Complex Relationships between Chromatin Accessibility, Sequence Divergence, and Gene Expression in Arabidopsis thaliana

Abstract

Variation in regulatory DNA is thought to drive phenotypic variation, evolution, and disease. Prior studies of regulatory DNA and transcription factors across animal species highlighted a fundamental conundrum: Transcription factor binding domains and cognate binding sites are conserved, while regulatory DNA sequences are not. It remains unclear how conserved transcription factors and dynamic regulatory sites produce conserved expression patterns across species. Here, we explore regulatory DNA variation and its functional consequences within Arabidopsis thaliana, using chromatin accessibility to delineate regulatory DNA genome-wide. Unlike in previous cross-species comparisons, the positional homology of regulatory DNA is maintained among A. thaliana ecotypes and less nucleotide divergence has occurred. Of the ∼50,000 regulatory sites in A. thaliana, we found that 15% varied in accessibility among ecotypes. Some of these accessibility differences were associated with extensive, previously unannotated sequence variation, encompassing many deletions and ancient hypervariable alleles. Unexpectedly, for the majority of such regulatory sites, nearby gene expression was unaffected. Nevertheless, regulatory sites with high levels of sequence variation and differential chromatin accessibility were the most likely to be associated with differential gene expression. Finally, and most surprising, we found that the vast majority of differentially accessible sites show no underlying sequence variation. We argue that these surprising results highlight the necessity to consider higher-order regulatory context in evaluating regulatory variation and predicting its phenotypic consequences.

Accelerating structure‐function mapping using the ViVa webtool to mine natural variation

“…Noncoding sequence variation is intentionally excluded from the analysis tools. This reflects the challenges both in alignment of noncoding sequences and the increased difficulty in assessing the effects of variation in these regions (Alexandre et al., ).…”

Dynamics of gene expression in single root cells ofA. thaliana

“…To address such possible differences, we focused on genes 481 that from bulk analyses have differential expression upon heat shock (1783 genes) or reside near 482 regulatory regions that change in accessibility upon heat shock (1730 genes) (Alexandre et al, 483 2018;Sullivan et al, 2014). Although these gene sets overlap (942 genes), they contain 484 complementary information, as changes in accessibility do not necessarily translate into altered 485 expression, and vice versa (Alexandre et al, 2018). In our single-cell expression analysis, we 486 identified 752 of 1783 heat-responsive genes as differentially expressed upon heat shock, and 487 (Supplemental Figure 12A, C), resulting in several gene clusters with distinct expression 490 patterns.…”

“…We asked whether we could identify transcription factors that may contribute to the cluster-218 specific expression patterns. To do so, we tested for transcription factor motif enrichments in the 219 proximal regulatory regions of genes with cluster-specific expression, examining 500 bp 220 upstream of the transcription start site (Alexandre et al, 2018;Sullivan et al, 2014) and a 221 comprehensive collection of A. thaliana transcription factor motifs (O'Malley et al, 2016). This 222 analysis revealed significant transcription factor motif enrichments among clusters and annotated 223 major tissues and cell types ( Figure 2B).…”

The role of chromatin accessibility in cis-regulatory evolution

“…Cis-regulatory evolution plays an important role in phenotypic diversity, including morphological (Prud'Homme, et al 2006), physiological (Siepel and Arbiza 2014) and behavioral (Saul, et al 2017;Wray 2007) evolution. Given its importance, many studies have examined cross-species changes in various aspects of gene regulation, including expression (Paris, et al 2013), enhancer activity (Khoueiry, et al 2017), transcription factor (TF)-DNA binding (Bradley, et al 2010;Carvunis, et al 2015;Stefflova, et al 2013;Wong, et al 2015), TF binding motifs (Bradley, et al 2010;Cheng, et al 2014;He, et al 2011;Moses, et al 2006;Naval-Sánchez, et al 2015), DNA accessibility (Alexandre, et al 2017) and chromatin states (Lesch, et al 2016). Changes have been observed to differing extents in these measurable aspects of gene regulation, leading to some emerging principles underlying their conservation and divergence (Cheng, et al 2014).…”