Regulation of transcriptional elongation by RNA polymerase II is a widely used mechanism for fine-tuning gene expression (1
). While progress has been made in understanding the mechanisms involved in RNA PolII elongation control, the role of promoter-specific factors in the repression of elongation is less well understood. In this study, we provide evidence that the zinc finger transcription factor Sp3 represses basal p21
expression, independently of SUMO modification, through a mechanism that limits the transition of stalled RNA PolII to productive elongation. Our studies suggest that modulation of the local balance of kinase and phosphatase activities is one mechanism by which Sp3 inhibits transcription by bound, stalled RNA PolII. These studies support the view that the transition of paused RNA PolII to productive elongation is an important step regulated by both promoter-specific activators and repressors to finely modulate mRNA expression levels.
Previous studies have shown that in the basal state, RNA PolII is paused at the promoter of the cyclin-dependent kinase inhibitor gene p21
and that in response to stimuli, including DNA damaging agents, p53 acts to stimulate elongation and increase the expression of this gene (12
). We observed that Sp3 contributes to keeping basal levels of p21
expression low. In agreement with the presence of an open chromatin structure and paused polymerase at p21
, we detected high levels of the active histone mark H3K4me3 at the promoter, and we observed much higher occupancy of RNA PolII at the 5′ end than across the body of the gene ( and ). Sp3 neither blocked nor generally enhanced RNA PolII binding at p21
or other Sp3-repressed promoters. These findings reveal the function of Sp3 to be distinct from that of GAGA factor in Drosophila melanogaster
, which promotes recruitment of RNA PolII that subsequently stalls (60
), but are consistent with a previous study showing that an Sp3 fusion protein targeted to a promoter-proximal RNA sequence repressed gene expression (62
). Furthermore, our studies have revealed a SUMO-independent mode of repression by Sp3, distinct from SUMO-Sp3-mediated heterochromatin silencing (22
). We suggest that promoters with paused RNA PolII are potentially sensitive to Sp3-dependent inhibition of elongation and that therefore, in the context of an open chromatin environment, the presence of paused RNA PolII is a key feature distinguishing targets of Sp3 activation and repression. Additional studies should provide further insights into how chromatin structure and factor binding at the promoter, together with SUMO modification, determine the context for Sp3-dependent activation, inhibition of elongation, and silencing.
Pausing of RNA PolII is a highly dynamic state that allows fine-tuning of gene expression in response to signals and changing cellular environments. Recent genomewide studies have revealed that paused RNA PolII appears to represent an active and tunable mechanism and that it may remain as a rate-limiting step even for highly transcribed genes (63
). Our finding that enrichment of RNA PolII at the 5′ end of the p21
locus was maintained regardless of Sp3 levels indicates that Sp3 does not modify RNA PolII pausing as a rate-limiting step for p21
transcription. The genomewide studies also showed that whereas the rate of RNA PolII escape can be up- and downregulated, paused PolII is rarely entirely blocked from transcribing the body of the gene (63
). In agreement with this, we observed P-TEFb binding, enriched CTD Ser2ph at the 3′ end of the gene, and a low level of p21
transcription even under conditions of Sp3-dependent repression ( and ). The relative enrichment of CTD Ser2ph at more 3′ positions of the p21
gene upon Sp3 knockdown, in addition to the increases in the transcription elongation marks H3K36me3 and H2Bub1 ( and and ), is consistent with the model that Sp3 acts to lower the rate of escape of paused RNA PolII into productive elongation. Although we have not observed Sp3-dependent effects on the splicing or mRNA stability of p21
or other repressed target genes (data not shown), cotranscriptional mRNA processing is coordinated with promoter-proximal pausing and is regulated by many common factors, including the negative elongation factor (NELF), P-TEFb, and CTD Ser2ph (53
), raising the possibility that Sp3-regulated pathways impact mRNA processing at some target genes.
