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ChIP-Seq with reference exogenous genome (ChIP-Rx) of MAX in U2OS-MYC-KO-MYC-Tet-On cells with exogenous wild-type and mutant MYC expression

ABSTRACT: Transcription factor (TF) MYC binds to thousands of gene regulatory elements in the genome such as promoters and enhancers and exerts an amplification effect on the expression of most genes, regardless of the E-box motif existence. In this study, we discovered that MYC is an RNA-binding protein with a high affinity to guanosine-rich RNAs. RNAs that bind to MYC increase MYC’s chromatin occupancy, which helps with MYC-mediated regulation of gene expression. Mechanistically, two highly conserved sequences, amino acids 355-357 and 364-367, in the basic region of MYC are essential for RNA binding. Notably, alanine substitution of Lys355, Arg356, and Arg357 of MYC abolishes its RNA binding function but does not influence its complex formation ability, and E-box binding capacity of MYC:MAX dimer in vitro. However, loss of RNA binding function alone decreases MYC recruitment at gene regulatory elements in vivo, followed by a decreased MYC:MAX chromatin binding at these regions. This process further attenuates MYC-mediated gene expression. Therefore, RNA-binding-deficient MYC is compromised in promoting cancer cell proliferation. By demonstrating the mechanism of MYC-RNA interaction and the fundamental role of RNA binding activity in MYC-centric functions, our study reveals a new paradigm to MYC function and highlights an essential and probably general role of RNA binding capacity of TFs for their gene regulatory function.

ORGANISM(S): Homo sapiens

PROVIDER: GSE273776 | GEO | 2025/05/08

REPOSITORIES: GEO

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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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ChIP-Seq with reference exogenous genome (ChIP-Rx) of MAX in U2OS-MYC-KO-MYC-Tet-On cells with exogenous wild-type and mutant MYC expression

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