Human Polymerase-Associated Factor complex (PAFc) connects the Super Elongation Complex (SEC) to RNA polymerase II on chromatin

Abstract

The Super Elongation Complex (SEC), containing transcription elongation activators/coactivators P-TEFb, ELL2, AFF4/1, ENL, and AF9, is recruited by HIV-1 Tat and mixed lineage leukemia (MLL) proteins to activate the expression of HIV-1 and MLL-target genes, respectively. In the absence of Tat and MLL, however, it is unclear how SEC is targeted to RNA polymerase (Pol) II to stimulate elongation in general. Furthermore, although ENL and AF9 can bind the H3K79 methyltransferase Dot1L, it is unclear whether these bindings are required for SEC-mediated transcription. Here, we show that the homologous ENL and AF9 exist in separate SECs with similar but nonidentical functions. ENL/AF9 contacts the scaffolding protein AFF4 that uses separate domains to recruit different subunits into SEC. ENL/AF9 also exists outside SEC when bound to Dot1L, which is found to inhibit SEC function. The YEATS domain of ENL/AF9 targets SEC to Pol II on chromatin through contacting the human Polymerase-Associated Factor complex (PAFc) complex. This finding explains the YEATS domain's dispensability for leukemogenesis when ENL/AF9 is translocated to MLL, whose interactions with PAFc and DNA likely substitute for the PAFc/chromatin-targeting function of the YEATS domain.A ccumulating evidence has implicated the elongation stage of RNA polymerase II (Pol II) transcription as a major ratelimiting step for the expression of a large number of metazoan genes, especially those that control cell growth, renewal, and differentiation (1-3). During elongation, the processivity of Pol II is regulated by a set of transcription factors, which had been thought to exist as separate entities and impact on the Pol II elongation complex independently of one another. However, recent data from us and others indicate that at least two well defined transcription elongation factors of different classes reside in a single multisubunit complex termed SEC [super elongation complex, (4)] to cooperatively activate transcription (4-6).The first elongation factor found in SEC is human positive transcription elongation factor b (P-TEFb). Consisting of CDK9 (Cyclin-dependent Kinase 9) and cyclin T1 (CycT1), P-TEFb functions by phosphorylating the C-terminal domain (CTD) of the largest subunit of Pol II and negative elongation factors DSIF and NELF. These events antagonize the actions of the negative factors, release Pol II from promoter-proximal pausing, and trigger the production of full-length mRNA transcripts (7,8). The second elongation factor in SEC is ELL2, which promotes elongation by keeping the 3′ OH of nascent mRNA in alignment with the catalytic site to prevent Pol II backtracking (9). Besides P-TEFb and ELL2, SEC also contains transcription factors/coactivators ENL, AF9, AFF4 (AF5q31), AFF1 (AF4), and probably others (4-6). Among these, AFF4 is known to mediate the interaction between ELL2 and P-TEFb and maintain the integrity of SEC (5). While AFF1 has been shown to interact with AFF4 (10), it remains to be seen whether the interaction between these two homologo...

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

“…AFF4 is a subunit of the super elongation complex that is recruited by mixed lineage leukaemia (MLL) proteins to activate the expression of MLL target genes [35][36][37][38] . The N-terminal 300 amino acids of AFF4 were shown to specifically interact with P-TEFb 39,40 . Again, the substrate specificity was tested for the consensus CTD [3] as well as serine pre-phosphorylated peptides and K7-CTD [3] .…”

HIV-1 Tat recruits transcription elongation factors dispersed along a flexible AFF4 scaffold

“…In vivo, AFF4 recruits SEC components through defined regions in the first 900 amino acids (23,29,30). AFF4 associates in vivo with P-TEFb, AFF4 300-600 recruits the C-terminus of ELL2, and AFF4 600-900 binds competitively to the C-terminal domain of homologs ENL and AF9.…”

“…S1), a positive regulator of P-TEFb (33,34). Because the AF9 and ENL C-terminal domains interact competitively with AFF4 (23,29,30), we analyzed one of the paralogs, ENL. To purify complexes of HIV-1 Tat and P-TEFb, we adapted baculovirus coexpression strategies (35).…”

“…Genetic and biochemical studies indicate that many factors function in large complexes or in conjunction with RNA-processing activities to stimulate transcription in vivo (22). For example, PAFc interacts with SII/TFIIS (20) and ENL/ AF9 both physically and functionally to stimulate elongation (23), highlighting the importance of coordination and cooperativity among different transcriptional regulators.…”

The essential role of acetyllysine binding by the YEATS domain in transcriptional regulation

“…3 AF9 is found to associate with several nuclear complexes, most notably the SEC, [14][15][16] and separately with the histone H3K79-specific methyltransferase DOT1L. 3,17,18 DOT1L-mediated H3K79 methylation is strongly linked to active gene expression, suggesting that interaction of AF9 with DOT1L may play a role in DOT1L recruitment to gene promoters and transcriptional activation. Significantly, Li et al showed that the YEATS domain of AF9 is required for targeting of DOT1L to H3K9ac-enriched chromatin and subsequent H3K79 methylation and for transcription of target genes such as MYC.…”