The identification of histone methyltransferases and demethylases has uncovered a dynamic methylation system needed to modulate appropriate levels of gene expression. Gene expression levels of various histone demethylases, such as the JARID1 family, show distinct patterns of embryonic and adult expression and respond to different environmental cues, suggesting that histone demethylase protein levels must be tightly regulated for proper development. In our study, we show that the protein level of the yeast histone H3 Lys 4 (H3 K4) demethylase Jhd2/ Kdm5 is modulated through polyubiquitination by the E3 ubiquitin ligase Not4 and turnover by the proteasome. We determine that polyubiquitin-mediated degradation of Jhd2 controls in vivo H3 K4 trimethylation and gene expression levels. Finally, we show that human NOT4 can polyubiquitinate human JARID1C/SMCX, a homolog of Jhd2, suggesting that this is likely a conserved mechanism. We propose that Not4 is an E3 ubiquitin ligase that monitors and controls a precise amount of Jhd2 protein so that the proper balance between histone demethylase and histone methyltransferase activities occur in the cell, ensuring appropriate levels of H3 K4 trimethylation and gene expression.[Keywords: Jhd2; chromatin; histone methylation; demethylase; ubiquitination; proteasome] Supplemental material is available at http://www.genesdev.org.

The proteasome regulates histone lysine methylation and gene transcription, but how it does so is poorly understood. To better understand this process, we used the epistatic miniarray profile (E-MAP) approach to identify factors that genetically interact with proteasomal subunits. In addition to members of the Set1 complex that mediate histone H3 lysine 4 methylation (H3K4me), we found that deleting members of the CCR4/NOT mRNA processing complex exhibit synthetic phenotypes when combined with proteasome mutants. Further biochemical analyses revealed physical associations between CCR4/NOT and the proteasome in vivo. Consistent with the genetic and biochemical interactions linking CCR4/NOT with proteasome and Set1-mediated methylation, we find that loss of Not4 decreases global and gene-specific H3K4 trimethylation (H3K4me3) and decreases 19S proteasome recruitment to the PMA1 gene. Similar to proteasome regulation of histone methylation, loss of CCR4/NOT members does not affect ubiquitinated H2B. Mapping of Not4 identified the RING finger domain as essential for H3K4me3, suggesting a role for ubiquitin in this process. Consistent with this idea, loss of the Not4-interacting protein Ubc4, a known ubiquitin-conjugating enzyme, decreases H3K4me3. These studies implicate CCR4/NOT in the regulation of H3K4me3 through a ubiquitin-dependent pathway that likely involves the proteasome.

In Saccharomyces cerevisiae, lysine 4 on histone H3 (H3K4) is methylated by the Set1 complex (Set1C or COMPASS). Besides the catalytic Set1 subunit, several proteins that form the Set1C (Swd1, Swd2, Swd3, Spp1, Bre2, and Sdc1) are also needed to mediate proper H3K4 methylation. Until this study, it has been unclear how individual Set1C members interact and how this interaction may impact histone methylation and gene expression. In this study, Bre2 and Sdc1 are shown to directly interact, and it is shown that the association of this heteromeric complex is needed for proper H3K4 methylation and gene expression to occur. Interestingly, mutational and biochemical analysis identified the C terminus of Bre2 as a critical protein-protein interaction domain that binds to the Dpy-30 domain of Sdc1. Using the human homologs of Bre2 and Sdc1, ASH2L and DPY-30, respectively, we demonstrate that the C terminus of ASH2L also interacts with the Dpy-30 domain of DPY-30, suggesting that this protein-protein interaction is maintained from yeast to humans. Because of the functionally conserved nature of the C terminus of Bre2 and ASH2L, this region was named the SDI (Sdc1 Dpy-30 interaction) domain. Finally, we show that the SDI-Dpy-30 domain interaction is physiologically important for the function of Set1 in vivo, because specific disruption of this interaction prevents Bre2 and Sdc1 association with Set1, resulting in H3K4 methylation defects and decreases in gene expression. Overall, these and other mechanistic studies on how H3K4 methyltransferase complexes function will likely provide insights into how human MLL and SET1-like complexes or overexpression of ASH2L leads to oncogenesis.In Saccharomyces cerevisiae, histone H3 lysine 4 (H3K4) 3 mono-, di-, and trimethylation is mediated by the Set1 histone methyltransferase (1, 2). Set1 and H3K4 methylation were first identified to be required for silencing at rDNA, telomeres, and mating-type loci (3-6). Set1 and H3K4 trimethylation are also found to be located at euchromatin and enriched in the 5Ј-ends of coding regions of transcriptionally active genes (7,8). In addition, Set1 and its human homologs, MLL1, MLL2, and hSET1, associate with the phosphorylated C-terminal domain of RNA polymerase II (7, 9 -11). More recently, it has been shown that various chromodomain and PHD zinc finger-containing proteins can bind to histones and H3K4 di-and trimethylated histone peptides and histones (12)(13)(14). Therefore, these and other studies suggest that Set1-mediated histone methylation plays a positive role in mediating gene expression through recruitment of effector proteins.Unlike other yeast histone methyltransferases, Set1 requires a multiprotein complex for its activity in vitro and in vivo (1,(15)(16)(17)(18). The yeast Set1 complex, also called Set1C or COMPASS (complex associated with Set1), consists of eight subunits (Set1, Bre2, Sdc1, Swd1, Swd2, Swd3, Spp1, and Shg1) (15,18,19). After the discovery of yeast Set1C, it was determined that the human H3K4 methyltransferases MLL1 ...

Background: Histone methyltransferases are key regulators in cell growth and gene expression. Results: We identified a charge-based protein-protein interaction within a histone H3K4 methyltransferase complex that is critical for protein stability and histone methylation. Conclusion: Charge-based protein-protein interactions are conserved among histone methyltransferases and are required for their function. Significance: This study helps determine how histone methyltransferase complexes are assembled and how they function.