Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.

Objective-Lipoprotein-derived phospholipid oxidation products have been implicated as candidate triggers of the inflammatory process in atherosclerosis. However, in vivo evidence regarding the impact of oxidized phospholipids on the artery wall thus far has been elusive. Therefore, the aim of this study was to investigate if structurally defined oxidized phospholipids induce expression of atherogenic chemokines and monocyte adhesion in intact murine arteries. Methods and Results-To model the accumulation of oxidized phospholipids in the arterial wall, oxidized 1-palmitoyl-2-arachidonoyl-sn-3-glycero-phosphorylcholine (OxPAPC) was topically applied to carotid arteries in mice using pluronic gel. Using quantitative reverse-transcriptase polymerase chain reaction (PCR) and immunohistochemistry, we show that OxPAPC induced a set of atherosclerosis-related genes, including monocyte chemotactic protein 1 (MCP-1) and keratinocyte-derived chemokine (KC), tissue factor (TF), interleukin 6 (IL-6), heme oxygenase 1 (HO-1), and early growth response 1 (EGR-1). OxPAPC-regulated chemokines were also expressed in atherosclerotic lesions of apolipoprotein E-deficient (ApoE Ϫ/Ϫ ) mice. In isolated perfused carotid arteries, OxPAPC triggered rolling and firm adhesion of monocytes in a P-selectin and KC-dependent manner. Conclusion-Oxidized phospholipids contribute to vascular inflammation in murine arteries in vivo, initiating atherogenic chemokine expression that leads to monocyte adhesion; therefore, they can be regarded as triggers of the inflammatory process in atherosclerosis. Key Words: atherosclerosis Ⅲ oxidized phospholipids Ⅲ inflammation Ⅲ chemokines Ⅲ leukocyte adhesion A therosclerosis is a chronic inflammatory disease involving accumulation of lipoproteins and mononuclear cells in the subendothelial space. Chemokines serve a vital role in supporting the inflammatory response of the arterial wall, leading to atherosclerotic plaque formation. In particular, genetic deletions of monocyte chemotactic protein 1 (MCP-1) or its receptor CCR2, as well as transplantation of bone marrow deficient in the IL-8 receptor homologue CXCR2, [1][2][3] have been shown to decrease monocyte accumulation and lesion formation in mice susceptible to atherosclerosis. Although our knowledge about the mechanisms underlying atherosclerosis and its complications has dramatically increased, the question about the initiating factors or triggers of atherogenesis remains unsolved. Accumulating evidence suggests retention of low-density lipoprotein (LDL) particles in the subendothelial space with subsequent oxidative modification as key steps in beginning atherosclerosis. Oxidized LDL has been shown to induce chemokines such as MCP-1 in vascular cells, but direct evidence from suitable animal models is scarce and it has been questioned if lipoproteins oxidized in vitro yield similar biological responses as lipoproteins oxidized in the arterial wall. 4 Recently, considerable advances have been made in dissecting the molecular components of oxidized LD...

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the sub-nuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.

Background: UHRF1-mediated targeting of DNMT1 to replicated DNA is essential for DNA methylation. Results: We show here that UHRF1 stimulates the activity and specificity of DNMT1 by an allosteric mechanism. Conclusion: UHRF1 has multiple roles that support DNA methylation including targeting and regulation of the activity and specificity of DNMT1. Significance: Regulation of DNMT1 is essential for the rapid and faithful remethylation of DNA after replication.

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