InteractiveFly: GeneBrief

Spt5: Biological Overview | References

Gene name - Spt5

Synonyms -

Cytological map position - 56D5-56D7

Function - chromatin factora

Keywords - DSIF, composed of Spt4 and Spt5, establishes the pause in transcription by recruiting NELF to the elongation complex - physically interacts with MYC oncoproteins and is essential for efficient transcriptional activation of MYC targets in cultured cells - Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation - Pho interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus - interacts directly with MSL1 and is required downstream of MSL complex for dosage compensation

Symbol - Spt5

FlyBase ID: FBgn0040273

Genetic map position - chr2R:19,437,483-19,441,898

NCBI classification - NGN_Euk: Eukaryotic N-Utilization Substance G (NusG) N-terminal (NGN) domain, KOW_Spt5_6

Cellular location - nuclear

NCBI links: EntrezGene, Nucleotide, Protein

GENE orthologs: Biolitmine

Promoter proximal pausing of RNA Polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires the transcription factor, DSIF (DRB sensitivity-inducing factor) and the negative elongation factor NELF. DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. This study used a highly purified in vitro system and Drosophila nuclear extract to investigate the role of DSIF in promoter proximal pausing. Two domains of Spt5 were identified, the KOW4 and NGN domains, that directly facilitate Pol II pausing. The KOW4 domain promotes pausing through its interaction with the nascent RNA while the NGN domain does so through a short helical motif that is in close proximity to the non-transcribed DNA template strand. Removal of this sequence in Drosophila has a male-specific dominant negative effect. The alpha helical motif is also needed to support fly viability. It was also shown that the interaction between the Spt5 KOW1 domain and the upstream DNA helix is required for DSIF association with the Pol II elongation complex. Disruption of the KOW1-DNA interaction is dominant lethal in vivo. Finally, the KOW2-3 domain of Spt5 was shown to mediate the recruitment of NELF to the elongation complex. In summary, these results reveal additional roles for DSIF in transcription regulation and identify specific domains important for facilitating Pol II pausing (Dollinger, 2023).

Eukaryotic transcription is a highly regulated process that depends on the precise spatiotemporal coordination of multiple interacting factors at each stage of the transcription cycle. Initiation, elongation, and termination have long been regarded as the primary canonical steps of this cycle. However, promoter proximal pausing of RNA polymerase II (Pol II) is now recognized as an additional critical post-initiation step in metazoan transcription. Promoter proximal pausing is characterized by an accumulation of Pol II ~30 to 60 nucleotides downstream of the transcription start site. This phenomenon was first observed as a concentration of transcriptionally engaged Pol II at the 5′ end of the beta-globin gene in nuclei from mature hen erythrocytes that were expected to be transcriptionally silent. Several subsequent studies led to the observation of similar phenomena on mammalian c-myc and HIV-1, as well as at non-induced Drosophila heat shock genes. The work by Gilmour and Lis on the Drosophila hsp70 gene established that a single Pol II molecule associates with the non-induced hsp70 gene ind the region between −12 and +65 and subsequent experiments demonstrated that this Pol II is transcriptionally engaged. Since then, genomic methods have provided overwhelming evidence that promoter proximal pausing is a ubiquitous step in the transcription cycle for most Drosophila and mammalian protein-coding genes. Pausing is associated with several critical regulatory functions, including developmental control and the maintenance of a nucleosome-free, permissive chromatin architecture around promoters (Dollinger, 2023).

Promoter proximal pausing requires Transcriptional inhibitor DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), two factors that function cooperatively to establish the pause. DSIF is a widely conserved eukaryotic transcription factor that associates with the elongation complex after the transcription of at least 18 nucleotides. The role of DSIF in pausing was first identified as an activity that rendered Pol II transcription sensitive to inhibition by the nucleoside analog 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). NELF was identified as an inhibitory factor that, together with DSIF, works to repress metazoan Pol II transcription. Release of the pause and the transition to productive elongation is thought to be mediated by the cyclin-dependent kinase positive transcription elongation factor b (P-TEFb; a dimer of Cyclin dependent kinase 9 and Cyclin T), which phosphorylates Pol II, DSIF, and NELF, resulting in the ejection of NELF from the elongation complex and the transformation of DSIF from a negative to a positive elongation factor (Dollinger, 2023).

A structure of the human paused elongation complex containing Pol II, DSIF, and NELF sheds light on the possible mechanisms by which NELF induces the pause . In this model, NELF stabilizes the formation of a half-translocated RNA-DNA duplex in the active site, preventing an incoming nucleotide from base pairing with the template. Furthermore, the interaction between NELF-C and the open Pol II trigger loop may interfere with trigger loop folding, which is needed to close off the active site and facilitate nucleotide addition. However, the role of DSIF in promoter proximal pausing has been less clear. DSIF is the lynchpin of the paused elongation complex because it is required to recruit NELF, but how the interactions between DSIF and the Pol II elongation complex contribute to pausing remains ambiguous. Several in vitro studies using highly purified systems indicate that on its own, DSIF either has no effect or a slight stimulatory effect on transcription. Hence, whether DSIF serves solely as an adapter that recruits regulators of elongation or itself contributes to pausing is an open question (Dollinger, 2023).

Of particular interest are the interactions between the Spt5 subunit and the nucleic acid scaffold. Spt5 has several domains, including unstructured N- and C-terminal regions, a NusG N-terminal (NGN) domain, and several Kyprides, Ouzounis, Woese (KOW) domains. Structures of the human elongation complex revealed that the NGN and KOW1 domains form part of the upstream DNA exit tunnel and that the KOW4 and KOW5 domains form a clamp around the nascent transcript. Comparison of the Spt5 conformations between cryo-EM structures of the paused and active elongation complexes highlights a repositioning of the KOW1 and KOW4 domains upon pause release, resulting in an opening of the nucleic acid clamps. Translocation of Pol II requires the movement of the nucleic acids through their respective exit channels. For Pol II to move along the DNA, the upstream DNA must be able to exit though the upstream DNA exit channel, the mouth of which is framed by the Spt5 DNA clamp, and the nascent transcript must exit through the Spt5 RNA clamp (Dollinger, 2023).

