Rad6 is recruited to promoters independently of the Paf1 complex but then requires this complex for entrance into the coding region of genes in a Pol II-associated manner. In support of a role for Rad6-dependent H2B ubiquitylation in transcription elongation, we find that ubH2B levels are dramatically reduced in strains bearing mutations of the Pol II C-terminal domain (CTD) and abolished by inactivation of Kin28, the serine 5 CTD kinase that promotes the transition from initiation to elongation. Furthermore, synthetic genetic array analysis reveals that the Rad6 complex interacts genetically with a number of known or suspected transcription elongation factors. Finally, we show that Saccharomyces cerevisiae mutants bearing defects in the pathway to H2B ubiquitylation display transcription elongation defects as assayed by 6-azauracil sensitivity. Collectively, our results indicate a role for Rad6 and H2B ubiquitylation during the elongation cycle of transcription and suggest a mechanism by which H3 methylation may be regulated.

scholarly journals Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

2006 ◽  
Vol 26 (3) ◽  
pp. 777-788 ◽  
Author(s):  
Yi Pei ◽  
Hongyan Du ◽  
Juliet Singer ◽  
Courtney St. Amour ◽  
Selena Granitto ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Growth Defect ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Elongation Complex ◽  
Pol Ii

ABSTRACT Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK—Mcs6, the kinase component of transcription factor IIH (TFIIH)—cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced ∼10-fold by csk1 deletion, and Cdk9 complexes from csk1Δ but not csk1 + cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1Δ strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9 T212A mcs6 S165A strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1—the third essential CDK-cyclin complex described in fission yeast—helps to target the capping apparatus to the transcriptional elongation complex.

scholarly journals LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Xiaodan Ji ◽  
Huasong Lu ◽  
Qiang Zhou ◽  
Kunxin Luo
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
Epithelial Mesenchymal Transition ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Breast Cancer Progression ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Pol Ii ◽  
Mesenchymal Transition

Transcriptional elongation by RNA polymerase (Pol) II is essential for gene expression during cell growth and differentiation. The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors. A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex. Here, we show that P-TEFb activity is important for the epithelial–mesenchymal transition (EMT) and breast cancer progression. Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis. Our data provide the first demonstration that the transcription elongation machinery plays a key role in promoting breast cancer progression by directly controlling the expression of upstream EMT regulators.

scholarly journals Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

2020 ◽  
Vol 117 (41) ◽  
pp. 25486-25493 ◽  
Author(s):  
Jun Xu ◽  
Wei Wang ◽  
Liang Xu ◽  
Jia-Yu Chen ◽  
Jenny Chong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Neurological Diseases ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Stable Complex ◽  
Loss Of Function ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

scholarly journals EXTH-46. ARTIFICIAL INTELLIGENCE-BASED IDENTIFICATION OF COMBINED VANDETANIB AND EVEROLIMUS IN THE TREATMENT OF ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii97-ii97
Author(s):  
Diana Carvalho ◽  
Peter Richardson ◽  
Nagore Gene Olaciregui ◽  
Reda Stankunaite ◽  
Cinzia Emilia Lavarino ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Cell Viability ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Serine Threonine Kinase ◽  
Orthotopic Xenografts ◽  
Extended Survival

Abstract Somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2 receptor, are found in a quarter of children with the currently incurable brain tumour diffuse intrinsic pontine glioma (DIPG). Treatment of ACVR1-mutant DIPG patient-derived models with multiple inhibitor chemotypes leads to a reduction in cell viability in vitro and extended survival in orthotopic xenografts in vivo, though there are currently no specific ACVR1 inhibitors licensed for DIPG. Using an Artificial Intelligence-based platform to search for approved compounds which could be used to treat ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an approved inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd=150nM) and reduce DIPG cell viability in vitro, but has been trialed in DIPG patients with limited success, in part due to an inability to cross the blood-brain-barrier. In addition to mTOR, everolimus inhibits both ABCG2 (BCRP) and ABCB1 (P-gp) transporter, and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination is well-tolerated in vivo, and significantly extended survival and reduced tumour burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Based on these preclinical data, three patients with ACVR1-mutant DIPG were treated with vandetanib and everolimus. These cases may inform on the dosing and the toxicity profile of this combination for future clinical studies. This bench-to-bedside approach represents a rapidly translatable therapeutic strategy in children with ACVR1 mutant DIPG.

scholarly journals Promoter-specific changes in initiation, elongation and homeostasis of histone H3 acetylation during CBP/p300 inhibition

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Emily Hsu ◽  
Nathan R Zemke ◽  
Arnold J Berk
Keyword(s):  
Rna Polymerase Ii ◽  
Control Point ◽  
Histone H3 ◽  
Airway Epithelial Cells ◽  
Proximal Region ◽  
Transferase Activity ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Histone H3 Acetylation ◽  
H3 Acetylation

Regulation of RNA Polymerase II (Pol2) elongation in the promoter proximal region is an important and ubiquitous control point for gene expression in metazoans. We report that transcription of the adenovirus 5 E4 region is regulated during the release of paused Pol2 into productive elongation by recruitment of the super elongation complex (SEC), dependent on promoter H3K18/27 acetylation by CBP/p300. We also establish that this is a general transcriptional regulatory mechanism that applies to ~6% of expressed protein-coding genes in primary human airway epithelial cells. We observed that a homeostatic mechanism maintains promoter, but not enhancer H3K18/27ac in response to extensive inhibition of CBP/p300 acetyl transferase activity by the highly specific small molecule inhibitor A-485. Further, our results suggest a function for BRD4 association at enhancers in regulating paused Pol2 release at nearby promoters. Taken together, our results uncover processes regulating transcriptional elongation by promoter region histone H3 acetylation and homeostatic maintenance of promoter, but not enhancer, H3K18/27ac in response to inhibition of CBP/p300 acetyl transferase activity.

scholarly journals The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions

2021 ◽  
Author(s):  
Rajaraman Gopalakrishnan ◽  
Fred Winston
Keyword(s):  
Mass Spectrometry ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Histone Chaperone ◽  
Mrna Processing ◽  
Wild Type ◽  
Elongation Complex ◽  
Protein Levels ◽  
Genome Wide ◽  
Capping Enzyme

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5 ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.

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