Modulation of histone acetyltransferase activity through interaction of epstein-barr nuclear antigen 3C with prothymosin alpha

Abstract

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for EBV-dependent immortalization of human primary B lymphocytes. Genetic analysis indicated that amino acids 365 to 992 are important for EBV-mediated immortalization of B lymphocytes. We demonstrate that this region of EBNA3C critical for immortalization interacts with prothymosin alpha (ProTalpha), a cellular protein previously identified to be important for cell division and proliferation. This interaction maps to a region downstream of amino acid 365 known to be involved in transcription regulation and critical for EBV-mediated transformation of primary B lymphocytes. Additionally, we show that EBNA3C also interacts with p300, a cellular acetyltransferase. This interaction suggests a possible role in regulation of histone acetylation and chromatin remodeling. An increase in histone acetylation was observed in EBV-transformed lymphoblastoid cell lines, which is consistent with increased cellular gene expression. These cells express the entire repertoire of latent nuclear antigens, including EBNA3C. Expression of EBNA3C in cells with increased acetyltransferase activity mediated by the EBV transactivator EBNA2 results in down-modulation of this activity in a dose-responsive manner. The interactions of EBNA3C with ProTalpha and p300 provide new evidence implicating this essential EBV protein EBNA3C in modulating the acetylation of cellular factors, including histones. Hence, EBNA3C plays a critical role in balancing cellular transcriptional events by linking the biological property of mediating inhibition of EBNA2 transcription activation and the observed histone acetyltransferase activity, thereby orchestrating immortalization of EBV-infected cells.

FIG. 1

ProTα was isolated from a yeast two-hybrid cDNA library screen as a cellular molecule interacting with EBNA3C. (A) The sequence of the cDNA obtained from screen was matched against the previously known ProTα sequence found with a BLAST search in GenBank. Shown is a schematic representation of the ProTα protein, showing the locations of the central acidic domain (AD) that may have a random coil conformation, the putative nuclear localization signal (NLS), and the amino-terminal basic region (Br) (16, 19, 26). (B) Diagram showing a schematic representation of the EBNA3C protein. The RBP-Jκ binding site is indicated by the open boxed region. The putative leucine zipper motif (L), the ADs, the NLS, the proline-rich repeats (P), and glutamine-rich regions (Q) are indicated on the diagram. The stop signal indicates position aa 365 where the amber codon was introduced in the genetic analysis of the EBNA3C gene (41, 69).

FIG. 2

ProTα is expressed in various tissue types and in EBV-infected cells. (A) Northern blot analysis of poly(A) RNA in EBV-infected LCLs and Burkitt's lymphoma cell lines shows ProTα. LCL1, IB4, and B/B958 are EBV-positive cell lines, while BL41 is an EBV-negative Burkitt's lymphoma cell line. The top panel shows Northern blot analysis using a ³²P-labeled ProTα DNA probe. The lower panel shows Northern blot analysis using a [³²P]GAPDH DNA probe. (B and C). Northern blot analysis of poly(A) RNA in Multiple Tissue Northern Blots (CLONTECH Laboratories Inc.). The top panels show Northern blot analysis using a ³²P-labeled ProTα DNA probe. The lower panels show Northern blot analysis using a ³²P-labeled human β-actin cDNA probe.

FIG. 3

EBNA3C and ProTα associate in cell lysates and interact in vitro. (A) BJAB cells converted with a eukaryotic expression vector containing EBNA3C (BJAB pZIP E3C) or no insert (BJAB pZIP) and an EBV-positive LCL (LCL1) were used in GST pull-down analysis. Cell lysates from 100 million cells were incubated with GST fusion protein coupled to glutathione-Sepharose beads followed by GST-ProTα fusion protein coupled to glutathione-Sepharose beads. Lysates (1%) were used as a control. Proteins were fractionated by SDS–7% PAGE, transferred to 0.45-μm-pore-size nitrocellulose membranes, and Western blotted for EBNA3C using the monoclonal antibody A10 (55). (B) GST and GST-ProTα fusion protein coupled to glutathione-Sepharose beads were incubated with ³⁵S-labeled in vitro-translated EBNA3C or luciferase (Luc). A 10% input of in vitro-translated proteins was run as a control. Bound proteins were fractionated by SDS–7% PAGE, dried, and analyzed on a PhosphorImager (Molecular Dynamics) using ImageQuant software.

FIG. 4

EBNA3C coimmunoprecipitates with ProTα in transiently transfected cells. 293 cells were transfected with pA3M, pA3M/ProTα, pA3M/ProTα and pSG5/EBNA3C, or no DNA (Mock). Anti-myc ascites antibodies were used to collect immunoprecipitates. (A) Proteins were fractionated by SDS–7% PAGE, transferred to 0.45-μm-pore-size nitrocellulose membranes, and Western blotted for EBNA3C (A10). Cell lysates (5%) were also analyzed by SDS-PAGE to serve as a control. (B) ProTα was analyzed on a 15% gel, transferred to 0.2-μm-pore-size nitrocellulose membranes, and Western blotted with rabbit anti-ProTα antibody (1:100). (C) EBNA3C was detected by Western analysis as described for panel A.

