Immuno-Depletion and Purification Strategies to Study Chromatin-Remodeling Factors In Vitro

Chalkley, Gillian E.; Verrijzer, C. Peter

doi:10.1016/s0076-6879(03)77028-1

Cited by 30 publications

(37 citation statements)

References 14 publications

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“…The following antigens were used: Mi2 amino acids (aa) 1290 to 1533, MTA1-like aa 132 to 476, MEP1 aa 681 to 800, TTK69 aa 333 to 546, and the full-length CG18292 (CDK2AP1/DOC1) protein. Immunization and affinity purification were carried out as described previously (7). Rabbit polyclonal antibodies against TTK69 (27) and ISWI (6) have been described previously.…”

Section: Methodsmentioning

confidence: 99%

“…Embryo nuclear extracts were prepared from 0-to 12-h-old Drosophila embryos. Immunopurification procedures using affinity-purified antibodies directed against Mi2 or MEP1 and mass spectrometric analysis were all performed as described previously (6,7). After affinity purification, beads were washed twice with HEMG buffer (25 mM HEPES-KOH [pH 7.6], 0.1 mM EDTA, 12.5 mM MgCl 2 , 10% glycerol, 0.1% NP-40, and a cocktail of protease inhibitors) containing 200 mM KCl (HEMG/200), 5 times with HEMG/500, once with HEMG/200, and finally once with HEMG/200 lacking NP-40.…”

Section: Methodsmentioning

confidence: 99%

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Drosophila Transcription Factor Tramtrack69 Binds MEP1 To Recruit the Chromatin Remodeler NuRD

Reddy¹,

Bajpe²,

Bassett

et al. 2010

Molecular and Cellular Biology

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ATP-dependent chromatin-remodeling complexes (remodelers) are essential regulators of chromatin structure and gene transcription. How remodelers can act in a gene-selective manner has remained enigmatic. A yeast two-hybrid screen for proteins binding the Drosophila transcription factor Tramtrack69 (TTK69) identified MEP1. Proteomic characterization revealed that MEP1 is a tightly associated subunit of the NuRD remodeler, harboring the Mi2 enzymatic core ATPase. In addition, we identified the fly homolog of human Deleted in oral cancer 1 (DOC1), also known as CDK2-associated protein 1 (CDK2AP1), as a bona fide NuRD subunit. Biochemical and genetic assays supported the functional association between MEP1, Mi2, and TTK69. Genomewide expression analysis established that TTK69, MEP1, and Mi2 cooperate closely to control transcription. The TTK69 transcriptome profile correlates poorly with remodelers other than NuRD, emphasizing the selectivity of remodeler action. On the genes examined, TTK69 is able to bind chromatin in the absence of NuRD, but targeting of NuRD is dependent on TTK69. Thus, there appears to be a hierarchical relationship in which transcription factor binding precedes remodeler recruitment.Chromatin is the natural template of the eukaryotic transcription machinery. Consequently, regulation of gene expression involves the interplay between sequence-specific transcription factors, the basal machinery, coregulators, and enzymes that modulate chromatin structure. ATP-dependent chromatin-remodeling factors (remodelers) constitute one class of enzymes that target chromatin. The basic biochemical activity of remodelers is to use the energy of ATP hydrolysis to move or eject nucleosomes (8). Although their in vitro activity might suggest that remodelers act in a generic way, it has become clear that different remodelers perform distinct, nonredundant functions. An early example of functional specialization was our finding that the Brahma (BRM) remodeling complexes, but not ISWI remodelers, act as chromatin-specific coactivators for the transcription factor ZESTE (15). Conversely, unlike ISWI, the BRM remodelers were unable to order a nucleosomal array. Moreover, several studies have demonstrated that different remodelers control distinct biological processes (4, 8, 24).Currently, four major classes of remodelers are recognized, based on their ATPase and accessory subunits (8). These comprise the SWI/SNF, ISWI, CHD, and INO80 families. Mi2 (also known as CHD4) is the founding member of the CHD family of remodelers, characterized by the presence of a tandem chromodomain in the ATPase subunit (9). A unique aspect of NuRD is its coupling of remodeling and histone deacetylase (HDAC) activities in one complex. Although there is some variability between the various vertebrate NuRD complexes described so far, the key subunits of NuRD are the Mi2 ATPase, the protein deacetylases HDAC1 and HDAC2, metastasis-associated proteins MTA1, MTA2, and MTA3, the retinoblastoma-associated histone binding proteins RBP46 and RBP48...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Drosophila Transcription Factor Tramtrack69 Binds MEP1 To Recruit the Chromatin Remodeler NuRD

Reddy¹,

Bajpe²,

Bassett

et al. 2010

Molecular and Cellular Biology

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“…Anti-Cut antibody (2B10) and Elav (7E8A10) were procured from the Developmental Studies Hybridoma bank. Anti-UBP64 antibodies were affinity purified using GST-UBP64 (amino acids 309 to 743) immobilized on an Affi-gel15 (Bio-Rad) matrix as described previously (8). UBP64 was purified using affinity-purified UBP64 antibodies coupled to protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) essentially as described previously (8).…”

