Mybbp1a is then known to associate with diverse transcriptional regula tors, acting largely as a repressor for Pol II transcription. In this capacity, Mybbp1a was recently identified as a component of several HDAC containing co repressor and ATP dependent chromatin remodeling complexes, including Ret CoR and esBAF complex. These enzymatic activities are intimately associated with the processes of differentiation and stem cell physiology, and mostly contain common constituents such as HDACs. Unlike other chromatin remodeling factors, no enzym atic activity has been described for Mybbp1a, despite the presence of several domains that are characteristic of transcriptional regulators, such as leucine zipper like, basic, and acidic motifs.

Intriguingly, the binding of Mybbp1a with histone H3 amino terminus that is dimethylated on the Lys9 residue was demonstrated pre viously. These findings thus imply that Mybbp1a may be capable of recognizing distinct chromatin domains, particularly the silenced regions, and consequently exert ing its regulatory functions. This mode of action may re semble that recently documented for the component of eNoRC, nucleomethylin. Together with the observa tions that abrogation of Mybbp1a impaired the rDNA promoter association of HDACs and DNA methylation, these attributes may pinpoint the repressor function of Mybbp1a temporally at a step subsequent to eNoRC but preceding or concomitant with the recruitment of HDACs and DNMT to the inactivated promoter.

Fur thermore, it is a formal possibility that Mybbp1a may serve as a structural adaptor for the co repressor com plexes, or an enhancer for the rDNA binding and activity of the various components. Methods Cell culture and synchronization HeLa cells were cultured in Dulbeccos modified Eagles medium supplemented with 10% heat inactivated fetal bovine serum and 100 U/ml penicillin and streptomycin solution. Mouse C2C12 myoblast cells were cultured similarly, except with supplementation of 20% heat inactivated FBS. Cells were maintained in a 5% CO2 hu midified incubator at 37 C. Cell cycle synchronization was done based on a previous report. For M phase synchronization, subconfluent cells were treated with 200 ng/ml nocodazole for 10 h. Mitotic cells were harvested through detachment by mitotic shake off. Cells were synchronized at the G1/S boundary by double thymidine block.

Cell cycle profiles were moni tored by FACS. For inhibition Cilengitide of HDAC and DNMT activity, cells were treated respectively with 200 nm of TSA and 5 uM of 5 AzaC for 24 hrs before gene ex pression analysis. RNAi and establishment of stable cell lines expressing the Mybbp1a targeting shRNA constructs Plasmid based shRNA expressing constructs targeting Mybbp1a and lucifer ase were transfected into HeLa cells using Lipofectamine 2000 reagent to obtain stable lines. Stable clones overexpressing Mybbp1a were estab lished using pcDNA3. 1 vector encoding Myc Mybbp1a.