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. 2019 Nov 14;10:1434.
doi: 10.3389/fpls.2019.01434. eCollection 2019.

Mapping and Dynamics of Regulatory DNA in Maturing Arabidopsis thaliana Siliques

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Free PMC article

Mapping and Dynamics of Regulatory DNA in Maturing Arabidopsis thaliana Siliques

Alessandra M Sullivan et al. Front Plant Sci. .
Free PMC article

Abstract

The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In Arabidopsis thaliana and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of polysaccharides. Here, we used DNase I-seq to generate regulatory landscapes of A. thaliana seeds at two critical time points in seed coat maturation (4 and 7 DPA), enriching for seed coat cells with the INTACT method. We found over 3,000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in A. thaliana, we compared our data to 11 other data sets generated with 7-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, plant regulatory landscapes from different tissues, cell types, or developmental stages were more dynamic than those generated from bulk tissue in response to environmental perturbations, highlighting the importance of extending studies of regulatory DNA to single tissues and cell types during development.

Keywords: Arabidopsis thaliana; open chromatin; regulatory DNA; seed coat maturation; seed development.

Figures

Figure 1
Figure 1
The chromatin landscape of maturing seed coat cells. (A) Distribution of log2(DNase I cut count in 7 DPA/DNase I cut count in 4 DPA) for all union DNase I-hypersensitive sites (DHSs) (gray) and dynamic DHSs, with DHSs more accessible at 4 DPA appearing on the left in blue and DHSs more accessible at 7 DPA appearing on the right in pink. Diagrams of 4 DPA (left) and 7 DPA seeds (right) are shown, with purple opacity indicating GL2 expression levels from Belmonte et al., 2013. (B) Examples showing a deactivated DHS, two examples of activated DHSs, and one example of a static DHS. A 5-kb region is shown in each window; all data tracks are read-depth normalized. (C) Distribution of the number of dynamic DHSs neighboring genes. Most genes reside next to one dynamic DHS; however, surprisingly many genes reside next to multiple dynamic DHSs. Genes neighboring dynamic DHSs are listed in Supplemental Table 13 . (D) The numbers of union DHSs (uDHSs) and dynamic DHSs (dDHSs) within each genomic context: TSS, intergenic, transposon, and intragenic.
Figure 2
Figure 2
Genes neighboring developmentally dynamic DNase I-hypersensitive sites (dDHSs) are often differentially expressed. (A) Overlap between the set of genes neighboring dDHSs and genes found to be differentially expressed in seed coat at stages 4 and 7 DPA in two different data sets (Dean et al., 2011; Belmonte et al., 2013). (B) Overlap of all four sets of genes. (C) Genes that are more highly expressed tend to be near more accessible DHSs and vice versa. P-values are calculated using the hypergeometric test. One asterisk (*) indicates p-value 0.01. Two asterisks (**) indicate p-value 10-20.
Figure 3
Figure 3
Term enrichment for genes nearest to dynamic DNase I-hypersensitive sites (DHSs). (A) Term enrichment for genes near DHSs that are deactivated (less accessible) or (B), activated (more accessible) at the 7 DPA time point.
Figure 4
Figure 4
Motif enrichments within dynamic DNase I-hypersensitive sites (DHSs). (A) Transcription factor motifs enriched in DHSs that are deactivated at the 7 DPA time point. (B) Transcription factor motifs enriched in DHSs that are activated at the 7 DPA time point. Dotted vertical line indicates adjusted p-values of 10-20 of 10-40, respectively. All transcription factor family members are displayed if at least one member is enriched with adjusted p-value of 10-20 or less [greater than -log10(10-20) or 20]. Transcription factor motifs derived using amplified (i.e., non-methylated) DNA have gray bars indicating enrichment p-value (O’Malley et al., 2016). Motifs derived from genomic (i.e., methylated) DNA have black bars indicating enrichment p-value.
Figure 5
Figure 5
Comparative analysis of DNase I-hypersensitive site (DHS) landscapes in diverse samples. (A) Dendrogram of thirteen samples using DNase I accessibility data. 4 DPA, 7 DPA denotes seed coat-enriched samples; auxin denotes 7-day-old seedlings treated with auxin (SRR8903039); seedling denotes 7-day-old control seedlings (Sullivan et al., 2014); heat shock denotes 7-day-old seedlings treated with heat shock (Sullivan et al., 2014); BRZ denotes 7-day-old seedlings treated with brassinazole (SRR8903038); dark+L24h, dark+L3h, dark+L30m denote 7-day-old seedlings which were grown in the dark and exposed to a long-day light cycle for the indicated amount of time, modeling development during photomorphogenesis (h, hours; m, minutes) (GSM1289351, GSM1289355, GSM1289353, respectively) (Sullivan et al., 2014); dark seedling denotes 7-day-old dark grown seedlings (GSM1289357) (Sullivan et al., 2014); root hair denotes root hair cell samples of 7-day old seedlings (SRR8903037); root nonhair denotes nonhair root cells of 7-day-old seedlings (GSM1821072) (Sullivan et al., 2014); root denotes whole root tissue (GSM1289374) (Sullivan et al., 2014). (B) Biplot of principal component analysis of 62,729 DHSs by 13-sample matrix. Numbers in gray represent union DHSs. Insets show dynamic accessibility for two DHSs that were highly informative for distinguishing the 13 samples (i.e. these DHSs were among the most differentially accessible across all 13 samples). The upper inset shows a DHS that appears to be specific to aerial tissue; the lower inset shows a DHSs that appears to be specific to dark-grown tissue as roots are typically not exposed to light.
Figure 6
Figure 6
Comparison of seed coat-enriched samples (4 and 7 DPA) results in the highest number of developmentally dynamic DNase I-hypersensitive sites (DHSs) identified among all pairs examined. Scatterplots of log10(cut counts per union DHS) for six pairwise comparisons. Dotted lines creating a cone capturing the majority of the dots are drawn in the same location on each graph. Gray boxes represents regions in which both samples have less than 50 [log10(50) = 1.69897] cleavage sites in that DHS. Numbers indicated above and below indicate the number of dots (DHSs) that lie above and below dotted lines. Screenshot insets in each graph showing an example dynamic DHSs above and below dotted lines are the following DHSs, respectively: {4 vs. 7 DPA: chr2:19,564,381–19,564,531, chr4:11,981,161–11,981,351; root hair vs. root nonhair: chr1:30,035,761–30,036,071, chr4:280,861–281,131; control vs. auxin-treated: chr1:10,320,801–10,321,131, chr1:5,204,361–5,204,551; dark-grown seedling vs. dark-grown seedling on BRZ: chr5:22,570,821–22,571,231, chr5:21,869,241–21,869,591; control vs. heat shocked seedling: chr4:7,338,681–7,342,041, chr2:18,374,201–18,374,371; dark-grown seedling vs. dark-grown seedling exposed to 24-h light cycle: chr3:6,023,601–6,023,871, chr5:5,968,041–5,968,291}.
Figure 7
Figure 7
Seed coat-enriched samples contribute the largest number of novel hypersensitive bases in a diverse set of samples. (A) Colored petals denote number of unique hypersensitive base pairs in each sample, gray circle denoted hypersensitive base pairs shared by two or more samples. Sample labels as in Figure 5 ; samples are grouped by seed coat-enriched samples, light-grown seedlings, dark-grown seedlings, and root samples. (B) Cumulative number of hypersensitive sites plateaus. Graph was generated by adding samples based on their number of unique hypersensitive base pairs, starting with the largest (4 DPA) and ending with the smallest (dark seedling).

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