Chromatin Structure Is Implicated in “Late” Elongation Checkpoints on the U2 snRNA and β-Actin Genes

Abstract

The negative elongation factor NELF is a key component of an early elongation checkpoint generally located within 100 bp of the transcription start site of protein-coding genes. Negotiation of this checkpoint and conversion to productive elongation require phosphorylation of the carboxy-terminal domain of RNA polymerase II (pol II), NELF, and DRB sensitivity-inducing factor (DSIF) by positive transcription elongation factor b (P-TEFb). P-TEFb is dispensable for transcription of the noncoding U2 snRNA genes, suggesting that a NELF-dependent checkpoint is absent. However, we find that NELF at the end of the 800-bp U2 gene transcription unit and RNA interference-mediated knockdown of NELF causes a termination defect. NELF is also associated 800 bp downstream of the transcription start site of the ␤-actin gene, where a "late" P-TEFbdependent checkpoint occurs. Interestingly, both genes have an extended nucleosome-depleted region up to the NELF-dependent control point. In both cases, transcription through this region is P-TEFb independent, implicating chromatin in the formation of the terminator/checkpoint. Furthermore, CTCF colocalizes with NELF on the U2 and ␤-actin genes, raising the possibility that it helps the positioning and/or function of the NELF-dependent control point on these genes.The negative elongation factors (N-TEFs) DSIF and NELF are key components of a polymerase II (pol II) checkpoint that occurs early in the transcription cycle of the human immunodeficiency virus (HIV) genome and many protein-coding genes (7,21,46,62). Release from an N-TEF-dependent block requires the activity of the cyclin-dependent kinase 9 (CDK9) subunit of positive transcription elongation factor b (P-TEFb), which phosphorylates serine (Ser) 2 in the YSPTSPS heptapeptide repeat of the pol II carboxy-terminal domain (CTD) and subunits of DSIF and NELF (7,21,46,62). Accordingly, CDK9 inhibitors effectively inhibit the elongation of pol II transcription both in vitro and in vivo (13,40). Once pol II has negotiated the early block, productive elongation can occur (37). Phosphorylation of Ser2 of the pol II CTD by CDK9 also activates cotranscriptional processing of transcripts from protein-coding genes and the mammalian noncoding small nuclear RNA (snRNA) genes (4,17,39,42), presumably through interactions between phospho-CTD and processing factors (17). In many Drosophila protein-coding genes, the NELF-dependent checkpoint is located within 100 bp downstream of the transcription start site, where paused polymerase is also located (30). These genes generally have a short promoter-proximal region of nucleosome depletion, with the first nucleosome mapping close to where NELF is found (31, 38).We have previously reported differences in elongation control between the short intronless pol II-transcribed human U2 snRNA genes with a transcription unit of less than 1 kb (40) and the ␤-actin protein-coding gene with a transcription unit of 5 kb (24). CDK9 inhibitors drastically affect the elongation of transcription of the ␤-actin gene bu...

CTCF Regulates Kaposi's Sarcoma-Associated Herpesvirus Latency Transcription by Nucleosome Displacement and RNA Polymerase Programming

“…CTCF has been implicated in programming RNAPII pausing at the mammalian tumor necrosis factor alpha (TNF-␣) gene (55) and at Hox gene insulators in Drosophila (41,56). CTCF was also found to localize with RNAPII elongation factors, including NELF, at the U2 and ␤-actin genes (57). In this study, we tested the role of CTCF binding sites in regulating RNAPII programming and RNAPII-accessory factor accumulation in the latency transcript control region.…”

Gene-specific repression of the p53 target gene PUMA via intragenic CTCF–Cohesin binding

“…We are left to ponder the function of the observed intragenic CTCF and Cohesin complex occupancy downstream from the transcriptional start sites on p21 and other p53 target genes. Interestingly, a recent study demonstrated that intragenic CTCF binding functions as a ''late'' checkpoint in early stages of elongation at the U2 snRNA and b-actin genes (Egloff et al 2009). This leaves open the possibility that CTCF and the Cohesin complex may function in a similar fashion at p21 and other p53 target genes.…”

The point of no return: The poly(A)-associated elongation checkpoint

“…For example, trimethylation of histone H3 lysine 36 (H3K36 m3 ) is linked to CTD Ser2 phosphorylation and CDK9 inhibition greatly reduces this histone modification. 3,33 We favor the hypothesis that CDK9-dependent phosphorylation of Spt5 plays a key role in negotiating poly(A)-associated checkpoints. 12 However, it seems likely that regulation of pol II elongation at these checkpoints involves more than one kinase and a range of kinase targets (Table 1).…”

“…Supporting this notion, CDK9 inhibitors cause loss of CDK9 from the poly(A) site region 12 but not from the EEC. 33 The Med18 subunit of Mediator is present at both the 5 0 and 3 0 ends in some yeast genes. 43 This protein has been shown to recruit CF1 and CPF complexes that are required for 3 0 -end processing and transcription termination.…”