Unknown,Transcriptomics,Genomics,Proteomics

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Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis

ABSTRACT: The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci. Recent work suggested that rather than up- and down-regulating selected groups of genes, Myc targets all active promoters and enhancers in the genome (a phenomenon termed "invasion") and acts as a general amplifier of transcription. However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B-cells and fibroblasts. Consistent with long-standing observations, we detected general increases in total RNA or mRNA copies per cell (hereby termed "amplification") when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, while having the potential to interact with all active/poised regulatory elements in the genome, Myc does not directly act as a global transcriptional amplifier. Instead, our results imply that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover. This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

ORGANISM(S): Homo sapiens

SUBMITTER: Marco Morelli

PROVIDER: E-GEOD-51011 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The Myc-Max heterodimer is a DNA binding protein that regulates expression of a large number of genes. Genome occupancy of Myc-Max is thought to be driven by E-boxes (CACGTG or variants) to which the heterodimer binds in vitro. By analyzing ChIP-Seq datasets, we demonstrated that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II (Pol II) transcription machinery better than with E-boxes. Metagene analyses showed that in promoter regions, Myc was uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 10 bp upstream of Myc. We re-evaluated the DNA binding properties of full length Myc-Max proteins using electrophoretic mobility shift assays (EMSA) and protein-binding microarrays (PBM). EMSA results demonstrated Myc-Max heterodimers have high affinity for both E-box containing and non-specific DNA. Quantification of the relative affinities of Myc-Max for all possible 8- mers using PBM assays showed that sequences surrounding core 6-mers significantly affect binding. Comparing to the in vitro sequence preferences, Myc-Max genomic occupancy measured by ChIP-Seq was largely, although not completely, independent of sequence specificity. Our results suggest that the transcription machinery and associated promoter accessibility play an important role in genomic occupancy of Myc. Two protein binding microarray (PBM) experiments were performed: one for the heterodimer of the human transcription factors c-Myc and Max, and one for the Max-Max homodimer. Briefly, 4x44K arrays (Agilent Technologies; AmadID 015681) containing the M-bM-^@M-^Xall 10-merM-bM-^@M-^Y universal PBM design were used. Arrays were incubated with a PBS buffer based protein mixture of wither 10nM His-tagged Myc-Max heterodimer or 10nM His-tagged Max-Max homodimer, 2% milk, 200ng/M-BM-5L BSA, 50ng/M-BM-5L Salmon Testes DNA, and 0.02% TX-100. Bound protein was tagged with 10ng/M-BM-5L anti-His antibody conjugated to Alexa 488 (Qiagen; 35310) in PBS with 2% milk. Data were analyzed to obtain fluorescence intensities for all 8mers. The PBM protocol is described in Berger et al., Nature Biotechnology 2006 (PMID 16998473).

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Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis

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