The human PAF1 complex acts in chromatin transcription elongation both independently and cooperatively with SII/TFIIS

Abstract

Genetic and cell-based studies have implicated the PAF1 complex (PAF1C) in transcription-associated events, but there has been no evidence showing a direct role in facilitating transcription of a natural chromatin template. Here, we demonstrate an intrinsic ability of human PAF1C (hPAF1C) to facilitate activator (p53)- and histone acetyltransferase (p300)-dependent transcription elongation from a recombinant chromatin template in a biochemically defined RNA polymerase II transcription system. This represents a PAF1C function distinct from its established role in histone ubiquitylation and methylation. Importantly, we further demonstrate a strong synergy between hPAF1C and elongation factor SII/TFIIS and an underlying mechanism involving direct hPAF1C-SII interactions and cooperative binding to RNA polymerase II. Apart from a distinct PAF1C function, the present observations provide a molecular mechanism for the cooperative function of distinct transcription elongation factors in chromatin transcription.

Figure 1. Characterization of Native and Reconstituted hPAF1Cs

(A) Silver staining and immunoblots of native hPAF1C purified from the FLAG-hPAF1 HeLa cell line. Non-specific proteins (marked by asterisks) are thought to be Hsp70, tubulin and actin (cf. Zhu et al., 2005). The indicated hRTF1 polypeptide is close in size to that encoded by residues 157–585 of the long hRTF1 open reading frame (Figure S1). (B) Immunoblots of purified native hPAF1C following Superose 6 gel filtration. Note that hRTF1 peaked separately from the circa 670 kDa complex and formed a stable trimeric complex with hPAF1 and hCDC73 through its C-terminus (Figure S1). (C) Coomassie blue staining and immunoblots of the purified, baculovirus-reconstituted hPAF1C. (D) Immunoblots of purified native baculovirus-reconstituted hPAF1C following Superose 6 gel filtration. (E) Multivalent hPAF1C subunit interactions. Arrows indicate direct interactions based on the analyses in Figure S2, with a weak interaction depicted by the dashed arrow.

Figure 2. Synergistic Effect of hPAF1C and SII on p53-Dependent Chromatin Transcription

(A) Schematic representation of the standard in vitro transcription assay. Transcription factors included TFIIA, TFIIB, TFIIE, TFIID, TFIIF, TFIIH, PC4, Mediator and Pol II. Chromatin-based assays also contained the components (ACF1, ISWI and NAP1) employed for chromatin assembly. (B) Independent and cooperative effects (with SII) of reconstituted hPAF1C in chromatin transcription. Reactions contained p300/acetyl-CoA (all lanes) and, as indicated, 10 ng p53, 10 ng SII and either 160 ng (lanes 3 and 8), 320 ng (lanes 4 and 9) or 640 ng (lanes 5 and 10) of reconstituted hPAF1C. (C) Independent and cooperative effects (with SII) of native hPAF1C on chromatin transcription. Reactions contained p53 and p300/acetyl-CoA (all lanes) and, as indicated, 10 ng SII and either 17.5 ng (lanes 2 and 6), 35 ng (lanes 3 and 7) or 70 ng (lanes 4 and 8) of native hPAF1C or 70 ng (lanes 9 and 10) of reconstituted hPAF1C. (D) p53- and p300/acetyl-CoA- dependent function of hPAF1C. Reactions contained 10 ng p53, 15 ng p300 and 320 ng baculovirus-reconstituted hPAF1C, as indicated, but no SII. (E) Independent and cooperative effects (with SII) of hPAF1C in NFκB(p65)-dependent chromatin transcription. Reactions contained chromatin assembled with pG₅ML plasmid, 10 ng Gal4-p65 and p300/acetyl-CoA (all lanes) and, as indicated, 10 ng SII and 280 ng reconstituted hPAF1C. (F) Titration of hPAF1C and SII in an in vitro chromatin transcription assay. Reactions contained p53 and p300/acetyl-CoA (all lanes) and, as indicated, either 5 ng (lanes 5–8), 10 ng (lanes 9–12) or 20 ng (lanes 13–16) of SII and either 70 ng (lanes 2, 6, 10 and 14), 140 ng (lanes 3, 7, 11 and 15) or 280 ng (lanes 4, 8, 12 and 16) of reconstituted hPAF1C. In (B), (C), (E) and (F), relative transcription levels were quantitated by phosphoimager and normalized to that of 10 ng SII alone. As shown in Figure S3, the effects of hPAF1C on transcription were still observed under saturating levels of Mediator and p300.

