The Myc binding partner Spt5 is essential for brain tumor growth in Drosophila
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE220110 | GEO | 2023/10/23
REPOSITORIES: GEO
Similar Datasets
Project description:The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and is expressed in a strictly growth factor-dependent manner in non-tumor cells. Here we show that MYC directly binds SPT5, a subunit of the RNA polymerase II (POL2) elongation factor DSIF. MYC recruits SPT5 to genes and enables the CDK7-dependent transfer of SPT5 onto POL2. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. In addition, MYC increases the directionality of promoters by stimulating sense transcription and suppressing the synthesis of antisense RNAs. Our results argue that MYC controls the productive assembly of POL2 with general elongation factors to form processive elongation complexes. The high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, slows transcription at growth-suppressive genes and promotes uncontrolled cellular growth.
2019-03-27 | GSE115365 | GEO
Project description:The Myc binding partner Spt5 is essential for brain tumor growth in Drosophila
| PRJNA908816 | ENA
Project description:The Spt4-Spt5 complex, and its human homolog DSIF (DRB sensitivity-inducing factor), is unique in its ability to regulate Pol II processivity. Previous studies have shown that Spt5 has the characteristics of a general transcription-elongation factor. However, mutagenesis of Spt5 showed specific phenotypes during development, which were far less severe than those of Pol II defects or TBP deficient embryos. It seems paradoxical that a mutation which alters a general elongation factor can cause rather specific developmental defects. By using Spt5 knockdown zebrafish embryos and microarrays, here we showed that transcript abundance for only a small subset of genes is altered by loss of Spt5. Further investigation of the down-regulated genes showed that the genes most intensely repressed by the knockdown were strongly activated during early development in untreated embryos. Thus, this study shows that gene activation levels may create different requirements for Pol II processivity. Active transcription requires Spt5 for efficient elongation through its stimulatory activity on Pol II processivity. Experiment Overall Design: The expression pattern of Spt5 knockdown embryos was compared to that of the control mopholino oligo injected embryos, both at 21 hours post-fertilization, to show the targets of Spt5. The activation levels of the Spt5-knockdown affected genes were measured by compareing the abundance of transcripts in 21 hours post-fertilization untreated embryos to that in 5 hours post-fertilization untreated embryos.
2008-06-14 | E-GEOD-6127 | biostudies-arrayexpress
Project description:The pause-release model of transcription proposes that pol II pauses 40-100 bases from the start site resulting in a pile-up that is relieved by subsequent release into productive elongation. Pause release is facilitated by PTEFb phosphorylation of the pol II elongation factor, Spt5. We mapped paused polymerases by eNETseq and found frequent pausing in zones that extend ~0.3-3kb into genes, even when PTEFb is inhibited. The fraction of paused polymerases or “pausiness” declines gradually over several kb, and not abruptly as predicted for a discrete pause release event. Spt5 depletion extends pausing zones suggesting that it promotes maturation of elongation complexes to a low-pausing state. Expression of mutants after Spt5 depletion showed that phosphomimetic substitutions in the Spt5 CTR1 domain diminished pausing throughout genes. In contrast mutants that prevent phosphorylation of the Spt5 RNA-binding domain strengthened pausing. Thus distinct Spt5 phospho-isoforms set the balance between pausing and elongation.
2022-10-01 | GSE202749 | GEO
Project description:The Spt4-Spt5 complex, and its human homolog DSIF (DRB sensitivity-inducing factor), is unique in its ability to regulate Pol II processivity. Previous studies have shown that Spt5 has the characteristics of a general transcription-elongation factor. However, mutagenesis of Spt5 showed specific phenotypes during development, which were far less severe than those of Pol II defects or TBP deficient embryos. It seems paradoxical that a mutation which alters a general elongation factor can cause rather specific developmental defects. By using Spt5 knockdown zebrafish embryos and microarrays, here we showed that transcript abundance for only a small subset of genes is altered by loss of Spt5. Further investigation of the down-regulated genes showed that the genes most intensely repressed by the knockdown were strongly activated during early development in untreated embryos. Thus, this study shows that gene activation levels may create different requirements for Pol II processivity. Active transcription requires Spt5 for efficient elongation through its stimulatory activity on Pol II processivity. Keywords: Spt5, microarray analysis, development, transcription, transcription elongation
2007-10-25 | GSE6127 | GEO
Project description:Activation induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes. Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these key transactions little is known about how AID finds its target sites. To examine AID regulation we performed an shRNA screen. We found that Spt5, a factor associated with paused RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for class switch recombination (CSR). Spt5 interacts with AID, it is required for the association of AID and Pol II, and for AID recruitment to switch regions. ChIP-seq experiments reveal that Spt5 co-localizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II pausing is predictive of AID-induced mutation. Our data suggest that Spt5 acts as an adaptor, which brings AID to Pol II on target DNA. PolII, AID, and Spt5 were immunoprecipitated from in vitro activated B cells. For Spt5 and AID, two different antibodies were used in separate, biological replicates
2010-10-05 | E-GEOD-24178 | biostudies-arrayexpress
Project description:Class-switch recombination (CSR), induced by activation-induced cytidine deaminase, can be divided into two phases: DNA cleavage of the switch (S) regions, and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in CH12F3-2A cells, and Spt4 and Spt5, carry out independent functions in CSR. Depleting Spt5 reduced the H3K4me3 levels and DNA cleavage at the S? region, whereas Spt4 knockdown did not perturb the H3K4me3 status or S region cleavage. Thus, we conclude that Spt5 plays a role similar to the histone chaperone FACT by regulating histone modification and DNA cleavage in CSR. Assay systems using synthetic repair substrates revealed that both Spt4 and Spt5 are required for non-homologous end joining and homologous recombination. Taken together, Spt4 and Spt5 are involved in multiple CSR steps, and can function independently. Spt4 or Spt5 were knocked down in CH12F3-2A cells in the presence of CD40L, IL4, TGFb (CIT) stimulation. RNA from each samples were extracted and relative difference in transcript level were compared with control RNAi-treated, CIT-stimulated samples.
