Recombinant Histone H3 Research Articles

Deciphering the Interactome of Histone Marks in Living Cells via Genetic Code Expansion Combined with Proximity Labeling.

Deciphering the endogenous interactors of histone post-translational modifications (hPTMs, also called histone marks) is essential to understand the mechanisms of epigenetic regulation. However, most of the analytical methods to determine hPTM interactomes are in vitro settings, lacking interrogating native chromatin. Although lysine crotonylation (Kcr) has recently been considered an important hPTM for the regulation of gene transcription, the interactors of Kcr still remain to be explored. Herein, we present a general approach relying upon a genetic code expansion system, APEX2 (engineered peroxidase)-mediated proximity labeling, and quantitative proteomics to profile interactomes of the selected hPTMs in living cells. We genetically fused APEX2 to the recombinant histone H3 with a crotonyl lysine inserted site specifically to generate APEX2-H3K9cr that incorporated into native chromatin. Upon activation, APEX2 triggered in vivo biotin labeling of H3K9cr interactors that can then be enriched with streptavidin beads and identified by mass spectrometry. Proteomic analysis further revealed the endogenous interactomes of H3K9cr and confirmed the reliability of the method. Moreover, DPF2 was identified as a candidate interactor, and the binding interaction of DPF2 to H3K9c was further characterized and verified. This study provides a novel strategy for the identification of hPTM interactomes in living cells, and we envision that this is key to elucidating epigenetic regulatory pathways.

Extracellular vesicles of P. gingivalis-infected macrophages induce lung injury

Periodontal diseases are common inflammatory diseases that are induced by infection with periodontal bacteria such as Porphyromonas gingivalis (Pg). The association between periodontal diseases and many types of systemic diseases has been demonstrated; the term “periodontal medicine” is used to describe how periodontal infection/inflammation may impact extraoral health. However, the molecular mechanisms by which the factors produced in the oral cavity reach multiple distant organs and impact general health have not been elucidated. Extracellular vesicles (EVs) are nano-sized spherical structures secreted by various types of cells into the tissue microenvironment, and influence pathophysiological conditions by delivering their cargo. However, a detailed understanding of the effect of EVs on periodontal medicine is lacking. In this study, we investigated whether EVs derived from Pg-infected macrophages reach distant organs in mice and influence the pathophysiological status. EVs were isolated from human macrophages, THP-1 cells, infected with Pg. We observed that EVs from Pg-infected THP-1 cells (Pg-inf EVs) contained abundant core histone proteins such as histone H3 and translocated to the lungs, liver, and kidneys of mice. Pg-inf EVs also induced pulmonary injury, including edema, vascular congestion, inflammation, and collagen deposition causing alveoli destruction. The Pg-inf EVs or the recombinant histone H3 activated the NF-κB pathway, leading to increase in the levels of pro-inflammatory cytokines in human lung epithelial A549 cells. Our results suggest a possible mechanism by which EVs produced in periodontal diseases contribute to the progression of periodontal medicine.

