A -galactosidase expression vector (pCMV-Sport-Gal, Life Technologies) was co-transfected as an internal control, and -galactosidase activity was used to normalize CAT activity as described . Coimmunoprecipitation HEK293 cells were transfected as described above with 15 g each of expression vectors encoding Flag-BCL11A and/or Myc-SIRT1. found to interact directly with BCL11A and was recruited to the promoter template in a BCL11A-dependent manner leading to transcriptional repression. These findings define a role for SIRT1 in transcriptional repression mediated by BCL11A in mammalian cells. promoter but upstream of the transcriptional start site. The resulting PCR product (327 bp) was analyzed by agarose gel electrophoresis and ethidium bromide staining. All experiments were performed three to five times. Transfection and reporter gene studies HEK293 cells were transfected and harvested as described above. Where indicated, TSA (100 ng/ml) or nicotinamide (15 mM) treatments were initiated 24 h after transfection, and cells were harvested 24 h later. A -galactosidase expression vector (pCMV-Sport-Gal, Life Technologies) was co-transfected as an internal control, and -galactosidase activity was used to normalize CAT activity as described . Coimmunoprecipitation HEK293 cells were transfected as described above with 15 g each of expression vectors encoding Flag-BCL11A and/or Myc-SIRT1. Forty-eight hours after transfection the cells were lysed in NET-N buffer (20 mM TrisCHCl, pH 8, made up of 150 mM NaCl, 0.5% NP-40, 10% glycerol, 1 mM EDTA, and a protease inhibitor cocktail) by agitation at 4 C for 30 min. After a brief sonication, lysates Dienogest were cleared by centrifugation, and immunoprecipitated as described previously  using the antibodies described above. Whole cell extracts from 70z/3 pre-B lymphocytes were prepared by using NET-N buffer as described above, and immunoprecipitated (10 mg of total protein per reaction) with purified anti-BCL11A (10C15 g) or anti-Sir2 (0.5C2.5 g) antibodies. Influenza B virus Nucleoprotein antibody All immunoprecipitates were analyzed by immunoblotting with appropriate antibodies. GST pulldown experiments GST pulldown experiments were conducted as described previously . Briefly, equivalent amounts of GST or GST-SIRT1 fusion proteins were bound to glutathioneCSepharose (Pharmacia) and incubated with [35S]methionine-labeled proteins (BCL11A or BCL11A truncation mutants) prepared using the TNT transcriptionCtranslation system (Promega). The reactions were washed five occasions with binding buffer (10 mM Na-Hepes made up of 10% glycerol, 1 mM EDTA, 1 mM DTT, 150 mM NaCl, and 0.05% NP-40) and bound proteins were eluted and resolved on denaturing SDSCPAGE gels for analysis by autoradiography. Results Transfection of BCL11A results in deacetylation of histone H3/H4 associated with the promoter region of a target gene Previous studies indicated that BCL11A represses transcription in a manner that was only partially reversed by TSA suggesting the lack of involvement of class I or II HDACs [1,5,8]. However, these results do not preclude the possibility that TSA-insensitive HDACs, such as class III HDACs  or novel, TSA-insensitive HDAC(s), may be involved in BCL11A-mediated transcriptional repression. Thus, chromatin immunoprecipitation (ChIP) studies were conducted to assess possible alterations in the extent of histone H3/H4 acetylation around the template of a reporter gene in cells transiently transfected with an expression vector encoding a Gal4-BCL11A fusion protein. The amount of acetylated histones H3 and/or H4 at the promoter region of a target gene (a multimerized 17-mer in the context of the thymidine kinase promoter upstream of chloramphenicol acetyl-transferase) was found to be decreased in Gal4-BCL11A-transfected cells (upper panel Dienogest of Fig. 1A, compare lane 4 with lane 6). Deacetylation of histone H3/H4 in BCL11A-transfected cells was unaffected by treatment with TSA (Fig. 1A, compare lanes 3C6 of the top and middle panels), consistent with previous studies that exhibited TSA-insensitive, BCL11A-mediated transcriptional repression [1,5]. However, deacetylation of promoter-associated histone H3/H4 in BCL11A-transfected cells was inhibited by nicotinamide (NAM), at least partially (Fig. 1A, compare lanes 3C6 of the Dienogest top and bottom panels), indicative of the possible involvement of a TSA-insensitive, NAD+ -sensitive, class III HDAC [8,9]. Open in a separate windows Fig. 1 Deacetylation of promoter-associated histones H3 and/or H4 in BCL11A-transfected cells and BCL11A-mediated transcriptional repression are partially reversed by nicotinamide. (A) HEK293 cells were transfected with 3 g (17-mer)4-BCL, B cell leukemia; bp, base pair; CAT, chloramphenicol acetyltransferase; COUP-TF, chicken ovalbumin upstream promoter transcription factor; CTIP 1 and 2, COUP-TF-interacting proteins 1 and 2; GST, glutathione em S /em -transferase; HA, hemagglutinin; HDAC, histone deacetylase; HEK293, human embryonic kidney 293 cells; IgG, immunoglobulin G; kDa, kilodalton; NAM, nicotinamide; Sir2, silent information regulator 2; SIRT1, Sir2-like protein 1 or sirtuin 1; TSA, trichostatin A; WT, wild type..