Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología (INCan)-Instituto de Investigaciones Biomédicas (IIB), Universidad Nacional Autónoma de México (UNAM), México, DF 14080 México.
Departamento de Investigación Básica, Dirección de Investigación, Instituto Nacional de Geriatría, Secretaría de Salud, México, DF 10200 México.
Cell Div. 2014 Dec 30;9:6. doi: 10.1186/s13008-014-0006-2. eCollection 2014.
Heterochromatin 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.
HCT116 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.
Our 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.
异染色质蛋白 1（HP1）在建立、传播和维持组成型异染色质中非常重要，尤其是在着丝粒区域。HP1 可能参与在有丝分裂间期将 Mis12 招募并指导到着丝粒，而 HP1 的破坏或消除可能导致 Mis12 无法整合到动粒中。因此，在缺乏 Mis12 的情况下促进的着丝粒结构和动粒松弛可能会通过降低动粒锚定微管的能力进一步导致染色体不稳定（CIN）。本研究的目的是确定 Trichostatin A（TSA）诱导的 HP1 蛋白定位改变是否会改变 Mis12 和着丝粒蛋白 A（CENP-A）向着丝粒的募集，以及 HP1 蛋白表达和组蛋白 H3K9 甲基化在着丝粒染色质上的改变是否会增加 HCT116 和 WI-38 细胞的 CIN。
培养 HCT116 和 WI-38 细胞并用 TSA 处理，以评估暴露 24 和 48 小时后 CIN 的变化。在这两种细胞系中进行免疫荧光、Western blot、ChIP 和 RT-PCR 检测，以评估 HP1α/β、Mis12 和 CENP-A 的定位和丰度，并评估间期和有丝分裂过程中以及 TSA 处理 24 和 48 小时后的染色质修饰。
我们的结果表明，TSA 诱导的着丝粒染色质上异染色质组蛋白标记的减少降低了非肿瘤性 WI-38 细胞着丝粒上的 HP1，这种减少与细胞周期停滞和 CIN 有关。然而，在 HCT116 细胞中，HP1 蛋白与 MIS12 和 CENP-A 一起，对 TSA 处理重新定位到着丝粒染色质，即使在着丝粒核小体中的 H3K9me3 耗尽后也是如此。HP1 的富集和 H3K9me3 的丢失与 CIN 的增加有关，这表明在着丝粒和着丝粒周围染色质中存在一种增强 HP1 蛋白在这些区域存在的反应机制，可能确保染色体分离，尽管存在严重的 CIN。我们的结果为着丝粒染色质的表观遗传景观和 HP1 蛋白在 CIN 中的作用提供了新的见解。