This study hypothesized that Spt5-nucleic acid interactions facilitate promoter proximal pausing by restricting the movement of the upstream DNA and nascent RNA through their exit channels. To test this hypothesis, DSIF mutants generated in which the charges of basic nucleic acid-interacting residues of Spt5 were reversed. To identify the pausing functions of the Spt5-nucleic acid contacts, a highly purified in vitro system was used to screen these mutants for Pol II binding and NELF recruitment. Each mutant's ability was tested to rescue promoter proximal pausing in Drosophila nuclear extract depleted of wild-type DSIF. It was found that the contacts between the KOW1 domain and the upstream DNA mediate the association of DSIF with the elongation complex; since DSIF binding to the elongation complex is a prerequisite for NELF recruitment, the KOW1-DNA interaction thus governs promoter proximal pausing indirectly. Furthermore, the expression of the Spt5 KOW1 mutant is lethal in Drosophila. In contrast, the interactions between the KOW4 domain and the nascent transcript directly facilitate promoter proximal pausing. A short helical motif in the NGN domain was identified that is critical to facilitating the pause. This sequence is highly conserved in eukaryotes that encode NELF but notably absent in eukaryotes that lack promoter proximal pausing and NELF. In flies, the replacement of this helical motif with homologous sequences from Saccharomyces cerevisiae and Caenorhabditis elegans results in a male-specific dominant negative effect. Spt5 NGN mutants also fail to support Drosophila viability when wild-type Spt5 has been depleted with RNAi. Taken together, these results provide a functional assessment of the various domains of Spt5 (Dollinger, 2023).

This work provides a functional assessment of the roles of various Spt5 domains in facilitating promoter proximal pausing. In addition to mediating interactions between NELF and the Pol II elongation complex, DSIF facilitates promoter proximal pausing through the KOW4 and NGN domains of the Spt5 subunit. The KOW4 domain interacts extensively with the nascent transcript; work from the Cramer group has shown that this domain switches from a 'closed' to an 'open' conformation when the elongation complex transitions from a paused state to an active state, suggesting that disengagement of the KOW4 domain from the RNA is a prerequisite for pause release. This work supports this hypothesis. Reversing the charge of KOW4 residues anticipated to interact with the RNA results in a pausing defect in Drosophila nuclear extract. Notably, this defect is accompanied by robust Pol II binding and NELF recruitment that is comparable to that of WT DSIF, indicating an effect mutations on Pol II pausing. Thus, the maintenance of the promoter proximal pause is likely dependent in part on the KOW4-RNA interaction, which is likely disrupted by the opening of the Spt5 RNA clamp (Dollinger, 2023).

The KOW4 domain's interaction with the nascent transcript may depend on the phosphorylation state of the linker region between the KOW4 and KOW5 domains. A previous study has shown that phosphorylation of this region by P-TEFb can act as a switch that determines whether Pol II enters productive elongation or prematurely terminates. Phosphorylation of the KOW4-5 linker on Ser666 by P-TEFb in human cells is associated with an increased proportion of Pol II in the gene body. This phosphorylation event may result in structuring of the flexible linker that forces the opening of the RNA clamp, allowing pause release. The KOW4-RNA interaction may also be mediated by NELF-E. The flexible NELF-E tentacle was shown to crosslink to the Spt5-KOW4 domain along the mouth of the RNA exit channel. Interaction with NELF may help stabilize the KOW4 domain in the 'closed' position, facilitating pausing (Dollinger, 2023).

Ectopic expression of the KOW4-Asp mutant in Drosophila did not have a dominant negative effect and the mutant was able to support viability in flies expressing Spt5 RNAi. This suggests that mutating the RNA-interacting residues of the KOW4 domain may not be sufficient to fully disrupt the promoter proximal pause in vivo. Additional contacts provided by the Spt5 NGN domain, NELF, and other factors such as nucleosomes present a much more complex regulatory context than the one reconstituted using Drosophila nuclear extract, which could account for the apparent discrepancy between the current in vitro and in vivo results (Dollinger, 2023).

The Spt5 NGN domain also plays a significant role in pausing. Replacement of a short helical motif in the Drosophila NGN domain with homologous unstructured loop regions from yeast or worms results in a severe pausing defect while leaving Pol II binding and NELF recruitment functions intact. This is the first report of a role for the NGN domain in transcriptional pausing in a eukaryotic system. The possible function was explored of a conserved arginine, hR246(dR283), that was oriented to interact with the non-transcribed template strand. Though no difference was observed in pausing activity between our NGN-K.p. and NGN-K.p._R mutants in nuclear extract, re-insertion of the arginine had a dramatic effect in flies. The NGN-K.p._R mutant had a less severe dominant negative effect than its NGN-K.p. counterpart, indicating that the conserved arginine residue is critical to the NGN domain's function. Neither the NGN-S.c., NGN-K.p., nor the NGN-K.p._R mutants were able to support Drosophila viability when expressed in the presence of Spt5 RNAi, indicating that the full NGN alpha-helical motif is necessary for proper fly development (Dollinger, 2023).

Experiments in Bacillus subtilis previously described RNA polymerase pausing mediated by the interaction of the NGN domain of the bacterial homolog NusG with the non-transcribed DNA in the transcription bubble. However, unlike in Drosophila, this process is dependent on the presence of a DNA sequence motif and does not involve a helical motif similar to what is described in this study. Indeed, the alpha helical motif appears to be exclusive to NELF-encoding eukaryotes, though the conserved hR246 (dR283) residue also appears in archaeal species. Available structures of archaeal Spt5 indicate that this arginine is located in a beta strand rather than the alpha helix found in metazoans. This beta strand is also present in E. coli and in B. subtilis, but both these species lack the conserved arginine found in NELF-encoding eukaryotes and archaea. Notably, in archaea, the NGN domain is required for stimulation of elongation, suggesting that the function of the conserved arginine is context-dependent (Dollinger, 2023).

The NGN domain is highly conserved across all domains of life and exhibits significant structural similarity from species to species. Paradoxically, the function of this domain is varied. In some cases, such as E. coli, archaea, and S. cerevisiae, the NGN domain stimulates elongation, but in B. subtilis and Drosophila, the NGN domain promotes pausing. It is proposed that the DNA-interacting region of the NGN domain is a subdomain that has evolved to serve different functions in various species. This may explain how the highly conserved NGN domain can serve as both a stimulator and a repressor of transcription. Ectopic expression of the NGN-S.c. and NGN-K.p. mutants greatly inhibited the development of adult male flies. In Drosophila, the NGN domain may also promote dosage compensation by stimulating the upregulation of genes on the single male X chromosome. Spt5 has been shown to interact with the dosage compensation factor male-specific lethal (MSL1) through the NGN domain as well as through the KOW domains. Though the mechanisms of this interaction are unknown, it is possible that mutations of the NGN domain described in this study disrupted either the association between Spt5 and MSL1 or their joint function, resulting in the male-specific dominant negative effect that was observed. The NGN domain's non-transcribed-DNA-interacting region is likely a hotspot for regulating Pol II processivity, making it a logical target for transcription regulation by MSL1 (Dollinger, 2023).

