CG methylated microarrays identify a novel methylated sequence bound by the CEBPB|ATF4 heterodimer that is active in vivo

By I. Mann, R. Chatterjee, Jianfei Zhao, Ximiao He, Matthew T. Weirauch, Timothy R. Hughes, C. Vinson. 2013

123 citations recorded by the source corpus.

To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Two Agilent DNA array designs were used. One contained 40,000 features using de Bruijn sequences where each 8-mer occurs 32 times in various positions in the DNA sequence. The second contained 180,000 features with each CG containing 8-mer occurring three times. The first design was better for identification of binding motifs, while the second was better for quantification. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD, and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. The electrophoretic mobility shift assay (EMSA) confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50× methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. This methodology can be used to identify new methylated DNA sequences preferentially bound by TFs, which may be functional in vivo.

Subfields: Molecular Biology

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Citations in the corpus

Impact of cytosine methylation on DNA binding specificities of human transcription factors (2017, methodology): “The 16× HK array design with 40,000 unique DNA features [as described in (11, 35)] were double stranded as described in (11).”
DNA methylation presents distinct binding sites for human transcription factors (2013, background): “Our approach also showed some false negatives because some known methylated DNA binding proteins were not identified in this study (Mann et al., 2013; Spruijt et al., 2013).”
High-resolution genome-wide DNA methylation maps of mouse primary female dermal fibroblasts and keratinocytes (2014, unknown): “We compared the fibroblast [31] and keratinocyte HMRs to identify overlapping and tissue-specific HMRs.”