Nucleolar Matrix Metalloproteinase‐2 Regulates rRNA Transcription

Gene expression in eukaryotic cells is a tightly regulated process, from pre‐transcriptional through post‐transcriptional stages. A major control point in gene expression is ribosome synthesis, since it executes mRNA translation and also regulates their stability. Genes encoding ribosomal proteins and ribosomal RNA (rRNA) are physically located and transcribed in specialized structures within the nucleus known as nucleoli. We previously reported that the protease matrix metalloproteinase‐2 (MMP‐2) was located inside the nucleus but its role there remains elusive.Confocal microscopy studies in our lab showed an accumulation of MMP‐2 in nucleoli in U‐2OS human osteosarcoma cells, suggesting a role in rRNA transcription. We found that MMP‐2 co‐localizes with nucleoli markers fibrillarin and nucleophosmin, and is located in active rRNA transcription sites within nucleoli of U‐2OS cells pulsed with 5‐fluorouridine (5‐FU).Chromatin immunoprecipitation (ChIP) assays showed an enrichment of MMP‐2 in the rRNA gene promoter region. We also found evidence of MMP‐2‐mediated proteolysis of purified histones (H1, H2A, H2B, H3 and H4), in a concentration‐dependent manner. Interestingly, we identified histone H3 to be the most susceptible to MMP‐2 activity and found at least one MMP‐ 2 cleavage site in the N‐terminal tail of histone H3.The N‐terminal tail of histone H3 is the location of a large number of known post‐translational modifications that are involved in the regulation of DNA relaxation and transcription, RNA splicing and DNA repair. Emerging evidence indicates that one of the mechanisms behind this is the proteolytic removal of the N‐terminal tail of histone H3 by a yet uncharacterized protease. Therefore, we sought to elucidate if nucleolar MMP‐2 has proteolytic activity on recombinant histone H3. We incubated MMP‐2 immunoprecipitated from isolated nucleoli with recombinant histone H3, in presence or absence of the MMP‐2 preferring inhibitor ARP‐100. Our data revealed that nucleolar MMP‐2 has proteolytic activity on histone H3 and is sensitive to ARP‐ 100.The involvement of MMP‐2 in rRNA gene expression in the nucleolus is a major novel mechanism by which intracellular may MMP‐2 regulate cell function via histone proteolysis.Support or Funding InformationCanadian Institutes of Health Research (CIHR) grants FDN‐143299 (to RS) and MOP‐119515 (to MH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intracellular Hmgb1 Inhibits Inflammatory Nucleosome Release and Limits Acute Pancreatitis in Mice

High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice after induction of acute pancreatitis. We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1(flox/flox) mice). Acute pancreatitis was induced in these mice (HMGB1(flox/flox) mice served as controls) after injection of l-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses. After injection of l-arginine or cerulein, Pdx1-Cre; HMGB1(flox/flox) mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1(flox/flox) mice after l-arginine or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA nuclear factor κB, degradation of inhibitor of κB, and phosphorylation of mitogen-activated protein kinase. Inhibitors of reactive oxygen species (N-acetyl-l-cysteine) blocked l-arginine-induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of nuclear factor κB in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1(flox/flox) and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival after l-arginine injection. In 2 mouse models of acute pancreatitis, intracellular HMGB1 appeared to prevent nuclear catastrophe and release of inflammatory nucleosomes to block inflammation. These findings indicate a role for the innate immune response in tissue damage.

An Adhesin-Like Protein, Lam29, fromLactobacillus mucosaeME-340 Binds to Histone H3 and Blood Group Antigens in Human Colonic Mucus

A cell-surface 29-kDa protein (Lam29, cysteine-binding protein of the ABC transporter) from Lactobacillus mucosae ME-340 showed an adhesin-like property for human ABO blood group antigens expressed on the gastrointestinal mucosa. In addition, here we report that Lam29 also bound to an 18-kDa protein on human colonic mucus. By ligand blot assay and N-terminal amino acid sequence of the protein, it was identified as human histone H3. By ligand blot and microplate binding assays with recombinant histone H3, binding between Lam29 and histone H3 was confirmed. The adhesion of ME-340 cells to histone H3 was significantly inhibited by 26% after the addition of 2.5 mg/mL Lam29 as compared to the absence of Lam29 (p<0.01). By GHCl extraction and transcription attenuation of ME-340 cells, binding reduction of ME340 cells against histone H3 was detected at 12% and 13% respectively, as compared to control cells by the BIACORE assay (p<0.01). These data indicate that Lam29 shows multiple binding activities to blood group antigens and histone H3 in human colonic mucus. This is the first report to indicate that lactobacilli expressing Lam29 adhere to histone H3 on gastrointestinal mucosa.

Prdm3 and Prdm16 are H3K9me1 Methyltransferases Required for Mammalian Heterochromatin Integrity

Heterochromatin serves important functions, protecting genome integrity and stabilizing gene expression programs. Although the Suv39h methyltransferases (KMTs) are known to ensure pericentric H3K9me3 methylation, the mechanisms that initiate and maintain mammalian heterochromatin organization remain elusive. We developed a biochemical assay and used in vivo analyses in mouse embryonic fibroblasts to identify Prdm3 and Prdm16 as redundant H3K9me1-specific KMTs that direct cytoplasmic H3K9me1 methylation. The H3K9me1 is converted in the nucleus to H3K9me3 by the Suv39h enzymes to reinforce heterochromatin. Simultaneous depletion of Prdm3 and Prdm16 abrogates H3K9me1 methylation, prevents Suv39h-dependent H3K9me3 trimethylation, and derepresses major satellite transcription. Most strikingly, DNA-FISH and electron microscopy reveal that combined impairment of Prdm3 and Prdm16 results in disintegration of heterochromatic foci and disruption of the nuclear lamina. Our data identify Prdm3 and Prdm16 as H3K9me1 methyltransferases and expose a functional framework in which anchoring to the nuclear periphery helps maintain the integrity of mammalian heterochromatin.