The NGN mutations described in this study may have also disrupted the function of RNA polymerase I (Pol I). Mass spectrometry and immunoprecipitation experiments in yeast demonstrated that Pol I is able to associate with Spt4/5 and later genetic studies demonstrated that Spt5 regulates Pol I transcription. This interaction is mediated at least in part by the Spt5 NGN domain. Thus, it is possible that replacing the NGN helical motif in vivo disrupted not only the processivity of Pol II but also the processivity of Pol I, dysregulating the synthesis of ribosomal RNA. Such a substantial disruption would account not only for the failure of the NGN mutants to support Drosophila viability but could also explain the dominant lethality of the KOW1-Asp mutant given that the KOW1 and NGN domains together form the DNA clamp (Dollinger, 2023).

This study also demonstrated that disrupting the interaction between the Spt5 KOW1 domain and the upstream DNA results in impaired binding of DSIF to the Pol II elongation complex. This is in agreement with previous work in yeast, which showed that deletion of this domain reduced the affinity of Spt5 for the elongation complex. The KOW1 domain is the only KOW domain conserved across all three domains of life, so its role in Pol II elongation complex binding is likely a conserved feature in Spt5 and Spt5 homologs. Ectopic expression of the KOW1-Asp mutant in Drosophila had a dominant lethal effect, highlighting the importance of this region. In addition to facilitating Spt5-Pol II interaction, the KOW1 domain also ensures physical separation of the upstream DNA and the transcript, potentially preventing the formation of irregular structures such as R-loops, which have been linked to genome instability (Dollinger, 2023).

Surprisingly, no elongation complex binding defect was observed in the KOW4-Asp mutant, suggesting that the interaction between the KOW4 domain and the nascent transcript is not necessary for DSIF-Pol II binding. This is in contrast to previous studies in yeast and Drosophila. In yeast, digestion of the nascent transcript with RNaseI nearly eliminated Spt5 binding to the elongation complex. Moreover, a prior study showed that DSIF failed to bind to Pol II elongation complexes that had transcripts shorter than 18 nucleotides. However, because varying the transcript length in these elongation complexes also resulted in varying the length of the upstream DNA, the decrease in DSIF binding could be attributed to reduced interaction between the DNA template and the Spt5 KOW1 domain rather than loss of the KOW4-transcipt interaction. Complexes with 18 nt transcripts only have ~4 base pairs of double-stranded upstream DNA extending out of the Pol II; based on the structures of the human elongation complex, association with the KOW1 domain requires at least ~10 bp of upstream DNA (Dollinger, 2023).

The mechanism of Pol II-DSIF association may nevertheless rely on multiple contact points. While this study has shown that the KOW1 domain is necessary for initial Pol II elongation complex-DSIF binding, recent structural experiments from the demonstrated that Spt5 can be retained on the elongation complex despite the displacement of the KOW1 and NGN domains and Spt4. This suggests that the RNA clamp formed by the KOW4 and KOW5 domains may function to preserve the Pol II-DSIF interaction after the initial association. Furthermore, though no effect of oSpt5 KOW2-3 domain mutations on Pol II binding was seen, it is possible that this region also plays a stabilizing role that helps maintain the association of DSIF with the elongation complex during various conformational transitions (Dollinger, 2023).

Of the nine DSIF mutants described in this study, all but one were able to bind NELF to a degree that was comparable to WT DSIF. This is perhaps unsurprising since the mutations in the Spt5 NGN and KOW1 domains are not located near the modeled paths of the NELF-A and NELF-E tentacles. Moreover, no effect was observed on NELF binding by the mutations in the Spt5 KOW4 domain. The NELF-E C-terminal tentacle is thought to stretch across the mouth of the RNA exit channel between the nascent transcript and the KOW4 domain, so it seemed likely that disrupting the contact between the Spt5 domain and the RNA would also disturb the NELF-E interaction. Nevertheless, the observation is in line with that of a previous study that deleted the NELF-E tentacle and failed to see an effect on pausing in vitro. Furthermore, mutating a pocket of residues (Spt4 R79, R82, K109) in close proximity to a putative contact point between Spt4 and NELF-A that was previously identified by crosslinking mass spectrometry had no effect on NELF recruitment, suggesting that crosslinking results must be interpreted with caution and followed up with biochemical analyses, particularly with regards to intrinsically disordered regions such as the NELF-A tentacle. It is possible that NELF recruitment is mediated in part by Spt4-NELF-A interaction, but verifying this will require careful and systematic biochemical assessment of both Spt4 and the NELF-A C-terminus. Previous biochemical data suggests that deletion of the NELF-A tentacle impairs Pol II pausing in vitro, so future work to interrogate the intrinsically disordered regions of this subunit will be necessary for a complete mechanistic description of NELF recruitment to the elongation complex (Dollinger, 2023).

Mutating the KOW2-3 domain of Spt5 reduced NELF binding in the in vitro system. The KOW2-3 domain is located near the modeled path of the NELF-E N-terminal region and has the greatest number of putative NELF-E contacts. Notably, NELF binding was not completely abolished and could be restored by adding greater quantities of NELF. Moreover, the KOW2-3-S.c. mutant exhibited no dominant negative effect when expressed in flies and was able to rescue the effects of RNAi knockdown of endogenous Spt5, suggesting that while the KOW2-3 domain contributes for NELF recruitment to the elongation complex, the mutations made in this study did not interfere with development. Recent work in human cells showed that the formation of biomolecular condensates mediated by the NELF-A tentacle enhances the recruitment of NELF to promoters. It is possible a similar phenomenon occurs at Drosophila promoters, resulting in a cellular concentration of NELF that is sufficient to overcome the defect of the KOW2-3-S.c. mutant. Interactions between NELF and Spt4, NELF and Pol II, as well as NELF and the nucleic acid scaffold likely serve as additional stabilizing contact points and may even drive the initial recruitment of the NELF complex (Dollinger, 2023).

This study performed an extensive analysis of the domains of the larger DSIF subunit, Spt5, and showed that the NGN and KOW4 domains facilitate pausing in a manner distinct from the role of DSIF as the mediator of NELF-Pol II interaction. It was also shown that the KOW1 domain facilitates DSIF binding to the Pol II elongation complex and that the KOW2-3 domain contributes to NELF recruitment (Dollinger, 2023).

Coregulators Reside within Drosophila Ecdysone-Inducible Loci before and after Ecdysone Treatment

Ecdysone signaling in Drosophila remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind ecdysone-response elements with or without the presence of a hormone. For years, EcR enhancers were thought to respond to ecdysone via recruiting coactivator complexes, which replace corepressors and stimulate transcription. However, the exact mechanism of transcription activation by ecdysone remains unclear. This study presents experimental data on 11 various coregulators at ecdysone-responsive loci of Drosophila S2 cells. The regulatory elements where coregulators reside within these loci are described and changes in their binding levels following 20-hydroxyecdysone treatment are assessed. In the current study, the presence was detected of some coregulators at the TSSs (active and inactive) and boundaries marked with CP190 rather than enhancers of the ecdysone-responsive loci where EcR binds. Minor changes were observed in the coregulators' binding level. Most were present at inducible loci before and after 20-hydroxyecdysone treatment. The findings suggest that: (1) coregulators can activate a particular TSS operating from some distal region (which could be an enhancer, boundary regulatory region, or inactive TSS); (2) coregulators are not recruited after 20-hydroxyecdysone treatment to the responsive loci; rather, their functional activity changes (shown as an increase in H3K27 acetylation marks generated by CBP/p300/Nejire acetyltransferase). Taken together, these findings imply that the 20-hydroxyecdysone signal enhances the functional activity of coregulators rather than promoting their binding to regulatory regions during the ecdysone response (Krasnov, 2023).

