An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes

Vermeulen, Michiel; Stunnenberg, Hendrik G.

doi:10.1251/bpo85

Cited by 3 publications

(3 citation statements)

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“…It was reconstituted into chromatin using purified recombinant histones as described above. Chromatin and naked DNA were adsorbed to Dynabeads (Dynal) as previously described (Vermeulen and Stunnenberg, 2004). Beads containing 400 ng of DNA or chromatin were incubated with 200 ng of His-tagged HNF4, 1 μg of His-tagged PRMT1, 50 μM SAM in 100 μl of binding buffer (70 mM KCl, 10 mM Hepes-KOH pH 7.8, 5% glycerol, 2 mM MgCl 2 , 5 mM DTT, 0.25 mg/ml BSA and 0.5 mM PMSF), washed with binding buffer containing 100 mM KCl, boiled in SDS sample buffer, resolved by SDS-PAGE and analyzed by western blot using anti-His antibodies.…”

Section: Immobilized Template Assaysmentioning

confidence: 99%

Two Functional Modes of a Nuclear Receptor-Recruited Arginine Methyltransferase in Transcriptional Activation

Barrero

Malik

2006

Molecular Cell

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Nuclear receptors, like other transcriptional activators, switch on gene transcription by recruiting a complex network of coregulatory proteins. Here, we have identified the arginine methyltransferase PRMT1 as a coactivator for HNF4, an orphan nuclear receptor that regulates the expression of genes involved in diverse metabolic pathways. Remarkably, PRMT1, whose methylation activity on histone H4 strongly correlates with induction of HNF4 target genes in differentiating enterocytes, regulates HNF4 activity through a bipartite mechanism. First, PRMT1 binds and methylates the HNF4 DNA-binding domain (DBD), thereby enhancing the affinity of HNF4 for its binding site. Second, PRMT1 is recruited to the HNF4 ligand-binding domain (LBD) through a mechanism that involves the p160 family of coactivators and methylates histone H4 at arginine 3. This, together with recruitment of the histone acetyltransferase p300, leads to nucleosomal alterations and subsequent RNA polymerase II preinitiation complex formation.

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Section: Immobilized Template Assaysmentioning

confidence: 99%

Two Functional Modes of a Nuclear Receptor-Recruited Arginine Methyltransferase in Transcriptional Activation

Barrero

Malik

2006

Molecular Cell

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“…Although much work has been devoted to antibody-based , , and mass spectrometry-based , , based indexing of histone modifications, relatively little effort has been devoted to understanding the consequences of the enormous combinatorial complexity achieved through multiple histone modifications , . Several isolated cases have illustrated how the interplay of histone modifications can regulate biological outcomes .…”

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confidence: 99%

A Combinatorial H4 Tail Library for Exploring the Histone Code

2008

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Histone modifications modulate chromatin structure and function. A posttranslational modificationrandomized, combinatorial library based on the first twenty-one residues of histone H4 was designed for systematic examination of proteins that interpret a histone code. The 800-member library represented all permutations of most known modifications within the N-terminal tail of histone H4. To determine its utility in a protein-binding assay, the on-bead library was screened with an antibody directed against phosphoserine 1 of H4. Among the hits, 59/60 sequences were phosphorylated at S1, while 30/30 of those selected from the non-hits were unphosphorylated. A 512-member version of the library was then used to determine the binding specificity of the double tudor domain of hJMJD2A, a histone demethylase involved in transcriptional repression. Global linear least squares fitting of modifications from the identified peptides (40 hits and 34 non-hits) indicated that methylation of K20 was the primary determinant for binding, but that phosphorylation/acetylation on neighboring sites attenuated the interaction. To validate the on-bead screen, isothermal titration calorimetry was performed with thirteen H4 peptides. Dissociation constants ranged from 1 mM -1μM and corroborated the screening results. The general approach should be useful for probing the specificity of any histone-binding protein. Keywordshistone code; chromatin; JMJD2A; tudor domain; posttranslational modifications; one-bead; onecompound; combinatorial peptide library Histone proteins package DNA into chromatin and regulate the accessibility of DNA in processes such as transcription, repair and replication (1). Control of chromatin structure and function is mediated by reversible posttranslational modifications (PTMs) of histones. The most prevalent histone modifications occur on the unstructured N-terminal "tails" and include acetylation, methylation and phosphorylation, but others such as citrullination, ubiquitylation, sumoylation, ADP-ribosylation and biotinylation have been described (1). Mounting evidence suggests that particular modification states modulate histone-histone, histone-DNA and histone-non-histone protein interactions.Recently, many specialized protein domains (histone-binding modules) have been identified and shown to display binding preferences for a particular modified amino acid side-chain. ‡ To whom correspondence should be addressed: University of Wisconsin, Dept. of Biomolecular Chemistry, 1300 University Ave. Madison, WI 53706-1532. Tel: (608) 265-1859 Fax: (608) 262-5253; Email: jmdenu@wisc.edu Author Contributions A.G. conceptualized the methodology, conducted the experiments, interpreted the data and wrote the paper. G.C. performed the linear least squares analysis and helped with the correlation matrices. J.D. interpreted the data and wrote the paper. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 August 5. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Man...

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“…Other widely used methods to study DNA-protein interactions involve DNA footprinting, EMSA ( E lectrophoretic M obility S hift A ssay), southwestern blotting (SW), DNA affinity chromatography and DNA-protein crosslinking in vitro (DPC) [ 23 , 24 ] DNA affinity precipitation is an efficient method for the analysis and purification of proteins displaying sequence specificity for DNA [ 25 , 26 ]. Such assays can be used to study binding affinity and recruitment specificity of either recombinant or cellular proteins [ 27 , 28 ]. Recently, important progress has been made in understanding the interactions of modified histone tails with transcriptional regulators [ 29 - 31 ].…”

Section: Introductionmentioning

confidence: 99%

Affinity-based enrichment strategies to assay methyl-CpG binding activity and DNA methylation in early Xenopus embryos

Bogdanović

Veenstra

2011

BMC Res Notes

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BackgroundDNA methylation is a widespread epigenetic modification in vertebrate genomes. Genomic sites of DNA methylation can be bound by methyl-CpG-binding domain proteins (MBDs) and specific zinc finger proteins, which can recruit co-repressor complexes to silence transcription on targeted loci. The binding to methylated DNA may be regulated by post-translational MBD modifications.FindingsA methylated DNA affinity precipitation method was implemented to assay binding of proteins to methylated DNA. Endogenous MeCP2 and MBD3 were precipitated from Xenopus oocyte extracts and conditions for methylation-specific binding were optimized. For a reverse experiment, DNA methylation in early Xenopus embryos was assessed by MBD affinity capture.ConclusionsA methylated DNA affinity resin can be applied to probe for MBD activity in extracts. This assay has a broad application potential as it can be coupled to downstream procedures such as western blotting, fluorimetric HDAC assays and quantitative mass spectrometry. Methylated DNA affinity capture by methyl-CpG binding proteins produces fractions highly enriched for methylated DNA, suitable for coupling to next generation sequencing technologies. The two enrichment strategies allow probing of methyl-CpG protein interactions in early vertebrate oocytes and embryos.

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An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes

Cited by 3 publications

References 19 publications

Two Functional Modes of a Nuclear Receptor-Recruited Arginine Methyltransferase in Transcriptional Activation

Two Functional Modes of a Nuclear Receptor-Recruited Arginine Methyltransferase in Transcriptional Activation

A Combinatorial H4 Tail Library for Exploring the Histone Code

Affinity-based enrichment strategies to assay methyl-CpG binding activity and DNA methylation in early Xenopus embryos

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