Enzymatic mechanism and product specificity of SET-domain protein lysine methyltransferases

Molecular dynamics and hybrid quantum mechanics/molecular mechanics have been used to investigate the mechanisms of (+)AdoMet methylation of protein-Lys-NH(2) catalyzed by the lysine methyltransferase enzymes: histone lysine monomethyltransferase SET7/9, Rubisco large-subunit dimethyltransferase, viral histone lysine trimethyltransferase, and the Tyr245Phe mutation of SET7/9. At neutrality in aqueous solution, primary amines are protonated. The enzyme reacts with Lys-NH(3)(+) and (+)AdoMet species to provide an Enz.Lys-NH(3)(+).(+)AdoMet complex. The close positioning of two positive charges lowers the pK(a) of the Lys-NH(3)(+) entity, a water channel appears, and the proton escapes to the aqueous solvent; then the reaction Enz.Lys-NH(2).(+)AdoMet --> Enz.Lys-N(Me)H(2)(+).AdoHcy occurs. Repeat of the sequence provides dimethylated lysine, and another repeat yields a trimethylated lysine. The sequence is halted at monomethylation when the conformation of the Enz.Lys-N(Me)H(2)(+).(+)AdoMet has the methyl positioned to block formation of a water channel. The sequence of reactions stops at dimethylation if the conformation of Enz.Lys-N(Me)(2)H(+).(+)AdoMet has a methyl in position, which forbids the formation of the water channel.     

Chromatin immunoprecipitation microarrays for identification of genes silenced by histone H3 lysine 9 methylation

Switching from acetylation to methylation at histone H3 lysine 9 (K9) has recently been shown to contribute to euchromatin gene silencing. To identify genes silenced by K9 modifications, we probed a human CpG island microarray with DNA obtained by chromatin immunoprecipitation (ChIP) in a cancer cell line using an anti-H3-K9 methylated antibody or an anti-H3-K9 acetylated antibody. Of the 27 clones with the highest signal ratio of K9 methylation over acetylation (Me/Ac), 13 contained repetitive sequences. Among 14 nonrepetitive clones, we identified 11 genes (seven known and four previously undescribed), one EST, and two unknown fragments. Using ChIP-PCR, all 18 examined clones showed higher ratios of H3-K9 Me/Ac than the active gene control, P21, thus confirming the microarray data. In addition, we found a strong correlation between the K9 Me/Ac ratio and CpG island DNA methylation (R = 0.92, P < 0.01), and five of seven genes examined (megalin, thrombospondin-4, KR18, latrophilin-3, and phosphatidylinositol-3-OH kinase P101 subunit) showed lack of expression by RT-PCR and reactivation by DNA methylation and/or histone deacetylase inhibition, suggesting that these genes are true targets of silencing through histone modifications. All five genes also showed significant DNA methylation in a cell line panel and in primary colon cancers. Our data suggest that CpG island microarray coupled with ChIP can identify novel targets of gene silencing in cancer. This unbiased approach confirms the tight coupling between DNA methylation and histone modifications in cancer and could be used to probe gene silencing in nonneoplastic conditions as well.     

DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin

Dynamic changes in histone modification are critical for regulating DNA double-strand break (DSB) repair. Activation of the Tip60 acetyltransferase by DSBs requires interaction of Tip60 with histone H3 methylated on lysine 9 (H3K9me3). However, how H3K9 methylation is regulated during DSB repair is not known. Here, we demonstrate that a complex containing kap-1, HP1, and the H3K9 methyltransferase suv39h1 is rapidly loaded onto the chromatin at DSBs. Suv39h1 methylates H3K9, facilitating loading of additional kap-1/HP1/suv39h1 through binding of HP1’s chromodomain to the nascent H3K9me3. This process initiates cycles of kap-1/HP1/suv39h1 loading and H3K9 methylation that facilitate spreading of H3K9me3 and kap-1/HP1/suv39h1 complexes for tens of kilobases away from the DSB. These domains of H3K9me3 function to activate the Tip60 acetyltransferase, allowing Tip60 to acetylate both ataxia telangiectasia-mutated (ATM) kinase and histone H4. Consequently, cells lacking suv39h1 display defective activation of Tip60 and ATM, decreased DSB repair, and increased radiosensitivity. Importantly, activated ATM rapidly phosphorylates kap-1, leading to release of the repressive kap-1/HP1/suv39h1 complex from the chromatin. ATM activation therefore functions as a negative feedback loop to remove repressive suv39h1 complexes at DSBs, which may limit DSB repair. Recruitment of kap-1/HP1/suv39h1 to DSBs therefore provides a mechanism for transiently increasing the levels of H3K9me3 in open chromatin domains that lack H3K9me3 and thereby promoting efficient activation of Tip60 and ATM in these regions. Further, transient formation of repressive chromatin may be critical for stabilizing the damaged chromatin and for remodeling the chromatin to create an efficient template for the DNA repair machinery.DNA double-strand breaks (DSBs) are toxic and must be repaired to maintain genomic stability. Detection of DSBs requires recruitment of the mre11–rad50–nbs1 (MRN) complex to the DNA ends (1). MRN then recruits and activates the ataxia telangiectasia-mutated (ATM) kinase (2, 3) through a mechanism that also requires the Tip60 acetyltransferase (3). Tip60 directly acetylates and activates ATM’s kinase activity (4–6) and functions, in combination with MRN, to promote ATM-dependent phosphorylation of DSB repair proteins (3), including histone H2AX. This process creates domains of phosphorylated H2AX (γH2AX) extending for hundreds of kilobases along the chromatin (7, 8). Mdc1 then binds to γH2AX, providing a landing pad for other DSB repair proteins, including the RNF8/RNF168 ubiquitin ligases (1, 3, 9, 10). Tip60 also plays a critical role in regulating chromatin structure at DSBs as part of the NuA4–Tip60 complex (11). NuA4-Tip60 catalyzes histone exchange (via the p400 ATPase subunit) and acetylation of histone H4 (by Tip60) at DSBs (12–15), leading to the formation of open, flexible chromatin domains adjacent to the break (12, 13). These open chromatin structures then facilitate histone ubiquitination, the loading of brca1 and 53BP1, and repair of the DSB (13, 16). The ordered acetylation and ubiquitination of the chromatin and loading of DNA repair proteins is therefore critical for DSB repair.Activation of Tip60’s acetyltransferase activity requires interaction between Tip60’s chromodomain and histone H3 methylated on lysine 9 (H3K9me3) on nucleosomes at the break (4, 6). This interaction, in combination with tyrosine phosphorylation of Tip60 (17), increases Tip60’s acetyltransferase activity and promotes acetylation of both the ATM kinase and histone H4 (4–6, 17). Consequently, loss of H3K9me2/3 leads to failure to activate the ATM signaling pathway, loss of H4 acetylation during DSB repair, disruption of heterochromatin, genomic instability, and defective DSB repair (4, 17–19). H3K9me3s therefore play a critical role in linking chromatin structure at DSBs to the activation of DSB signaling proteins such as Tip60 and ATM.How Tip60 gains access to H3K9me3 and how H3K9me3 levels at DSBs are regulated is not known. H3K9me3 is concentrated in heterochromatin domains, where it recruits HP1, kap-1, and H3K9 methyltransferases (20, 21) to maintain the silent, compact conformation of heterochromatin (20). This implies that Tip60’s acetyltransferase activity can only be activated at DSBs in regions of high H3K9me3 density, such as heterochromatin. Alternatively, H3K9 methylation may be actively increased at DSBs in regions of low H3K9me3 density to allow for Tip60 activation and efficient DSB repair in euchromatin. Understanding the dynamics of H3K9 methylation at DSBs is therefore critical to understanding how Tip60 activity is regulated by the local chromatin architecture. Here, we show that the suv39h1 methyltransferase is recruited to DSBs in euchromatin as part of a larger kap-1/HP1/suv39h1 complex. Suv39h1 increases H3K9me3 at DSBs, activating Tip60’s acetyltransferase activity and promoting the subsequent acetylation and activation of ATM. Further, loss of inducible H3K9me3 at DSBs leads to defective repair and increased radiosensitivity. Finally, loading of the kap-1/HP1/suv39h1 complex is transient, and the complex is rapidly released from the chromatin through a negative feedback loop driven by ATM-dependent phosphorylation of the kap-1 protein.     

