Abstract
Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus) domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene) finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs) YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases) respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3 ubiquitin ligases to ensure excess histone degradation and thus contribute to the maintenance of genomic stability.
Highlights
Histones are essential basic proteins that package the genomic DNA of all eukaryotes into nucleosomes to form chromatin [1,2,3]
Identification of four RING finger containing predicted E3 ligases that are sensitive to histone overexpression
The tom1 mutant was not as sensitive to genotoxic agents as the ubc4 ubc5 double deletion strain that lacks both the E2 enzymes responsible for the degradation related ubiquitylation of histones in yeast [7]. These data strongly suggested that additional E3 ligases may be involved in the degradation of excess histones in budding yeast
Summary
Histones are essential basic proteins that package the genomic DNA of all eukaryotes into nucleosomes to form chromatin [1,2,3]. In organisms such as the Xenopus that undergo rapid embryonic cell cycles in the absence of transcription, large quantities of histones are stored bound to chaperone proteins such as Nucleoplasmin in their oocytes to provide adequate quantities of histones for chromatin assembly during DNA synthesis [9,10]. To avoid the problems associated with excess histone accumulation, most eukaryotic cells rely largely on the strict regulation of their histone protein levels via transcriptional, posttranscriptional, translational and posttranslational mechanisms [5,17]
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