Modification of Histones by Sugar β-N-Acetylglucosamine (GlcNAc) Occurs on Multiple Residues, Including Histone H3 Serine 10, and Is Cell Cycle-regulated

The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked β-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1-14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017-1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G(1) cells, declines throughout the S phase, increases again during late S/early G(2), and persists through late G(2) and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.

DrosophilaSet1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription

Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. Here, we report that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation.

A chemical method for labeling lysine methyltransferase substrates.

Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.

Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [ 3H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.

Top‐down analysis of recombinant histone H3 and its methylated analogs by ESI/FT‐ICR mass spectrometry

Detailed characterization of the amino acid (aa) compositions of recombinant histone H3 (Xenopus laevis) and its Lys-9 mono-, di-, and trimethylated isoforms was carried out by ESI/FT-ICR-MS. The measured molecular masses of these four proteins did not agree with those predicted from the published wild-type histone H3 sequence. Using MS/MS with both collision-activated dissociation and electron capture dissociation, three aa substitutions (Gly102Ala, Cys110Ala, and Gly111Ala) were unambiguously identified in each protein by protein database searching and de novo peptide tag sequencing. In addition, it was determined through accurate mass measurement and elemental composition generation that Lys-9, to which the methyl groups are attached, is not in fact a lysine. Instead, it was identified as a chemical analog of lysine--aminoethylcysteine--in the methylated proteins. After incorporation of these three aa substitutions and the aminoethylcysteine into the protein sequences, the re-calculated molecular masses of four proteins were completely consistent with the measured values within 1 ppm. This work demonstrates the power of top-down MS for a detailed structural confirmation of recombinant proteins, even without prior information on aa substitutions or modifications.

An Irreversible Modification of Histone H3 ‐ Identification and Characterization of Histone H3‐specific Protease from Chicken Liver

Regulated removal of N‐terminal tails of histones would constitute an irreversible type of modification. A novel histone H3 specific modifying enzyme from chicken liver nuclei was purified that specifically cleaved histone H3 in vitro and generated a faster migrating histone H3 band. The enzyme named H3ase was sequenced with the help of MALDI‐MS &amp; PSD and was found to align with a known NAD+‐dependent dehydrogenase (NDH). The peptide substrates against the N‐terminus histone H3 could compete with H3ase activity, building the possibility that probably, H3ase modified histone H3 in the N‐terminus. Site of proteolysis was mapped using bacterially expressed recombinant histone H3. The cleaved products corresponded to polypeptides lacking 2, 4 and 8 amino acid residues from the N‐terminus, respectively. The mechanism of proteolysis is still unknown. Efforts are being taken to evaluate the status of H3 proteolysis in chromatin level and to find out its physiological importance.This research work was supported by research grants from Department of Science and Technology, Government of India, New Delhi (Grant no. SR/SO/BB‐38/2002), and University of Delhi, India.

Identification and Characterization of Propionylation at Histone H3 Lysine 23 in Mammalian Cells

Propionylation has been identified recently as a new type of protein post-translational modification. Bacterial propionyl-CoA synthetase and human histone H4 are propionylated at specific lysine residues that have been known previously to be acetylated. However, other proteins subject to this modification remain to be identified, and the modifying enzymes involved need to be characterized. In this work, we report the discovery of histone H3 propionylation in mammalian cells. Propionylation at H3 lysine Lys(23) was detected in the leukemia cell line U937 by mass spectrometry and Western analysis using a specific antibody. In this cell line, the propionylated form of Lys(23) accounted for 7%, a level at least 6-fold higher than in other leukemia cell lines (HL-60 and THP-1) or non-leukemia cell lines (HeLa and IMR-90). The propionylation level in U937 cells decreased remarkably during monocytic differentiation, indicating that this modification is dynamically regulated. Moreover, in vitro assays demonstrated that histone acetyltransferase p300 can catalyze H3 Lys(23) propionylation, whereas histone deacetylase Sir2 can remove this modification in the presence of NAD(+). These results suggest that histone propionylation might be generated by the same set of enzymes as for histone acetylation and that selection of donor molecules (propionyl-CoA versus acetyl-CoA) may determine the difference of modifications. Because like acetyl-CoA, propionyl-CoA is an important intermediate in biosynthesis and energy production, histone H3 Lys(23) propionylation may provide a novel epigenetic regulatory mark for cell metabolism.

