Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of post-translational modifications

Johnson, Lianna M.; Mollah, Sahana; Garcia, Benjamin A.; Muratore, Tara L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Jacobsen, Steven E.

doi:10.1093/nar/gkh992

Cited by 224 publications

(221 citation statements)

References 61 publications

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“…All ChIP assays were performed at least three times from at least two chromatin samples prepared from biological replicates. It should be noted that unlike ChIP using antibodies against dimethyl H3K9 and dimethyl H3K27, Ta3 sequences were not well amplified from ChIP samples using antibodies against trimethyl H3K9 and trimethyl H3K27, consistent with previous reports (Johnson et al 2004;Mathieu et al 2005). Thus Ta3 lanes in these cases only serve as background controls.…”

Section: Chromatin Immunoprecipitation (Chip) Analysissupporting

confidence: 87%

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A PHD finger protein involved in both the vernalization and photoperiod pathways in Arabidopsis

Sung¹,

Schmitz²,

Amasino³

2006

Genes Dev.

218

261

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The proper timing of flowering is critical for successful reproduction. The perception of the seasonal cues of daylength changes and exposure to cold influences flowering time in many plant species through the photoperiod and vernalization pathways, respectively. Here we show that a plant homeodomain (PHD) finger-containing protein, VIN3-LIKE 1 (VIL1), participates in both the photoperiod and vernalization pathways in Arabidopsis thaliana by regulating expression of the related floral repressors FLOWERING LOCUS C (FLC) and FLOWERING LO-CUS M (FLM). In the vernalization pathway, VIL1, along with VERNALIZATION INSENSITIVE 3 (VIN3), is necessary for the modifications to FLC and FLM chromatin that are associated with an epigenetically silenced state and with acquisition of competence to flower. In addition, VIL1 regulates FLM independently of VIN3 in a photoperiod-dependent manner.Supplemental material is available at http://www.genesdev.org.Received September 13, 2006; revised version accepted October 13, 2006. The transition to flowering is a critical developmental event in the plant life cycle. In many plants, the perception of seasonal changes affects the timing of this transition. Two environmental cues that many plant species monitor for flowering time control are the prolonged cold of winter and changing day length; these cues promote flowering through the vernalization and photoperiod pathways, respectively. Most accessions of Arabidopsis thaliana exhibit a facultative response to these signals: In the absence of either environmental cue, flowering is delayed but nevertheless eventually occurs.In Arabidopsis, vernalization results in the mitotically stable repression of the potent floral repressor, FLOW-ERING LOCUS C (FLC) (Michaels and Amasino 1999;Sheldon et al. 1999). Vernalization-mediated repression of FLC is associated with histone modifications such as methylation of histone H3 Lys 9 (H3K9) and Lys 27 (H3K27) as well as H3 deacetylation (Bastow et al. 2004;Sung and Amasino 2004). A plant homeodomain (PHD) finger-containing protein, VERNALIZATION , is required for FLC repression by vernalization and the associated modifications to FLC chromatin (Sung and Amasino 2004). Here we show that a related PHD finger-containing protein, VIN3-LIKE 1 (VIL1), identified in a screen for proteins that interact with VIN3, cooperates with VIN3 in the vernalizationmediated repression of FLC. Furthermore, independently of VIN3 activity, VIL1 mediates the photoperiod-specific repression of another member of the FLC clade. Thus, VIL1 is involved in the regulation of flowering by two environmental-sensing pathways. Results and DiscussionBecause VIN3 is essential for the vernalization response (Sung and Amasino 2004) and PHD finger-containing proteins are often members of multisubunit chromatin remodeling complexes (Bienz 2006), we searched for potential components of a VIN3 complex using the yeast two-hybrid system. This screen revealed two independent VIN3-interacting clones encoding C-terminal regions of At3g24440. In...

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Section: Chromatin Immunoprecipitation (Chip) Analysissupporting

confidence: 87%

“…S2) are correlated with regions at which LHP1 is also enriched by vernalization (Sung et al 2006). Thus, although at a genome-wide level trimethylation of H3K9 and H3K27 exists at low levels in Arabidopsis (Johnson et al 2004), these modifications may play a key role in the repression of developmentally regulated genes in Arabidopsis.…”

Section: Resultsmentioning

confidence: 99%

A PHD finger protein involved in both the vernalization and photoperiod pathways in Arabidopsis

Sung¹,

Schmitz²,

Amasino³

2006

Genes Dev.