According to previous results, the Spt5 subunit of DSIF was found at TSSs of ecdysone-inducible genes before and after S2 cells were treated with 20-hydroxyecdysone. Spt5 binding levels at EcR-bound enhancers was not detected. Presumably, Spt5 recruits to promoters directly by RNA polymerase II binding. In the eip74ef locus, it was found that Spt5 was present both at induced and uninduced TSSs, indicating that its presence at the promoter is not sufficient to activate transcription (Krasnov, 2023).

PAF1 and cdk8 showed a similar binding pattern to ecdysone-dependent regulatory regions. They were present at both TSSs and enhancers and only slightly increased the binding level after induction. Similar to Spt5, PAF1 intensively binds to uninducible TSSs at the eip74ef locus, showing possible involvement in its suppression. In addition to TSSs and enhancers, cdk8 demonstrates substantial binding levels at the CP190 boundary of the eip74ef locus. Therefore, it is possible to incorporate a new member into the boundary-associated complex of coregulators involved in the ecdysone response. Transcription is activated with ecdysone by stimulating elongation; however, all studied coregulatory complexes involved in elongation were found to be stably associated with ecdysone-responsive loci. This leaves two options: to find an as-of-yet unknown elongation regulator that is recruited to ecdysone-responsive loci upon activation or to accept that ecdysone stimulates not recruitment but changes in the functional activity of already associated complexes, causing transcriptional elongation (Krasnov, 2023).

Spt5 interacts genetically with Myc and is limiting for brain tumor growth in Drosophila

The transcription factor SPT5 physically interacts with MYC oncoproteins and is essential for efficient transcriptional activation of MYC targets in cultured cells. This study used Drosophila to address the relevance of this interaction in a living organism. Spt5 displays moderate synergy with Myc in fast proliferating young imaginal disc cells. During later development, Spt5-knockdown has no detectable consequences on its own, but strongly enhances eye defects caused by Myc overexpression. Similarly, Spt5-knockdown in larval type 2 neuroblasts has only mild effects on brain development and survival of control flies, but dramatically shrinks the volumes of experimentally induced neuroblast tumors and significantly extends the lifespan of tumor-bearing animals. This beneficial effect is still observed when Spt5 is knocked down systemically and after tumor initiation, highlighting SPT5 as a potential drug target in human oncology (Hofstetter, 2024).

The negative elongation factor NELF promotes induced transcriptional response of Drosophila ecdysone-dependent genes
For many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in Drosophila development by controlling spatiotemporal properties of their activation and repression. But the exact proteins that cooperate to stall Pol II in promoter-proximal regions of developmental genes are still largely unknown. The current work describes the molecular mechanism employed by the Negative ELongation Factor (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to the current data, the NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with observed interaction between the NELF-A subunit and the ecdysone receptor (EcR). NELF depletion causes a significant decrease in transcription induced by 20E, which is associated with the disruption of Pol II elongation complexes. A considerable reduction in the promoter-bound level of the Spt5 subunit of transcription elongation factor DSIF was observed at the 20E-dependent genes upon NELF depletion. It is presumed that an important function of NELF is to participate in stabilizing the Pol II-DSIF complex, resulting in a significant impact on transcription of its target genes. In order to directly link NELF to regulation of 20E-dependent genes in development, this study shows the presence of NELF at the promoters of 20E-dependent genes during their active transcription in both embryogenesis and metamorphosis. This study also demonstrates that 20E-dependent promoters, while temporarily inactive at the larval stage, preserve a Pol II paused state and bind NELF complex (Mazina, 2021).

Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination

Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, it was reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. This study reports that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases (Huang, 2020).

Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus

Numerous target genes of the Polycomb group (PcG) are transiently activated by a stimulus and subsequently repressed. However, mechanisms by which PcG proteins regulate such target genes remain elusive. This study employed the heat shock-responsive hsp70 locus in Drosophila to study the chromatin dynamics of PRC1 and its interplay with known regulators of the locus before, during and after heat shock. Mutually exclusive binding patterns were detected for HSF and PRC1 at the hsp70 locus. Pleiohomeotic (Pho), a DNA-binding PcG member, dynamically was found to interact with Spt5, an elongation factor. The dynamic interaction switch between Pho and Spt5 is triggered by the recruitment of HSF to chromatin. Mutation in the protein-protein interaction domain (REPO domain) of Pho interferes with the dynamics of its interaction with Spt5. The transcriptional kinetics of the heat shock response is negatively affected by a mutation in the REPO domain of Pho. It is proposed that a dynamic interaction switch between PcG proteins and an elongation factor enables stress-inducible genes to efficiently switch between ON/OFF states in the presence/absence of the activating stimulus (Pereira, 2017).

Identification of Regions in the Spt5 Subunit of DRB Sensitivity-inducing Factor (DSIF) That Are Involved in Promoter-proximal Pausing

DRB sensitivity-inducing factor (DSIF or Spt4/5) is a conserved transcription elongation factor that both inhibits and stimulates transcription elongation in metazoans. In Drosophila and vertebrates, DSIF together with negative elongation factor (NELF) associates with RNA polymerase II during early elongation and causes RNA polymerase II to pause in the promoter-proximal region of genes. The mechanism of how DSIF establishes pausing is not known. Spt5 mutant forms of DSIF were constructed and their capacity to restore promoter-proximal pausing to DSIF-depleted Drosophila nuclear extracts was tested. The C-terminal repeat region of Spt5, which has been implicated in both inhibition and stimulation of elongation, is dispensable for promoter-proximal pausing. A region encompassing KOW4 and KOW5 of Spt5 is essential for pausing, and mutations in KOW5 specifically shift the location of the pause. RNA cross-linking analysis reveals that KOW5 directly contacts the nascent transcript, and deletion of KOW5 disrupts this interaction. These results suggest that KOW5 is involved in promoter-proximal pausing through contact with the nascent RNA (Qiu, 2017).

CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription

CTCF is a versatile transcription factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of elongation by RNA polymerase (pol) II. This study shows that CTCF knockdown abrogates pol II pausing at the early elongation checkpoint of c-myc by affecting recruitment of DRB-sensitivity-inducing factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of negative elongation factor (NELF). In addition, CTCF is required for recruitment of positive elongation factor b (P-TEFb), which phosphorylates NELF, DSIF and Ser2 of the pol II CTD to activate elongation of transcription of c-myc and recognition of the snRNA gene-specific 3' box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling pol II transcription through the elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes (Laitem, 2015).