Figure 3. hPAF1C Independently and Cooperatively (with SII) Stimulates Transcription Elongation

(A) Schematic representation of the in vitro transcription assay in (B). (B) Independent and cooperative hPAF1C and SII functions after PIC assembly. Preinitiation complexes were formed prior to chromatin assembly by incubation of DNA with p53 and transcription factors. After subsequent chromatin assembly, sequential incubations with p300/acetyl-CoA, hPAF1C (320 ng), and SII (10 ng), as indicated, were carried out. Relative transcription levels were quantitated by phosphoimager and normalized to that of SII alone. (C) Schematic representation of the in vitro transcription elongation assay in (D). (D) Independent and cooperative hPAF1C and SII functions at the transcription elongation stage. Radiolabeled, chromatin-associated early elongation complexes were prepared according to the protocol in (C) and allowed to elongate in the presence of hPAF1C (320 ng) and/or SII (30 ng) as indicated.

Figure 4. Direct Physical and Functional Interactions of hPAF1C with Pol II and SII

(A and B) Binding to Pol II of (A) purified hPAF1Cs that lack indicated subunits (Figure S4C) or (B) purified hPAF1C subunits (Figure S4B). (C) Chromatin transcription with SII and indicated hPAF1Cs. SII (10 ng) and reconstituted hPAF1Cs (320 ng for intact complex) containing equivalent amounts of FLAG-hCTR9 were assayed as indicated. p53 and p300/acetyl-CoA were added to all reactions. (D, F and G) Binding of (D) purified hPAF1C (Figure 1C), (F) purified hPAF1C subunits (Figure S4B) or (G) purified hPAF1Cs that lack indicated subunits (Figure S4C) to purified GST versus GST-SII (Figure S4A). (E) Intracellular binding of hPAF1C to SII. HeLa nuclear extracts were immunoprecipitated with anti-SII antibody and bound proteins were visualized by immunoblots with indicated antibodies (hRTF1 and hCDC73 are not shown because of their comigration with IgG heavy chain).

Figure 5. Cooperative Binding of hPAF1C and SII to Pol II

Binding of purified SII and hPAF1C to Pol II. HeLa nuclear extracts (A) or purified Pol II (B and C) were incubated with GST-SII or reconstituted hPAF1Cs (containing FLAG-hCTR9) in the presence and in the absence, respectively, of purified reconstituted hPAF1Cs or SII as indicated. hPAF1Cs in (C) were reconstituted with subunit omissions as indicated.

Figure 6. Distribution of hPAF1C and SII on the p21 Locus during p53-Dependent Transcription

(A) Doxorubicin induction of p53 and the p21 target gene. HCT116 cells were treated with 0.5 μM doxorubicin for the indicated times, and protein and mRNA levels were analyzed by immunoblot and RT-PCR, respectively. (B) Schematic representation of the p21 locus indicating the six amplicons used for real-time PCR. (C) ChIP analyses on the p21 locus. Cells were treated as in (A) and ChIP analyses were performed with indicated antibodies. Error bars denote standard deviations from three independent PCR reactions from a single ChIP that is representative of several that were performed. (D–F) HCT116 cells were treated with control and hPAF1-targeted siRNAs as indicated. Cell extracts were analyzed by immunoblots with indicated antibodies (D). p21 mRNA levels were measured by quantitative PCR analysis and normalized to GAPDH mRNA. Error bars represent standard deviations from three independent PCR reactions (E). (F) After siRNA transfection, cells were treated with doxorubicin as in (A) and ChIP analyses were performed as in (C). [Note that the data in (C) are part of a comprehensive ChIP analysis and limited parts (e.g., p53 and p21 expression data and p53 and Pol II ChIP data) were recently published (Kim et al., 2009), for cross reference, with more extensive data on ubiquitylated H2B and H2B ubiquitylation factor analyses.] Additional data presented in Figure S5 document corresponding increases in H3 acetylation and H3K4 methylation on the p21 locus during induction by doxorubicin.

Figure 7. Models of Independent and Cooperative Functions of hPAF1C and SII in Transcription Elongation

Human PAF1C and SII independently facilitate transcription elongation through direct interactions with Pol II. In the case of hPAF1C, this involves a strong hPAF1-Pol II interaction (solid arrow) and a weak hLEO1-Pol II interaction (dashed arrow). While not shown, cooperative inter-subunit interactions within hPAF1C potentiate its interaction with Pol II relative to the independent interactions of individual subunits with Pol II. A direct interaction between SII and hPAF1C (via hPAF1 and hLEO1) results in their cooperative binding to Pol II (depicted by thick arrows) and a strong synergistic effect on transcription elongation.