Genome-Wide Identification of Open Chromatin in Plants Using MH-Seq.

Functional cis-regulatory elements (CREs) act as precise transcriptional switches for fine-tuning gene transcription. Identification of CREs is critical for understanding regulatory mechanisms of gene expression associated with various biological processes in eukaryotes. It is well known that CREs reside in open chromatin that exhibits hypersensitivity to enzyme cleavage and physical shearing. Currently, high-throughput methodologies, such as DNase-seq, ATAC-seq, and FAIRE-seq, have been widely applied in mapping open chromatin in various eukaryotic genomes. More recently, differential MNase (micrococcal nuclease) treatment has been successfully employed to map open chromatin in addition to profiling nucleosome landscape in both mammalian and plant species. We have developed a MNase hypersensitivity sequencing (MH-seq) technique in plants. The MH-seq procedure includes plant nuclei fixation and purification, differential treatments of purified nuclei with MNase, specific recovery of MNase-trimmed small DNA fragments within 20~100 bp in length, and MH-seq library construction followed by Illumina sequencing and data analysis. MH-seq has been successfully applied for global identification of open chromatin in both Arabidopsis thaliana and maize. It has been proven to be an attractive alternative for profiling open chromatin. Thus, MH-seq is expected to be valuable in probing chromatin accessibility on a genome-wide scale for other plants with sequenced genomes. Moreover, MHS data allow to implement footprinting assays to unveil binding sites of transcription factors.

Characterization of Transposon-Derived Accessible Chromatin Regions in Rice (Oryza Sativa).

Growing evidence indicates that transposons or transposable elements (TEs)-derived accessible chromatin regions (ACRs) play essential roles in multiple biological processes by interacting with trans-acting factors. However, the function of TE-derived ACRs in the regulation of gene expression in the rice genome has not been well characterized. In this study, we examined the chromatin dynamics in six types of rice tissues and found that ~8% of ACRs were derived from TEs and exhibited distinct levels of accessibility and conservation as compared to those without TEs. TEs exhibited a TE subtype-dependent impact on ACR formation, which can be mediated by changes in the underlying DNA methylation levels. Moreover, we found that tissue-specific TE-derived ACRs might function in the tissue development through the modulation of nearby gene expression. Interestingly, many genes in domestication sweeps were found to overlap with TE-derived ACRs, suggesting their potential functions in the rice domestication. In addition, we found that the expression divergence of 1070 duplicate gene pairs were associated with TE-derived ACRs and had distinct distributions of TEs and ACRs around the transcription start sites (TSSs), which may experience different selection pressures. Thus, our study provides some insights into the biological implications of TE-derived ACRs in the rice genome. Our results imply that these ACRs are likely involved in the regulation of tissue development, rice domestication and functional divergence of duplicated genes.

Single-Cell Transcriptome and Network Analyses Unveil Key Transcription Factors Regulating Mesophyll Cell Development in Maize.

BACKGROUND: Maize mesophyll (M) cells play important roles in various biological processes such as photosynthesis II and secondary metabolism. Functional differentiation occurs during M-cell development, but the underlying mechanisms for regulating M-cell development are largely unknown. RESULTS: We conducted single-cell RNA sequencing (scRNA-seq) to profile transcripts in maize leaves. We then identified coregulated modules by analyzing the resulting pseudo-time-series data through gene regulatory network analyses. WRKY, ERF, NAC, MYB and Heat stress transcription factor (HSF) families were highly expressed in the early stage, whereas CONSTANS (CO)-like (COL) and ERF families were highly expressed in the late stage of M-cell development. Construction of regulatory networks revealed that these transcript factor (TF) families, especially HSF and COL, were the major players in the early and later stages of M-cell development, respectively. Integration of scRNA expression matrix with TF ChIP-seq and Hi-C further revealed regulatory interactions between these TFs and their targets. HSF1 and COL8 were primarily expressed in the leaf bases and tips, respectively, and their targets were validated with protoplast-based ChIP-qPCR, with the binding sites of HSF1 being experimentally confirmed. CONCLUSIONS: Our study provides evidence that several TF families, with the involvement of epigenetic regulation, play vital roles in the regulation of M-cell development in maize.

