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Figures and Figure Supplements RESEARCH ARTICLE Figures and figure supplements Cooperative interactions enable singular olfactory receptor expression in mouse olfactory neurons Kevin Monahan et al Monahan et al. eLife 2017;6:e28620. DOI: https://doi.org/10.7554/eLife.28620 1 of 30 Research article Genes and Chromosomes Lhx2 ChIP Ebf ChIP A 2 2 BCGenome Wide OR clusters Lhx2 Sites mOSN 1 1 Lhx2 Sites Ebf Sites bits 16,311 Ebf Sites 114 65 C C G C A AT T T A CGG C G G CC GTA G C T T G A CA G 0 TAATTA 0 CTT AAG 9,024 5 5 10 2 2 2 11,519 4,792 4,232 51 63 Known 1 1 bits G T A A CG C GC C C AT CT GTGA CT CT AT GA GA 0 AAT 0 C C G G 5 5 10 p = 5.7e-09 p = 1.6e-15 D E ATAC ChIP Lhx2 ChIP Ebf chr2:36,107,000-37,507,000 Crete 300 kb 25 - 8 40 20 ATAC 0_ 4 - 20 10 90 Signal ChIP Lhx2 0 0 0 0_ -4kb Island 4 -4kb Island 4 -4kb Island 4 40 - ChIP Ebf 0_ 9 - ChIP H3K9me3 -2 _ Genes / ORs Greek Islands chr6:42,161,494-43,561,494 300 kb - Anafi Sfaktiria 35 0.5 6 0.5 27 0.5 14 ATAC _ 0 - 115 F ATAC ChIP Lhx2 ChIP Ebf ChIP Lhx2 8 40 20 0_ 85 - ChIP Ebf 4 20 10 0_ Signal - 9 0 0 0 ChIP H3K9me3 -2kbTSS TES 2 -2kbTSS TES 2 -2kbTSS TES 2 -2 _ Genes / ORs chr2:36,107,000-37,507,000 300 kb Lipsi 20 - ATAC 0_ 55 - ORs ChIP Lhx2 0 _ 30 - ChIP Ebf 0_ 9- ChIP H3K9me3 -2 _ 0.5 6 0.5 27 0.5 14 Genes / ORs Figure 1. Greek Islands represent Lhx2 and Ebf co-bound regions residing in heterochromatic OR clusters. (A) The top sequence motif identified for mOSN ChIP-seq peaks is shown above sequence motifs generated from previously reported Lhx2 (Folgueras et al., 2013) and Ebf (Lin et al., 2010) ChIP-seq data sets. mOSN ChIP-seq peaks were identified using HOMER and motif analysis was run on peaks present in both biological replicates. (B) Overlap between mOSN Lhx2 and Ebf bound sites genome-wide. See Figure 1—figure supplement 2 for analysis of ChIP-seq signal on Ebf and Lhx2 Co-bound sites within OR clusters. (C) Overlap between mOSN Lhx2 and Ebf bound sites within OR clusters. For each factor, co-bound sites are À À significantly more frequent within OR clusters than in the rest of the genome (p=5.702e 9 for Lhx2, p=1.6e 15 for Ebf, Binomial test). See Figure 1— figure supplement 2 for gene ontology analysis of peaks bound by Lhx2 and Ebf. (D) mOSN ATAC-seq and ChIP-seq signal tracks for three representative OR gene clusters. Values are reads per 10 million. Below the signal tracks, OR genes are depicted in red and non-OR genes are depicted in blue. Greek Island locations are marked. Anafi is a newly identified Greek Island, located in a small OR cluster upstream of the Sfaktiria cluster. See also Figure 1—figure supplement 3 and Supplementary file 1. For ATAC-seq, pooled data is shown from 4 biological replicates, for ChIP-seq, pooled data is shown from 2 biological replicates. For H3K9me3 ChIP-seq, input control signal is subtracted from ChIP signal prior to plotting. (E) mOSN ATAC-seq or ChIP-seq signal across 63 Greek Islands. Each row of the heatmap shows an 8 kb region centered on a Greek Island. Regions of high signal are shaded red. Mean signal across all elements is plotted above the heatmap, values are reads per 10 million. All heatmaps are Figure 1 continued on next page Monahan et al. eLife 2017;6:e28620. DOI: https://doi.org/10.7554/eLife.28620 2 of 30 Research article Genes and Chromosomes Figure 1 continued sorted in the same order, based upon ATAC-seq signal. See also Figure 1—figure supplement 3 and Supplementary file 1. For ATAC-seq, pooled data is shown from 4 biological replicates, for ChIP-seq, pooled data is shown from 2 biological replicates. See Figure 1—figure supplement 4 for a comparison of newly and previously identified Greek Islands, and Figure 1—figure supplement 5 for RNA-seq analysis of ORs with Greek Islands near the TSS. (F) mOSN ATAC-seq and ChIP-seq signal tracks on OR genes. Each row of the heatmap shows an OR gene scaled to 4 kb as well as the 2 kb regions upstream and downstream. Plots and heatmap are scaled the same as in Figure 1E. DOI: https://doi.org/10.7554/eLife.28620.002 Monahan et al. eLife 2017;6:e28620. DOI: https://doi.org/10.7554/eLife.28620 3 of 30 Research article Genes