Epigenetic histone H3 lysine 9 methylation in metabolic memory and inflammatory phenotype of vascular smooth muscle cells in diabetes

Diabetic patients continue to develop inflammation and vascular complications even after achieving glycemic control. This poorly understood "metabolic memory" phenomenon poses major challenges in treating diabetes. Recent studies demonstrate a link between epigenetic changes such as chromatin histone lysine methylation and gene expression. We hypothesized that H3 lysine-9 tri-methylation (H3K9me3), a key repressive and relatively stable epigenetic chromatin mark, may be involved in metabolic memory. This was tested in vascular smooth muscle cells (VSMC) derived from type 2 diabetic db/db mice. These cells exhibit a persistent atherogenic and inflammatory phenotype even after culture in vitro. ChIP assays showed that H3K9me3 levels were significantly decreased at the promoters of key inflammatory genes in cultured db/db VSMC relative to control db/+ cells. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase Suv39h1 were also reduced in db/db VSMC. Furthermore, db/db VSMC were hypersensitive to TNF-alpha inflammatory stimulus, which induced dramatic and sustained decreases in promoter H3K9me3 and Suv39h1 occupancy. Recruitment of corepressor HP1alpha was also reduced under these conditions in db/db cells. Overexpression of SUV39H1 in db/db VSMC reversed this diabetic phenotype. Conversely, gene silencing of SUV39H1 with shRNAs in normal human VSMC (HVSMC) increased inflammatory genes. HVSMC cultured in high glucose also showed increased inflammatory gene expression and decreased H3K9me3 at their promoters. These results demonstrate protective roles for H3K9me3 and Suv39h1 against the preactivated state of diabetic VSMC. Dysregulation of epigenetic histone modifications may be a major underlying mechanism for metabolic memory and sustained proinflammatory phenotype of diabetic cells.     

Methylation of SUV39H1 by SET7/9 results in heterochromatin relaxation and genome instability

Suppressor of variegation 3–9 homolog 1 (SUV39H1), a histone methyltransferase, catalyzes histone 3 lysine 9 trimethylation and is involved in heterochromatin organization and genome stability. However, the mechanism for regulation of the enzymatic activity of SUV39H1 in cancer cells is not yet well known. In this study, we identified SET domain-containing protein 7 (SET7/9), a protein methyltransferase, as a unique regulator of SUV39H1 activity. In response to treatment with adriamycin, a DNA damage inducer, SET7/9 interacted with SUV39H1 in vivo, and a GST pull-down assay confirmed that the chromodomain-containing region of SUV39H1 bound to SET7/9. Western blot using antibodies specific for antimethylated SUV39H1 and mass spectrometry demonstrated that SUV39H1 was specifically methylated at lysines 105 and 123 by SET7/9. Although the half-life and localization of methylated SUV39H1 were not noticeably changed, the methyltransferase activity of SUV39H1 was dramatically down-regulated when SUV39H1 was methylated by SET7/9. Consequently, H3K9 trimethylation in the heterochromatin decreased significantly, which, in turn, led to a significant increase in the expression of satellite 2 (Sat2) and α-satellite (α-Sat), indicators of heterochromatin relaxation. Furthermore, a micrococcal nuclease sensitivity assay and an immunofluorescence assay demonstrated that methylation of SUV39H1 facilitated genome instability and ultimately inhibited cell proliferation. Together, our data reveal a unique interplay between SET7/9 and SUV39H1—two histone methyltransferases—that results in heterochromatin relaxation and genome instability in response to DNA damage in cancer cells.     

