Downregulation of Histone H3 Lysine 9 Methyltransferase G9a Induces Centrosome Disruption and Chromosome Instability in Cancer Cells

Abstract

BackgroundModifications of the histone amino-terminal tails affect access of regulatory factors and complexes to chromatin and thereby influence biological processes. Cancer cells are characterized by prominent epigenetic dysregulation, including histone modifications. However, the functional roles of the histone methyltransferases (HMT) in cancer remain unclear.Methodology/Principal FindingsWe studied RNAi-based inhibition (knockdown, KD) of 2 different H3K9 HMTs, SUV39H1 and G9a. Knockdown of the 2 HMTs in PC3 cancer cell line markedly inhibited cell growth and caused profound morphological changes with loss of telomerase activity and shortened telomeres. SUV39H1 KD cells showed substantial increase in G2/M fraction. G9a KD cells showed increased DNA content (1.7-fold in 2 independent clones) compared with FACS analyses to control. Karyotype analyses showed that this was due to an increased number of chromosomes (from 61 to 102) in G9a KD cells compared to parental PC3. Intriguingly, we found abnormal centrosome morphology and number in about 25% of the G9a KD cells, while centrosomes were morphologically normal in control cells. Microarray analyses after KD of SUV39H1 or G9a showed very few genes up-regulated among the 39,000 genes. The silenced tumor-suppressor genes p16 and RASSF1A were not activated in KD cells.Conclusions/SignificanceThese data suggest that the 2 HMTs, SUV39H1 and G9a are required to perpetuate the malignant phenotype. Furthermore, G9a plays a critical role in regulating centrosome duplication presumably through chromatin structure rather than through affecting gene expression in cancer cells. Targeting these histone methyltransferases may be of therapeutic benefit in cancers.

Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease

“…We speculate that this effect could be mediated by the binding of EVI1 to G9a, a H3K9-specific histone methyltransferase. EVI1−G9a complexes have reduced methytransferase activity, and downregulation of G9a activity has been linked to centrosome disruption and chromosome instability in cancer cells 50,52 .Although we observed increased EVI1 and MDS1−EVI1 expression in the bulk population of expanded hematopoietic clones, two clones, one in each subject, dominated gene-marked hematopoiesis at later time points. Both clones, 76776G11 (subject 1) and 87429F02 (subject 2), © 2010 Nature America, Inc. All rights reserved.…”

“…We speculate that this effect could be mediated by the binding of EVI1 to G9a, a H3K9-specific histone methyltransferase. EVI1−G9a complexes have reduced methytransferase activity, and downregulation of G9a activity has been linked to centrosome disruption and chromosome instability in cancer cells 50,52 .…”

“…In our study, we observed profound clonal expansions driven by the insertional activation of EVI1, MDS1−EVI1 and PRDM16, a homolog of MDS1−EVI1, in multiple clones of both subjects 5 months after gene therapy. We detected no abnormalities in blood counts, bone marrow morphology or karyotype during this period, indicating that this insertional event was probably the driving event for progression toward clonal dominance.In addition to the role of EVI1 and MDS1−EVI1 in development, transcriptional regulation, self-renewal and cell cycle progression [44][45][46][47] , EVI1 has been shown to interact with proteins involved in the chromatin-remodeling process, including BRG1, SUV39H1, G9a and MBD3B, and may thereby influence proper centrosome replication, chromatin assembly and genomic stability [48][49][50][51][52] . During mitosis, centrosomes function as spindle poles, directing the formation of bipolar spindles, a process essential for accurate chromosome segregation 53 .…”

The expanding role of the Ehmt2/G9a complex in neurodevelopment

“…There have been recent reports suggesting a requirement of Ehmt2 for genome integrity. 81,89 How a loss of Ehmt2 dependant methylation leads to chromosome instability is still not well understood; One intriguing possibility is suggested in the recent report that Ehmt2 participates in a multi-HMT complex that is required for maintaining pericentromeric chromatin, [90][91][92] a structure that is essential for proper spindle attachment and chromosome segregation during mitosis (reviewed in ref. 93).…”

“…EHMT2 knockdown resulted in disruptions in centrosome biogenesis leading to increased genome instability in several cancer cell lines. 89 Interestingly, disruptions in centrosome biogenesis have been shown to lead to cell cycle exit defects, genome instability and increases in apoptosis reminiscent of the Ehmt2 loss-of-function phenotype in vivo. 94,95 Whether Ehmt2 is required to regulate the expression or activity of individual genes in the centrosome biogenesis pathway has not been studied, and thus the mechanism of centrosome disruption is not clearly defined.…”