SETD6 Regulates E2-Dependent Human Papillomavirus Transcription

Abstract

Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal domain (BET) family of proteins. Brd4 regulates human papillomavirus (HPV) transcription, genome replication, and segregation by binding to the E2 protein. The SETD6 methyltransferase binds to and methylates Brd4 at lysine 99. We investigated the interactions of SETD6 and Brd4 with E2 and their role in HPV transcription. SETD6 coimmunoprecipitated with the E2 transactivation domain, and its depletion in CIN612 episomal cells reduced human papillomavirus type 31 (HPV-31) transcription, whereas depletion of SETD6 in integrated HPV cell lines had no effect on viral gene expression. The mutant Brd4 K99R (bearing a change of K to R at position 99), which cannot be methylated by SETD6, displayed decreased binding to HPV-31 E2, suggesting that SETD6 methylation of Brd4 also influences E2 association with the Brd4 protein. Using chromatin immunoprecipitation, SETD6 was detected at the enhancer region of the HPV long control region. We propose that methylation of Brd4 at K99 by SETD6 is an important mechanism for E2-Brd4 association and HPV transcriptional activation. IMPORTANCE Human papillomaviruses (HPV) cause cervical, anogenital, and oral cancers. Brd4 plays an important role in the HPV life cycle. SETD6 was recently shown to methylate Brd4. The current study demonstrates that methylation of Brd4 by SETD6 in HPV-episomal cells is required for the activation of viral transcription. This study illustrates a novel regulatory mechanism involving E2, Brd4, and SETD6 in the HPV life cycle and provides insight into the multiple roles of Brd4 in viral pathogenesis.

Keywords: Brd4; HPV; SETD6; transcriptional regulation.

FIG 1

SETD6 immunoprecipitates with HPV-31 and HPV-16 E2 protein. (A) HPV-31 E2 is in complex with Brd4 and SETD6. HEK293TT cells were transfected with FLAG-Brd4, hemagglutinin (HA)-SETD6, or FLAG-31 E2 constructs. Brd4 was immunoprecipitated with Brd4 antibodies and blotted with FLAG and HA antibodies. IgG was used as a negative control. (B) HEK293TT cells were transfected with FLAG-Brd4, HA-SETD6, or FLAG-31 E2 constructs. Brd4 was used as a positive control for SETD6 association. Brd4 and HPV-31 E2 were immunoprecipitated with FLAG antibodies and blotted with FLAG and HA antibodies. (C) HEK293TT cells were transfected with HA-SETD6 and FLAG-16 E2 constructs. HPV-16 E2 was immunoprecipitated with FLAG antibodies and blotted with FLAG and HA antibodies. Mouse anti-FLAG (M2) and HA (HA-7) antibodies were incubated and exposed on the same immunoblot.

FIG 2

HPV-31 E2 associates with SETD6 independently of Brd4. (A) HEK293TT cells were treated with 250 nM ARV-825 for 42 h. Cells lysates were probed with Brd4 and β-actin antibodies. (B) HEK293TT cells were transfected with FLAG-31 E2 and HA-SETD6 constructs. Cells were treated with vehicle or 250 nM ARV-825 6 h posttransfection until cells were harvested at 48 h posttransfection. HPV-31 E2 was immunoprecipitated with M2 agarose and blotted with FLAG (M2) and HA (HA-7) antibodies. Inputs were blotted with HA antibodies. (C) HEK293TT cells were transfected with HA-SETD6 and FLAG-31 E2 truncated constructs. HPV-31 E2 proteins were immunoprecipitated with FLAG antibodies. HA (HA-7) antibodies were incubated and exposed, followed by mouse anti-FLAG (M2) Ab incubation and exposure on the same immunoblot. FL, full length.

FIG 3

SETD6 associates with the HPV-31 enhancer region in CIN612 cells. (A) HEK293TT cells were transfected with HA-SETD6. SETD6 was immunoprecipitated with SETD6 antibodies and detected with HA (HA-7) antibodies. IgG was used as a negative control. (B) Diagram illustrating primer regions used for ChIP assays in this study. PP, proximal promoter. Black boxes indicate E2 binding sites. (C and D) ChIP experiments were performed in CIN612 cells using EE epitope tag supernatant (nonspecific IgG control) or HPV-31 E2 (C) or BRD4 or SETD6 (D) antibodies. Real-time PCR was performed with primers described in Materials and Methods. Cycle threshold (C_T) values were normalized to input and EE was set to equal 1. Values are expressed as means ± standard errors of the means (SEM) (n = 6). *, P ≤ 0.05 compared to EE; #, P ≤ 0.1 compared to EE.

