Chromatin remodeling of the Kaposi's sarcoma-associated herpesvirus ORF50 promoter correlates with reactivation from latency

Abstract

The switch from latent to lytic infection of Kaposi's sarcoma-associated herpesvirus is initiated by the immediate early transcriptional activator protein Rta/open reading frame 50 (ORF50). We examined the transcriptional regulation of the ORF50 core promoter in response to lytic cycle stimulation. We show that the ORF50 promoter is highly responsive to sodium butyrate (NaB) and trichostatin A (TSA), two chemicals known to inhibit histone deacetylases. The NaB and TSA responsive element was mapped to a 70-bp minimal promoter containing an essential GC box that binds Sp1/Sp3 in vitro and in vivo. Micrococcal nuclease mapping studies revealed that a nucleosome is positioned over the transcriptional initiation and the Sp1/3 binding sites. Stimulation with NaB or TSA increased histone acetylation and restriction enzyme accessibility of the ORF50 promoter transcription initiation site. Chromatin immunoprecipitation assay was used to demonstrate that the ORF50 promoter is associated with several different histone deacetylase proteins (including HDAC1, 5, and 7) in latently infected cells. NaB treatment led to the rapid association of Ini1/Snf5, a component of the Swi/Snf family of chromatin remodeling proteins, with the ORF50 promoter. Ectopic expression of the CREB-binding protein (CBP) histone acetyltransferase (HAT) stimulated plasmid-based ORF50 transcription in a HAT-dependent manner, suggesting that CBP recruitment to the ORF50 promoter can be an initiating event for transcription and viral reactivation. Together, these results suggest that remodeling of a stably positioned nucleosome at the transcriptional initiation site of ORF50 is a regulatory step in the transition from latent to lytic infection.

FIG. 1.

Chemical induction of ORF50 promoter. (A) BCBL-1 cells were treated with TSA, NaB, or TPA and analyzed by RT-PCR for expression levels of ORF50 (top panel) or GAPDH (lower panel). Controls lacking reverse transcriptase (−RT) are indicated for ORF50. (B) The ORF50 promoter fused to luciferase (p50) or the pGL3-Basic control plasmid were transfected into BCBL-1 cells and treated with TSA, NaB, or TPA. (C) BCBL-1 cells were transfected with luciferase reporter plasmid pGL3-Basic or promoters derived from BZLF1, ORF50 (p50), the K8 immediate early promoter (K8-IE), or the K8 delayed early promoter (K8-DE). (D) p50 and pGL3-Basic control plasmids were transfected into JSC-1, BCBL-1, 293, or DG75 cells. Luciferase activity was measured as induction (n-fold) upon treatment with 3 mM NaB for 24 h.

FIG. 2.

Mutational analysis of ORF50 promoter response to NaB. (A) 5′ and 3′ deletion mutants of ORF50 were transfected into BCBL-1 cells and assayed for luciferase activity in the presence or absence of NaB. Luciferase activity is displayed as activation (n-fold) in response to NaB. Schematic diagrams of deletion mutants are shown to the left. The ATG start site is indicated as +1 and corresponds to position 71598 in the KSHV genome. (B) Substitution mutants of ORF50 (−298/+1) were assayed for NaB induction in JSC1 cells. Substitution mutations were indicated by underlined sequences in the schematic to the left.

FIG. 3.

Sp1/3 binds to the ORF50 promoter in vitro and in vivo. (A) BCBL-1-derived nuclear extracts from NaB-treated (+) or untreated (−) cells were subject to DNA affinity purification with DNA derived from pBKSII (BKS), ORF50 (−298/+1), or the GC-box substitution mutant of ORF50 (−114) (ORF50mt). Bound proteins were assayed by Western blotting of SDS-polyacrylamide gel electrophoresis gels with antibodies specific for Sp1 (αSp1), Sp3 (αSp3), or Rb (αRb) as indicated to the left of each panel. (B) NaB-treated (+) or untreated (−) cells were analyzed by ChIP with antibodies specific for Sp1 or control IgG. Promoter fragments from the ORF50 promoter (left panel) or for the coding sequences of GAPDH (right panel) were generated by PCR. Input DNA is indicated in the lower panel. Threefold dilutions of input DNA were used to determine the linearity of PCRs. (C) 293 cells transfected with ORF50-Luc plasmid were assayed by ChIP for association with Sp1 before and after NaB treatment.

