Differential distribution of HP1 proteins after trichostatin a treatment influences chromosomal stability in HCT116 and WI-38 cells

Abstract

BackgroundHeterochromatin protein 1 (HP1) is important in the establishment, propagation, and maintenance of constitutive heterochromatin, especially at the pericentromeric region. HP1 might participate in recruiting and directing Mis12 to the centromere during interphase, and HP1 disruption or abrogation might lead to the loss of Mis12 incorporation into the kinetochore. Therefore, the centromere structure and kinetochore relaxation that are promoted in the absence of Mis12 could further induce chromosome instability (CIN) by reducing the capacity of the kinetochore to anchor microtubules. The aim of this study was to determine whether alterations in the localization of HP1 proteins induced by trichostatin A (TSA) modify Mis12 and Centromere Protein A (CENP-A) recruitment to the centromere and whether changes in the expression of HP1 proteins and H3K9 methylation at centromeric chromatin increase CIN in HCT116 and WI-38 cells.MethodsHCT116 and WI-38 cells were cultured and treated with TSA to evaluate CIN after 24 and 48 h of exposure. Immunofluorescence, Western blot, ChIP, and RT-PCR assays were performed in both cell lines to evaluate the localization and abundance of HP1α/β, Mis12, and CENP-A and to evaluate chromatin modifications during interphase and mitosis, as well as after 24 and 48 h of TSA treatment.ResultsOur results show that the TSA-induced reduction in heterochromatic histone marks on centromeric chromatin reduced HP1 at the centromere in the non-tumoral WI-38 cells and that this reduction was associated with cell cycle arrest and CIN. However, in HCT116 cells, HP1 proteins, together with MIS12 and CENP-A, relocated to centromeric chromatin in response to TSA treatment, even after H3K9me3 depletion in the centromeric nucleosomes. The enrichment of HP1 and the loss of H3K9me3 were associated with an increase in CIN, suggesting a response mechanism at centromeric and pericentromeric chromatin that augments the presence of HP1 proteins in those regions, possibly ensuring chromosome segregation despite serious CIN. Our results provide new insight into the epigenetic landscape of centromeric chromatin and the role of HP1 proteins in CIN.Electronic supplementary materialThe online version of this article (doi:10.1186/s13008-014-0006-2) contains supplementary material, which is available to authorized users.

Calcium depletion destabilises kinetochore fibres by the removal of CENP-F from the kinetochore

“…H3K9me3 signals were detected at pericentromeric regions (and also some telomeric regions) of chromosomes, consistent with previous studies (Fig. 3a ) 18 – 20 . Moreover, there was no significant difference in the centromere-to-arm H3K9me3 intensity ratio between control and Ca 2+ -depletion (Fig.…”

“…Heterochromatin protein 1α (HP1α) localises at the centromere by recognising and binding to trimethylated histone H3 lysine 9 (H3K9me3), and provides itself as a mark to anchor other inner kinetochore proteins 19 . Mislocalisation of H3K9me3 due to chromosome decondensation may prevent the localisation of other centromeric proteins, including HP1α, to the centromere 18 , 20 . H3K9me3 signals were detected at pericentromeric regions (and also some telomeric regions) of chromosomes, consistent with previous studies (Fig.…”

“…H3K9me3 is lost from the centromeric regions of the cells with chromosome instability. Reduction of H3K9me3 and HP1 from chromatin centromeric regions was detected after TSA treatment, which caused global chromatin decompaction in HTC116 nuclei 20 . Pericentromeric localisation of H3K9me3 could be regulated by altering SUV39H1 activity.…”

Super-resolution microscopy reveals the nanoscale organization of the DEK protein cancer biomarker

“…This finding nicely demonstrates that upon the histone hyperacetylation, and increased accessibility to the nucleosomal DNA, chromatin-associated proteins can reorganize into bigger nanostructures. This observation stays in line with other works in which a redistribution of nuclear organization of chromatin-associated proteins has been observed (for example heterochromatin protein 1 (HP1)) (48). Furthermore, single-molecule localization imaging demonstrated the association of RNA Polymerase II to nucleosomes clutches of smaller size and fewer number of nucleosomes, which correspond to the clutches similar to the ones generated after TSA treatment (34).…”

Heterochromatin Protein 1: A Multiplayer in Cancer Progression

“…HDAC inhibitor induces hyperacetylated environments of centromeres and repositions HP1 proteins into the internal part of the nucleus. After treating trichostatin A (TSA), which is a pan-HDAC inhibitor, HP1 relocates to the centromeric chromatin in CRC HCT116 cells but not in non-tumoral cells [47]. This difference is associated with chromosome instability caused by TSA-induced hyperacetylation and reduced H3K9me3.…”