H3K9 methyltransferase G9a negatively regulates UHRF1 transcription during leukemia cell differentiation.
Bottom Line: Here, we provide evidence that UHRF1 is transcriptionally downregulated by H3K9 HMTase G9a.Finally, we showed that G9a regulates UHRF1-mediated H3K23 ubiquitination and proper DNA replication maintenance.Therefore, we propose that H3K9 HMTase G9a is a specific epigenetic regulator of UHRF1.
Affiliation: Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756.Show MeSH
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Mentions: We further analyzed the UHRF1 promoter sequence to identify possible transcription factor binding sites. Among them, we found that 5 YY1 binding sites (−12, −40, −840, −1097 and −1175 sites in the UHRF1 promoter) were clustered between the −1921 and +145 sequences in the UHRF1 promoter region, indicating that YY1 could be involved in UHRF1 transcription. As a ubiquitous and multifunctional Polycomb-group protein family transcription factor, YY1 plays critical roles in hematopoiesis and cell cycle control (34). We determined whether YY1 had a synergistic effect with G9a on the negative regulation of UHRF1 expression. As expected, the luciferase reporter assay showed that G9a and YY1 alone each repressed UHRF1 transcription. G9a cotransfection and an increase in YY1 further repressed UHRF1 transcription, suggesting that YY1 functions as a mediator for G9a recruitment to the UHRF1 promoter and as a direct repressor of transcription (Figure 2A). Cotransfection of HDAC1 (but not HDAC2) with G9a further repressed G9a-mediated UHRF1 repression, indicating that HDAC1 plays a role in corepression (Figure 2B). Adding Trichostatin A (TSA) restored UHRF1 transcriptional repression by G9a, strongly suggesting that HDAC1 was involved in G9a-mediated UHRF1 transcriptional repression. However, adding nicotinamide (NIA) did not affect G9a-mediated UHRF1 transcriptional repression, indicating that the sirtuin class of histone deacetylases was not involved in this process (Figure 2B). The role of YY1 in G9a-mediated transcriptional repression of UHRF1 was further investigated by performing the UHRF1-luc reporter assay in the presence of siYY1 RNA. Interestingly, YY1 knockdown by two independent siYY1 RNAs abolished the UHRF1 transcriptional repression induced by G9a (Figure 2C). These data strongly suggest that negative regulation of UHRF1 transcription by G9a is dependent on the presence of YY1. We used real-time PCR and western analysis to determine whether UHRF1 expression was influenced by the presence or absence of YY1. Downregulation of UHRF1 was dependent on YY1, as shown by increased UHRF1 expression under two different YY1 knockdown conditions (Figure 2D). Next, we used ChIP and real-time PCR analysis to evaluate whether YY1 played a role in G9a recruitment to the UHRF1 promoter. The data shown in Figure 2E indicate that G9a was highly recruited to the UHRF1 promoter, and that YY1 knockdown by two independent siYY1 RNAs reduced G9a recruitment along with YY1. These findings consistently demonstrate that G9a can repress UHRF1 via binding of the YY1 transcription factor to the UHRF1 promoter. Previously, we reported that G9a and YY1 interact to mediate transcriptional repression of JAK2 (35). The finding that the level of H3K9-me2 on the UHRF1 promoter is downregulated in the absence of YY1 confirms that G9a-mediated UHRF1 transcriptional repression is YY1 dependent (Figure 2E). Taken together, these results suggest that G9a mediates transcriptional repression of UHRF1 in a YY1-dependent manner.
Affiliation: Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756.