Abstract 4866: The dynamic interaction of nuclear Smads with peri- and centromeric heterochromatin is involved in the modulation of histone modification and chromatin structure

Smads are intracellular signaling transducers of TGF-β family ligands. Through TGF-β activation, phosphorylated R-Smads (Smad2/3) form active heteromeric complexes with co-Smad (Smad4) and translocate to the nucleus where they interact with chromatin to assemble the transcriptional machinery of various target genes. Evidence also indicates that Smads are involved in chromatin modulation via interactions with histone modifiers. The role of pre-existing nuclear Smads in chromatin modulation, however, has not been described, nor have the molecular mechanisms of their dynamic interaction with heterochromatin been elucidated. Here we report that nuclear Smads directly bind to specific sites on heterochromatin, altering chromatin architecture upon TGF-β activation. Subcellular fractionation indicates that both active and inactive nuclear Smad 2/3 remains in the biochemically insoluble fraction. We found that only non-phosphorylated Smad3 interacted with HP1α, a key component of heterochromatin under transcriptionally inactive conditions. The level of HP1α, but not HP1β and γ, was regulated in a TGF-β dependent manner in both Smad 2/3 KO MEFs and mouse mammary epithelial cells. Localization of the Smad3 in the pericentromeric heterochromatin and centromere regions of the human chromosomes suggests Smad3 may function as an organizer of chromatin architecture and chromosome assembly during mitosis. ChIP assay indicated that pericentromeric and centromeric heterochromatin bound Smads were dissociated by TGF-β activation and translocated to target gene promoter regions to activate gene transcription. Interestingly, TGF-β enhanced pericentromeric heterochromatin binding of Smad3 in a Smad3 overexpressed human breast cancer cell line, MCF10Ca1, but dramatically decreased in a cell line with deletion of MH2 domain in Smad3. Furthermore, deletion of MH2 domain markedly increased by more than 100 folds both H3 lys4 dimethylation and lys9 dimethylation in centromeric heterochromatin but diminished them in pericentromeric heterochromatin. This suggests that the MH2 domain of the Smad3 may play a critical role on modulation of heterochromatin structure in a manner that activates or represses genome wide gene transcription activity. In conclusion, these results indicate that nuclear Smads are involved in the modulation of chromatin structure through the regulation of HP1α and their direct interaction with peri- and centromeric heterochromatin. Therefore dysregulation of nuclear Smad expression may increase genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4866.