Transcription factor LSF facilitiates lysine methylation of α-tubulin by microtubule-associated SET8

Microtubules are critical for mitosis, cell motility, and protein and organelle transport, and are a validated target for anticancer drugs. However, tubulin regulation and recruitment in these cellular processes is less understood. Post-translational modifications of tubulin are proposed to regulate microtubule functions and dynamics. Although many such modifications have been investigated, tubulin methylations and enzymes responsible for methylation have only recently begun to be described. Here we report that N-lysine methyl transferase KMT5A (SET8/PR-Set7), which methylates histone H4K20, also methylates α-tubulin. Furthermore, the transcription factor LSF binds both tubulin and SET8, and enhances α-tubulin methylation in vitro, countered by FQI1, a specific small molecule inhibitor of LSF. Thus, the three proteins SET8, LSF, and tubulin, all essential for mitotic progression, interact with each other. Overall, these results point to dual functions for both SET8 and LSF not only in chromatin regulation, but also for cytoskeletal modification.First author draf

Automethylation of G9a and its implication in wider substrate specificity and HP1 binding.

Methylation of lysine residues on histones participates in transcriptional gene regulation. Lysine 9 methylation of histone H3 is a transcriptional repression signal, mediated by a family of SET domain containing AdoMet-dependent enzymes. G9a methyltransferase is a euchromatic histone H3 lysine 9 methyltransferase. Here, G9a is shown to methylate other cellular proteins, apart from histone H3, including automethylation of K239 residue. Automethylation of G9a did not impair or activate the enzymatic activity in vitro. The automethylation motif of G9a flanking target K239 (ARKT) has similarity with histone H3 lysine 9 regions (ARKS), and is identical to amino acids residues in EuHMT (ARKT) and mAM (ARKT). Under steady-state kinetic assay conditions, full-length G9a methylates peptides representing ARKS/T motif of H3, G9a, mAM and EuHMT efficiently. Automethylation of G9a at ARKT motif creates a binding site for HP1 class of protein and mutation of lysine in the motif impairs this binding. In COS-7 cells GFP fusion of the wild-type G9a co-localized with HP1alpha and HP1gamma isoforms whereas the G9a mutant with K239A displayed poor co-localization. Thus, apart from transcriptional repression and regulatory roles of lysine methylation, the non-histone protein methylation may create binding sites for cellular protein-protein interactions

PKC isoforms interact with and phosphorylate DNMT1

<p>Abstract</p> <p>Background</p> <p>DNA methyltransferase 1 (DNMT1) has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC) may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization.</p> <p>Results</p> <p>Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. <it>In vitro </it>phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, <it>in vitro </it>phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity.</p> <p>Conclusions</p> <p>Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.</p

Functional analysis of the N- and C-terminus of mammalian G9a histone H3 methyltransferase

Methylation of lysine 9 (K9) in the N-terminus tail of histone H3 (H3) in chromatin is associated with transcriptionally silenced genes and is mediated by histone methyltransferases. Murine G9a is a 1263 amino acid H3-K9 methyltransferase that possesses characteristic SET domain and ANK repeats. In this paper, we have used a series of green fluorescent protein-tagged deletion constructs to identify two nuclear localization signals (NLS), the first NLS embedded between amino acids 24 and 109 and the second between amino acids 394 and 401 of murine G9a. Our data show that both long and short G9a isoforms were capable of entering the nucleus to methylate chromatin. Full-length or N-terminus-deleted G9a isoforms were also catalytically active enzymes that methylated recombinant H3 or synthetic peptides representing the N-terminus tail of H3. In vitro methylation reactions using N-terminus tail peptides resulted in tri-methylation of K9 that remained processive, even in G9a enzymes that lacked an N-terminus region by deletion. Co-expression of G9a and H3 resulted in di- and tri-methylation of H3-K9, while siRNA-mediated knockdown of G9a in HeLa cells resulted in reduction of global H3-K9 di- and tri-methylation. A recombinant deletion mutant enzyme fused with maltose-binding protein (MBP-G9aΔ634) was used for steady-state kinetic analysis with various substrates and was compared with full-length G9a (G9aFL). Turnover numbers of MBP-G9aΔ634 for various substrates was ∼3-fold less compared with G9aFL, while their Michaelis constants (K(m)) for recombinant H3 were similar. The [Formula: see text] for MBP-G9aΔ634 was ∼2.3–2.65 μM with various substrates. Catalytic efficiencies (k(cat)/K(m)) for both MBP-G9aΔ634 and G9aFL were similar, suggesting that the N-terminus is not essential for catalysis. Furthermore, mutation of conserved amino acids R1097A, W1103A, Y1120A, Y1138A and R1162A, or the metal binding C1168A in the catalytic region, resulted in catalytically impaired enzymes, thereby confirming the involvement of the C-terminus of G9a in catalysis. Thus, distinct domains modulate nuclear targeting and catalytic functions of G9a

UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells

UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is a multi-domain protein associated with cellular proliferation and epigenetic regulation. The UHRF1 binds to methylated CpG dinucleotides and recruits transcriptional repressors DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) through its distinct domains. However, the molecular basis of UHRF1-mediated transcriptional regulation via chromatin modifications is yet to be fully understood. Here we show that UHRF1 binds histone lysine methyltransferase G9a, and both are co-localized in the nucleus in a cell-cycle-dependent manner. Concurrent with the cell-cycle progression, gradual deposition of UHRF1 and G9a was observed, which mirrored H3K9me2 accumulation on chromatin. Murine Uhrf1-null embryonic stem (ES) cells displayed a reduced amount of G9a and H3K9me2 on chromatin. UHRF1 recruited and cooperated with G9a to inhibit the p21 promoter activity, which correlated with the elevated p21 protein level in both human UHRF1 siRNA-transfected HeLa cells and murine Uhrf1-null ES cells. Furthermore, endogenous p21 promoter remained bound to UHRF1, G9a, DNMT1 and HDAC1, and knockdown of UHRF1 impaired the association of all three chromatin modifiers with the promoter. Thus, our results suggest that UHRF1 may serve as a focal point of transcriptional regulation mediated by G9a and other chromatin modification enzymes

Functional cooperation between HP1 and DNMT1 mediates gene silencing

Mammalian euchromatic gene silencing results from the combined repressive effects of histone and DNA methyltransferases. Little is known of the mechanism by which these enzymes cooperate to induce silencing. Here we show that mammalian HP1 family members mediate communication between histone and DNA methyltransferases. In vitro, methylation of histone 3 Lys 9 by G9a creates a binding platform for HP1α, β, and γ. DNMT1 interacts with HP1 resulting in increased DNA methylation on DNA and chromatin templates in vitro. The functional and physical interaction can be recapitulated in vivo. Binding of GAL4-HP1 to a reporter construct is sufficient to induce repression and DNA methylation in DNMT1 wild-type but not DNMT1-null cells. Additionally, silencing of the Survivin gene coincides with recruitment of G9a and HP1 in DNMT1 wild-type but not null cells. We conclude that direct interactions between HP1 and DNMT1 mediate silencing of euchromatic genes

UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells.

UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is a multi-domain protein associated with cellular proliferation and epigenetic regulation. The UHRF1 binds to methylated CpG dinucleotides and recruits transcriptional repressors DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) through its distinct domains. However, the molecular basis of UHRF1-mediated transcriptional regulation via chromatin modifications is yet to be fully understood. Here we show that UHRF1 binds histone lysine methyltransferase G9a, and both are co-localized in the nucleus in a cell-cycle-dependent manner. Concurrent with the cell-cycle progression, gradual deposition of UHRF1 and G9a was observed, which mirrored H3K9me2 accumulation on chromatin. Murine Uhrf1-null embryonic stem (ES) cells displayed a reduced amount of G9a and H3K9me2 on chromatin. UHRF1 recruited and cooperated with G9a to inhibit the p21 promoter activity, which correlated with the elevated p21 protein level in both human UHRF1 siRNA-transfected HeLa cells and murine Uhrf1-null ES cells. Furthermore, endogenous p21 promoter remained bound to UHRF1, G9a, DNMT1 and HDAC1, and knockdown of UHRF1 impaired the association of all three chromatin modifiers with the promoter. Thus, our results suggest that UHRF1 may serve as a focal point of transcriptional regulation mediated by G9a and other chromatin modification enzymes

MBD4 cooperates with DNMT1 to mediate methyl-DNA repression and protects mammalian cells from oxidative stress

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