Pleiohomeotic interacts with the core transcription elongation factor Spt5 to regulate gene expression in Drosophila

The early elongation checkpoint regulated by Positive Transcription Elongation Factor b (P-TEFb) is a critical control point for the expression of many genes. Spt5 interacts directly with RNA polymerase II and has an essential role in establishing this checkpoint, and also for further transcript elongation. This study demonstrates that Drosophila Spt5 interacts both physically and genetically with the Polycomb Group (PcG) protein Pleiohomeotic (Pho), and the majority of Pho binding sites overlap with Spt5 binding sites across the genome in S2 cells. These results indicate that Pho can interact with Spt5 to regulate transcription elongation in a gene specific manner (Harvey, 2013).

Mutations in the transcription elongation factor SPT5 disrupt a reporter for dosage compensation in Drosophila

In Drosophila, the MSL (Male Specific Lethal) complex up regulates transcription of active genes on the single male X-chromosome to equalize gene expression between sexes. One model argues that the MSL complex acts upon the elongation step of transcription rather than initiation. In an unbiased forward genetic screen for new factors required for dosage compensation, this study found that mutations in the universally conserved transcription elongation factor Spt5 lower MSL complex dependent expression from the miniwhite reporter gene in vivo. SPT5 interacts directly with MSL1 in vitro and is required downstream of MSL complex recruitment, providing the first mechanistic data corroborating the elongation model of dosage compensation (Prabhakaran, 2012).

Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex

Negative elongation factor (NELF) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) are involved in pausing RNA Polymerase II (Pol II) in the promoter-proximal region of the hsp70 gene in Drosophila, before heat shock induction. Such blocks in elongation are widespread in the Drosophila genome. However, the mechanism by which DSIF and NELF participate in setting up the paused Pol II remains unclear. The interactions were analyzed among DSIF, NELF, and a reconstituted Drosophila Pol II elongation complex to gain insight into the mechanism of pausing. The results show that DSIF and NELF require a nascent transcript longer than 18 nt to stably associate with the Pol II elongation complex. Protein-RNA cross-linking reveals that Spt5, the largest subunit of DSIF, contacts the nascent RNA as the RNA emerges from the elongation complex. Taken together, these results provide a possible model by which DSIF binds the elongation complex via association with the nascent transcript and subsequently recruits NELF. Although DSIF and NELF were both required for inhibition of transcription, no NELF-RNA contact is detected when the nascent transcript was between 22 and 31 nt long, which encompasses the region where promoter-proximal pausing occurs on many genes in Drosophila. This raises the possibility that RNA binding by NELF is not necessary in promoter-proximal pausing (Missra, 2010).

DSIF and NELF are key factors in pausing Pol II in the promoter-proximal region of genes in Drosophila and human cells. To gain insight into the mechanism by which DSIF and NELF contribute to promoter-proximal pausing, a system was developed in which the physical interaction of DSIF and NELF with a Pol II elongation complex could be monitored using a native gel electrophoresis assay. Previously, it was demonstrated that DSIF alone could associate with the Pol II elongation complex. In this study a method was developed to purify Drosophila NELF, thus allowing exploration of the interplay of DSIF and NELF with the elongation complex (Missra, 2010).

The results show that the association of NELF with the elongation complex is dependent on the presence of DSIF. Previous work provided evidence that NELF associated with preformed complexes of DSIF and Pol II in nuclear extracts but the interaction of DSIF and Pol II was not dependent on NELF. These interactions were likely occurring outside the context of an elongation complex and were relatively weak because the bulk of DSIF, NELF, and Pol II exist independent of each other in nuclear extracts. In contrast, the current results show that NELF can significantly influence the binding of DSIF to Pol II within the context of an elongation complex when limiting amounts of DSIF are present. Since Pol II, DSIF, and NELF have been shown to interact individually with each other, it is likely that this network of interactions contributes to stable association of these proteins in the context of the elongation complex (Missra, 2010).

The binding assays show that the length of the nascent transcript affects the association of DSIF and NELF with the elongation complex. While binding of DSIF alone or in combination with NELF to the elongation complex was evident for an elongation complex with a nascent transcript of 22 nt, no binding was detected when the nascent transcript was 18 nt long. These results are consistent with the finding that human DSIF and NELF require transcripts ≥18 nt long to inhibit transcription, and also a recent study showed human DSIF preferentially bound elongation complexes containing transcripts that were at least 25 nt long. The 5′ end of an 18-nt-long nascent transcript just begins to emerge from the surface of Pol II. Exposure of four additional nucleotides appears to be sufficient for binding of DSIF alone or with NELF. Notably, the association of DSIF with the elongation complex is not simply due to nonspecific interaction with the RNA or DNA because previous experiments show that binding of DSIF to the elongation complex requires specific contacts with Pol II (Missra, 2010).

One way in which nascent transcript length could affect the association of DSIF and NELF is by providing an additional binding site in the elongation complex. Previous results have directed attention at an RRM in NELF-E. Mutations in this RRM impair the capacity of NELF to inhibit elongation in the presence of DSIF. However, these experiments focused on elongation over distances greater than 100 nt. The finding that DSIF associates with elongation complex containing a 22-nt-long radioactive transcript (EC22) but not EC18 suggests that DSIF rather than NELF might be interacting with the nascent transcript, and RNA-protein cross-linking data support this hypothesis. The Spt5 subunit of Drosophila and human DSIF contains five Kyprides, Ouzounis, Woese (KOW) domains. An isolated KOW domain from Aquifex aeolicus NusG has been shown to associate with RNA, so it is possible that one of these domains in Spt5 is contacting the nascent transcript as it emerges from the elongation complex (Missra, 2010).

Cross-linking analysis detected contact between NELF-E and the nascent transcript in EC70 but not in EC31. The 5′ end of the nascent transcript contacts Rpb7 when its length is between 26 to 32 nt. Therefore it is possible that a longer nascent transcript is required to allow contact with NELF. Given that promoter-proximal pausing can occur before Pol II transcribes 30 nt, it is proposed that the RRM of NELF-E is not involved in promoter-proximal pausing. Its role could be limited to processes involving longer nascent transcripts such as regulation that appears to involve the transactivation response element of HIV or 3′ end formation of histone mRNAs (Missra, 2010).

The finding that DSIF and NELF associate with EC22 but not with EC18 is very relevant to the process of promoter-proximal pausing. Permanganate genomic footprinting of over 60 different promoters reveals that Pol II pauses in the promoter-proximal region 20 to 50 nt downstream from the transcription start site. Those cases where the Pol II appeared to be pausing closer to a transcription start site were found to have the start sites inaccurately mapped. Thus, the promoter-proximal limit for the range where Pol II pauses is likely to be dictated by the minimum length of RNA required for DSIF to associate with the elongation complex (Missra, 2010).

