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1.
PLoS One ; 11(11): e0166438, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27875550

RESUMO

Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.


Assuntos
Proteínas de Drosophila/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/metabolismo , Animais , Imunoprecipitação da Cromatina , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Inibidores Enzimáticos/farmacologia , Estudo de Associação Genômica Ampla , Histonas/genética , Humanos , Metilação/efeitos dos fármacos , Análise de Sequência de DNA
2.
Genome Res ; 24(11): 1842-53, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25301795

RESUMO

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.


Assuntos
Anticorpos/metabolismo , Histonas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Anticorpos/imunologia , Sítios de Ligação/genética , Western Blotting , Imunoprecipitação da Cromatina , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Células HEK293 , Histonas/imunologia , Humanos , Lisina/metabolismo , Metilação , Peptídeos/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Análise Serial de Proteínas/métodos , Ligação Proteica , Reprodutibilidade dos Testes
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