Ru Cao

Purification and Functional Characterization of a Histone H3-Lysine 4-Specific Methyltransferase

Methylation of histone H3 at lysine 9 by SUV39H1 and subsequent recruitment of the heterochromatin protein HP1 has recently been linked to gene silencing. In addition to lysine 9, histone H3 methylation also occurs at lysines 4, 27, and 36. Here, we report the purification, molecular identification, and functional characterization of an H3-lysine 4-specific methyltransferase (H3-K4-HMTase), SET7. We demonstrate that SET7 methylates H3-K4 in vitro and in vivo. In addition, we found that methylation of H3-K4 and H3-K9 inhibit each other. Furthermore, H3-K4 and H3-K9 methylation by SET7 and SUV39H1, respectively, have differential effects on subsequent histone acetylation by p300. Thus, our study provides a molecular explanation to the differential effects of H3-K4 and H3-K9 methylation on transcription.

Purification and Functional Characterization of SET8, a Nucleosomal Histone H4-Lysine 20-Specific Methyltransferase

BACKGROUND Covalent modifications of histone N-terminal tails play fundamental roles in regulating chromatin structure and function. Extensive studies have established that acetylation of specific lysine residues in the histone tails plays an important role in transcriptional regulation. Besides acetylation, recent studies have revealed that histone methylation also has significant effects on heterochromatin formation and transcriptional regulation. Histone methylation occurs on specific arginine and lysine residues of histones H3 and H4. Thus far, only 2 residues on histone H4 are known to be methylated. While H4-arginine 3 (H4-R3) methylation is mediated by PRMT1, the enzyme(s) responsible for H4-lysine 20 (H4-K20) methylation is not known. RESULTS To gain insight into the function of H4-K20 methylation, we set out to identify the enzyme responsible for this modification. We purified and cloned a novel human SET domain-containing protein, named SET8, which specifically methylates H4 at K20. SET8 is a single subunit enzyme and prefers nucleosomal substrates. We find that H4-K20 methylation occurs in a wide range of higher eukaryotic organisms and that SET8 homologs exist in C. elegans and Drosophila. We demonstrate that the Drosophila SET8 homolog has the same substrate specificity as its human counterpart. Importantly, disruption of SET8 in Drosophila reduces levels of H4-K20 methylation in vivo and results in lethality. Although H4-K20 methylation does not correlate with gene activity, it appears to be regulated during the cell cycle. CONCLUSIONS We identified and characterized an evolutionarily conserved nucleosomal H4-K20-specific methyltransferase and demonstrated its essential role in Drosophila development.

Role of hPHF1 in H3K27 Methylation and Hox Gene Silencing

ABSTRACT Polycomb group (PcG) proteins are required for maintaining the silent state of the homeotic genes and other important developmental regulators. The silencing function of the PcG proteins has been linked to their intrinsic histone modifying enzymatic activities. The EED-EZH2 complex, containing the core subunits EZH2, EED, SUZ12, and RbAp48, functions as a histone H3K27-specific methyltransferase. Here we describe the identification and characterization of a related EED-EZH2 protein complex which is distinguished from the previous complex by the presence of another PcG protein, hPHF1. Consistent with the ability of hPHF1 to stimulate the enzymatic activity of the core EED-EZH2 complex in vitro, manipulation of mPcl1, the mouse counterpart of hPHF1, in NIH 3T3 cells and cells of the mouse male germ cell line GC1spg results in global alteration of H3K27me2 and H3K27me3 levels and Hox gene expression. Small interfering RNA-mediated knockdown of mPcl1 affects association of the Eed-Ezh2 complex with certain Hox genes, such as HoxA10, as well as Hox gene expression concomitant with an alteration on the H3K27me2 levels of the corresponding promoters. Therefore, our results reveal hPHF1 as a component of a novel EED-EZH2 complex and demonstrate its important role in H3K27 methylation and Hox gene silencing.

The MES-2/MES-3/MES-6 Complex and Regulation of Histone H3 Methylation in C. elegans

The C. elegans proteins MES-2 and MES-6, orthologs of the Polycomb group (PcG) chromatin repressors E(Z) and ESC, exist in a complex with their novel partner MES-3. The MES system participates in silencing the X chromosomes in the hermaphrodite germline. Loss of maternal MES function leads to germline degeneration and sterility. We report here that the MES complex is responsible for di- and trimethylation of histone H3 Lys27 (H3-K27) in the adult germline and in early embryos and that MES-dependent H3-K27 marks are concentrated on the Xs. Another H3-K27 HMT functions in adult somatic cells, oocytes, and the PGCs of embryos. In PGCs, the MES complex may specifically convert dimethyl to trimethyl H3-K27. The HMT activity of the MES complex appears to be dependent on the SET domain of MES-2. MES-2 thus joins its orthologs Drosophila E(Z) and human EZH2 among SET domain proteins known to function as HMTs (reviewed in ). Methylation of histones is important for long-term epigenetic regulation of chromatin and plays a key role in diverse processes such as X inactivation and oncogenesis. Our results contribute to understanding the composition and roles of E(Z)/MES-2 complexes across species.

MES-4: an autosome-associated histone methyltransferase that participates in silencing the X chromosomes in the C. elegans germ line

Germ cell development in C. elegans requires that the X chromosomes be globally silenced during mitosis and early meiosis. We previously found that the nuclear proteins MES-2, MES-3, MES-4 and MES-6 regulate the different chromatin states of autosomes versus X chromosomes and are required for germline viability. Strikingly, the SET-domain protein MES-4 is concentrated on autosomes and excluded from the X chromosomes. Here, we show that MES-4 has histone H3 methyltransferase (HMT) activity in vitro, and is required for histone H3K36 dimethylation in mitotic and early meiotic germline nuclei and early embryos. MES-4 appears unlinked to transcription elongation, thus distinguishing it from other known H3K36 HMTs. Based on microarray analysis, loss of MES-4 leads to derepression of X-linked genes in the germ line. We discuss how an autosomally associated HMT may participate in silencing genes on the X chromosome, in coordination with the direct silencing effects of the other MES proteins.

Identification and functional characterization of the p66/p68 components of the MeCP1 complex.

ABSTRACT Methylation of cytosine at CpG dinucleotides is a common feature of many higher eukaryotic genomes. A major biological consequence of DNA methylation is gene silencing. Increasing evidence indicates that recruitment of histone deacetylase complexes by methyl-CpG-binding proteins is a major mechanism of methylated DNA silencing. We have previously reported that the MeCP1 protein complex represses transcription through preferential binding, remodeling, and deacetylation of methylated nucleosomes. To understand the molecular mechanism of the functioning of the MeCP1 complex, the individual components of the MeCP1 complex need to be characterized. In this paper, we report the identification and functional characterization of the p66 and p68 components of the MeCP1 complex. We provide evidence that the two components are different forms of the same zinc finger-containing protein. Analysis of the p66 homologs from different organisms revealed two highly conserved regions, CR1 and CR2. While CR1 is involved in the association of p66 with other MeCP1 components, CR2 plays an important role in targeting p66 and MBD3 to specific loci. Thus, our study not only completes the identification of the MeCP1 components but also reveals the potential function of p66 in MeCP1 complex targeting. The identification and characterization of all the MeCP1 components set the stage for reconstitution of the MeCP1 complex.