Section: C405amentioning

confidence: 99%

“…Anti-UBP64 antibodies were affinity purified using GST-UBP64 (amino acids 309 to 743) immobilized on an Affi-gel15 (Bio-Rad) matrix as described previously (8). UBP64 was purified using affinity-purified UBP64 antibodies coupled to protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) essentially as described previously (8). Briefly, 80 l of beads coated with anti-UBP64 antibodies was incubated with 1 ml of embryo nuclear extract (10-mg/ml protein concentration) in 50 mM HEPES-KOH-0.2 MEDTA-25 mM MgCl 2 -20% glycerol (HEMG) buffer containing 100 mM KCl (HEMG/100), 0.1% NP-40, 0.1 mg/ml insulin, and a cocktail of protease inhibitors (8).…”

Section: C405amentioning

confidence: 99%

“…UBP64 was purified using affinity-purified UBP64 antibodies coupled to protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) essentially as described previously (8). Briefly, 80 l of beads coated with anti-UBP64 antibodies was incubated with 1 ml of embryo nuclear extract (10-mg/ml protein concentration) in 50 mM HEPES-KOH-0.2 MEDTA-25 mM MgCl 2 -20% glycerol (HEMG) buffer containing 100 mM KCl (HEMG/100), 0.1% NP-40, 0.1 mg/ml insulin, and a cocktail of protease inhibitors (8). After 2 h at 4°C, the beads were washed extensively with HEMG/600-0.1% NP-40.…”

Section: C405amentioning

confidence: 99%

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Deubiquitylating Enzyme UBP64 Controls Cell Fate through Stabilization of the Transcriptional Repressor Tramtrack

Bajpe

Knaap

Demmers

et al. 2008

Molecular and Cellular Biology

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Protein ubiquitylation plays a central role in multiple signal transduction pathways. However, the substrate specificity and potential developmental roles of deubiquitylating enzymes remain poorly understood. Here, we show that the Drosophila ubiquitin protease UBP64 controls cell fate in the developing eye. UBP64 represses neuronal cell fate but promotes the formation of nonneuronal cone cells. Using a proteomics approach, we identified the transcriptional repressor Tramtrack (TTK) as a primary UBP64 substrate. In common with TTK, reduced UBP64 levels lead to a loss of cone cells, supernumerary photoreceptors, and mechanosensory bristle cells. Previously, it was demonstrated that the blockade of neuronal cell fate was relieved by SINAdependent ubiquitylation and degradation of TTK. We found that UBP64 counteracts SINA function by deubiquitylating TTK, leading to its stabilization and thereby promoting a nonneuronal cell fate. Mass spectrometric mapping revealed that SINA ubiquitylates multiple sites dispersed throughout TTK, which are duly deubiquitylated by UBP64. This observation suggests that both E3 SINA and UBP64 use a scanning mechanism to (de)ubiquitylate TTK. We conclude that the balance of TTK ubiquitylation by SINA and deubiquitylation by UBP64 constitutes a specific posttranslational switch controlling cell fate.

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Global quantitative proteomics reveals novel factors in the ecdysone signaling pathway inDrosophila melanogaster

et al. 2015

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The ecdysone signaling pathway plays a major role in various developmental transitions in insects. Recent advances in the understanding of ecdysone action have relied to a large extent on the application of molecular genetic tools in Drosophila. Here, we used a comprehensive quantitative SILAC MS-based approach to study the global, dynamic proteome of a Drosophila cell line to investigate how hormonal signals are transduced into specific cellular responses. Global proteome data after ecdysone treatment after various time points were then integrated with transcriptome data. We observed a substantial overlap in terms of affected targets between the dynamic proteome and transcriptome, although there were some clear differences in timing effects. Also, downregulation of several specific mRNAs did not always correlate with downregulation of their corresponding protein counterparts, and in some cases there was no correlation between transcriptome and proteome dynamics whatsoever. In addition, we performed a comprehensive interactome analysis of EcR, the major target of ecdysone. Proteins copurified with EcR include factors involved in transcription, chromatin remodeling, ecdysone signaling, ecdysone biosynthesis, and other signaling pathways. Novel ecdysone-responsive proteins identified in this study might link previously unknown proteins to the ecdysone signaling pathway and might be novel targets for developmental studies. To our knowledge, this is the first time that ecdysone signaling is studied by global quantitative proteomics. All MS data have been deposited in the ProteomeXchange with identifier PXD001455 (http://proteomecentral.proteomexchange.org/dataset/PXD001455).

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Immuno-Depletion and Purification Strategies to Study Chromatin-Remodeling Factors In Vitro

Cited by 30 publications

References 14 publications

Drosophila Transcription Factor Tramtrack69 Binds MEP1 To Recruit the Chromatin Remodeler NuRD

Drosophila Transcription Factor Tramtrack69 Binds MEP1 To Recruit the Chromatin Remodeler NuRD

Deubiquitylating Enzyme UBP64 Controls Cell Fate through Stabilization of the Transcriptional Repressor Tramtrack

Global quantitative proteomics reveals novel factors in the ecdysone signaling pathway inDrosophila melanogaster

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