From the results presented in this study, it is proposed that the first step in promoter-proximal pausing involves binding of DSIF to the nascent transcript. NELF subsequently associates to form a stable complex. Importantly, this complex alone is not sufficient to stably pause the Pol II as the results show that elongation is slowed but not halted in reactions involving only these three proteins. Hence other factors that remain to be identified are likely to act in concert with this core complex of DSIF, NELF, and Pol II to stably pause Pol II in the promoter-proximal region of genes. Since transcription in vivo occurs on chromatin, nucleosomes may cooperate with DSIF and NELF in setting up the paused polymerase. The experimental approach described in this study could serve as a way to identify additional factors involved in pausing (Missra, 2010).

Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo

Spt5 is a conserved essential protein that represses or stimulates transcription elongation in vitro. Immunolocalization studies on Drosophila polytene chromosomes suggest that Spt5 is associated with many loci throughout the genome. However, little is known about the prevalence and identity of Spt5 target genes in vivo during development. This study identifies direct target genes of Spt5 using fogsk8 zebrafish mutant, which disrupts the foggy/spt5 gene. fogsk8 and their wildtype siblings differentially express less than 5% of genes examined. These genes participate in diverse biological processes from stress response to cell fate specification. Up-regulated genes exhibit shorter overall gene length compared to all genes examined. Through chromatin immunoprecipitation in zebrafish embryos, a subset of developmentally critical genes that are bound by both Spt5 and RNA polymerase II. The protein occupancy patterns on these genes are characteristic of both repressive and stimulatory elongation regulation. Together these findings establish Spt5 as a dual regulator of transcription elongation in vivo and identify a small but diverse set of target genes critically dependent on Spt5 during development (Krishnan. 2008).

NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila

Transcriptional elongation regulators NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF (FlyBase term: Spt5) from a Drosophila nuclear extract reduces the level of polymerase that pauses in the promoter proximal region of hsp70. Depletion of one Negative elongation factor E (NELF) subunit in salivary glands using RNA interference also reduces the level of paused polymerase. In vivo protein-DNA cross-linking shows that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborates the cross-linking result and shows that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF are strongly recruited to chromosomal puffs harboring the hsp70 genes. It is proposed that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation (Wu, 2003).

It is proposed that promoter proximal pausing occurs when the nascent transcript emerges from the RNA exit channel of the Pol II and is grabbed by the NELF-E subunit. Tethering of the NELF-E to the elongation complex would generate a rigid body that could restrict the movement of the Pol IIa. This model is supported by several observations. The paused polymerase is in the Pol IIa state, and NELF and DSIF only inhibit elongation by Pol IIa. In vitro transcription analysis indicates that the elongation complex is not receptive to inhibition by NELF and DSIF until the nascent transcript is ~30 nucleotides long. This length coincides approximately to the distance polymerase elongates on hsp70 before it pauses. In vitro transcription analyses indicate that DSIF and NELF associate with polymerase shortly after initiation but probably before the polymerase reaches the region of pausing. Finally, NELF-E has an RNA-binding motif that is essential for its inhibitory action (Wu, 2003 and references therein).

Although NELF and DSIF are sufficient to slow the elongation rate of purified Pol IIa, it is suspected that additional proteins are involved in stably pausing Pol II on the hsp70 promoter. In cell-free transcription reactions done with other promoters, the pausing caused by DSIF and NELF appears to be transient -- the polymerase eventually moves forward if given enough time. In contrast, several observations indicate that the Pol II on hsp70 is stably paused. The paused Pol II remains associated with the hsp70 promoter when nuclei are isolated from uninduced cells, and sarkosyl or high salt must accompany addition of nucleotides to cause the Pol II to resume elongation. In a cell-free system, Pol II remains stably paused on the hsp70 promoter for at least 30 min. GAGA factor might be involved in stabilizing the pause because mutations in the GAGA element result in a loss of paused Pol II (Wu, 2003).

Heat shock rapidly induces transcription as a result of the association of HSF with sites located upstream from the TATA element. The data suggest that HSF may activate transcription in part by causing NELF to dissociate from the Pol II. How HSF might cause the release of NELF is unclear. Phosphorylation of Pol IIa is likely to be an important step because the Pol II found in the body of the gene during heat shock is hyperphosphorylated. Phosphorylation of DSIF is another possibility as this has been observed to occur early in elongation in vitro. It is also unclear which kinase might be responsible for phosphorylating the Pol II. P-TEFb (see Cdk9) is a candidate because it associates with the hsp70 gene during heat shock induction, and HSF can be bypassed by directing a Gal4/P-TEFb fusion protein to the hsp70 promoter. No interaction, however, has been detected between P-TEFb and HSF. Recent results show that HSF associates with the mediator. Drosophila mediator contains a kinase that phosphorylates the CTD, and phosphorylation can occur synergistically with the TFIIH kinase. Perhaps HSF recruits the mediator and in turn the mediator releases the paused polymerase by phosphorylating the CTD (Wu, 2003).

The strong immunofluorescence staining observed for DSIF at heat shock loci during heat shock indicates that DSIF is associated with many of the polymerase molecules transcribing the gene. RNA polymerase initiates at a rate of once every few seconds during heat shock resulting in a train of elongation complexes traversing the gene. In the absence of NELF, DSIF might act as a positive elongation factor. Shortly after DSIF was discovered, another investigation identified DSIF as a cofactor required for reconstituting tat-dependent transcription. In this situation, DSIF appears to be stimulating elongation. DSIF has been found in a complex with another positive elongation factor called Tat-SF1. Tat-SF1 was first identified as a stimulatory factor for Tat, but subsequent results indicate that Tat-SF1 may promote elongation on cellular genes. In yeast, DSIF appears to act as either a positive or negative regulator of elongation depending on circumstances. A hypothesis that unites the positive and negative activities of DSIF considers this factor an adaptor that connects other modulators to the elongation complex. In this regard, DSIF has been shown to bind on its own to Pol II, whereas the stable association of NELF with Pol II requires the presence of DSIF (Wu, 2003 and references therein).

NELF and DSIF appear to associate with several hundred interbands in polytene chromosomes. Each interband could contain many genes. The weak staining of interbands by Hoecsht suggests that the DNA in the interbands is in a decondensed state. Residing in these decondensed regions could be genes whose primary control mechanism does not involve a disruption of chromatin structure or even assembly of the initiation complex. Instead, alleviating repression by NELF and DSIF could underlie the mechanism of activation (Wu, 2003).

