Lid2 is required for coordinating H3K4 and H3K9 methylation of heterochromatin and euchromatin

In most eukaryotes, histone methylation patterns regulate chromatin architecture and function: methylation of histone H3 lysine-9 (H3K9) demarcates heterochromatin, whereas H3K4 methylation demarcates euchromatin. We show here that the S. pombe JmjC-domain protein Lid2 is a trimethyl H3K4 demethylase responsible for H3K4 hypomethylation in heterochromatin. Lid2 interacts with the histone lysine-9 methyltransferase, Clr4, through the Dos1/Clr8-Rik1 complex, which also functions in the RNA interference pathway. Disruption of the JmjC domain alone results in severe heterochromatin defects and depletion of siRNA, whereas overexpressing Lid2 enhances heterochromatin silencing. The physical and functional link between H3K4 demethylation and H3K9 methylation suggests that the two reactions act in a coordinated manner. Surprisingly, crossregulation of H3K4 and H3K9 methylation in euchromatin also requires Lid2. We suggest that Lid2 enzymatic activity in euchromatin is regulated through a dynamic interplay with other histone-modification enzymes. Our findings provide mechanistic insight into the coordination of H3K4 and H3K9 methylation.     

The trithorax-group gene in Drosophila little imaginal discs encodes a trimethylated histone H3 Lys4 demethylase

Histone H3 Lys4 (H3K4) is methylated by yeast Set1-COMPASS and its mammalian homolog, the MLL complex. Human JARID1d can demethylate trimethyl-H3K4 (H3K4me3). We identified Drosophila melanogaster little imaginal discs (Lid) as the JARID1d homolog. We report that Lid knockdown using RNA interference results in a specific genome-wide increase in H3K4me3 levels without affecting other patterns of H3 methylation, and results in an altered distribution of the chromo-helicase protein Chd1.     

Unbiased proteomic screen for binding proteins to modified lysines on histone H3

We report a sensitive peptide pull‐down approach in combination with protein identification by LC‐MS/MS and qualitative abundance measurements by spectrum counting to identify proteins binding to histone H3 tail containing dimethyl lysine 4 (H3K4me2), dimethyl lysine 9 (H3K9me2), or acetyl lysine 9 (H3K9ac). Our study identified 86 nuclear proteins that associate with the histone H3 tail peptides examined, including seven known direct binders and 16 putative direct binders with conserved PHD finger, bromodomain, and WD40 domains. The reliability of our proteomic screen is supported by the fact that more than one‐third of the proteins identified were previously described to associate with histone H3 tail directly or indirectly. To our knowledge, the results presented here are the most comprehensive analysis of H3K4me2, H3K9me2, and H3K9ac associated proteins and will provide a useful resource for researchers studying the mechanisms of histone code effector proteins.     

Molecular recognition of histone H3 by the WD40 protein WDR5

The WD40-repeat protein WDR5 is a conserved subunit of Trithorax (TRX) histone methyltransferase complexes. WDR5 has been reported to selectively bind dimethylated Lys4 (K4me2) in histone H3 to promote K4 trimethylation by TRX. To elucidate the basis of this binding specificity, we have determined the crystal structure of WDR5 bound to a histone H3 peptide bearing K4me2. The structure reveals that the N terminus of histone H3 binds as a 3(10)-helix in the central depression formed by the WD40 repeats. R2 in histone H3 is bound in the acidic channel in the protein's core, whereas K4me2 is solvent exposed and does not engage in direct interactions with WDR5. Functional studies confirm that WDR5 recognizes A1, R2 and T3 in histone H3 but has virtually identical affinities for the unmodified and mono-, di- and trimethylated forms of K4, demonstrating that it does not discriminate among different degrees of methylation of this residue.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules

The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.     

组蛋白去甲基化酶PHD锌指蛋白8与神经发育

PHD锌指蛋白8(PHF8)是一种Fe2+和α-酮戊二酸依赖的组蛋白赖氨酸去甲基化酶.PHF8属于包含JmjC结构域蛋白家族,在N端还含有一个PHD(planthomeodomain)锌指结构域.人的PHF8基因突变往往破坏组蛋白去甲基化酶活性,从而引发遗传性X-连锁智力迟滞(XLMR)并伴发唇裂的发生.PHF8一方面可催化H3K9me2/1、H4K20me1和H3K27me2的去甲基化,另一方面还通过N端PHD锌指结构域与H3K4me3结合而发挥转录共激活作用.PHF8可调节rRNA和多个涉及神经发育的蛋白质编码基因如JARID1C的表达.这些研究显示,PHF8是一种重要的神经发育调节因子,从而拓宽了对组蛋白甲基化与基因表达关联的理解,同时为XLMR疾病的理解提供了新的线索.