Profiling of H3K4me3 and H3K27me3 and Their Roles in Gene Subfunctionalization in Allotetraploid Cotton.

Cotton is an excellent model for studying crop polyploidization and domestication. Chromatin profiling helps to reveal how histone modifications are involved in controlling differential gene expression between A and D subgenomes in allotetraploid cotton. However, the detailed profiling and functional characterization of broad H3K4me3 and H3K27me3 are still understudied in cotton. In this study, we conducted H3K4me3- and H3K27me3-related ChIP-seq followed by comprehensively characterizing their roles in regulating gene transcription in cotton. We found that H3K4me3 and H3K27me3 exhibited active and repressive roles in regulating the expression of genes between A and D subgenomes, respectively. More importantly, H3K4me3 exhibited enrichment level-, position-, and distance-related impacts on expression levels of related genes. Distinct GO term enrichment occurred between A/D-specific and homeologous genes with broad H3K4me3 enrichment in promoters and gene bodies, suggesting that broad H3K4me3-marked genes might have some unique biological functions between A and D subgenome. An anticorrelation between H3K27me3 enrichment and expression levels of homeologous genes was more pronounced in the A subgenome relative to the D subgenome, reflecting distinct enrichment of H3K27me3 in homeologous genes between A and D subgenome. In addition, H3K4me3 and H3K27me3 marks can indirectly influence gene expression through regulatory networks with TF mediation. Thus, our study provides detailed insights into functions of H3K4me3 and H3K27me3 in regulating differential gene expression and subfunctionalization of homeologous genes, therefore serving as a driving force for polyploidization and domestication in cotton.

Molecular cytogenetic identification of a wheat - Thinopyrum ponticum substitution line with stripe rust resistance.

Thinopyrum ponticum (Th. ponticum) (2n = 10x = 70) is an important breeding material with excellent resistance and stress tolerance. In this study, we characterized the derivative line CH1113-B13-1-1-2-1 (CH1113-B13) through cytological, morphological, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), expressed sequence tag (EST), and PCR-based landmark unique gene (PLUG) marker analysis. The GISH analysis revealed that CH1113-B13 contained 20 pairs of common wheat chromosomes and one pair of JSt genomic chromosomes. Linkage analysis of Th. ponticum using seven EST and seven PLUG markers indicated that the pair of alien chromosomes belonged to the seventh homeologous group. Nulli-tetrasomic and FISH analysis revealed that wheat 7B chromosomes were absent in CH1113-B13; thus, CH1113-B13 was identified as a 7JSt (7B) substitution line. Finally, adult-stage CH1113-B13 exhibited immunity to wheat stripe rust. This substitution line is therefore a promising germplasm resource for wheat breeding.

Molecular cytogenetics identification of a wheat - Leymus mollis double disomic addition line with stripe rust resistance.

Leymus mollis (Trin.) Pilg. (2n = 4x = 28, NsNsXmXm) possesses a number of valuable genes against biotic and abiotic stress, which could be transferred into common wheat background for wheat improvement. In the present study, we determined the karyotypic constitution of a wheat - L. mollis double disomic addition line, M11003-4-4-1-1, selected from the F5 progeny of a stable wheat - L. mollis derivative M39 (2n = 56) × Triticum aestivum cultivar 7182, by morphological and cytogenetic identification, GISH (genomic in situ hybridization), FISH (fluorescent in situ hybridization), molecular markers analysis, and stripe rust resistance evaluation. Cytological studies demonstrated that M11003-4-4-1-1 had a chromosome karyotype of 2n = 46 with 23 bivalents, while GISH and FISH analysis indicated that this line contained 42 common wheat chromosomes and two pairs of L. mollis chromosomes. DNA markers showed that the alien chromosomes from L. mollis belonged to homoeologous groups 5 and 6. Evaluation of the agronomic traits revealed that M11003-4-4-1-1 was resistant to stripe rust at the adult stage, while the plant height was reduced and the 1000-grain weight was increased significantly. Therefore, the new line M11003-4-4-1-1 could be exploited as an important bridge material in chromosome engineering and wheat breeding.