High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation

Recent studies have demonstrated roles for Spt4, Spt5, and Spt6 in the regulation of transcriptional elongation in both yeast and humans. This study showed that Drosophila Spt5 and Spt6 colocalize at a large number of transcriptionally active chromosomal sites on polytene chromosomes and are rapidly recruited to endogenous and transgenic heat shock loci upon heat shock. Costaining with antibodies to Spt6 and to either the largest subunit of RNA polymerase II or cyclin T, a subunit of the elongation factor P-TEFb, reveals that all three factors have a similar distribution at sites of active transcription. Crosslinking and immunoprecipitation experiments show that Spt5 is present at uninduced heat shock gene promoters, and that upon heat shock, Spt5 and Spt6 associate with the 5' and 3' ends of heat shock genes. Spt6 is recruited within 2 minutes of a heat shock, similar to heat shock factor (HSF); moreover, this recruitment is dependent on HSF. These findings provide support for the roles of Spt5 in promoter-associated pausing and of Spt5 and Spt6 in transcriptional elongation in vivo (Andrulis, 2000).

Functions of Spd5 orthologs in other species

RBM22 regulates RNA polymerase II 5' pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5

Splicing factors are vital for the regulation of RNA splicing, but some have also been implicated in regulating transcription. The underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood. This paper describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity, and provokes transcriptional readthrough genome-wide, coupled with production of transcripts containing sequences from downstream of the gene. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level. These results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control (Du, 2024).

Structural basis of exoribonuclease-mediated mRNA transcription termination

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II). This study reports two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. The structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. These results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes (Zeng, 2024).

Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription

Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements. The Restrictor complex, composed of the Pol II-interacting protein WDR82 and the RNA-binding protein ZC3H4, suppresses processive transcription at thousands of extragenic sites in mammalian genomes. Restrictor-driven termination does not involve nascent RNA cleavage, and its interplay with other termination machineries is unclear. This study shows that efficient termination at Restrictor-controlled extragenic transcription units involves the recruitment of the protein phosphatase 1 (PP1) regulatory subunit PNUTS, a negative regulator of the SPT5 elongation factor, and Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent and phosphatase-dependent termination of noncoding RNAs in yeast. PNUTS and Symplekin act synergistically with, but independently from, Restrictor to dampen processive extragenic transcription. Moreover, the presence of limiting nuclear levels of Symplekin imposes a competition for its recruitment among multiple transcription termination machineries, resulting in mutual regulatory interactions. Hence, by synergizing with Restrictor, Symplekin and PNUTS enable efficient termination of processive, long-range extragenic transcription (Russo, 2023).

How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes

Interactions between RNA guanylyltransferase (GTase) and the C-terminal domain (CTD) repeats of RNA polymerase II and elongation factor Spt5 are thought to orchestrate cotranscriptional capping of nascent mRNAs. The crystal structure of a fission yeast GTase*Pol2 CTD complex reveals a unique docking site on the nucleotidyl transferase domain for an 8-amino-acid Pol2 CTD segment, S5PPSYSPTS5P, bracketed by two Ser5-PO4 marks. Analysis of GTase mutations that disrupt the Pol2 CTD interface shows that at least one of the two Ser5-PO4-binding sites is required for cell viability and that each site is important for cell growth at 37 degrees C. Fission yeast GTase binds the Spt5 CTD at a separate docking site in the OB-fold domain that captures the Trp4 residue of the Spt5 nonapeptide repeat T(1)PAW(4)NSGSK. A disruptive mutation in the Spt5 CTD-binding site of GTase is synthetically lethal with mutations in the Pol2 CTD-binding site, signifying that the Spt5 and Pol2 CTDs cooperate to recruit capping enzyme in vivo. CTD phosphorylation has opposite effects on the interaction of GTase with Pol2 (Ser5-PO4 is required for binding) versus Spt5 (Thr1-PO4 inhibits binding). It is proposed that the state of Thr1 phosphorylation comprises a binary 'Spt5 CTD code' that is read by capping enzyme independent of and parallel to its response to the state of the Pol2 CTD (Doamekpor, 2014).

Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes
Earlier work has shown that siRNA-mediated reduction of the SUPT4H (Drosophila Spt4) or SUPT5H (Drosophila Spt5) proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, this study identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction, and then their effects were investigated on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (HTT), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). This study reports that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila 'rough eye' phenotype associated with degeneration of photoreceptor neurons in vivo were observed. These findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions (Deng, 2022).

Expansion of the number of contiguous nucleotide repeats that normally exist within certain human genes is the cause of multiple human diseases. Earlier work has shown that expression of alleles containing nucleotide repeat expansions can be reduced differentially by inhibiting production of SUPT4H or SUPT5H, highly conserved cellular proteins that interact to form the transcription elongation complex, DSIF (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor). DSIF assists in the elongation of mRNA molecules by attaching to RNA polymerase II (RNAPII) via an SUPT5H binding site and forming a structural clamp that maintains RNAPII occupancy of template DNA as the polymerase proceeds along the template . A decrease in production or function of SUPT4H or SUPT5H has been found to decrease synthesis of transcripts encoded by genes containing nucleotide repeat expansions including HTT, the gene that causes Huntington's Disease, the C9orf72 locus associated with amyotrophic lateral sclerosis and frontotemporal dementia, and NOP56, the gene associated with spinocerebellar atrophy type 36 (SCA36), and it has been suggested that SUPT4H or SUPT5H may be a target for treatment of certain diseases caused by nucleotide repeat expansions. As interaction between SUPT4H and SUPT5H to form the DSIF complex is required for these proteins to form the structural clamp that maintains RNAPII on DNA template, this study sought to identify compounds that interfere with the SUPT4H-SUPT5H interaction and to elucidate their effects on mutant HTT gene products. This study describes the results of experiments aimed at: 1) identifying chemicals that can interfere with the SUPT4H/5H interaction, 2) determining whether chemical interference with the interaction recapitulates the effects of decreasing SUPT4H or SUPT5H on expression of genes containing expanded nucleotide repeats, and 3) determining whether chemical interference with the interaction has phenotypic effects (Deng, 2022).

Decreasing the expression of the SUPT4H or SUPT5H components of the DSIF complex can lower production of mRNAs encoded by mutant gene alleles containing nucleotide repeat expansions, and also can modify phenotypes associated with repeat expansions. These findings have led to proposals that that chemical or genetic targeting of SUPT4H or SUPT5H may be useful therapeutically. The results reported in this study indicate that chemical interference with the interaction of SUPT4H and SUPT5H is achievable, that such interference -which has been confirmed by two independent reporter assays and a direct biochemical assay-can lower the abundance of mutant HTT gene products in cultured cells and an HD animal model, and that chemical targeting of DSIF complex formation can mitigate phenotypic effects of repeat expansions. However, the broad and essential biochemical functions of DSIF, raise the prospect that therapeutic targeting of DSIF may be challenging. As SUPT4H and SUPT5H can act individually, as well as in complex with each other, the effects of targeting DSIF also may differ from the effects of targeting its individual components (Deng, 2022).

