Fig 2: Effect of RbAp48 on HIV-1 production in 293T cells. pRbAp48 at concentrations of 100, 200, 300, 400, 500, 600 and 700 ng were transfected into 293T cells after HIV-1 infection (multiplicity of infection of 1 by spinoculation at 1,200 x g for 2 h at 25˚C). The cell supernatants were collected after 48 h of pRbAp48 transfection to examine HIV-1 genome expression by reverse transcription-quantitative PCR. *P<0.05, **P<0.02 and ***P<0.01 vs. pCTL-transfected cells. RbAp48, retinoblastoma binding protein 4; HIV-1, human immu-nodeficiency virus type 1.

Fig 3: Effect of TEP and pRbAp48 on LTR activity. (A) Cells were treated with 100, 200, 300, 400, 500, 600 and 700 ng pRbAp48 to analyze the LTR activity after 48 h. (B) TNF-α and PMA-stimulated 293T cells were incubated with pRbAp48 (700 ng) and TEP (2.5 µM) for determination of the luciferase activity. The experiments were performed in triplicates. The 0 ng pRbAp48 group was transfected with the negative control, pCTL. *P<0.05, **P<0.02 and ***P<0.01 vs. respective control cells. TEP, thieno[3,4-d]pyrimidine; TNF-α, tumor necrosis factor-α; RbAp48, retinoblastoma binding protein 4; LTR, long terminal repeat; PMA, phorbol 12-myristate 13-acetate.

Fig 4: Two conserved residues from minimal MBD2IDR are necessary to bind the histone deacetylase core of NuRD. (a) Mutating five residues from the first region of minimal MBD2IDR (P244G, R246E, P255A, V256A and R246E/T248A) does not affect immunoprecipitation of the histone deacetylase core complex. Similarly, mutating four residues from the second ordered region of minimal MBD2IDR (P278G, Q280A, R286E and L287A) does not affect immunoprecipitation of the histone deacetylase core complex. However, combined mutation of two adjacent residues (R286E/L287A) significantly abrogates the ability of MBD2IDR to recruit the histone deacetylase core components. Strikingly, the individual mutants R286E and L287A have no effect on interaction of the MBD2IDR with the histone deacetylase core. Three additional mutations (T248A, W283A and L290A) do not affect binding of MBD2IDR to the histone deacetylase core complex (data not shown). (b) Full length flag-tagged MBD2 carrying the double mutation R286E/L287A (Double Mutant) also displays a disrupted interaction with RbAp48, HDAC2 and MTA2. The interaction of Double Mutant MBD2 with MTA2 is completely lost whereas its interaction with RbAp/HDAC shows significant reduction.

Fig 5: The MBD2IDR binds the histone deacetylase core complex of NuRD. (a) The MBD2IDR with scMBD2-p66α was transfected in high-transfection-efficiency HEK 293T cells. Immunoprecipitation and western blot analysis of the transfected cells indicates that the MBD2IDR binds the histone deacetylase core components RbAp48, HDAC2 and MTA2, whereas the scMBD2-p66α construct does not. The flag-IP lane shows pull down of the histone deacetylase core components by immunoprecipitation using an anti-flag antibody directed against the flag-tagged MBD2IDR. IgG and expression vector pCMV serve as negative controls while the input lane shows 2% of the input. (b) The MBD2IDR was divided into three sub-regions to test whether we could isolate critical binding region(s) for the core complex components. (c) The three sub-regions of MBD2IDR were expressed individually in HEK 293T cells and immunoprecipitated, but failed to bind to either of the histone deacetylase core complex components. Note that a non-specific band appears in the IgG lane when blotted with anti-MTA2 that runs just below the MTA2 protein in the input and flag IP lanes. (d) The region of MBD2IDR from amino acids 212–316 comprising the first and second ordered sub-regions in combination can bind to RbAp48, HDAC2 and MTA2, although a weaker interaction with MTA2 was observed.