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. 2005 Oct;25(19):8415–8429. doi: 10.1128/MCB.25.19.8415-8429.2005

FIG. 8.

FIG. 8.

Histone modifications at the cyclin D1 promoter locus. (A) HDAC activity was determined in Flag-immunoprecipitated fractions obtained from Flag or Flag-SMAR1 overexpressed 293 cells upon incubation with labeled histones. (B) In ChIP assays, chromatin was immunoprecipitated by using antibodies against H3K9, H4K8, and H3 p-Ser-10. The probe II and probe III region of the cyclin D1 promoter was amplified from pulled chromatin. For every PCR, 1 and 0.5 μl of the template was used for amplification. (C) Densitometric analysis of the acetylation status of the cyclin D1 promoter locus (probe II). (D) Endogenous SMAR1 was depleted using SMAR1 siRNA (RS) and studied for recruitment of SMAR1, HDAC1, Sin3A/SinB, p107, and p130. Further histone modifications were studied upon depletion of SMAR1 as indicated using the probe II and probe III region of the cyclin D1 promoter. (E) Densitometric analysis of the acetylation status of the cyclin D1 promoter locus (probe II) upon depletion of endogenous SMAR1. (F) MAR β was amplified as the positive control from SMAR1-pulled samples in siRNA (RS) and scrambled siRNA-treated cells. No amplification of MARβ in siRNA (RS)-treated cells further confirms the depletion of SMAR1. (G) An additional SMAR1 binding site and associated repressor molecule binding were checked on the probe VI region of the cyclin D1 promoter in 293 cells (0 h) and 293 cells transfected with Flag-SMAR1 (24 h) (left and middle panel, respectively), while the status of histone modifications on probe VI were studied in 293 cells (0 h) and cells overexpressing Flag-SMAR1 (24 h) (left panel). (H) An additional SMAR1 binding site and associated repressor molecule binding were checked on the probe VII (a MAR-like region 5 kb upstream) region of the cyclin D1 promoter in 293 cells (0 h) and 293 cells transfected with Flag-SMAR1 (24 h) (left and middle panels, respectively), while the status of histone modifications on probe VI were studied in 293 cells (0 h) and cells overexpressing Flag-SMAR1 (24 h). Histone modifications were studied at the probe VII (5 kb upstream) region of the cyclin D1 promoter in 293 cells (0 h) and 293 cells transfected with Flag-SMAR1 (24 h) (left panel). (I) Histone modifications and recruitment of SMAR1 and HDAC1 were checked at probe VII (5 kb upstream) of the cyclin D1 promoter upon depletion of endogenous SMAR1 from 293 cells. (J) Densitometric analysis of acetylation status of the cyclin D1 promoter locus (probe VII) upon depletion of endogenous SMAR1 from 293 cells. Autorad., autoradiography; Scr, scrambled siRNA.