FIG 4

GST-CTM associates with SETD6. (A) HEK293TT cells were transfected with GST-CTM or HA-SETD6. Brd4 CTM was immunoprecipitated with glutathione Sepharose and blotted with GST and HA-7 antibodies. (B) HEK293TT cells were transfected with the FLAG-Brd4 construct, the FLAG-31 E2 construct, or increasing amounts of the HA-SETD6 construct. HPV-31 E2 was immunoprecipitated with M2 agarose and blotted with FLAG and GST antibodies. Inputs were blotted with FLAG (M2) and HA (HA-7) antibodies.

FIG 5

Inhibition of Brd4 methylation at K99 decreases E2-Brd4 association. (A) HEK293TT cells were transfected with FLAG-31 E2 and HA-SETD6 constructs. Cells were lysed in 2% SDS containing 50 mM NaCl and 10 mM Tris-HCl, pH 8.0. Immunoblots were probed with FLAG, pan-methyl lysine antibodies, and HA-7 antibodies. (B) HEK293TT cells were transfected with FLAG-HPV 31 E2 and FLAG-BRD4 WT or Brd4 K99R constructs. Brd4 was immunoprecipitated with Brd4 antibodies and blotted with FLAG (M2) antibodies. IgG was used as a negative control. (C) The percentages of E2 binding to BRD4 were quantified using ImageLab software. WT = 100%. Values are expressed as means ± SEM (n = 3). *, P < 0.05 by two-way t test.

FIG 6

SETD6 depletion decreases expression of HPV-31 transcripts. CIN612 (HPV-31-episomal) cells were treated with 15 nM control or SETD6 siRNA for 48 h. (A) SETD6 protein levels by Western blotting. (B) RNA was harvested and converted to cDNA. HPV early transcripts were measured using qPCR. KD, knockdown. Control = 1; n = 8. *, P < 0.05 by 1-way t test. (C) DNA was harvested, and HPV-31 DNA content was measured using qPCR and normalized to the amount of β-actin DNA. Control = 1; n = 8. *, P < 0.05 by two-way t test. (D) Lysates were blotted with phospho-RNA polymerase II (S5) and β-actin antibodies. (E) RNA was harvested and converted to cDNA. HPV early transcripts were measured using qPCR. Control = 1; n = 9. *, P < 0.05 by two-way t test. Values are expressed as means ± SEM.

FIG 7

Brd4 activates HPV transcription. CIN612 cells were treated with DMSO or 250 nM ARV-825 for 24 h. (A) Lysates were probed with Brd4 and β-actin antibodies. (B) RNA was harvested and converted to cDNA. HPV transcripts were measured using qPCR. Control = 1. Values are expressed as means ± SEM (n = 9 to 12). *, P < 0.05 by one-way t test.

FIG 8

SETD6 depletion has no effect on HPV-31 transcripts in HPV-integrated cell lines. (A and B) CIN612 HPV-integrated cells were treated with 15 nM control or SETD6 siRNA for 48 h. (A) SETD6 protein levels by Western blotting. (B) RNA was harvested and converted to cDNA. HPV-31 E6 and E7 transcripts were measured using qPCR. Control = 1; n = 9. *, P < 0.05 by two-way t test. (C and D) HeLa cells were treated with 15 nM control or SETD6 siRNA for 48 h. (C) SETD6 protein levels by Western blotting. (D) RNA was harvested and converted to cDNA. HPV-31 E6 and E7 transcripts were measured using qPCR. Control = 1; n = 6. *, P < 0.05 by two-way t test. Values are expressed as means ± SEM.

FIG 9

Model for SETD6 regulation of HPV transcription. During transcriptional activation, SETD6 binds the Brd4 CTM and methylates Brd4 at K99 on chromatin. Methylated Brd4 associates with E2 at the distal E2 binding sites (E2BS) on the viral episome. SETD6 also binds to E2. The Brd4 CTM interacts with the E2 TAD and the Brd4 NPS associates with the E2 DBD to enhance HPV transcription.