Compounds of multiple chemical classes potentially may interfere with the SUPT4H-SUPT5H interaction. Among the compounds identified by the screening assays was 6-azauridine, a previously studied nucleoside inhibitor of de novo uridine-5'-monophosphate productive pathway and consequently of nucleic acid synthesis and cell division. Addition of uridine to cell cultures reversed the effects of approximately equimolar amounts of 6-AZA on global nucleic acid synthesis without affecting mutant HTT expression, demonstrating the distinctness of these two effects of the compound (Deng, 2022).

Loss of medium spiny neurons (MSNs) in the striatum is a characteristic feature of HD and other neurodegenerative diseases. This study used CRISPR/Cas9 gene editing methodology to shorten the number of HTT gene CAG repeats in HD patient MSNs to a nonpathological length, and found that shortening of repeats in these congenic cells was associated with diminished sensitivity to H2O2 exposure. Treatment with 6-AZA partially reversed the incremental sensitivity of cells containing expanded repeats, but did not affect H2O2 sensitivity in cells containing shorter repeats (Deng, 2022).

Analogous partial reversal of phenotypic effects of mutant HTT expression was observed also in the adult Drosophila compound eye, which has been widely used as a model for Huntington's Disease and other human neurodegenerative disorders. No loss of Drosophila larval viability was detected at a 6-AZA concentration that rescued animals displaying the rough eye phenotype. However, the ability of uridine supplementation to reverse the global effects of 6-AZA on nucleic acid synthesis in cell culture raises the possibility that such supplementation may prove useful also in mammalian models during in vivo studies (Deng, 2022).

Whereas the pathogenic effects of repeat expansions in HD and certain other diseases have been observed most clearly in neuronal cells, they are also evident in non-CNS tissues. In the current experiments, they were observed in MSNs, in neuronal cells, in blood cells, and in photoreceptor cells of the eye-and in replicating and nonreplicating cells. Whereas chemical interference with the SUPT4H-SUPT5H interaction has the potential for affecting multiple tissues simultaneously, differences in the length of repeats as well as tissue-specific factors unrelated to DSIF may influence the results of such interference (Deng, 2022).


Search PubMed for articles about Drosophila

Andrulis, E. D., Guzman, E., Doring, P., Werner, J., Lis, J. T. (2000). High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation. Genes Dev, 14(20):2635-2649 PubMed ID: 11040217

Deng, N., Wu, Y. Y., Feng, Y., Hsieh, W. C., Song, J. S., Lin, Y. S., Tseng, Y. H., Liao, W. J., Chu, Y. F., Liu, Y. C., Chang, E. C., Liu, C. R., Sheu, S. Y., Su, M. T., Kuo, H. C., Cohen, S. N. and Cheng, T. H. (2022). Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes. Proc Natl Acad Sci U S A 119(32): e2204779119. PubMed ID: 35914128

Doamekpor, S. K., Sanchez, A. M., Schwer, B., Shuman, S., Lima, C. D. (2014). How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes. Genes Dev, 28(12):1323-1336 PubMed ID: 24939935

Dollinger, R., Deng, E. B., Schultz, J., Wu, S., Deorio, H. R. and Gilmour, D. S. (2023). Assessment of the roles of Spt5-nucleic acid contacts in promoter proximal pausing of RNA polymerase II. J Biol Chem: 105106. PubMed ID: 37517697

Du, X., Qin, W., Yang, C., Dai, L., San, M., Xia, Y., Zhou, S., Wang, M., Wu, S., Zhang, S., Zhou, H., Li, F., He, F., Tang, J., Chen, J. Y., Zhou, Y., Xiao, R. (2024). RBM22 regulates RNA polymerase II 5' pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5. Genome Biol, 25(1):102 PubMed ID: 38641822

Harvey, R., Schuster, E., Jennings, B. H. (2013). Pleiohomeotic interacts with the core transcription elongation factor Spt5 to regulate gene expression in Drosophila. PLoS One, 8(7):e70184 PubMed ID: 23894613

Hofstetter, J., Ogunleye, A., Kutschke, A., Buchholz, L. M., Wolf, E., Raabe, T., Gallant, P. (2024). Spt5 interacts genetically with Myc and is limiting for brain tumor growth in Drosophila. Life science alliance, 7(1) PubMed ID: 37935464

Huang, K. L., Jee, D., Stein, C. B., Elrod, N. D., Henriques, T., Mascibroda, L. G., Baillat, D., Russell, W. K., Adelman, K., Wagner, E. J. (2020). Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination. Mol Cell, 80(2):345-358 e349 PubMed ID: 32966759

Krasnov, A. N., Evdokimova, A. A., Mazina, M. Y., Erokhin, M., Chetverina, D., Vorobyeva, N. E. (2023). Coregulators Reside within Drosophila Ecdysone-Inducible Loci before and after Ecdysone Treatment. Int J Mol Sci, 24(14) PubMed ID: 37511602

Krishnan, K., Salomonis, N., Guo, S. (2008). Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo. PLoS One, 3(11):e3621 PubMed ID: 18978947

Laitem, C., Zaborowska, J., Tellier, M., Yamaguchi, Y., Qingfu, C., Egloff, S., Handa, H. and Murphy, S. (2015). CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription. Transcription: 0. PubMed ID: 26399478

Missra, A. and Gilmour, D. S. (2010). Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex. Proc. Natl. Acad. Sci. 107(25): 11301-6. PubMed ID: 20534440

Pereira, A., Paro, R. (2017). Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus. Epigenetics & chromatin, 10(1):57 PubMed ID: 29208012

Prabhakaran, M., Kelley, R. L. (2012). Mutations in the transcription elongation factor SPT5 disrupt a reporter for dosage compensation in Drosophila. PLoS Genet, 8(11):e1003073 PubMed ID: 23209435

Qiu, Y., Gilmour, D. S. (2017). Identification of Regions in the Spt5 Subunit of DRB Sensitivity-inducing Factor (DSIF) That Are Involved in Promoter-proximal Pausing. J Biol Chem, 292(13):5555-5570 PubMed ID: 28213523

Russo, M., Piccolo, V., Polizzese, D., Prosperini, E., Borriero, C., Polletti, S., Bedin, F., Marenda, M., Michieletto, D., Mandana, G. M., Rodighiero, S., Cuomo, A., Natoli, G. (2023). Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription. Genes Dev, 37(21-24):1017-1040 PubMed ID: 38092518

Wu, C.-H., et al. (2003). NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes Dev. 17: 1402-1414. PubMed ID: 12782658

Zeng, Y., Zhang, H. W., Wu, X. X., Zhang, Y. (2024). Structural basis of exoribonuclease-mediated mRNA transcription termination. Nature, 628(8009):887-893 PubMed ID: 38538796

Biological Overview

date revised: 4 May, 2024

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