FIG. 5

ProTα interacts with EBNA3C 3′ to aa 365. GSTΔEBNA3C fusion proteins were incubated with in vitro-translated ProTα. Bound proteins were fractionated by SDS–12% PAGE, dried, and then exposed to a PhosphorImager. (A) EBNA3C fragments fused to GST in pGEX vector. The appropriate amino acids for each fragment are indicated, and the names of each fragment begin with R for RsaI fragments and with A for AluI fragments. (B) Results of binding experiment indicating that the EBNA3C fragment from aa 260 to 509 interacts with ProTα. Another fragment, aa 393 to 641, does not interact, indicating that the region of interaction lies before aa 393. Lane 1, input in vitro-translated ProTα; lane 4, ProTα bound to R2 fragment.

FIG. 6

ProTα coimmunoprecipitates with EBNA3C and histone H1 in B-lymphoblastoid cells. BJAB cells converted with the pZIP eukaryotic expression vector containing EBNA3C (BJAB pZIP E3C) or no insert (BJAB pZIP control) were lysed in RIPA buffer and precleared with either protein A-Sepharose beads (ProA) or GST rabbit polyclonal serum (GST). Immunoprecipitates were collected with anti-ProTα rabbit polyclonal antibody. Cell lysates (1%) were fractionated as a control. (A) Immunoprecipitates fractionated by SDS–7% PAGE, transferred to 0.45-μm-pore-size nitrocellulose membranes, and Western blotted for EBNA3C using the monoclonal antibody A10. EBNA3C coimmunoprecipitated with ProTα in EBNA3C-expressing BJAB cells (lane 6) but not in control B cell lines (lane 3). (B) Immunoprecipitates were fractionated by SDS–15% PAGE, transferred to 0.2-μm-pore-size nitrocellulose membranes, and Western blotted for ProTα using the rabbit polyclonal antiserum. ProTα coimmunoprecipitated with EBNA3C seen in the immunoprecipitate lane 6, similar to lysate in lane 4, but not in the EBNA3C-negative BJAB cell lines. (C) Immunoprecipitates fractionated SDS–12% PAGE, transferred to 0.45-μm-pore-size nitrocellulose membranes, and Western blotted for histone H1 using the monoclonal antibody from Upstate Biotechnology. Histone H1 was detected as a doublet in the immunoprecipitate lanes 3 and 6, similar to that seen in the lysate lanes 1 and 4. (D) Immunoprecipitation performed as described above except 40 million BJAB (EBV-negative) and LCL1 (EBV-positive) cells were used and 5% cell lysates were used as a control. Cell lysates were precleared with anti-GST rabbit serum. Immunoprecipitates were fractionated by SDS–8% PAGE, transferred to 0.45-μm-pore-size nitrocellulose membranes, and Western blotted for EBNA3C using monoclonal antibody A10. EBNA3C immunoprecipitated with ProTα in the EBV-infected LCL (lanes 4 and 6), but not in the control EBV-negative cells (lanes 1 and 3). Quantitative analysis of the signals for changes in immunoprecipitation was done using SCION, NIH Image software.

FIG. 7

The acetyltransferase p300 coimmunoprecipitates with ProTα and EBNA3C in B-lymphoblastoid cells. BJAB cells converted with the pZIP eukaryotic expression vector containing EBNA3C (BJAB pZIP E3C) or no insert (BJAB pZIP control) were lysed in RIPA buffer and precleared with either protein A-Sepharose beads (ProA) or GST rabbit polyclonal serum (GST). Immunoprecipitates were collected with anti-ProTα rabbit polyclonal antibody. Cell lysates (1%) were fractionated as a control. (A) Immunoprecipitates were fractionated by SDS–6% PAGE, transferred to 0.45-μm-pore-size nitrocellulose membranes, and Western blotted for p300. The p300 signal seen in the immunoprecipitate lane 6 is barely detectable in the EBNA3C-negative cell line in lane 3. (B) Immunoprecipitates were fractionated by SDS–6% PAGE, transferred to 0.45-μm-pore-size nitrocellulose membranes, and Western blotted for p300. Similarly, the p300 signal was seen in the EBNA3C-expressing cell line (lane 6) but not in the EBNA3C-negative cell line (lane 3). (C) Immunoprecipitates were fractionated by SDS–6% PAGE, transferred to 0.45-μm-pore-size nitrocellulose membranes, and Western blotted for EBNA3C. EBNA3C signal was clearly observed in lane 6 where immunoprecipitates from EBNA3C-expressing cells were fractionated.