Prognostic value of lymphocyte-to-monocyte ratio and histone methyltransferase G9a histone methyltransferase in patients with double expression lymphoma: A retrospective observational study

In patients with diffuse large B-cell lymphoma, MYC combined with Bcl2 and/or Bcl6-based protein expression is called double expression lymphoma (DEL). R-DA-EPOCH program chemotherapy is typically recommended because these patients often have a poor prognosis. Although numerous factors affect survival of patients with DEL, the roles of the tumor biomarker histone methyltransferase G9a (G9a) and the lymphocyte-to-monocyte ratio (LMR) are unknown.We performed a retrospective analysis of data from 51 patients. These patients were newly diagnosed with DEL and treated with R-DA-EPOCH at Taizhou People’ s Hospital and Northern Jiangsu People''s Hospital between June 2014 and December 2019. Receiver operator characteristic curve results were used to calculate the LMR cutoff value. We used an immunohistochemical analysis to examine G9a expression in DEL tissues. The Kaplan–Meier method was used to determine progression-free survival (PFS) and overall survival (OS) characteristics. Cox proportional-hazards models were constructed for univariate and multivariate analyses to examine the prognostic values of LMRs and G9a in patients with DEL.The cutoff value for LMR was 2.18. The 5-year PFS rate was 35.3%, and the 5-year OS rate was 39.2%. Patients with DEL with lower LMRs and who were G9a-positive predicted inferior PFS and OS. Univariate analysis revealed that patients with elevated LDH levels, high National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) scores, LMRs ≤2.18, and G9a-positive results had relatively poorer PFS and OS. The multivariate analysis revealed that LMRs ≤2.18 and a G9a-positive result were independent prognostic factors for PFS and OS in patients with DEL treated with R-DA-EPOCH.The study results suggested that peripheral blood LMRs were an important marker for evaluation of prognosis in patients with DEL. High expression of G9a was associated with worse outcomes, indicating that G9a may serve as a prognostic biomarker for patients with DEL who undergo R-DA-EPOCH program chemotherapy.     

Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation.

The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.     

Epigenetic regulation of the Drosophila chromosome 4 by the histone H3K9 methyltransferase dSETDB1

The polytene chromosomes of Drosophila melanogaster consist of condensed heterochromatin regions most of which are in the chromocenter, telomeres, and the fourth chromosome. Whereas suppressor of variegation 3-9 [SU(VAR)3-9], a histone methyltransferase, is mainly responsible for lysine 9 of histone H3 (H3K9) methylation of the chromocenter and consequent binding of the heterochromatin-protein HP1, the enzyme for painting of the fourth chromosome by H3K9 methylation has been elusive. We show here that dSETDB1, the Drosophila ortholog of the mammalian SETDB1, is an authentic H3K9 methyltransferase and a pleiotropic regulator of the fly's development. Drosophila mutants hypomorphic or null in dSETDB1 expression lose most of the H3K9 methylation as well as HP1-binding on the fourth chromosome. We also show that binding of painting of fourth (POF), a known fourth chromosome-specific protein, and the dSETDB1-controlled H3K9 methylation of this chromosome are interdependent. Furthermore, POF and dSETDB1 interact with each other in vivo. The deregulation of H3K9 methylation, HP1-binding, and POF-binding resulted in, on the average, a global reduction of gene expression from the fourth chromosome but not the other chromosomes. Deficiency of dSETDB1 also up-regulated the expression of HP1. These results have suggested an interactive network, as controlled in part by dSETDB1, regulating the epigenetic modification and gene expression of Drosophila chromosome 4.     

Relationship between the structure of SET/TAF-Ibeta/INHAT and its histone chaperone activity

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Ibeta/INHAT at a resolution of 2.3 A. We found that SET/TAF-Ibeta/INHAT formed a dimer that assumed a "headphone"-like structure. Each subunit of the SET/TAF-Ibeta/INHAT dimer consisted of an N terminus, a backbone helix, and an "earmuff" domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Ibeta/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by approximately 40 degrees . Our biochemical analyses of mutants revealed that the region of SET/TAF-Ibeta/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Ibeta/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.     

