Adenovirus E1A recruits the human Paf1 complex to enhance transcriptional elongation

Abstract

During infection by human adenovirus (HAdV), the proteins encoded by the early region 1A (E1A) gene bind and appropriate components of the cellular transcriptional machinery to activate the transcription of viral early genes. Previously, we identified roles for the human Bre1 (hBre1) and hPaf1 complexes in E1A-mediated transcriptional activation of HAdV early genes. Here we show that E1A binds hBre1 directly and that this complex targets the hPaf1 complex via the Rtf1 subunit. Depletion of hPaf1 reduces E1A-dependent activation of transcription from the E2e, E3, and E4 viral transcription units, and this does not result from a reduced ability of RNA polymerase II to be recruited to the promoter-proximal regions of these genes. In contrast, depletion of hPaf1 reduces the occupancy of RNA polymerase II across these transcription units. This is accompanied by reductions in the level of H3K36 trimethylation, a posttranslational histone modification associated with efficient transcriptional elongation, and the number of full-length transcripts from these genes. Together, these results indicate that E1A uses hBre1 to recruit the hPaf1 complex in order to optimally activate viral early transcription by enhancing transcriptional elongation.

Importance: This work provides the mechanism by which the hPaf1 complex contributes to E1A-dependent activation of early gene transcription. The work also demonstrates that E1A induces gene expression by stimulating transcriptional elongation, in addition to its better-characterized effects on transcriptional initiation.

FIG 1

hPaf1 interacts with residues 4 to 25 of E1A. A549 cells were infected with wild-type (WT) HAdV or with HAdV expressing the indicated E1A deletion mutants for 16 h at an MOI of 5. E1A was immunoprecipitated (IP), and Western blotting (IB) was performed using antibodies specific for hPaf1.

FIG 2

hBre1 interacts with E1A and is required for interaction with the Rtf1 component of the Paf1 complex. (A) hBre1 and E1A interact directly. GST-hBre1 and His-tagged 289R E1A were purified from Escherichia coli BL21. Ten micrograms of purified GST-hBre1 and 5 μg of His-E1A were mixed in BSA buffer. Interaction was then detected via GST pulldown assays. Stars indicate the positions of the various GST-hBre1 fusions. (B) Rtf1 is sufficient to bridge hBre1 and E1A with the hPaf1 complex. Sf21 insect cells were infected with baculoviruses expressing the indicated FLAG-tagged hPaf1 complex components, E1A, or hBre1. Portions equivalent to 0.05 mg of lysates of cells infected with baculoviruses expressing both hBre1 and E1A were then mixed with 0.05 mg of a cell lysate expressing an hPaf1 complex component as indicated, and immunoprecipitation for hPaf1 complex components was performed with a FLAG-specific antibody. Western blotting was then performed using an anti-E1A antibody (M73). (C) Rtf1 is necessary to bridge hBre1 and E1A with the hPaf1 complex. Specific proteins were produced in Sf21 insect cells and were combined as described above. Immunoprecipitation was performed with an anti-E1A antibody (M73), and Western blotting was performed with an hPaf1-specific antibody, to determine whether the hPaf1 complex was coprecipitated. (D) Expression of hBre1, E1A, and hPaf1 components. Extracts from Sf21 cells infected with recombinant baculoviruses were subjected to Western blotting to confirm the expression of the indicated proteins. The star indicates the position of hPaf1.

FIG 3

hPaf1 is required for the efficient production of full-length transcripts from the E2e, E3, and E4 transcription units. (Top) The HAdV5 genome and relevant transcription units. Primer pairs used in the analyses are indicated by asterisks. (A through D) A549 cells were treated with hPaf1-specific siRNA for 48 h. Cells were then infected with the WT or ΔE1A virus for 16 h. mRNA was then extracted, and cDNA was produced and was probed via qRT-PCR using primer sets specific for the 5′ and 3′ ends of the E1A and E1B (A), E2A (B), E3 (C), and E4 (D) transcription units. UI, uninfected. (Insets) The locations of primers are indicated. Numbers correspond to locations in the HAdV5 genome.

FIG 4

Knockdown of hBre1 and hPaf1 results in decreased expression of the E2e and E4 proteins. Cells were treated with Ctrl siRNA, hBre1 siRNA, or hPaf1 siRNA and were infected with the WT virus as described in the text. Cells were lysed, and protein levels were determined via Western blotting with the indicated antibodies.

FIG 5

hPaf1 knockdown reduced the occupancy of RNA polymerase II at the 3′ ends of the viral E2e, E3, and E4 transcription units. A549 cells were treated with Ctrl or hPaf1 siRNA for 48 h. Cells were then infected with the WT or ΔE1A virus for 16 h. ChIP analyses were performed to determine the occupancy of RNA polymerase II at the 5′ and 3′ ends of the HAdV E1A and E1B (A), E2A (B), E3 (C), and E4 (D) transcription units. Primer pairs are the same as those shown in Fig. 3.

FIG 6

E1A is present across the chromatin of all early transcription units. A549 cells were treated with Ctrl or hPaf1 siRNA for 48 h. Cells were then infected with the WT or ΔE1A virus for 16 h. ChIP analyses were performed to determine the occupancy of E1A at the 5′ and 3′ ends of the HAdV E1A and E1B (A), E2A (B), E3 (C), and E4 (D) transcription units. Primer pairs are the same as those shown in Fig. 3.

FIG 7

hPaf1 is required for efficient H3K36me3 of chromatin at the 3′ ends of the viral E2A, E3, and E4 transcription units. A549 cells were treated with Ctrl or hPaf1 siRNA for 48 h. Cells were then infected with the WT or ΔE1A virus for 16 h. ChIP analyses were performed to determine the frequency of H3K36me3, a mark of transcriptional elongation, at the 5′ and 3′ ends of the HAdV E1A and E1B (A), E2A (B), E3 (C), and E4 (D) transcription units. Primer pairs are the same as those shown in Fig. 3.

FIG 8

hBre1 is required for efficient occupancy by RNA pol II, E1A, and H3K36me3 of chromatin at the 3′ ends of the viral E2A, E3, and E4 transcription units. A549 cells were treated with Ctrl or hBre1 siRNA for 48 h. Cells were then infected with the WT or ΔE1A virus for 16 h. ChIP analyses were performed to determine the frequency of occupancy by RNA pol II (A), E1A (B), and H3K36me3 (C) at the 5′ and 3′ ends of HAdV genes E2A, E3, and E4. Primer pairs are the same as those shown in Fig. 3.