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Histone methyltransferases and demethylases possess key chromatin remodeling activities that are needed to control proper gene expression in eukaryotes. Importantly, the recent discovery of histone demethylases has revealed that histone methylation is a dynamic process and is likely to be regulated. How histone demethylases are regulated is not thoroughly understood and is still currently being investigated. It has been suggested that post-translational modifications may play a role but how modifications regulate histone demethylase activity or how the balance of histone methyltransferases and demethylases is maintained have not been fully explored.1 To address these issues, we turned to S. cerevisiae as our model organism of choice. In S. cerevisiae, it was recently discovered that deletion of NOT4 leads to decreased global histone H3 lysine 4 (H3 K4) trimethylation. However, until our study, the mechanism by which Not4 was regulating histone H3 K4 trimethylation was not clear.2,3 We determined that expression of Jhd2, a histone H3 K4 demethylase, in wild-type yeast cells is tightly regulated by Not4, which in turns allows for proper H3 K4 trimethylation and gene expression levels (Fig. 1A).4 How, then, does Not4 control Jhd2 protein levels and histone methylation? We demonstrate for the first time that Not4, a known E3 ubiquitin ligase, can polyubiquitinate Jhd2 and target it for degradation by the proteasome.4 Furthermore, in the absence of Not4 or upon proteasome inhibition, Jhd2 protein levels increase leading to demethylation of trimethylated H3 K4 even in the presence of the H3 K4 methyltransferase, Set1.4 This, in turn, results in decreased gene expression of the GMP Synthetase, GUA1. We also show that the human homolog of Jhd2, JARID1C, is ubiquitinated by human NOT4 in vitro, suggesting that this may be a conserved mechanism by which histone demethylase activity is regulated.4 Our study also raises additional questions to be considered and addressed.
Besides Jhd2, Egd2, a member of the nascent-associated polypeptide (NAC-EGD) complex which is a complex associated with the ribosomal exit tunnel needed to ensure nascent protein targeting, is one of the few identified substrates for the E3 activity of Not4.5 Interestingly, ubiquitination of Egd2 appeared to only affect its cellular localization.5 More recently, it was shown that Not4's ubiquitin ligase activity is needed for the degradation of nascent protein products arrested during translation.6 Therefore it is likely that Not4 has additional targets. Given that our work and the work of others show that Not4 is also important in positively and negatively regulating gene expression, it would be interesting to determine if Not4 targets other transcriptional regulators such as activators/repressors, other histone modifying activities or elongation factors (Fig. 1A).7 The identification of additional Not4 substrates might clarify why Not4 can act to promote or inhibit transcription.
It has been previously noted that deletion of Not4 leads to increased monoubiquitination of histone H2B.2 H2B ubiquitination is important for wild-type levels of H3 K4 and H3 K79 methylation and it is known that, in Drosophila, GMP Synthetase, GMPS, can help stimulate deubiquitination of H2B via association with USP-7, an ubiquitin protease.8,9 Intriguingly, we find that loss of NOT4 results in a 2.5-fold decrease in expression of the yeast GMP Synthetase, GUA1.4 Keeping in mind the observations found in Drosophila, this result suggests that the increase in H2B ubiquitination observed in a not4Δ strain could be due to lower expression of GUA1 which would result in less ubiquitin protease activity. Therefore, it would be interesting to determine if yeast GMP Synthetase associates with and/or regulates known H2B ubiquitin proteases in yeast such as Ubp8 or Ubp10.10,11 If this process is conserved, it raises the possibility that other basic metabolic enzymes are likely regulating chromatin-mediated processes.
The fact that Jhd2 protein is being transcriptionally produced and, at the same time, is kept at an extremely low level in the cell through proteasome-mediated degradation is not energy efficient for the cell. Therefore, we favor the idea that this is a regulated process rather than just a housekeeping function. Why would a cell maintain this futile cycle given the cost in cellular energy and materials? We propose that this futile cycle exists until a rapid response is needed for modulating gene repression via removing histone H3 K4 trimethylation and effector binding proteins. We would predict that Not4's activity and Jhd2 protein levels would most likely respond to different environmental queues/stimuli or as a result of progression through the cell cycle (Fig. 1B). Although it is unclear what these environmental conditions are, if similar mechanisms occur in humans, this could give vital insight into how human demethylases are regulated.
The JARID1 family of histone demethylases contains human homologs of Jhd2, including JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX and JARID1D/SMCY, and has been linked to a wide variety of human disorders from X-linked mental retardation to breast cancer.12 PLU-1/JARID1B shows restricted and low expression in most human adult tissues and is found to be highly upregulated in breast cancer.13 The mechanism behind PLU-1 overexpression in breast cancer is not yet known, but our study may shed some light on this observation. We show that the human demethylase JARID1C/SMCX can be polyubiquitinated in vitro by human NOT4.4 In addition, protein levels of human LSD1, a non-JARID1 family member histone demethylase, have been shown to be influenced by the proteasome, suggesting that ubiquitination and degradation of demethylases may be a common mechanism for regulating their activity.14 Because of these observations and the conserved nature of these enzymes, we are currently determining if members of the JARID1 family are ubiquitinated in vitro and in vivo. If this is the case, we predict that defects in ubiquitination and degradation of PLU-1/JARID1B by human NOT4 and/or the proteasome could be a mechanism behind increased PLU-1 expression in breast cancer (Fig. 1C). It is also quite possible that NOT4 expression itself is affected in human cancers which could explain why PLU-1 is upregulated. Finally, if regulating histone demethylases by NOT4 is conserved and the reason for various human cancers, this could lead to a new therapeutic target.
Given the importance of the JARID1 family of histone demethylases in human diseases, the questions above will be exciting for us and others to explore. Ultimately, we hope our current and future studies will provide new insights for basic and cancer research.
We thank members of the Briggs laboratory for helpful comments on the manuscript. This work was supported by a NIH grant to S.D.B. (GM74183).