FIG. 8

The cellular acetylase-coactivator p300 interacts with ProTα. GST and GST-ProTα fusion protein coupled to glutathione-Sepharose beads were incubated with ³⁵S-labeled in vitro-translated 3′ p300, 5′ p300, or luciferase (Luc). Bound proteins as well as 10% input protein were fractionated by SDS–7% PAGE, dried, and analyzed on a PhosphorImager (Molecular Dynamics). (A) GST-ProTα binds the 3′ p300 polypeptide at a level approximately 20-fold (lane 5) over GST (lane 3). (B) The 5′ polypeptide of p300 bound at a level only about 1.2-fold over GST alone. No signal was seen with luciferase nonspecific control polypeptide. (C) GST (lane 1) and GST-ProTα fusion protein coupled to glutathione-Sepharose beads (lanes 2 to 5) were incubated with ³⁵S-labeled 3′ p300 and 2, 5, and 10 μl, respectively, of nonlabeled in vitro-translated EBNA3C. Bound proteins were fractionated by SDS–7% PAGE, dried, and analyzed on a PhosphorImager (Molecular Dynamics). (D) Histogram representing arbitrary counts corresponding to the in vitro binding analysis in panel C analyzed by ImageQuant software (Molecular Dynamics). Column numbers refer to lane numbers in panel C. Numbers indicate actual values obtained. Addition of increasing amounts of EBNA3C to the reaction results in reduction of 3′ p300 signal.

FIG. 9

Immunofluorescence analysis demonstrates nuclear colocalization of EBNA3C and ProTα. Cells from LCL1, a nascently derived EBV-immortalized cell line, were fixed in methanol-acetone (1:1) and incubated with antibodies against ProTα (rabbit polyclonal at 1:500 dilution) and against EBNA3C (mouse monoclonal at 1:1,000). Secondary antibodies to detect primary rabbit polyclonal antibodies against ProTα were goat anti-rabbit Texas red (A), and secondary antibodies to detect primary monoclonal antibodies against EBNA3 were goat anti-mouse FITC (B). (C) Overlay of panels A and B demonstrating colocalization in perinuclear regions. These data indicate that EBNA3C, ProTα, and p300 colocalize in the nucleus.

FIG. 10

EBV increases histone acetylation in nascently transformed human LCLs. (A) Results of HAT assay in cell lines expressing the EBV nuclear antigens in recently transformed LCLs, in B-cell lines overexpressing EBNA3C, and in a B-cell line that does not express any of the EBNA proteins. Immunoprecipitates from an anti-ProTα immunoprecipitation demonstrated that a significant HAT activity was seen in the two isogenic B-cell lines expressing all the EBNA proteins compared to the control (compare lanes 1 and 2 with lane 5), whereas this activity was much lower (approximately fourfold) in isogenic B-cell lines overexpressing EBNA3C (lanes 3 and 4), similar to that of the EBNA-negative isogenic B-cell line control (lane 5). A histogram of the results of counts obtained from HAT assays performed using ProTα immunoprecipitates is shown in panel A. (B) Histogram of the radioactive counts obtained from the HAT assay performed using p300 immunoprecipitates. (C) Western blot analysis showing levels of EBNA3C expression in cells used in HAT assays. Lanes correspond to the numbered columns in panels A and B. Lanes 1 and 2 represent assays done with two isogenic B-cell lines infected with EBV, lanes 3 and 4 also contain isogenic B-cell lines overexpressing EBNA3C, and lane 5 contains cells from the same isogenic B cell line used as a control which does not express any of the EBV EBNA proteins. (D) Western blot analysis to determine p300 levels as a control for each cell line used in HAT assays. (E) Internal protein loading controls on the SDS-PAGE gel, transferred to nitrocellulose and stained with Ponceau S.

FIG. 11

EBNA3C down-modulates HAT activity mediated by EBNA2 and acetyltransferases. In a representative experiment, 293T cells were transfected with expression constructs containing EBNA2 or EBNA2 and EBNA3C. Transfected cells were incubated for 24 h and then harvested and lysed in RIPA buffer. Complexes were collected by immunoprecipitation using the 5′ p300 polyclonal antibody from Santa Cruz. HAT activity was tested as described in Materials and Methods. Addition of EBNA3C results in depression of the HAT activity in a dose-responsive manner.

FIG. 12

Schematic diagram illustrating a possible model for EBNA3C association with ProTα and p300. ProTα may recruit the acetylase p300 and other viral or cellular transactivators denoted as protein X (PX). It is possible that corepressor complexes are also displaced by ProTα as the coactivator-acetylase p300 is recruited to promoters (38). This leads to acetylation of histone and upregulation of transcription. The expression of EBNA3C then displaces the megacoactivator complex, which may include p300, ProTα, and other coactivators like PX, leading to down-modulation of the histone acetylation activity. The net result of this activity leads to modulation of transcriptional activity.