Protein phosphorylation is a key regulatory mechanism in circadian systems. TIMING OF CAB EXPRESSION 1 (TOC1) is a core transcriptional repressor in the plant circadian system that is phosphorylated near its N terminus. Phenotype testing of TOC1 phosphosite mutants shows incomplete rescue of the short period toc1 mutant. We establish that TOC1 phosphorylation (particularly at S175) is necessary for optimal interaction with FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and PHYTOCHROME INTERACTING FACTOR 5 (PIF5) at the CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) promoter to down-regulate CCA1 expression. Expression of the closely related LATE ELONGATED HYPOCOTYL (LHY) also requires TOC1 but is independent of TOC1 phosphorylation, suggesting different TOC1-dependent gene repression mechanisms. We additionally show that TOC1 phosphorylation-dependent interactions at specific clock gene promoters selectively regulate these circadian system components more acutely than nonrhythmic genes. Our genome-wide analysis demonstrates that the TOC1 phosphostate is important for optimal chromatin presence and robust rhythmic gene expression.

Eukaryotic genomes are extensively transcribed into various types of RNAs, many of which are physically associated with chromatin in cis at their transcription sites or in trans to other genomic loci. Emerging roles have been uncovered for these chromatin-associated RNAs (caRNAs) in gene regulation and genome organization, yet they remain challenging to interrogate. Here, we present TaDRIM-seq, a technique employing Protein G (PG)-Tn5-targeted DNA elements and in situ proximity ligation to concurrently probe caRNAs across diverse genomic regions as well as global RNA-RNA interactions within intact nuclei. Notably, this approach diminishes required cell inputs, minimizes hands-on time compared to established methodologies, and is compatible in both mammalian cells and plants. Using this technique, we identify extensive caRNAs at DNA anchor regions associated with chromatin loops and reveal diurnal variation in RNA-DNA and RNA-RNA connectivity networks within rice.

Background Epigenetic modifications that exhibit circadian oscillations also promote circadian oscillations of gene expression. Brassica napus is a heterozygous polyploid species that has undergone distant hybridization and genome doubling events and has a young and distinct species origin. Studies incorporating circadian rhythm analysis of epigenetic modifications can offer new insights into differences in diurnal oscillation behavior among subgenomes and the regulation of diverse expressions of homologous gene rhythms in biological clocks.Results In this study, we created a high-resolution and multioscillatory gene expression dataset, active histone modification (H3K4me3, H3K9ac), and RNAPII recruitment in Brassica napus. We also conducted the pioneering characterization of the diurnal rhythm of transcription and epigenetic modifications in an allopolyploid species. We compared the evolution of diurnal rhythms between subgenomes and observed that the Cn subgenome had higher diurnal oscillation activity in both transcription and active histone modifications than the An subgenome. Compared to the A subgenome in Brassica rapa, the An subgenome of Brassica napus displayed significant changes in diurnal oscillation characteristics of transcription. Homologous gene pairs exhibited a higher proportion of diurnal oscillation in transcription than subgenome-specific genes, attributed to higher chromatin accessibility and abundance of active epigenetic modification types. We found that the diurnal expression of homologous genes displayed diversity, and the redundancy of the circadian system resulted in extensive changes in the diurnal rhythm characteristics of clock genes after distant hybridization and genome duplication events. Epigenetic modifications influenced the differences in the diurnal rhythm of homologous gene expression, and the diurnal oscillation of homologous gene expression was affected by the combination of multiple histone modifications.Conclusions Herein, we presented, for the first time, a characterization of the diurnal rhythm characteristics of gene expression and its epigenetic modifications in an allopolyploid species. Our discoveries shed light on the epigenetic factors responsible for the diurnal oscillation activity imbalance between subgenomes and homologous genes' rhythmic expression differences. The comprehensive time-series dataset we generated for gene expression and epigenetic modifications provides a valuable resource for future investigations into the regulatory mechanisms of protein-coding genes in Brassica napus.

DNA methylation plays an important role in gene regulation and genomic stability. However, large DNA hypomethylated regions known as DNA methylation valleys (DMVs) or canyons have also been suggested to serve unique regulatory functions, largely unknown in rice (Oryza sativa). Here, we describe the DMVs in rice seedlings, which were highly enriched with developmental and transcription regulatory genes. Further detailed analysis indicated that grand DMVs (gDMVs) might be derived from nuclear integrants of organelle DNA (NORGs). Furthermore, Domains Rearranged Methylase 2 (OsDRM2) maintained DNA methylation at short DMV (sDMV) shores. Epigenetic maps indicated that sDMVs were marked with H3K4me3 and/or H3K27me3, although the loss of DNA methylation had a negligible effect on histone modification within these regions. In addition, we constructed H3K27me3-associated interaction maps for homozygous T-DNA insertion mutant of the gene (osdrm2) and wild type (WT). From a global perspective, most (90%) compartments were stable between osdrm2 and WT plants. At a high resolution, we observed a dramatic loss of long-range chromatin loops in osdrm2, which suffered an extensive loss of non-CG (CHG and CHH, H = A, T, or C) methylation. From another viewpoint, the loss of non-CG methylation at sDMV shores in osdrm2 could disrupt H3K27me3-mediated chromatin interaction networks. Overall, our results demonstrated that DMVs are a key genomic feature in rice and are precisely regulated by epigenetic modifications, including DNA methylation and histone modifications. OsDRM2 maintained DNA methylation at sDMV shores, while OsDRM2 deficiency strongly affected three-dimensional (3D) genome architectures.

