Genome-wide DNase I-hypersensitive site assay reveals distinct genomic distributions and functional features of open chromatin in autopolyploid sugarcane.

The characterization of cis-regulatory DNA elements (CREs) is essential for deciphering the regulation of gene expression in eukaryotes. Although there have been endeavors to identify CREs in plants, the properties of CREs in polyploid genomes are still largely unknown. Here, we conducted the genome-wide identification of DNase I-hypersensitive sites (DHSs) in leaf and stem tissues of the auto-octoploid species Saccharum officinarum. We revealed that DHSs showed highly similar distributions in the genomes of these two S. officinarum tissues. Notably, we observed that approximately 74% of DHSs were located in distal intergenic regions, suggesting considerable differences in the abundance of distal CREs between S. officinarum and other plants. Leaf- and stem-dependent transcriptional regulatory networks were also developed by mining the binding motifs of transcription factors (TFs) from tissue-specific DHSs. Four TEOSINTE BRANCHED 1, CYCLOIDEA, and PCF1 (TCP) TFs (TCP2, TCP4, TCP7, and TCP14) and two ethylene-responsive factors (ERFs) (ERF109 and ERF03) showed strong causal connections with short binding distances from each other, pointing to their possible roles in the regulatory networks of leaf and stem development. Through functional validation in transiently transgenic protoplasts, we isolate a set of tissue-specific promoters. Overall, the DHS maps presented here offer a global view of the potential transcriptional regulatory elements in polyploid sugarcane and can be expected to serve as a valuable resource for both transcriptional network elucidation and genome editing in sugarcane breeding.

Genome-wide chromatin accessibility landscape and dynamics of transcription factor networks during ovule and fiber development in cotton.

BACKGROUND: The development of cotton fiber is regulated by the orchestrated binding of regulatory proteins to cis-regulatory elements associated with developmental genes. The cis-trans regulatory dynamics occurred throughout the course of cotton fiber development are elusive. Here we generated genome-wide high-resolution DNase I hypersensitive sites (DHSs) maps to understand the regulatory mechanisms of cotton ovule and fiber development. RESULTS: We generated DNase I hypersensitive site (DHS) profiles from cotton ovules at 0 and 3 days post anthesis (DPA) and fibers at 8, 12, 15, and 18 DPA. We obtained a total of 1185 million reads and identified a total of 199,351 DHSs through ~ 30% unique mapping reads. It should be noted that more than half of DNase-seq reads mapped multiple genome locations and were not analyzed in order to achieve a high specificity of peak profile and to avoid bias from repetitive genomic regions. Distinct chromatin accessibilities were observed in the ovules (0 and 3 DPA) compared to the fiber elongation stages (8, 12, 15, and 18 DPA). Besides, the chromatin accessibility during ovules was particularly elevated in genomic regions enriched with transposable elements (TEs) and genes in TE-enriched regions were involved in ovule cell division. We analyzed cis-regulatory modules and revealed the influence of hormones on fiber development from the regulatory divergence of transcription factor (TF) motifs. Finally, we constructed a reliable regulatory network of TFs related to ovule and fiber development based on chromatin accessibility and gene co-expression network. From this network, we discovered a novel TF, WRKY46, which may shape fiber development by regulating the lignin content. CONCLUSIONS: Our results not only reveal the contribution of TEs in fiber development, but also predict and validate the TFs related to fiber development, which will benefit the research of cotton fiber molecular breeding.

Structure and cis-regulatory analysis of a Drosophila grainyhead neuroblast enhancer.

Evolutionary analysis of cis-regulatory DNA reveals that enhancers consist of clusters of conserved sequence blocks (CSBs) that are made up of both unique and repeated sequence elements. This study seeks to address the basis for spatial and temporal regulation of neuroblas. A search for temporally restricted CNS NB enhancers identified one within the transcription factor grainyhead (grh) gene locus. The intronic enhancer, grh-15, contains two separable semi-autonomous activities, one that drives expression predominantly within the developing brain NBs and another in ventral cord NBs. To gain insight into the function of the CSBs constituting the brain-specific enhancer, we have systematically deleted each CSB and compared the activity of the altered enhancer to that of the full brain-specific enhancer. While our results indicate that information regulating enhancer activity is highly redundant, we have found that individual CSBs convey expression in subsets of larval lineages that are generated from either Type I or Type II NBs. These studies also highlight how evolutionary sequence conservation can be used as a guide the functional analysis of cis-regulatory DNA.

En masse DNA Electroporation for in vivo Transcriptional Assay in Ascidian Embryos.

Ascidian embryos are powerful models for functional genomics, in particular, due to the ease of generating a large number of transgenic embryos by electroporation. In addition, the small size of their genome makes them an attractive model for studying cis-regulatory elements that control gene expression during embryonic development. Here, I describe the adaptation of the seminal method developed 25 years ago in Ciona robusta for en masse DNA electroporation for in vivo transcription to additional species belonging to three genera. It is likely that similar optimizations would make electroporation successful in other ascidian species, where in vitro fertilization can be performed on a large number of eggs.

Genome-Wide Identification of DNase I Hypersensitive Sites in Plants.

DNase I hypersensitive site (DHS) mapping combined with high-throughput sequencing (DNase-seq) enables the identification of cis-regulatory DNA elements (CREs) genome wide. However, despite the wide applications of DNase-seq in plants, its application to the highly repetitive genomes of plants has lagged. Here, we describe a modified DNase-seq method, making it more practical for application to plants with genomes enriched with repetitive DNA. This approach adopts a double-hit-based strategy, in which small (<250-bp) DNA fragments digested by DNase I are selected and used for sequencing library construction. Using these protocols, we have conducted DNase-seq in plants with high content of repetitive DNA, including maize, sugarcane, and tetraploid cotton. Genome-wide maps of DHS and CREs have been created using these DNase-seq datasets. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation Basic Protocol 2: DNase I digestion Basic Protocol 3: Target DNA isolation Basic Protocol 4: Library construction and validation.