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. 2013 Jun;23(6):988-97.
doi: 10.1101/gr.146654.112. Epub 2013 Apr 16.

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

Ishminder K Mann  1 , Raghunath Chatterjee, Jianfei Zhao, Ximiao He, Matthew T Weirauch, Timothy R Hughes, Charles Vinson
Affiliations

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

Ishminder K Mann et al. Genome Res. 2013 Jun.

Abstract

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.

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Figures

Figure 1.
Figure 1.
The effects of methylation on DNA binding properties of B-ZIP proteins. (A,B) Validation of CG methylation using methylation-sensitive (HpaII) endonuclease. The 40K feature microarray is scanned at 570 nm to detect Cy3-cytosine spiked into the DNA double-stranding reactions. Fluorescence intensities before and after methylation were normalized. DNA features containing CCGG are in red. Fluorescence intensities on (A) unmethylated and (B) methylated arrays before and after HpaII digestion. (C–H) Z-scores for all 32,896 8-mers from unmethylated and methylated 40K feature microarray. Lines are fitted to the non-CG 8-mers, which show no change in Z-scores between unmethylated and methylated arrays and serve as an internal control. The 8-mers are color-coded: CG (gray), non-CG (black), TTGC|G (green), TGAC|G (red), CGAT|G (blue), and chimeric CRE|CEBP sequence TGAC|GCAA is shown in brown. The best-bound 8-mers are indicated by arrows. (C) CEBPA-GST. (D) CEBPB-GST. (E) CEBPD-GST. (F) CREB1-GST. (G) ATF4-GST. (H) CEBPB-GST|ATF4.
Figure 2.
Figure 2.
Validation of CG methylation and CEBPA binding on 180K array. (A,B) Digestion of (A) unmethylated and (B) methylated 180K feature microarray using methylation sensitive (HpaII) endonuclease. Fluorescence intensities at 570 nm are plotted for all features, CCGG-containing features are colored in gray, and the remaining features are in black. (C) Scatter plot of CEBPA-GST binding to 180K array showing fluorescence intensities at 660 nm for all 65,536 8-mers in the context TNNNNNNNNA before and after methylation. The 8-mers are coded: CG (gray dots), non-CG (black dots), NTGAC|GNN (square), NTTGC|GNN (triangle). The gray line is fitted to the CG 8-mers, and the black line is fitted to the non-CG 8-mers. Binding to non-CG 8-mers did not change following methylation of the array.
Figure 3.
Figure 3.
The effect of methylation on DNA binding properties of CEBPB|ATF4 heterodimer. Fluorescence intensities at 660 nm from the 180K feature microarray are plotted for all of the 65,536 8-mers in the background TNNNNNNNNA. The 8-mers are color-coded as in Figure 1. Black lines are fitted to the non-CG 8-mers, and colored lines are fitted to the respective 64 8-mers containing the indicated 5-mers with NCGAT|GNN in blue. Effect of methylation on DNA binding of (A) CEBPB-GST and (B) ATF4-GST. (C,D) Comparison of DNA binding of CEBPB-GST|ATF4 heterodimer to the CEBPB-GST homodimer on (C) unmethylated and (D) methylated arrays. (E) Effect of methylation on DNA binding of CEBPB-GST|ATF4.
Figure 4.
Figure 4.
(A) EMSA showing CEBPB|ATF4 heterodimer preferentially binds to methylated CGAT|GCAA. Purified CEBPB, ATF4, and CEBPB|ATF4 B-ZIP domain dimers were mixed with unmethylated, hemi-methylated, or methylated CGAT|GCAA containing DNA probes. Protein dimer concentrations are indicated. Asterisks mark lanes with the same protein concentration. (B,C) EMSA showing CEBPB preferentially binds to ATTGC|GCAAT 10-mer. (B) Purified CEBPB was mixed with four palindromic methylated DNA probes containing the same consensus TTGC|GCAA 8-mer but different nucleotides at the 5′ and 3′ end of the 8-mer. EMSA showing CEBPB preferentially binds to methylated ATTGC|GCAAT. (C) Purified CEBPB B-ZIP domain dimers were mixed with methylated or unmethylated DNA probes with the same sequences as used in B. Both the acrylamide gel and the binding reactions contained 10 mM Mg2+. Protein dimer concentrations are indicated.
Figure 5.
Figure 5.
(A) Motif identified by MEME motif-finding algorithm from CEBPB ChIP-seq peaks before ATF4 induction. (B) Percent of methylated 8-mers with one CG dinucleotide in CEBPB ChIP-seq peaks in dermal fibroblasts plotted against Z-scores obtained from protein binding microarrays of CEBPB-GST binding to methylated 8-mers. The 8-mers are color coded: CG (gray dots), TGAC|G (square), TTGC|G (black dots). (C) Enriched GO terms for genes bound by CEBPB in dermal fibroblasts with methylated and unmethylated CEBP canonical motif (TTGC|GCAA) within −10 kbp to 1 kbp of the TSS. (D) CEBPB binding to unmethylated and methylated CEBP 10-mer palindromes in primary female mouse dermal fibroblasts. (E) Western blot showing induction of ATF4 in mouse dermal fibroblasts after 3 h of treatment with 2 μM thapsigargin (Tg). (F) UCSC Browser shot of CEBPB and ATF4 ChIP-seq read coverage before and after ATF4 induction along with the percent methylation at each CG dinucleotide in primary mouse dermal fibroblasts. CEBPB and ATF4 are preferentially localized in the methylated regions only after ATF4 induction. (G) Motif identified by MEME from new CEBPB ChIP-seq peaks after treatment with Tg.
Figure 6.
Figure 6.
(A) Number of reads normalized to the total number of reads in the CEBPB ChIP-seq peaks before and after ATF4 induction by Tg in mouse primary dermal fibroblasts. (Green) Peaks containing methylated canonical CEBP 8-mer; (blue) CGAT|GCA-containing peaks; (yellow) TGAT|GCAA-containing peaks. (B) Number of reads normalized to the total number of reads in the CEBPB ChIP-seq peaks vs. ATF4 ChIP-seq peaks after ATF4 induction by Tg in mouse primary dermal fibroblasts. Peaks containing methylated canonical CEBP 8-mer are colored as in Figure 6A. (C) Enrichment of selected k-mers in CEBPB ChIP-seq peaks before and after thapsigargin treatment and ATF4 ChIP-seq peaks after thapsigargin treatment. (D) Motif identified using RSAT from ATF4 ChIP-seq peaks after treatment with Tg. (E) Transcript abundances as determined using RNA-seq were plotted for dermal fibroblasts before and after ATF4 induction. Transcript abundances were reported in fragments per kilobase of transcript per million fragments mapped (FPKM). The peaks are color-coded: all peaks (gray); TGAT|GCAA (yellow)–commonly bound CEBPB and ATF4 ChIP-seq peaks (in promoters); CGAT|G (blue)–(nearest gene to the ATF4 ChIP-seq peaks). Approximately 50% of the promoters had no signal and are not shown.
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