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 HP1α and HP1γ 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.
DNA cytosine methylation is one of the major epigenetic gene silencing marks in the human genome facilitated by DNA methyltransferases. DNA cytosine-5 methyltransferase 1 (DNMT1) performs maintenance methylation in somatic cells. In cancer cells, DNMT1 is responsible for the aberrant hypermethylation of CpG islands and the silencing of tumor suppressor genes. Here we show that the catalytically active recombinant DNMT1, lacking 580 amino acids from the amino terminus, binds to unmethylated DNA with higher affinity than hemimethylated or methylated DNA. To further understand the binding domain of enzyme, we have used gel shift assay. We have demonstrated that the CXXC region (C is cysteine; X is any amino acid) of DNMT1 bound specifically to unmethylated CpG dinucleotides. Furthermore, mutation of the conserved cysteines abolished CXXC mediated DNA binding. In transfected COS-7 cells, CXXC deleted DNMT1 (DNMT1 ∆CXXC ) localized on replication foci. Both point mutant and DNMT1 ∆CXXC enzyme displayed significant reduction in catalytic activity, confirming that this domain is crucial for enzymatic activity. A permanent cell line with DNMT1 ∆CXXC displayed partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme. Thus, the CXXC domain encompassing the amino terminus region of DNMT1 cooperates with the catalytic domain for DNA methyltransferase activity.
N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.
RNA modifications have recently emerged as an important layer of gene regulation. N6-methyladenosine (m 6 A) is the most prominent modification on eukaryotic messenger RNA and has also been found on noncoding RNA, including ribosomal and small nuclear RNA. Recently, several m 6 A methyltransferases were identified, uncovering the specificity of m 6 A deposition by structurally distinct enzymes. In order to discover additional m 6 A enzymes, we performed an RNAi screen to deplete annotated orthologs of human methyltransferaselike proteins (METTLs) in Drosophila cells and identified CG9666, the ortholog of human METTL5. We show that CG9666 is required for specific deposition of m 6 A on 18S ribosomal RNA via direct interaction with the Drosophila ortholog of human TRMT112, CG12975. Depletion of CG9666 yields a subsequent loss of the 18S rRNA m 6 A modification, which lies in the vicinity of the ribosome decoding center; however, this does not compromise rRNA maturation. Instead, a loss of CG9666-mediated m 6 A impacts fly behavior, providing an underlying molecular mechanism for the reported human phenotype in intellectual disability. Thus, our work expands the repertoire of m 6 A methyltransferases, demonstrates the specialization of these enzymes, and further addresses the significance of ribosomal RNA modifications in gene expression and animal behavior.
Summary Tandem zinc finger (ZF) proteins are the largest and most rapidly diverging family of DNA-binding transcription regulators in mammals. ZFP568 represses a transcript of placental-specific insulin like growth factor 2 (Igf2-P0) in mice. ZFP568 binds a 24-base pair sequence-specific element upstream of Igf2-P0 via the eleven-ZF array. Both DNA and protein conformations deviate from the conventional one finger-three bases recognition, with individual ZFs contacting 2, 3, or 4 bases and recognizing thymine on the opposite strand. These interactions arise from a shortened minor groove caused by an AT-rich stretch, suggesting adaptability of ZF arrays to sequence variations. Despite conservation in mammals, mutations at Igf2 and ZFP568 reduce their binding affinity in Chimpanzee and humans. Our studies provide important insights into the evolutionary and structural dynamics of ZF-DNA interactions that play a key role in mammalian development and evolution.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.