NELF is the major factor associated with the early pausing of RNA PolII, and it has been suggested previously that NELF may control RNA PolII pausing at the p21
). We observed strong binding of NELF to the promoter-proximal regions of p21
and other Sp3-repressed genes, correlating with the density profile of RNA PolII and supporting the presence of paused polymerase at these promoters. After Sp3 knockdown, NELF occupancy was significantly reduced at p21
and several other promoters, and this likely contributed to the observed increase in the levels of transcription of these genes. NELF-independent pausing mechanisms have also been described (2
), and we consider it possible that these play a more significant role at those genes where Sp3 depletion increased transcription without reducing NELF occupancy. Several studies suggest a surprising complexity of steps and factors that contribute to the regulation of elongation. Recent investigations have demonstrated that cohesin, a factor essential for sister chromatid cohesion but also implicated in DNA repair and transcription, selectively binds genes with paused polymerase and inhibits the transition of paused polymerase to elongation at a step distinct from those regulated by NELF and DSIF (65
). Thus, while Sp3 may also regulate additional mechanisms, our data show that Sp3 promotes NELF binding at the p21
The kinase subunit of P-TEFb, CDK9, phosphorylates multiple substrates, including DSIF, the NELF-E subunit of the negative elongation factor complex, and Ser2 of the RNA PolII CTD, to promote the release of paused RNA PolII. P-TEFb also plays an important role in the cotranscriptional regulation of histone modifications, such as H3K36me3 and H2Bub1 (56
). CDK9 was present at Sp3-repressed genes, and its binding did not increase upon Sp3 knockdown (), indicating that Sp3 does not inhibit transcription by blocking P-TEFb recruitment. The finding that reduced occupancy of NELF upon Sp3 knockdown was blocked by inhibition of CDK9 activity () is consistent with the view that, in the presence of Sp3, the activity of promoter-bound CDK9 in releasing NELF is inhibited. Taken together, our data are most consistent with the view that Sp3 acts to downregulate the transition to productive elongation via mechanisms that include antagonizing P-TEFb activity on specific substrates.
Reduced levels of H3S10 phosphorylation showed a strong correlation with Sp3-dependent repression at the p21
promoter and at the promoters of many other Sp3-repressed genes. These findings are consistent with a previous study supporting a role for a dual H3S10ph/K14Ac mark in the activation of p21 transcription (57
). H3S10ph has been reported to act through a cascade of histone modifications and protein interactions to promote Brd4-dependent recruitment of P-TEFb (49
). In our studies, increased H3S10ph upon Sp3 RNAi was not correlated with increased recruitment of P-TEFb (), highlighting the context-dependent functions of this histone modification. Brd4 has been shown to regulate the recruitment of additional elongation factors (67
), and other pathways for H3S10ph-mediated activation have been proposed, including a role for H3S10ph in maintaining the active state of a gene, in part by preventing the spreading of H3K9me2 and HP1 and thus counteracting heterochromatin formation (51
). Thus, while we have demonstrated that Sp3 inhibits the phosphorylation of H3S10 at the p21
promoter and have shown a correlation between H3S10 phosphorylation and derepression, the function of this histone modification is currently unclear.
Levels of protein phosphorylation are determined by the balance of opposing kinase and phosphatase activities. We found that both PP1 and PP2A interact with Sp3 and, furthermore, that PP1 is recruited to the p21
promoter in an Sp3-dependent manner (). PP1 is an important regulator of transcription and posttranscriptional events, and in some cases, levels of PP1 at the promoter have been shown to correlate inversely with gene expression (69
). PP2A has also been shown to localize to the p21
promoter and to antagonize the phosphorylation of H3S10 mediated by MSK1 and MSK2 (57
). In agreement with these reports, the addition of the phosphatase inhibitor okadaic acid increased p21
mRNA levels. Dephosphorylation of several substrates by PP1 and/or PP2A could contribute to inhibiting the expression of p21
. Several studies support a role for PP1 and/or PP2A as the H3S10 phosphatase (69
), and our data suggest that Sp3-dependent binding of PP1 contributes to keeping H3Ser10ph levels low at the p21
promoter. Furthermore, PP1 and/or PP2A at the promoter could antagonize other stimulatory phosphorylation events, such as P-TEFb-dependent phosphorylation of NELF and DSIF, or the phosphorylation of CTD Ser2 by P-TEFb or other kinases. In fact, CDK9 itself can be phosphorylated, and although the functions of this modification are not resolved, PP1 has been shown to dephosphorylate CDK9 in vivo
). Thus, our data suggest that modulation of the balance of kinase and phosphatase activities is one of several mechanisms by which Sp3 inhibits productive transcriptional elongation.
The data presented here suggest that transcription factor Sp3 acts to reduce the expression of many genes with Sp3 binding sites in their promoters by inhibiting the transition of paused RNA PolII to productive elongation. Our data further suggest that recruitment of phosphatases by promoter-specific transcription factors may help maintain paused RNA polymerase. Thus, local antagonism of the activity of promoter-bound kinases such as P-TEFb may provide an additional level of regulation that contributes to the precise regulation of gene expression.