Stabilization of N-Myc Is a Critical Function of Aurora A in Human Neuroblastoma

In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. In a shRNA screen of genes that are highly expressed in MYCN-amplified tumors, we have identified AURKA as a gene that is required for the growth of MYCN-amplified neuroblastoma cells but largely dispensable for cells lacking amplified MYCN. Aurora A has a critical function in regulating turnover of the N-Myc protein. Degradation of N-Myc requires sequential phosphorylation by cyclin B/Cdk1 and Gsk3. N-Myc is therefore degraded during mitosis in response to low levels of PI3-kinase activity. Aurora A interacts with both N-Myc and the SCF(Fbxw7) ubiquitin ligase that ubiquitinates N-Myc and counteracts degradation of N-Myc, thereby uncoupling N-Myc stability from growth factor-dependent signals.

Identification of Small Molecule Inhibitors of the Mitotic Kinase Haspin by High-Throughput Screening Using a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assay

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at K(m) concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC( 50) determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC( 50) values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics.

14-3-3 Proteins recognize a histone code at histone H3 and are required for transcriptional activation

Interphase phosphorylation of S10 at histone H3 is linked to transcriptional activation of a specific subset of mammalian genes like HDAC1. Recently, 14-3-3 proteins have been described as detectors for this phosphorylated histone H3 form. Here, we report that 14-3-3 binding is modulated by combinatorial modifications of histone H3. S10 phosphorylation is necessary for an interaction, but additional H3K9 or H3K14 acetylation increases the affinity of 14-3-3 for histone H3. Histone H3 phosphoacetylation occurs concomitant with K9 methylation in vivo, suggesting that histone phosphorylation and acetylation can synergize to overcome repressive histone methylation. Chromatin immunoprecipitation experiments reveal recruitment of 14-3-3 proteins to the HDAC1 gene in an H3S10ph-dependent manner. Recruitment of 14-3-3 to the promoter is enhanced by additional histone H3 acetylation and correlates with dissociation of the repressive binding module HP1gamma. Finally, siRNA-mediated loss of 14-3-3 proteins abolishes the transcriptional activation of HDAC1. Together our data indicate that 14-3-3 proteins are crucial mediators of histone phosphoacetylation signals.

Proline Isomerization of Histone H3 Regulates Lysine Methylation and Gene Expression

The cis-trans isomerization of proline serves as a regulatory switch in signaling pathways. We identify the proline isomerase Fpr4, a member of the FK506 binding protein family in Saccharomyces cerevisiae, as an enzyme which binds the amino-terminal tail of histones H3 and H4 and catalyses the isomerization of H3 proline P30 and P38 in vitro. We show that P38 is necessary for methylation of K36 and that isomerization by Fpr4 inhibits the ability of Set2 to methylate H3 K36 in vitro. These results suggest that the conformational state of P38, controlled by Fpr4, is important for methylation of H3K36 by Set2. Consistent with such an antagonistic role, abrogation of Fpr4 catalytic activity in vivo results in increased levels of H3K36 methylation and delayed transcriptional induction kinetics of specific genes in yeast. These results identify proline isomerization as a novel noncovalent histone modification that regulates transcription and provides evidence for crosstalk between histone lysine methylation and proline isomerization.

