Abstract: Gene expression and complex phenotypes are determined by the activity of cis-regulatory elements. However, an understanding of how extant genetic variants affect cis regulation remains limited. Here, we investigated the consequences of cis-regulatory diversity using single-cell genomics of more than 0.7 million nuclei across 172 Zea mays (maize) inbreds. Our analyses pinpointed cis-regulatory elements distinct to domesticated maize and revealed how historical transposon activity has shaped the cis-regulatory landscape. Leveraging population genetics principles, we fine-mapped about 22,000 chromatin accessibility–associated genetic variants with widespread cell type–specific effects. Variants in TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR–binding sites were the most prevalent determinants of chromatin accessibility. Finally, integrating chromatin accessibility–associated variants, organismal trait variation, and population differentiation revealed how local adaptation has rewired regulatory networks in unique cellular contexts to alter maize flowering. Editor's summary: A transcription factor's ability to act requires access to the DNA to which it binds, which is called chromatin accessibility. However, identifying variants that causally affect chromatin accessibility is complicated by many factors, including linkage disequilibrium between variants. Taking advantage of maize's low levels of linkage disequilibrium, Marand et al. examined single-cell chromatin accessibility from 172 inbred maize lines. They found about 22,000 variants associated with chromatin accessibility, many of which were specific to cell types and overlapped transposable elements. Many of these variants were linked to traits such as flowering time and affected binding sites for the transcription factor TCP. These results provide insight not only into important agricultural traits, but also regulatory element turnover during adaptation. —Corinne Simonti INTRODUCTION: Noncoding genetic variants are a major driver of phenotypic diversity. In Zea mays (maize), genetic variation within cis-regulatory regions accounts for ~40% of phenotypic variability in agronomically important traits. Missing from past studies, however, is the role of cell context in shaping regulatory variant effects and a clear definition of the molecular mechanisms underlying phenotypic variation. Thus, resolving the genetic and molecular principles that give rise to phenotypic diversity in a cell state–aware framework is paramount to advancing crop improvement efforts. RATIONALE: Single-cell genomic methods offer a powerful approach for understanding the genetic sources of gene expression and chromatin accessibility variation. We generated and analyzed single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) and single-nuclei RNA sequencing (snRNA-seq) data across 172 genetically and phenotypically diverse inbred maize lines for insight into the regulatory mechanisms underlying phenotypic variability. RESULTS: Our single-cell dataset comprises >700,000 nuclei from 33 distinct cell states. We first used this resource to investigate the extent of cis-regulatory variation among diverse genetic backgrounds, identifying binding sites for specific transcription factor (TF) families as being critical determinants of regulatory sequence conservation and functional activity. Through comparisons with 21 teosinte (a progenitor of modern maize) genomes, we identified 1587 accessible chromatin regions that were unique and fixed in the domesticated maize lineage. These accessible chromatin regions were enriched for hAT and PIF/Harbinger transposons, implicating co-option of transposon cis-regulatory elements as a major source of new regulatory sequences specific to domesticated maize. By applying the principles of population genetics, we identified 107,623 cis chromatin accessibility quantitative trait loci (cis-caQTL) within accessible chromatin regions and validated their effects on enhancer activity using self-transcribing active regulatory region sequencing (STARR-seq). We found that cell state–specific cis-caQTL are common and often overlap with phenotype-associated variants identified by genome-wide association studies (GWASs). Deep investigation of caQTL indicated that variants within TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP)–binding sites are strong determinants of chromatin accessibility. Moreover, all caQTL variants with decreased TCP-binding affinity were concomitant with loss of chromatin accessibility. Construction of cell state–specific gene-regulatory networks indicated that TCP TFs are highly cell state specific and controlled by master cell identity regulators. Analysis of caQTL affecting distal accessible chromatin regions further implicated TCP TFs as being major contributors toward chromatin accessibility variation and, as a result, chromatin interactions. Through transcriptome-wide association mapping, chromatin accessibility–wide association mapping, and integration of caQTL with expression QTL and GWAS variants, we found that caQTL were commonly associated with flowering-related phenotypes. We hypothesized that the transition of modern maize from tropical to temperate climates may have been a significant contributor of extant chromatin accessibility variation. Indeed, we found that caQTL are associated with signatures of population differentiation, and that these population-divergent variants occur within binding sites for TFs previously implicated in flowering time and floral morphology nonuniformly among cell states. CONCLUSIONS: These analyses advance our understanding of how cell context and molecular diversity contribute to innovations in phenotype, providing the blueprints for future crop improvement efforts. Single-nuclei caQTL mapping and investigation of cell context–specific effects.: scATAC-seq profiles from >700,000 single nuclei across 172 maize inbred lines were used to identify >100,000 cis-caQTL within accessible chromatin regions. This study uncovered TF–binding site perturbations with effects on chromatin accessibility, caQTL colocalized with GWAS and eQTL, regulatory evolution associated with maize domestication, and local adaptation–associated regulatory rewiring at cell type resolution. [ABSTRACT FROM AUTHOR]
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