p16INK4a in cellular senescence

senescence is the activation of the INK4/ARF locus, which is epigenetically regulated and under tight control of the Polycomb group (PcG) Trithorax group (TrxG) proteins [1]. In proliferating cells, the locus is silenced by Polycomb repressive complexes (PRCs), and the chromatin is enriched in H3K27me3 [2]. Upon senescence triggers, PRCs are displaced and the repressive H3K27me3 mark is removed. Instead, the Trithorax MLL1 complex is recruited and leads to deposition of H3K4me3 and transcriptional activation of the locus [1]. Correspondingly, it was shown that overexpression of PcG proteins delayed the onset of replicative senescence, while deletion of MLL1 bypassed OIS by repressing the INK4/ARF locus [1,3]. Interestingly, we recently discovered that transcriptional regulation of p16INK4a can occur independently of H3K4me3 and H3K27me3 and is instead dependent on the proper function of transcription factors [4]. While characterizing DPY30, a common member of all H3K4 histone methyltransferase (HMTase) complexes (including MLL1), we found that DPY30 is a crucial regulator of cell proliferation. Depletion of DPY30 in human fibroblasts, as well as in transformed cells, led to severe impairment of cell cycle progression and a senescence-like phenotype [4]. This included a flattened and enlarged morphology, elevated levels of reactive oxygen species (ROS), activation of DNA damage response pathways (DDR), increased SA-β-galactosidase activity, formation of senescence-associated heterochromatin foci (SAHFs) and increased p16INK4a expression levels. Although activation of INK4/ARF was in accordance with the observed senescence-like phenotype, it immediately raised the question how this activation takes place in the absence of an intact DPY30-MLL/Set1 complex. We wondered whether a H3K4-specific HTMase complex might still be active in the absence of DPY30. However, H3K4me3 levels were significantly reduced on the p16INK4a promoter. These results indicate that DPY30 is essential for an active HMTase complex, but that its action is not strictly required to activate p16INK4a expression. Nevertheless, we observed an active chromatin conformation at the p16INK4a promoter in DPY30 Editorial knockdown cells that was characterized by hyperacetylation of histones H3 and H4, as well as increased Ets1/2 occupancy, a transcription factor required for activation of the INK4/ARF locus [5]. To better understand how the loss of DPY30 can lead to a senescence-like phenotype, we screened for direct DPY30 target genes using a genome-wide approach that combined ChIP sequencing for DPY30 and H3K4me3 with expression arrays in wild-type and DPY30 knockdown cells. Interestingly, DPY30 target genes are predominantly involved in cell cycle and proliferation regulation. For one …