FIG. 4.

Nuclease mapping of ORF50 promoter chromatin. (A) Partial Mnase digestion of JSC1 cells treated with (+) or without (−) NaB was assayed by Southern blotting and probed with ORF50 sequences (positions 71301 to 71598). Hypersensitive sites are indicated by the arrows, and molecular size standards are indicated to the right. (B) Endonuclease sensitivity assay of ORF50 promoter region in JSC1 cells treated with (+) or without (−) NaB. Nuclei were incubated with Asp718 (left panel) or HincII (right panel), and then isolated DNA was cleaved with NcoI/EcoRV and analyzed by Southern blotting with an ORF50-specific probe. Asp718 (left panel) and HincII (right panel) cleavage products are indicated. A schematic of ORF50 restriction sites, GC box, and presumptive nucleosomes is indicated below. (C) Schematic indicating restriction sites and probe used for indirect end-labeling.

FIG. 5.

Primer extension mapping of Mnase I nuclease cleavage sites in ORF50. (A) Mononucleosomal fragments from Mnase-treated nuclei were gel purified and analyzed by primer extension with primers Pr1 and Pr2, as indicated above the gel. Sequence reactions were generated with Pr1, and the GC box is indicated to the left. Numbered arrows indicate the major primer extension products that correlate with Mnase I cleavage sites and nucleosome boundaries. (B) Primer extension with Pr2 was used to compare the mononucleosomal fragments derived from BCBL-1, JSC-1, and 293 cells and 293 cells transfected with ORF50-Luc plasmid DNA. Sequencing lanes were generated with Pr1. The schematic at the bottom indicates the nucleosome positions mapped by the above primer extension and analysis.

FIG. 6.

Chromatin modifications and modifying enzymes at ORF50. (A) A ChIP assay with antibodies specific for hyperacetylated histone H3 (αacetylH3) or H4 (αacetylH4) was used to compare histone acetylation in untreated (−) or NaB-treated (+) BCBL-1 cells at the ORF50 promoter (left panel) or the GAPDH coding sequence (right panel). Control IgG ChIP and input DNA are indicated in the lower panels. (B) The ORF50 promoter was compared to the latency-associated ORF72 and ORF73 promoters by ChIP assay with antibodies to acetylated histone H3 and H4. (C) 293 cells transfected with ORF50-Luc plasmid were assayed by ChIP for association of acetylated histones. Quantification of ethidium-stained DNA in agarose gels is indicated by the bar graphs to the right.

FIG. 7.

Recruitment of chromatin-modifying activities at ORF50. (A) JSC1 cells were transfected with ORF50 (−298/+1) luciferase and CMV-driven expression vectors for CBP, CBP-ΔHAT, p300, P/CAF, hGCN5, or TIP60. Luciferase activity is indicated in relative light units. (B) ORF50-Luc (−298/+1), the GC-box mutant (ORF50/−114), and pGL2-Basic were compared for their responsiveness to CMV-FLAG, CMV-CBP, or CMV-CBPΔHAT. (C) Antibodies to HDACs 7, 6, 5, 4, 3, and 1 were used in a ChIP assay to determine association with the ORF50 promoter (top panel) in BCBL-1 untreated cells. The GAPDH coding sequence is indicated in the lower panel. IgG control ChIP, input DNA, and marker DNA (M) are indicated. (D) Western blotting with HDAC antibodies was used to measure protein levels in total cell extracts from untreated (−) or NaB-treated (+) BCBL1 cells. (E) ChIP assay with antibodies specific for the Ini1/Snf5 protein in untreated (−) or NaB-treated (+) BCBL-1 cells comparing ORF50 (top panel) and GAPDH (lower panel) DNA amplification. Control IgG ChIP and input DNA are indicated. (F) 293 cells transfected with ORF50-Luc were assayed by ChIP for association with Ini1/Snf5 in the absence (−) and presence (+) of NaB.

FIG. 8.

Nucleosome modification and chromatin remodeling can regulate the ORF50 promoter during reactivation signals. Sp1 binds ORF50 near the transcription initiation site and within the boundary of the nucleosome. Histone acetylation caused by NaB, TSA, or ectopic expression of CBP leads to nucleosome remodeling and transcription activation. Contributions from unknown additional factors are represented by question marks.