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein

The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.The emergence of multidrug-resistant bacteria is a mounting global health crisis. In particular, multidrug-resistant Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and is resistant to most antibiotics commonly used for patient treatment (1). Hospital intensive care units in many countries, including the United States, now report methicillin-resistant S. aureus infection rates exceeding 50% (2, 3). Antibiotic resistance in contemporary infectious S. aureus strains, such as in hospitals, is often encoded by plasmids that can be transmitted between strains via horizontal DNA transfer mechanisms. These plasmids are typically classified as small (<5 kb) multicopy plasmids, which usually encode only a single resistance gene; medium-sized (8–40 kb) multirestance plasmids that confer resistance to multiple antibiotics, disinfectants, and/or heavy metals; and large (>40 kb) conjugative multiresistance plasmids that additionally encode a conjugative DNA transfer mechanism (4–6). Importantly, sequence analyses have shown that most staphylococcal conjugative and nonconjugative multiresistance plasmids encode a highly conserved replication initiation protein, denoted RepA_N (5–15). RepA_N proteins are also encoded by plasmids from other Gram-positive bacteria as well as by some phage, underscoring their ubiquitous nature (10). These RepA proteins are essential for replication of multiresistance plasmids, and hence plasmid carriage and dissemination, yet the mechanisms by which these proteins function in replication are currently unknown.The DNA replication cycle can be divided into three stages: initiation, elongation, and termination. Replication initiation proteins (RepA) mediate the crucial first step of initiation. Bacterial chromosome replication is initiated by the chromosomal replication initiator protein, DnaA, which binds the origin and recruits the replication components known as the primosome (16). In Gram-negative bacteria the primosome includes DnaG primase, the replicative helicase (DnaB), and DnaC (17). Replication initiation in Gram-positive bacteria involves DnaG primase and helicase (DnaC) and the proteins DnaD, DnaI, and DnaB (18–22). DnaD binds first to DnaA at the origin. This is followed by binding of DnaI/DnaB and DnaG, which together recruit the replicative helicase (23, 24). Instead of DnaA, plasmids encode and use their own specific replication initiator binding protein. Structures are only available for RepA proteins (F, R6K, and pPS10 Rep) harbored in Gram-negative bacteria. These proteins contain winged helix-turn-helix (winged HTH) domains and bind iteron DNA as monomers to, in some still unclear manner, drive replicon assembly (25–27).Replication mechanisms used by plasmids harbored in Gram-positive bacteria are less well understood and are distinct from their Gram-negative counterparts. Indeed, most plasmid RepA proteins in Gram-negative and Gram-positive bacteria show no sequence homology and seem to be unrelated. The multiresistance RepA proteins are arguably among the most abundant of plasmid Rep proteins, yet how they function is not known. Data suggest that these proteins are composed of three main regions: an N-terminal domain (NTD) consisting of ∼120 aa, a long and variable linker region (∼30–50 residues), and a C-terminal domain (CTD) of ∼120 residues (28–31). The NTD and CTD are both essential for replication. The NTD exhibits the highest level of sequence conservation, which has resulted in the designation of plasmids that encode these proteins as the RepA_N replicon family (10). Although not as well conserved as the NTD, RepA CTD regions show homology between plasmids found in genus-specific clusters, suggesting that this domain may perform a host-specific role (28–32). Although the function of the RepA CTD remains enigmatic, recent studies have indicated that the NTD mediates DNA binding and interacts with iterons that reside within the plasmid origin (30). The essential roles played by RepA proteins in multiresistance plasmid retention marks them as attractive targets for the development of specific chemotherapeutics. However, the successful design of such compounds necessitates structural and mechanistic insight. Here, we describe a detailed dissection of the RepA proteins from the multiresistance plasmids pSK41 and pTZ2126. The combined data reveal the molecular underpinnings of a minimalist replication assembly mediated by multiresistance RepA proteins.