Ent. CIN occurred after TSA treatment, especially in HCT116 cells, in which very low levels of cell cycle arrest were promoted compared with those in WI38 cells. These results suggest that the epigenetic landscape of centromeric and pericentromeric chromatin leads to the differential promotion of CIN upon TSA treatment in tumoral and non-tumoral cell lines.Gonz ez-Barrios et al. Cell Division (2014) 9:Page 11 ofFigure 7 The effect of TSA exposure on HP1/ localization and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28298493 centromeric and pericentromeric chromatin modifications, as well as on the relationship with CIN in WI-38 and HCT116 cells. Upper panel: Untreated WI-38 and HCT116 cells presented a similar localization of HP1 and HP1 along centromeric (CC) and pericentromeric chromatin (PC). At PC, H3K9me2/3 were enriched together with HP1 proteins; at CC, CENP-A was enriched during mitosis, whereas H3K4me2, H3K9me2/3 and H3K9ac modifications, as well as HP1 and HP1 and Mis12 proteins, fluctuate through interphase and mitosis. Lower panel: After treatment of HCT116 cells with TSA, H3K9me3 was significantly reduced at PC and CC, resulting in increased H3K4me2 and H3K9ac at PC satellite-2 regions; HP1/ were initially significantly reduced. However, they later recovered by an unknown mechanism that could include other proteins or could involve an ncRNA PC transcript [43-45]. CC was enriched with H3K9ac and CENP-A, together with HP1/ and Mis12 proteins. HCT116 cells proliferated with low levels of cell arrest and exhibited CIN in 50 of the cells. In both WI-38 and HCT116 cells, PC presented reduced H3K9me3, whereas H3K9ac and HP1 were enriched; moreover, CC was DM-3189 web depleted of CENP-A and HP1, and no significant reduction in H3K9me3 was observed, even though H3K9ac was increased. While CIN was still generated, it was reduced compared with HCT116 cells.Conclusions The data presented here provide new insight into the epigenetic landscape of centromeric chromatin, as well as into the role of HP1 and HP1 proteins in chromosome segregation and, by extension, cell division stability. We also present evidence of differences in the organization of centromeric chromatin and HP1 localization in response to TSA in the WI-38 and HCT116 cell lines. These differences are associated with CIN resulting from a chromatin disturbance caused by reduced H3K9me3 levels and TSA-induced hyperacetylation. The effects of TSA weresubstantially more pronounced in the malignant, transformed HCT116 cells than in WI-38 cells, leading to more significant chromosome mis-segregation and CIN. In addition, we believe that one cause underlying the effects of TSA-induced CIN and cell cycle arrest might be the deregulation of centromeric and pericentromeric chromatin regions, leading to the possibility that epigenetic regulation of the centromere might alter the response of tumor cell lines to TSA treatment. Nonetheless, many questions remain regarding the nature of centromere epigenetics, how these epigenetic modifications regulate kinetochoreGonz ez-Barrios et al. Cell Division (2014) 9:Page 12 ofassembly and their role in chromosome segregation, as well as how communication is established between centromeric and pericentromeric chromatin.Materials and methodsAntibodiesThe following antibodies were used: anti-ACA (immunofluorescence (IF) dilution 1:200; Antibodies Incorporated, Davis, CA, USA 15-235-F); anti-Mis12 (C-13; IF dilution 1:80, ChIP 4 g; Santa Cruz, Santa Cruz, CA, USA sc107750); anti-H3K4me2 (IF dilution 1:200, ChIP 3.