2013-10-22 | E-GEOD-33206 | biostudies-arrayexpress
Project description:The timely termination of RNA polymerase II (Pol II) transcription is essential for recycling of this enzyme and preventing transcriptional interference. While 5'-3' exonuclease Xrn2 is known to degrade nascent RNA after poly(A) site cleavage to promote Pol II release, its molecular mechanism remains not fully understood. Spt5 is one of highly conserved transcription factors that controls and regulates multiple aspects of Pol II transcription. Here, using mass spectrometry analysis of immunoprecipitated Flag-tagged Spt5-WT and Spt5-mutant complexes from fission yeast cells, we show that Xrn2 is highly enriched in these samples. Through in vitro BS3 crosslinking experiments with purified components, we further demonstrated that Xrn2 associates with transcription machinery and we probed interfaces between different subunits of transcription machinery
2024-11-22 | PXD058168 | Pride
Project description:The regulation of gene expression by RNA polymerase II (Pol II) is a multistep process requiring the concerted action of diverse transcription factors. Very little is known about in vivo function of transcription factor SPT5 as its deletion results in loss of viability. To circumvent this issue and define in vivo mechanism of action for SPT5, we employed acute degradation of SPT5 and studied its consequence on transcription. We find that SPT5 loss triggers degradation of the core Pol II subunit RPB1, a process which we show to be evolutionarily conserved from yeast to human. This RPB1 degradation requires the E3 ubiquitin ligase Cullin 3, the unfoldase VCP/p97 and a novel form of CDK9 kinase complex. SPT5 specifically stabilizes Pol II at promoter proximal regions, permitting Pol II release from promoters to gene bodies. Our findings provide mechanistic insight into the in vivo function of SPT5 in stabilization of promoter-proximal Pol II prior to release into gene bodies for safeguarding accurate gene expression.
2021-08-31 | GSE168827 | GEO
Project description:Many proteins associate with elongating RNA polymerase II (RNAPII), although the functions of most of them are still not well understood. Spt5 is an essential transcription elongation factor that binds directly to RNA polymerase and is conserved in prokaryotes, archaea, and eukaryotes1. In eukaryotes, evidence suggests that Spt5 functions in transcription elongation by both RNA polymerases I and II, mRNA capping, mRNA splicing, and mRNA 3’ end formation2. However, the genome-wide requirement for Spt5 in transcription has not been extensively studied. To address this issue, we have comprehensively analyzed the consequences of Spt5 depletion on transcription by RNAPII in Schizosaccharomyces pombe using four genome-wide approaches: ChIP-seq, NET-seq, 4tU-seq, and RNA-seq. Our results demonstrate that, in the absence of Spt5, RNAPII accumulates at a high level over the first ~500 bp of transcription units, suggesting that Spt5 is globally required to elongate past a barrier to transcription. This possibility is strongly supported by results showing that Spt5 is required for a normal level of RNA synthesis. In addition to these effects on sense-strand transcription, Spt5 depletion results in widespread convergent antisense transcription that initiates at approximately the same position as the sense-strand barrier. While the role of these antisense transcripts is unknown, the two that were tested control the distribution of RNAPII. Our results also show that Spt5 represses divergent antisense transcription, explaining why it has not been previously observed in S. pombe. Taken together, our results reveal a global and critical role for Spt5 in multiple classes of transcription by RNAPII.
2017-03-30 | GSE85181 | GEO