Cohesins localize with CTCF at the KSHV latency control region and at cellular c-myc and H19/Igf2 insulators

Abstract

Cohesins, which mediate sister chromatin cohesion, and CTCF, which functions at chromatin boundaries, play key roles in the structural and functional organization of chromosomes. We examined the binding of these two factors on the Kaposi's sarcoma-associated herpesvirus (KSHV) episome during latent infection and found a striking colocalization within the control region of the major latency transcript responsible for expressing LANA (ORF73), vCyclin (ORF72), vFLIP (ORF71), and vmiRNAs. Deletion of the CTCF-binding site from the viral genome disrupted cohesin binding, and crippled colony formation in 293 cells. Clonal instability correlated with elevated expression of lytic cycle gene products, notably the neighbouring promoter for K14 and vGPCR (ORF74). siRNA depletion of RAD21 from latently infected cells caused an increase in K14 and ORF74, and lytic inducers caused a rapid dissociation of RAD21 from the viral genome. RAD21 and SMC1 also associate with the cellular CTCF sites at mammalian c-myc promoter and H19/Igf2 imprinting control region. We conclude that cohesin subunits associate with viral and cellular CTCF sites involved in complex gene regulation and chromatin organization.

Figure 1

KSHV genome-wide ChIP analysis: colocalization of CTCF and cohesins upstream of ORF73. (A) A 384-well array of PCR primers spanning ∼400-bp intervals across the KSHV genome was used to analyse ChIP DNA isolated with antibodies specific for LANA, ORC2, CTCF, SMC1, SMC3, RAD21, or control IgG. The KSHV coordinates are indicated below each column of graphs. •LANA- and ORC2-binding sites at TR; ^*location of the major CTCF–cohesin-binding site upstream of ORF73. (B) Schematic of the region of KSHV genome (nt 116 000–138 000) containing the major latency transcript (leftward ORF73/72/71, vmiRNA, and K12), the rightward-oriented K14/ORF74 lytic transcript, and the TR. (C) Western blot of BCBL1 cell extracts with antibodies to CTCF, SMC1, SMC3, and RAD21 used for ChIP in (A). (D) Co-immunoprecipitation assay with antibodies to CTCF, SMC1, RAD21, or IgG control. IPs were immunoblotted with anti-CTCF.

Figure 2

Mapping CTCF-binding sites upstream of ORF73. (A) DNA affinity purification assay with biotinylated DNA from KSHV 126 841–127 840 or a control 1000-bp fragment from pBKS. BCBL1 nuclear extract was incubated with biotinylated DNA and bound proteins were identified by western blotting with antibody specific for CTCF (top panel) or SMC1 (lower panel). (B) Smaller DNA fragments were used to fine map the CTCF-binding site upstream of ORF73 as indicated by coordinates above each lane. Bound proteins were detected by western blot with antibodies specific for CTCF (top panel). A nonspecific, cross-reacting protein is shown in lower panel as a loading control (CTRL). (C) DNase I footprinting of KSHV DNA with purified recombinant CTCF protein. KSHV coordinates are indicated to the right of each footprint boundary. G and A are chemical sequencing ladders. ORF73p⁺ is the sense strand (left panel) and ORF73p⁻ is the antisense (right panel). (D) Schematic showing the location of the cluster of three CTCF-binding sites relative to ORF73 (LTi and LTc) and K14/ORF74 (rightward arrow) transcription start sites. CSL/Rta indicates the sites in K14/ORF74 promoter stimulated during lytic activation by Rta.

Figure 3

CTCF sites are required for cohesin binding. (A) Schematic diagram of KSHV bacmid mutations generated to substitute the CTCF-binding site (CBS) cluster at positions 127 331–127 549 with LoxP recognition site. (B) BAC36 WT, MT1, and MT2 were analysed by ethidium bromide (EtBr) staining of agarose gels for insertion of novel EcoRI site (left) and by Southern blot of the same gel probed with LoxP-specific probe (right). (C) RT–PCR was used to measure LANA mRNA levels in WT, MT1, MT2, or a control deletion in the viral miRNAs (CTRL). bacmid-expressed GFP mRNA was used as an internal standard for genome copy number. (D) LANA protein expression was analysed by western blotting of immunoprecipitates with two different LANA-specific antibodies. Extracts were derived from identical number of cells for WT, MT1, MT2, untransfected 293, and latently infected BCBL1 cells. (E) ChIP assay of 293 cells transfected with WT, MT1, or MT2 after selection with hygromycin for 2 weeks. Antibodies for ChIP included CTCF, SMC1, RAD21, and LANA. ChIP DNA was analysed by real-time PCR with primers specific for the CBS or the terminal repeat (TR).