Substrate Preferences of the EZH2 Histone Methyltransferase Complex

Histone methylation plays an important role in chromatin dynamics and gene expression. Methylation of histone H3-lysine 27 by the EZH2 complex has been linked to the silencing of homeotic genes and the inactivation of the X chromosome. Here we report a characterization of the substrate preferences of the enzyme complex using a reconstituted chromatin and enzyme system. We found that the linker histone H1, when incorporated into nucleosomes, stimulates the enzymatic activity toward histone H3. This stimulatory activity may be explained by protein-protein interactions between H1 and components of the EZH2 complex. In addition, we found that the EZH2 complex exhibits a dramatic preference for dinucleosomes when compared with mononucleosomes and that the stimulation of H3 methylation by H1 requires dinucleosomes or oligonucleosome substrates. Furthermore, in contrast with a recent study suggesting that Embryonic Ectoderm Development EED isoforms may affect substrate specificity, we found that EZH2 complexes reconstituted with different EED isoforms exhibit similar substrate preference and specificity. Our work supports the hypothesis that linker histone H1 and chromatin structure are important factors in determining the substrate preference of the EZH2 histone methyltransferase complex.

CpG-binding Protein (CXXC Finger Protein 1) Is a Component of the Mammalian Set1 Histone H3-Lys4 Methyltransferase Complex, the Analogue of the Yeast Set1/COMPASS Complex

CpG-binding protein (CXXC finger protein 1 (CFP1)) binds to DNA containing unmethylated CpG motifs and is required for mammalian embryogenesis, normal cytosine methylation, and cellular differentiation. Studies were performed to identify proteins that interact with CFP1 to gain insight into the molecular function of this protein. Immunoprecipitation and mass spectrometry reveal that human CFP1 associates with a approximately 450-kDa complex that contains the mammalian homologues of six of the seven components of the Set1/COMPASS complex, the sole histone H3-Lys4 methyltransferase in yeast. In vitro assays demonstrate that the human Set1/CFP1 complex is a histone methyltransferase that produces mono-, di-, and trimethylated histone H3 at Lys4. Confocal microscopy reveals that CFP1 and Set1 co-localize to nuclear speckles associated with euchromatin. A Set1 complex of reduced mass persists in murine embryonic stem cells lacking CFP1. These cells carry elevated levels of methylated histone H3-Lys4 and reduced levels of methylated histone H3-Lys9. Together with the previous finding of reduced levels of cytosine methylation, these data indicate that cells lacking CFP1 contain reduced levels of heterochromatin. Furthermore, ES cells lacking CFP1 exhibit a 4-fold excess of histone H3-Lys4 methylation following induction of differentiation, indicating that CFP1 restricts the activity of the Set1 histone methyltransferase complex. These results reveal a mammalian counterpart to the yeast Set1/COMPASS complex. The presence of CFP1 in this complex implicates this protein as a critical epigenetic regulator of histone modification in addition to cytosine methylation and reveals one mechanism by which this protein intersects with the epigenetic machinery.

Substrate Specificity and Kinetic Mechanism of Mammalian G9a Histone H3 Methyltransferase

Lysine-specific murine histone H3 methyltransferase, G9a, was expressed and purified in a baculovirus expression system. The primary structure of the recombinant enzyme is identical to the native enzyme. Enzymatic activity was favorable at alkaline conditions (>pH 8) and low salt concentration and virtually unchanged between 25 and 42 degrees C. Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un- or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9). In vitro methylation of the H3 tail peptide resulted in trimethylation of Lys-9 and the reaction is processive. The turnover number (k(cat)) for methylation was 88 and 32 h(-1) on the wild type and K4AK9 histone H3 tail, respectively. The Michaelis constants for wild type and K4AK9 ((K(m)(pep))) were 0.9 and 1.0 microM and for S-adenosyl-L-methionine (K(m)(AdoMet)) were 1.8 and 0.6 microM, respectively. Comparable kinetic constants were obtained for recombinant histone H3. The conversion of K4AK9 di- to trimethyl-lysine was 7-fold slower than methyl group addition to unmethylated peptide. Preincubation studies showed that G9a-AdoMet and G9a-peptide complexes are catalytically active. Initial velocity data with peptide and S-adenosyl-L-methionine (AdoMet) and product inhibition studies with S-adenosyl-L-homocysteine were performed to assess the kinetic mechanism of the reaction. Double reciprocal plots and preincubation studies revealed S-adenosyl-L-homocysteine as a competitive inhibitor to AdoMet and mixed inhibitor to peptide. Trimethylated peptides acted as a competitive inhibitor to substrate peptide and mixed inhibitor to AdoMet suggesting a random mechanism in a Bi Bi reaction for recombinant G9a where either substrate can bind first to the enzyme, and either product can release first.