Fig 1.
Flowchart summarizes the five key steps that allow for genome-wide, high-throughput and -resolution transcription factor footprinting and candidate motif discovery. Crosslinked, purified nuclei are subjected to light digestion with MNase (Step 1), producing fragments that range from large nucleosome-sized (grey dsDNA) to small, protein-bound (green dsDNA) DNA-footprints. High salt is included during DNA purification and adapter ligation (Step 2) is performed prior to size selection (Step 3). These steps serve to increase the recovery of desired small footprint fragments while efficiently eliminating unwanted nucleosome-sized fragments, reducing the required sequencing depth. After MOA-seq fragments are aligned to the genome (Step 4) as illustrated around the na1 gene [63]. The resolution of the putative protein/DNA interaction sides can be further enhanced to approximately 30 bp wide footprints by defining fragment centers (MOA-footprints, orange). Sequences underlying MOA-footprints were used as input for de novo motif discovery (Step 5), helping to define the maize earshoot cistrome.
Fig 2.
Reproducibility of MOA-seq coverage and footprints from bio-replicate analysis.
Comparison of the two bio-replicates, rep1 and rep2, each from ABCD combined (Methods). (A) MOA-seq read coverage (Mc, light orange), MOA footprint coverage (Mfc, dark orange), MNase control coverage (CTRL-c), and MNAse control footprint coverage (CTRL-fc) are shown for two representative areas around the sdg118 gene (left, Zm00001eb363830) and a DUF-like gene (right, Zm00001eb364010). For each example, the peaks for the combined datasets (Mp and Mfp) are shown. (B) Venn diagram of rep1 and rep2 shows the 1-to-1 quantification of overlapping bases within overlapping peaks. The total shared base pairs (7.42 Mbp) represent 69.7% of Rep1 and 67.8% of Rep2 bases in their respective peaks. (C) Biological replicate correlation analysis for replicate 1 (rep1), replicate 2 (rep2) and the MNase control (CTRL).
Fig 3.
MOA-seq identifies shared and unique regions in open chromatin.
Comparison of MOA-seq to open chromatin profiling techniques DNS-Seq and ATAC-Seq. (A) Genome-wide distribution of MOA-seq footprints relative to genomic annotations. (B) MOA-seq read coverage (CPM) is non-randomly distributed around genes. Genes were scaled (metagene) to 2kb and average coverage is shown within the metagene body and adjacent genomic regions +/- 1.5kb from the nearest metagene boundary (grey dash). (C) Comparative analysis, shown as bar graphs, of MOA-seq peaks overlapping (shared, blue, %) or non-overlapping (unique, yellow) with ATAC-seq (MOA/ATAC), and the reciprocal intersections (ATAC/MOA). (D) and (E) Distribution of previously published DNS-seq (Positive light-heavy digest = blue, negative = red), ATAC-Seq (grey) coverage, MOA-seq (light orange) coverage (Mc) and peaks (Mp), and MF (dark orange) coverage (Mfc) and peaks (Mfp), surrounding example genes, vp14 (D) and dwf4/brs1 (E), with direction of transcription and the TSS indicated (arrow).
Fig 4.
MOA-seq overlaps with TF-binding sites and correlates at promoters with mRNA levels.
Average earshoot MOA-seq coverage was plotted around the midpoints for FEA4 tassel ChIP-seq peaks (A), Knotted1 ChIP-seq peaks subdivided into Ear only, Ear & Tassel, or Tassel only (B), and conserved non-coding sequences (CNS) showing all, position-shuffled control (Shuffled), or an intergenic subset classified as being more than 1 kb from genes (C). For each plot, the average peak size was used to define a single metapeak size (metapeak, indicated in each plot) for fitting the data within the peak region; metapeak flanking regions are in real bp relative to the metapeak region. (D) MOA-seq coverage is positively correlated with gene regulation. Mean MOA-seq coverage for genes divided into quintiles (1st highest mRNA to 5th lowest mRNA level) according to mRNA levels in earshot tissue.
Fig 5.
Global summary of maize earshot motifs discovered by MOA-seq.
(A-B) Radial similarity-cluster tree of the 140 oligo (A) and 75 dyad (B) consensus motifs identified within MOA-seq footprints. Circular sectors underlying tree branches are colored to highlight the frequency at which each motif occurred (high > 2000, medium 2000–1000, low < 1000). The inner (red dash) colored ring indicates similarity threshold for motif clustering at 90% similarity, delineating different motif clusters (alternating light and dark gray ring) flanked by motif names and consensus sequence logos at the inner and outer circles, respectively. (C) Median distance to the nearest transcription start site (TSS) plotted for each motif found in regions annotated as within repeats (IR, grey) or in non-repetitive regions (NR, red).
Fig 6.
MOA-seq footprints improve the spatial resolution of TF binding event prediction.
(A-F) ChIP-seq binding peaks of (A-B) FEA4 or (E-F) TB1, ATAC-Seq coverage (Ac) and peaks (Ap), MOA-seq footprint coverage (Mfc) and peaks (Mfp), and MOA-seq motif om015 in the promoter region of example genes (A) zw18-like (GRMZM2G039505), (B) bx9 (GRMZM2G161335), (E) nactf49 (GRMZM2G347043), and (F) d8 (GRMZM2G144744). (C, G) MOA-seq motif (C) om015 (G) dym33, aligned to their best database hit (footprintdb) of bzip and TCP family, respectively. The asterisk for (G) dym33 indicates that only a portion of the larger dym33 logo is shown. (D, H) Enrichment of motifs (D) om015 and a control motif om006 within FEA4 ChIP-seq peaks, or (H) dym33 and control motif dym36 with TB1 ChIP-seq peaks, relative to the mappable B73 genome (gen.), and the 3 kb, non-ORF overlapping, region upstream of B73 gene models (prom.). (I) Genome-wide overlap analysis of base pairs shared between MOA (MOA, MFs) and ChIP-seq of 104 TFs [27] or the MNase control (CTRL). (J) Representative example of the complex landscape of TF and MFs profiles surrounding tip1 (Zm00001eb00373). TF tracks shown: MYB, TF55 rep1 GSM4095564; C2C2 TF235 rep2 GSM4095532,; bHLH, TF6 rep2 GSM4095589; TCP, TF237 rep1 GSM4095534.
Fig 7.
MOA-seq footprints highlight known long-distance chromatin interaction.
Genome-wide overlap analysis of base pairs shared between MOA and previously defined enhancers (husk, IST, from [13]) and long-range interaction sites (seedling, from [41]). (B-C) Examples of MOA-seq coverage and peaks (Mfc, Mfp, dark orange) at known long-range interaction sites compared to ATAC-seq coverage and peaks (Ac, Ap, gray) at the (B) UB-KRN4 region and the (C) TB1 region. (B) Panel shows chromatin interaction (CI, black lines) loops inferred from HiC, UB2 ChIP-seq, and H3K27ac- and H3K4me3-mediated ChIA-PET-seq data for the KRN4 and UB3 region, three high-confidence remote sites (A1, A2 and A3; yellow columns), and the locations of KRN43.1 (light green column), and UB3 promoter (light blue column) [45]. (C) Interaction region of the -65 Kb distal enhancer region (left zoom-in panel) and promoter region (right zoom-in panel) of TB1. Motif names, positions (Mo) and consensus logos, identified within MOA-seq footprints, are indicated. Dashed lines connect consensus motifs identified in both interacting regions.The best database hit (footprintdb) with the similarity p-value are indicated.