and Chromosomes -log10(Binomial p value) GO Biological Process 0 5 10 15 positive regulation of axonogenesis positive regulation of axon extension regulation of calcium ion transport regulation of extent of cell growth regulation of cell size semaphorin-plexin signaling pathway regulation of mRNA processing regulation of axon extension MSigDB Pathway Olfactory transduction Ca++/ Calmodulin-dependent Protein Kinase Activation Figure 1—figure supplement 1. Gene Ontology terms associated with Ebf and Lhx2 co-bound sites. Top Gene Ontology terms from the Biological Process and MSigDB Pathway categories associated with genes proximal to sites bound by both Ebf and Lhx2. DOI: https://doi.org/10.7554/eLife.28620.003 Monahan et al. eLife 2017;6:e28620. DOI: https://doi.org/10.7554/eLife.28620 4 of 30 Research article Genes and Chromosomes ChIP Lhx2 ChIP Ebf A B ChIP Peak Strength by Peak Type C ChIP Peak Strength by Peak Type OR Cluster Sites 40 20 0.6 Ebf+Lhx2 Lhx2-only Ebf+Lhx2 Ebf or Lhx2 OR-cluster Lhx2-only 0.6 Ebf-only 20 10 Ebf+Lhx2 Greek Islands Greek Islands 0 0 0.4 -4kb Peak 4 -4kb Peak 4 0.4 Density Density 0.2 (Ebf+Lhx2) 0.2 Greek Islands 0.0 0.0 (Ebf or Lhx2) Singly Bound 0.5 27 0.5 14 5.0 7.5 10.0 4 6 8 10 12 Log2(Lhx2 ChIP Signal) Log2(Ebf ChIP Signal) Figure 1—figure supplement 2. Co-binding of Ebf and Lhx2 within OR clusters. (A) mOSN Lhx2 and Ebf ChIP-seq signal on OR Cluster Ebf +Lhx2 peaks (Greek Islands) compared to OR-cluster singly bound (Ebf or Lhx2) sites. Mean signal for each group is plotted above the heatmap, values are reads per 10 million. Both heatmaps are sorted in the same order, based upon ATAC-seq signal. Pooled data is shown from 2 biological replicates. (B) Density plot of the distribution of peaks over Lhx2 ChIP-seq peak strength (normalized number of reads in each peak) for different categories of peaks. ChIP signal is calculated by averaging normalized peak counts from two biological replicates. (C) Density plot of the distribution of peaks over Ebf ChIP-seq peak strength (normalized number of reads in each peak) for different categories of peaks. OR-cluster Ebf-only peaks are not included because there are only two peaks in this category. ChIP signal is calculated by averaging normalized peak counts from two biological replicates. DOI: https://doi.org/10.7554/eLife.28620.004 Monahan et al. eLife 2017;6:e28620. DOI: https://doi.org/10.7554/eLife.28620 5 of 30 Research article Genes and Chromosomes chr6:42,865,500-42,874,499 chr11:74,031,930-74,040,929 chr2:37,123,665-37,132,664 A 2 kb 2 kb 2 kb Sfaktiria Crete Lipsi 9 - 9 - 9 - ChIP H3K9me3 0 _ 0 _ 0 _ 11 - 11 - 10 - ChIP H3K79me3 0 _ -0.5 _ 0 _ 1 - 1 - 1 - ChIP H3K27ac 0 _ 0 _ 0 _ 35 - 25 - 20 - ATAC 0 _ 0 _ 0 _ 115 - 90 - 55 - ChIP Lhx2 0 _ 0 _ 0 _ 85 - 40 - 30 - ChIP Ebf 0 _ 0 _ 0 _ B ChIP H3K9me3 ChIP H3K79me3 ChIP H3K27ac 0.5 4 4 0.3 2 2 0.1 Signal 0 0 í0.1 -4 Island 4 -4 Island 4 -4kb Island 4 Greek Islands 0.2 6.4 0.2 7.6 0.1 0.7 Figure 1—figure supplement 3. Histone modifications proximal to Greek Islands. (A) ATAC-seq and ChIP-seq signal tracks for three Greek Islands, Sfaktiria, Crete and Lipsi. Greek Island position is highlighted in yellow. For heterochromatin modifications (H3K9me3 and H3K79me3), input control signal is subtracted from ChIP signal. Pooled data is shown from 4 biological replicates for ATAC-seq, 2 biological replicates for Lhx2, Ebf, H3K9me3, and H3K27ac, and one replicate for HeK79me3. (B) ChIP-seq signal for histone modifications associated with heterochromatin and active enhancers in the vicinity of Greek Islands. Pooled data is shown from 2 biological replicates for H3K9me3 and H3K27ac, and one replicate for HeK79me3. DOI: https://doi.org/10.7554/eLife.28620.005 Monahan et al. eLife 2017;6:e28620. DOI: https://doi.org/10.7554/eLife.28620 6 of 30 Research article Genes and Chromosomes ATAC ChIP-Lhx2 ChIP-Ebf ChIP H3K9me3 ChIP H3K27ac ChIP H3K79me3 14 60 30 5 0.7 6 Original Islands 0.6 12 50 25 5 New Islands 4 10 0.5 40 20 4 8 3 0.4 30 15 3 0.3 Signal 6 2 20 10 2 4 0.2 1 1 2 10 5 0.1 0 0 0.0 -4kb Peak 4 -4 Peak 4 -4 Peak 4 -4 Peak 4 -4 Peak 4-4 Peak 4 Figure 1—figure supplement 4.
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