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

Abstract

To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a highthroughput 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 503 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.

5-Hydroxymethylcytosine in E-box motifs ACAT|GTG and ACAC|GTG increases DNA-binding of the B-HLH transcription factor TCF4

“…This results in a hemi-methylated or hemi-hydroxymethylated state. DNA double-stranding was performed as previously described 1, 27 .…”

“…Protein binding reactions, image quantification, and calculation of Z-scores were performed as described previously 27 . The Z-scores for 8-mers were calculated by two different approaches: for each 8-mer, either the reverse complementary 8-mers was count as the same (32,896 8-mers, e.g.…”

Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape

“…However, these assays employ synthetic DNA that lacks genomic DNA properties known to impact TF binding, including primary sequence context and chemical modifications, such as the widespread and tissue-specific 5-methylcytosine found in plants and animals. Efforts have been made to build synthetic oligomer pools that reflect relevant cis-element sequence (Levo and Segal, 2014) or incorporate methylation (Mann et al, 2013), but complex variation in nucleotide sequence and DNA methylation patterns ) makes it extremely challenging to fully reproduce native nuclear DNA patterns by synthesis.…”

C/EBPγ Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4

“…In stressed cells, ATF4 or, in some cases, ATF2 or ATF5 is recruited together with a C/EBP subunit to CARE-containing promoters (13). C/EBP␤ has been suggested to serve as an ATF4 partner that regulates transcription of certain ISR genes (14,15) and is capable of heterodimerizing with ATF4 and other ATF family members (16,17). However, cells lacking C/EBP␤ remain capable of activating many genes induced by ER stress or amino acid deficiency and, indeed, display increased or prolonged expression of some ATF4 targets (15,18).…”