218

261

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“…The highresolution, high mass accuracy mass spectrometer should be operated in positive mode using a data-dependent MS/MS mode setting as described previously (see refs. [13][14][15][16][17].…”

Section: Reagent Setupmentioning

confidence: 99%

“…The described protocols have been applied for the successful identification and quantification of histone PTMs in cells from various organisms and under various external stimuli [13][14][15][16][17] , but can clearly be extended to histones extracted from any cellular source.…”

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confidence: 99%

Chemical derivatization of histones for facilitated analysis by mass spectrometry

et al. 2007

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Histone post-translational modifications have been recently intensely studied owing to their role in regulating gene expression. Here, we describe protocols for the characterization of histone modifications in both qualitative and semiquantitative manners using chemical derivatization and tandem mass spectrometry. In these procedures, extracted histones are first derivatized using propionic anhydride to neutralize charge and block lysine residues, and are subsequently digested using trypsin, which, under these conditions, cleaves only the arginine residues. The generated peptides can be easily analyzed using online LC-electrospray ionization-tandem mass spectrometry to identify the modification site. In addition, a stable isotope-labeling step can be included to modify carboxylic acid groups allowing for relative quantification of histone modifications. This methodology has the advantage of producing a small number of predicted peptides from highly modified proteins. The protocol should take approximately 15-19 h to complete, including all chemical reactions, enzymatic digestion and mass spectrometry experiments.

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“…The sequence information and the exact sites of methylation are usually determined by tandem mass spectrometry (MS/MS) analysis of these peptides. [117][118][119][120] "Topdown" methods directly measure the masses of full length histones, and the methylation level and their relative stoichiometry can be obtained by MS profiling and protein fragmentation. 121,122 Because a significant number of PTMs are located in the N-terminal region of the core histones, an alternative version of top-down method, "middle-town" approach has been applied to characterize large peptides that usually contain less than 50 Nterminal amino acid residues of histone tails.…”

Section: Analysis Of Histone Methylation By Mass Spectrometrymentioning

confidence: 99%

Chemical and biochemical approaches in the study of histone methylation and demethylation

Luo

Wang

et al. 2010

Med. Res. Rev.

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Histone methylation represents one of the most critical epigenetic events in DNA function regulation in eukaryotic organisms. Classic molecular biology and genetics tools provide significant knowledge about mechanisms and physiological roles of histone methyltransferases and demethylases in various cellular processes. In addition to this stream line, development and application of chemistry and chemistry-related techniques are increasingly involved in biological study, and provide information otherwise difficulty to obtain by standard molecular biology methods. Herein, we review recent achievements and progress in developing and applying chemical and biochemical approaches in the study of histone methylation, including chromatin immunoprecipitation (ChIP), chemical ligation, mass spectrometry (MS), biochemical assays, and inhibitor development. These technological advances allow histone methylation to be studied from genome-wide level to molecular and atomic levels. With ChIP technology, information can be obtained about precise mapping of histone methylation patterns at specific promoters, genes or other genomic regions. MS is particularly useful in detecting and analyzing methylation marks in histone and nonhistone protein substrates. Chemical approaches that permit site-specific incorporation of methyl groups into histone proteins greatly facilitate the investigation of the biological impacts of methylation at individual modification sites. Discovery and design of selective organic inhibitors of histone methyltransferases and demethylases provide chemical probes to interrogate methylation-mediated cellular pathways. Overall, these chemistry-related technological advances have greatly improved our understanding of the biological functions of histone methylation in normal physiology and diseased states, and also are of great potential to translate basic epigenetics research into diagnostic and therapeutic application in the clinic.

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Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of post-translational modifications

Cited by 224 publications

References 61 publications

A PHD finger protein involved in both the vernalization and photoperiod pathways in Arabidopsis

A PHD finger protein involved in both the vernalization and photoperiod pathways in Arabidopsis

Chemical derivatization of histones for facilitated analysis by mass spectrometry

Chemical and biochemical approaches in the study of histone methylation and demethylation

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