Retinoid-dependent in vitro transcription

Valcárcel, Rafael; Stunnenberg, Hendrik G.

doi:10.1016/s0076-6879(96)74014-4

Cited by 3 publications

(2 citation statements)

References 42 publications

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“…HeLa cell nuclear extract was prepared as described previously (8). Approximately 5 g of protein was loaded onto a P11 phosphocellulose column (Whatman) as described previously (36). The majority of the N-CoR protein was found in the 300 mM KCl fraction, whereas the majority of the Sin3a protein was found in the 500 mM KCl fraction.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Targeting Reveals Intrinsic Histone Tail Specificity of the Sin3/Histone Deacetylase and N-CoR/SMRT Corepressor Complexes

Vermeulen

Carrozza

Lasonder

et al. 2004

Molecular and Cellular Biology

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The histone code is among others established via differential acetylation catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). To unambiguously determine the histone tail specificity of HDAC-containing complexes, we have established an in vitro system consisting of nucleosomal templates reconstituted with hyperacetylated histones or recombinant histones followed by acetylation with native SAGA or NuA4. Selective targeting of the mammalian Sin3/HDAC and N-CoR/SMRT corepressor complexes by using specific chimeric repressors created a near physiological setting to assess their histone tail specificity. Recruitment of the Sin3/HDAC complex to nucleosomal templates preacetylated with SAGA or NuA4 resulted in deacetylation of histones H3 and H4, whereas recruitment of N-CoR/SMRT resulted in deacetylation of histone H3 only. These results provide solid evidence that HDAC-containing complexes display distinct, intrinsic histone tail specificities and hence may function differently to regulate chromatin structure and transcription.

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Section: Methodsmentioning

confidence: 99%

In Vitro Targeting Reveals Intrinsic Histone Tail Specificity of the Sin3/Histone Deacetylase and N-CoR/SMRT Corepressor Complexes

Vermeulen

Carrozza

Lasonder

et al. 2004

Molecular and Cellular Biology

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“…Briefly, approximately 5 g of protein was loaded on a P11 phosphocellulose column (Whatman) as described (6). Bound proteins were eluted stepwise with 300 mM, 500 mM and 1000 mM KCl.…”

Section: Methodsmentioning

confidence: 99%

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

Vermeulen

Stunnenberg

2004

Biol. Proced. Online

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Post-translational modifications of core histones play an important role in regulating fundamental biological processes such as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The assay can be used to study the histone substrate specificity of chromatin modifying enzymes as well as whether and how certain enzymes affect each other's histone modifying activities. As such the assay can help to understand how a certain histone code is established and interpreted.

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Human Lymphoblastoid Proteome Analysis Reveals a Role for the Inhibitor of Acetyltransferases Complex in DNA Double-Strand Break Response

Dirksen

Cloos²,

Braakhuis³

et al. 2006

Cancer Research

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A DNA double-strand break (DSB) is highly cytotoxic; it emerges as the type of DNA damage that most severely affects the genomic integrity of the cell. It is essential that DNA DSBs are recognized and repaired efficiently, in particular, prior to mitosis, to prevent genomic instability and eventually, the development of cancer. To assess the pathways that are induced on DNA DSBs, 14 human lymphoblastoid cell lines were challenged with bleomycin for 30 and 240 minutes to establish the fast and more prolonged response, respectively. The proteomes of 14 lymphoblastoid cell lines were investigated to account for the variation among individuals. The primary DNA DSB response was expected to occur within the nucleus; therefore, the nuclear extracts were considered. Differential analysis was done using two-dimensional difference in gel electrophoresis; paired ANOVA statistics were used to recognize significant changes in time. Many proteins whose nuclear levels changed statistically significantly showed a fast response, i.e., within 30 minutes after bleomycin challenge. A significant number of these proteins could be assigned to known DNA DSB response processes, such as sensing DSBs (Ku70), DNA repair through effectors (high-mobility group protein 1), or cell cycle arrest at the G 2 -M phase checkpoint (14-3-3 Z). Interestingly, the nuclear levels of all three proteins in the INHAT complex were reduced after 30 minutes of bleomycin challenge, suggesting that this complex may have a role in changing the chromatin structure, allowing the DNA repair enzymes to gain access to the DNA lesions.

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Retinoid-dependent in vitro transcription

Cited by 3 publications

References 42 publications

In Vitro Targeting Reveals Intrinsic Histone Tail Specificity of the Sin3/Histone Deacetylase and N-CoR/SMRT Corepressor Complexes

In Vitro Targeting Reveals Intrinsic Histone Tail Specificity of the Sin3/Histone Deacetylase and N-CoR/SMRT Corepressor Complexes

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

Human Lymphoblastoid Proteome Analysis Reveals a Role for the Inhibitor of Acetyltransferases Complex in DNA Double-Strand Break Response

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