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Quadruple 9-mer-based protein binding microarray analysis confirms AACnG as the consensus nucleotide sequence sufficient for the specific binding of AtMYB44.

ABSTRACT: AtMYB44 is a member of the R2R3 MYB subgroup 22 transcription factors and regulates diverse cellular responses in Arabidopsis thaliana. We performed quadruple 9-merbased protein binding microarray (PBM) analysis, which revealed that full-size AtMYB44 recognized and bound to the consensus sequence AACnG, where n represents A, G, C or T. The consensus sequence was confirmed by electrophoretic mobility shift assay (EMSA) with a truncated AtMYB44 protein containing the N-terminal side R2R3 domain. This result indicates that the R2R3 domain alone is sufficient to exhibit AtMYB44 binding specificity. The sequence AACnG is the type I binding site for MYB transcription factors, including all members of the subgroup 22. EMSA showed that the R2R3 domain protein binds in vitro to promoters of randomly selected Arabidopsis genes that contain the consensus binding sequence. This implies that AtMYB44 binds to any promoter region that contains the consensus sequence, without determining their functional activity or specificity. The C-terminal side transcriptional activation domain of AtMYB44 contains an asparagine-rich fragment, NINNTTSSRHNHNN (aa 215-228), which, among the members of subgroup 22, is unique to AtMYB44. A transcriptional activation assay in yeast showed that this fragment is included in a region (aa 200-240) critical for the ability of AtMYB44 to function as a transcriptional activator. We hypothesize that the C-terminal side of the protein, but not the N-terminal side of the R2R3 domain, contributes to the functional activity and specificity of AtMYB44 through interactions with other regulators generated by each of a variety of stimuli.

SUBMITTER: Jung C

PROVIDER: S-EPMC3887824 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Quadruple 9-mer-based protein binding microarray analysis confirms AACnG as the consensus nucleotide sequence sufficient for the specific binding of AtMYB44.

Jung Choonkyun C Kim Yeon-Ki YK Oh Nam Iee NI Shim Jae Sung JS Seo Jun Sung JS Choi Yang Do YD Nahm Baek Hie BH Cheong Jong-Joo JJ

Molecules and cells 20121115 6

AtMYB44 is a member of the R2R3 MYB subgroup 22 transcription factors and regulates diverse cellular responses in Arabidopsis thaliana. We performed quadruple 9-merbased protein binding microarray (PBM) analysis, which revealed that full-size AtMYB44 recognized and bound to the consensus sequence AACnG, where n represents A, G, C or T. The consensus sequence was confirmed by electrophoretic mobility shift assay (EMSA) with a truncated AtMYB44 protein containing the N-terminal side R2R3 domain. T ...[more]

PMID: 23161171

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Project description:Recent genome-scale ChIP-chip studies of transcription factors have shown that a low percentage of experimentally determined binding sites contain the consensus motif for the immunoprecipitated factor. In most cases, differences between in vivo target sites that contain or lack a consensus motif have not been explored. We have previously shown that most sites to which E2F family members are bound in vivo do not contain E2F consensus motifs. The main purpose of this study was to develop an understanding of how E2F binding specificity is achieved in vivo. In particular, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using promoter and ENCODE arrays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are a) the site must be in a core promoter and b) the promoter region must be utilized as a promoter by the transcriptional machinery in that particular cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including a) indirect recruitment, b) looping to the core promoter mediated by an E2F bound to a distal consensus motif, and c) assisted binding of E2F to a site that weakly resembles an E2F consensus motif within the core promoter. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated promoter fragments. Our findings suggest that in vivo a) the presence of a consensus motif is not sufficient to recruit E2Fs, b) E2Fs can bind to isolated regions that lack a consensus motif, and c) binding can require regions other than the best match to the E2F PWM in the core promoter. Keywords: E2F, ChIP-chip, transcription factor binding, consensus motifs 37 ChIP-chip arrays (of these, 14 array sets are biological duplicates). 22 samples are included in this series, the rest can be found in supplementary info to the following papers: Xu 2007, Jin 2006, Komashko 2008

Dataset Information

Quadruple 9-mer-based protein binding microarray analysis confirms AACnG as the consensus nucleotide sequence sufficient for the specific binding of AtMYB44.

Publications

Quadruple 9-mer-based protein binding microarray analysis confirms AACnG as the consensus nucleotide sequence sufficient for the specific binding of AtMYB44.

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