Epigenomic regulation of human T-cell leukemia virus by chromatin-insulator CTCF

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes an aggressive T-cell malignancy and a variety of inflammatory conditions. The integrated provirus includes a single binding site for the epigenomic insulator, CCCTC-binding protein (CTCF), but its function remains unclear. In the current study, a mutant virus was examined that eliminates the CTCF-binding site. The mutation did not disrupt the kinetics and levels of virus gene expression, or establishment of or reactivation from latency. However, the mutation disrupted the epigenetic barrier function, resulting in enhanced DNA CpG methylation downstream of the CTCF binding site on both strands of the integrated provirus and H3K4Me3, H3K36Me3, and H3K27Me3 chromatin modifications both up- and downstream of the site. A majority of clonal cell lines infected with wild type HTLV-1 exhibited increased plus strand gene expression with CTCF knockdown, while expression in mutant HTLV-1 clonal lines was unaffected. These findings indicate that CTCF binding regulates HTLV-1 gene expression, DNA and histone methylation in an integration site dependent fashion.

Fig 1. HTLV-1 molecular clone with mutations in CTCF binding site (vCTCF-BS).

The top panel shows a schematic of the HTLV-1 proviral genome, and the positions of the pX domain (nucleotides 6619-8281), and the vCTCF-BS. Positions of sense-strand spliced tax and p12 transcripts and the predominant antisense hbz transcript are depicted. The bottom panel shows the consensus CTCF binding sequence, and the adjacent nucleotide sequence in wild type HTLV-1. A control virus was constructed with a termination codon in p12 (blue) in place of alanine residue 77, designated HTLV-1p12Stop. A mutant virus was constructed with mutations (red) in the vCTCF-BS, as well as the p12 termination codon, designated HTLV-1ΔCTCF. The positions of mutations introduced in the sense reading frame of p12 and antisense reading frame of HBZ are shown at the bottom.

Fig 2. Deletion of the CTCF binding site of HTLV-1 (vCTCF-BS) results in expansion of DNA methylation in the pX region of the provirus.

DNA methylation of the HTLV-1 provirus is presented as the percentage of methylated CpG (A) or fold change (B) (Y-axis) at the indicated locations of the viral DNA (X-axis). The schematic diagram of HTLV-1 provirus indicates the regions examined by bisulfite treatment and DNA sequencing as described in the Materials and Methods. Upper panel: HTLV-1 immortalized bulk population of PBMCs; lower panel: HTLV-1 infected bulk population of JET cells. CTCF binding site: 7041-7052 as indicated by an arrow. * Lost CpG sites, ** New CpG site due to the introduced mutations in vCTCF-BS. Nine CpG sites (5649-6076), 19 CpG sites (6503-7014), 8 CpG sites (7398-7615) are not shown because of unsuccessful PCR amplification in these regions after bisulfite treatment.

Fig 3. Histone methylation is increased in HTLV-1ΔCTCF.

Plots show fold enrichment over input for the indicated chromatin modifications on HTLV-1 provirus DNA in the bulk populations of JET cells (left) or PBMCs (right). Cells infected with HTLV-1ΔCTCF (orange), HTLV-1-p12stop (blue), or WT HTLV-1 (green) virus were subjected to chromatin immunoprecipitation for H3K4Me3, H3K36Me3 or H3K27Me3. Called peaks in HTLV-1ΔCTCF that are significantly different from both HTLV-1p12Stop and WT HTLV-1 are indicated by horizontal red lines.

Fig 4. Effect of CTCF-BS Mutation on HTLV-1 Integration Sites.

A) Clonality of HTLV-1 and HTLV-1ΔCTCF integration sites in JET cells and PBMCs, as measured by the Gini index. Inconsistent results were obtained with HTLV-1 WT infected cells. B-D). Frequency distribution of observed WT HTLV-1, HTLV-1p12stop, and HTLV-1ΔCTCF integration sites compared to random expectation within indicated window sizes (base pairs) in JET cells and PBMCs. B) Frequency of integration positions relative to upstream or downstream active transcription start sites (ATSS). The inset shows the transcriptional frequency of active genes in normal CD4+ T lymphocytes, with the shaded gene showing that for Fox P3. C) Frequency of HTLV integration sites relative to cellular CTCF sites. D) Frequency of HTLV integration sites relative to CpG islands.

Fig 5. Inhibition of CTCF binding to HTLV-1 provirus affects proviral gene transcription.

Clonal HTLV-1 (A-a and B) or HTLV-1ΔCTCF (A-c and C) infected JET cell lines were generated, each carrying a single, latent provirus at a unique integration site. The infected JET cells from panels B, and C were collected and cellular CTCF was detected by Western blotting using anti-CTCF antibody. Histone H3 was also blotted as a loading control. The amount of CTCF was measured by densitometry quantification using BioRad image lab software, normalized to the value of histone H3 and is indicated under each lane. A-b). The JET cells from Fig 5B were cocultured with Jurkat cells expressing firefly luciferase driven by the HTLV-1 LTR (LTR-Luc) in the presence or absence of PMA/Ionomycin for 48 hr and viral infectivity was determined by measuring luciferase activity and presented as a fold change compared with the values of untreated cells (t test: *p < 0.05; ns: not significant). B & C). Viral gene expression was activated with PMA/Ionomycin and monitored by measuring Tax mediated RFP production using the IncuCyte live cell image system. The levels of tax and hbz mRNA were also measured by qRT-PCR. Total copy number of each mRNA was determined using plasmid DNA standards and normalized to 10⁶ copies of GAPDH mRNA. Cell clone number of HTLV-1 infected and HTLV-1ΔCTCF cells are indicated in each panel. Each experiment was repeated three times and a representative result with three replicates is shown with standard errors.

Fig 6. CTCF knock down by shRNA in clonal JET cell lines results in expansion of DNA methylation in the pX region of the provirus.

DNA methylation of the HTLV-1 provirus is presented as fold change, CTCF vs control shRNA (Y-axis) at the indicated locations of the viral DNA (X-axis). Average fold increase is calculated from nt 7029 to 9615. The schematic diagram of the HTLV-1 provirus indicates the regions examined by bisulfite treatment and DNA sequencing as described in the Materials and Methods. Upper panel: Type I clonal cell lines. Lower panel: Type II clonal cell lines.

Fig 7. Latency establishment and reactivation of HTLV-1 does not depend on CTCF binding.

JET cells were infected by coculturing with lethally γ-irradiated 729B/HTLV-1, 729B/HTLV-1p12stop or 729B/HTLV-1 ΔCTCF and single infected cell clones were selected via limiting dilution. RFP (the indicator of Tax expression) positive or negative cells were examined and percentages of latent (RFP negative) and active (RFP positive) HTLV-1 infected cells are shown in panel A. RFP negative cells were activated with PMA/Ionomycin for 48hr. RFP positive and negative cells were counted and the result from three experiments is presented with standard errors in panel B.