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, 3 (2), 141-6

MEF2-mediated Recruitment of Class II HDAC at the EBV Immediate Early Gene BZLF1 Links Latency and Chromatin Remodeling

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MEF2-mediated Recruitment of Class II HDAC at the EBV Immediate Early Gene BZLF1 Links Latency and Chromatin Remodeling

H Gruffat et al. EMBO Rep.

Abstract

In B lymphocytes induced to proliferate in vitro by the Epstein-Barr virus (EBV), extra-chromosomal viral episomes packaged in chromatin persist in the nucleus, and there is no productive cycle. A switch from this latency to the productive cycle is observed after induced expression of the EBV BZLF1 gene product, the transcription factor EB1. We present evidence that, during latency, proteins of the myocyte enhancer binding factor 2 (MEF2) family are bound to the BZLF1 promoter and recruit class II histone deacetylases. Furthermore, we propose that latency is determined primarily by a specific and local recruitment of class II histone deacetylase (HDAC) by MEF2D to the BZLF1 gene promoter. The switch from latency to the productive cycle could be due in part to post-translational modification of MEF2 proteins and changes in the local acetylation state of the chromatin.

Figures

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Fig. 1. VP16-HDAC4/5 are specifically recruited at the BZLF1 gene promoter in vivo and activate the switch from latency to the productive cycle. (A) Schematic representation of the BZLF1 gene promoter. Binding sites for cellular (MEF2, Sp1/Sp3, CREB) and viral (ZRE: EB1 responsive element) proteins are represented along the promoter region by light-gray and dark-gray boxes, respectively. (B) Schematic representation of class II HDACs (HDAC4 and HDAC5) and their modified versions, VP16-HDAC4 and VP16-HDAC5. (C) Western blots from Raji cell extracts. The equivalent of 2 × 106 cells was loaded onto each lane of a 10% SDS–PAGE. Membranes were incubated with either the anti-BRLF1, the anti-BMRF1 or the anti-BZLF1 Mab. Lane 1, Raji cells treated with TPA/BA; lane 2, untreated Raji cells; lane 3, Raji cells transfected with VP16-HDAC4 expression plasmid; lane 4, Raji cells transfected with VP16-HDAC5 expression plasmid. (D) Left: the dark-gray boxes represent the EB1 responsive elements present on these promoters, and the light-gray boxes represent the MEF2 sites on the Zp promoter. Right: histogram of representative results from CAT ELISA made following co-transfection of DG75 cells with the reporter plasmids and the transactivator expression plasmids as indicated.
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Fig. 2. MEF2 family proteins mediate the effect of class II HDAC on the BZLF1 gene promoter. (A) Schematic representation of the HDAC4 protein and its modified versions, HDAC4ΔMEF2, VP16-HDAC4 and VP16-HDAC4ΔMEF2. The MEF2 interacting domain is shown in black and the deleted region is represented by a thin line. (B) Left: schematic representation of the Zp-CAT reporter constructs co-transfected with the VP16-HDAC4 expression plasmid. Right: histogram of the results of a representative CAT ELISA made following co-transfection of DG75 cells with the reporter constructs indicated on the left of the figure and an VP16-HDAC4 expression plasmid. (C) Histogram of the results of a representative CAT ELISA made following co-transfection of DG75 cells with the Zp-CAT reporter construct and expression plasmids for VP16-HDAC4 and VP16-HDAC4ΔMEF2 as indicated. (D) Histogram of the results of a representative CAT ELISA made following co-transfection of DG75 cells with the reporter plasmids Zp-CAT or Nap-CAT and the expression plasmids (coding for EB1, HDAC4, HDAC4ΔMEF2 or HDAC1) as indicated.
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Fig. 3. (A) Effect of the induction of the EBV productive cycle in Akata cells upon acetylation at the Zp promoter. H3 and H4 acetylation of the Zp, Cp and Mp promoters was determined by CHIP assay using anti-acetylated H3 or anti-acetylated H4 antibodies in untreated Akata cells and in Akata cells treated for 3 h with anti-IgG. (B) Model of transcriptional regulation at the BZLF1 gene promoter. During latency, MEF2-D proteins are present on the BZLF1 gene promoter and they mediate local histone deacetylation on this promoter by recruiting class II HDAC. This contributes to the absence of expression of the BZLF1 gene and maintenance of latency. After reactivation of the productive cycle by different inducers, the MEF2-D proteins are modified so that they do not interact with class II HDAC but do interact with HATs and/or other cellular factors such as NFAT.

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