Figure 4

Host cell growth defects and plasmid instability in KSHV genomes lacking CTCF sites. (A) WT-, MT1-, and MT2-transfected 293 cells were cell sorted for GFP expression and assayed for hygromycin-resistant colony formation. After 10–15 days of selection, the plates were photographed with a 35 mm camera and the colony number was quantitated using Image-Pro Plus software. (B) Example of colony size and density for WT-, MT1-, and MT2-formed colonies. (C) WT-, MT1-, and MT2-transfected 293 cells were sorted for GFP and then cultured in hygromycin-containing media. Cells were then assayed at 4, 8, 12, and 16 days for proliferation using fluorosphere beads and FACS analysis to count cell number. (D) Plasmid stability was assayed by monitoring GFP expression of hygromycin-resistant cell colonies. Hygromycin-resistant cell colony derived with either WT (a, c) or MT1 (b, d); KSHV bacmid was assayed by fluorescence microscopy for GFP (a, b) or a merge of fluorescence with phase-contrast microscopy (c, d). (E) Plasmid stability was assayed by FACS analysis of GFP-positive cells. Identical numbers of WT- and MT1-transfected 293 cells were sorted 4 days after transfection. The percentage of cells retaining GFP-positive signals was then determined 4 or 8 days after initial sorting.

Figure 5

K14 and ORF74 transcription are elevated in CBS deletion mutants. (A) WT-, MT1-, and MT2-transfected 293 cells were sorted by GFP and selected in hygromycin for 4 days. RNA was isolated and analysed by RT–qPCR for primers specific for ORF50, ORFK12, ORF71, ORF73, ORFK14, ORF74, and GFP. The values were reported as fold over the internal control of GFP, which is a common component of the bacmid. (B) Latently infected BCBL1 cells were transfected with siRAD21 or control siLucif, sorted by FACS, and then assayed by RT–qPCR for RNA expression levels of RAD21, ORF50, ORF73, ORFK14, and ORF74 with normalization to cellular actin. The relative change between siRAD21 and siLucif is indicated above each pair of bars. (C) RNA samples from experiment described in (B) were evaluated for expression of Cyclin A2, Cyclin B, and Cyclin E using RT–qPCR.

Figure 6

Dissociation of RAD21, SMC1, and CTCF upon lytic cycle induction. (A) BCBL1 cells were treated with sodium butyrate for 0, 1 or 24 h and then assayed by ChIP with antibodies to CTCF, SMC1, or RAD21. ChIP DNA was analysed by real-time PCR with primers specific for the KSHV CBS or a control region between ORFK11 and K2. ChIP data are presented as fold over IgG for each primer set and antibody. (B) RNA levels were monitored at 0, 1, or 24 h after sodium butyrate treatment and then assayed by RT–qPCR with primers specific for ORF50, ORFK12, ORF71, ORF73, ORFK14, or ORF74. RNA was quantified as fold increase over time zero relative to cellular actin. (C) FACS analysis of propidium iodide-stained BCBL1 cells at 0, 1, or 24 h after treatment with sodium butyrate (3 mM) was used for cell cycle profiling.

Figure 7

Colocalization of cohesin subunits at cellular CTCF sites. (A–D) Analysis of CTCF and cohesin binding at the human c-myc locus. (A) Schematic of the CTCF-binding sites and other chromatin features of the c-myc promoter. Location of primer pairs A–D are indicated. Primer pairs C and E amplify the known CTCF sites. (B–D) ChIP assays with antibodies for CTCF, RAD21, or IgG control were analysed by real- time PCR with primer pairs A–E specific for different regions of the c-myc promoter. HeLa (B), KSHV-positive BCBL1 (C), or Burkitt lymphoma Raji (D) cells were used for ChIP assays. (E, F) Analysis of CTCF and cohesin binding at the H19/Igf2-imprinted locus. (E) Schematic of the H19/Igf2 locus and primer location. Rep1&2 and Rep3 indicate the first three CTCF-binding sites in the differentially methylated domain (DMD). The H19 transcription unit is designated by a filled box and enhancers by filled circles. (F) Pattern of CTCF and cohesin binding across the H19 locus was analysed by real-time PCR relative to the input DNA. The fraction of precipitated DNA relative to input was normalized to the value obtained at Rep1&2 region, which typically had the highest level in the H19 locus. Four regions as indicated in (E) were analysed and c-myc locus was included as positive control. The graph shows the average and s.d of the normalized values. ^*P<0.01, CTCF, SMC1 and RAD21 binding is significantly different between Rep1&2 and other regions. ^**P>0.05, CTCF, SMC1, and RAD21 binding is not significantly different between Rep1&2 and c-myc regions. (G) Allele-specific ChIPs were carried out with antibodies against CTCF, SMC1, RAD21, or no antibody (no Ab). F1 hybrid B × P12X mouse embryonic fibroblast cells and neonatal liver cells from H19^DMD−ΔR/+ mice isolated and examined by ChIP. DNA from control (B, C, and B × C genomic DNA alone, left-hand panels), input and precipitated samples were subjected to quantitative-PCR for the indicated region of H19 (Rep3 and Ex5) and then digested with a restriction enzyme that is polymorphic between B and C (Tsp45I for Rep3 and BglI for Ex5). The genotypes of cells and tissues are indicated at the top. In all experiments shown here, the B allele corresponds to the maternal allele, while the C allele is the paternal allele. The numbers underneath each panel indicate the percentage of B allele (maternal) relative to total. Note that ChIP assays were performed multiple times and the allele-specific assays were conducted on each ChIP sample but only one representative experiment is shown for each antibody and region.