Photoperiods integrate with the circadian clock to coordinate gene expression rhythms and thus ensure plant fitness to the environment. Genome-wide characterization and comparison of rhythmic genes under different light conditions revealed delayed phase under constant darkness (DD) and reduced amplitude under constant light (LL) in rice. Interestingly, ChIP-seq and RNA-seq profiling of rhythmic genes exhibit synchronous circadian oscillation in H3K9ac modifications at their loci and long non-coding RNAs (lncRNAs) expression at proximal loci. To investigate how gene expression rhythm is regulated in rice, we profiled the open chromatin regions and transcription factor (TF) footprints by time-series ATAC-seq. Although open chromatin regions did not show circadian change, a significant number of TFs were identified to rhythmically associate with chromatin and drive gene expression in a time-dependent manner. Further transcriptional regulatory networks mapping uncovered significant correlation between core clock genes and transcription factors involved in light/temperature signaling. In situ Hi-C of ZT8-specific expressed genes displayed highly connected chromatin association at the same time, whereas this ZT8 chromatin connection network dissociates at ZT20, suggesting the circadian control of gene expression by dynamic spatial chromatin conformation. These findings together implicate the existence of a synchronization mechanism between circadian H3K9ac modifications, chromatin association of TF and gene expression, and provides insights into circadian dynamics of spatial chromatin conformation that associate with gene expression rhythms.

Interspecific hybridization drives the evolution of angiosperms and can be used to introduce novel alleles for important traits or to activate heterosis in crop breeding. Hybridization brings together gene expression networks from two different species, potentially causing global alterations of gene expression in the F1 plants which is called 'transcriptome shock'. Here, we explored such a transcriptome shock in allotriploid Brassica hybrids. We generated interspecific F1 allotriploid hybrids between the allotetraploid species Brassica napus and three accessions of the diploid species Brassica rapa. RNA-seq of the F1 hybrids and the parental plants revealed that 26.34-30.89% of genes were differentially expressed between the parents. We also analyzed expression level dominance and homoeolog expression bias between the parents and the F1 hybrids. The expression-level dominance biases of the Ar, An, and Cn subgenomes was genotype and stage dependent, whereas significant homoeolog expression bias was observed among three subgenomes from different parents. Furthermore, more genes were involved in trans regulation than in cis regulation in allotriploid F1 hybrids. Our findings provide new insights into the transcriptomic responses of cross-species hybrids and hybrids showing heterosis, as well as a new method for promoting the breeding of desirable traits in polyploid Brassica species.

Interspecific hybridization is the intrinsic forces behind genome evolution. Long non-coding RNAs (lncRNAs) are important for plant biological processes regulation. However, it is unclear that these non-coding fractions are impacted by interspecific hybridization. Here we examined the profiles of lncRNAs by comparing them with coding genes in Brassica napus, three accessions of Brassica rapa, and their F1 hybrids. 6206 high-confidential lncRNAs were identified in F 1 hybrids and their parentals, and the lncRNAs transcriptome in the F1 hybrids was reprogrammed by the genome shock. Notably, genome-wide unbalanced of lncRNAs were observed between An and Ar subgenomes, ELD (Expression Level Dominance) was biased toward the An -genome in F1 hybrids, and ELD of non-conserved lncRNAs was more than conserved lncRNAs. Our findings demonstrate that the reprogramed lncRNAs acts as important role in enhancing plant plasticity, leading to the acquisition of desirable traits in polyploid Brassica species.

Muscle growth and fat deposition are the two important biological processes in the development of pigs which are closely related to the pig production performance. Long intergenic noncoding RNAs (lincRNAs), with lack of coding potential and the length of at least 200nt, have been extensively studied to play important roles in many biological processes. However, the importance and molecular regulation mechanism of lincRNAs in the process of muscle growth and fat deposition in pigs are still to be further studied comprehensively. In our study, we used the data, including liver, abdominal fat, and longissimus dorsi muscle of 240 days' age of two F2 full-sib female individuals from the white Duroc and Erhualian crossbreed, to identify 581 putative lincRNAs associated with pig muscle growth and fat deposition. The 581 putative lincRNAs shared many common features with other mammalian lincRNAs, such as fewer exons, lower expression levels, and shorter transcript lengths. Cross-tissue comparisons showed that many transcripts were tissue-specific and were involved in the important biological processes in their corresponding tissues. Gene ontology and pathway analysis revealed that many potential target genes (PTGs) of putative lincRNAs were involved in pig muscle growth and fat deposition-related processes, including muscle cell proliferation, lipid metabolism, and fatty acid degradation. In Quantitative Trait Locus (QTLs) analysis, some PTGs were screened from putative lincRNAs, MRPL12 is associated with muscle growth, GCGR and SLC25A10 were associated with fat deposition, and PPP3CA, DPYD, and FGGY were related not only to muscle growth but also to fat deposition. Therefore, it implied that these lincRNAs might participate in the biological processes related to muscle growth or fat deposition through homeostatic regulation of PTGs, but the detailed molecular regulatory mechanisms still needed to be further explored. This study lays the molecular foundation for the in-depth study of the role of lincRNAs in the pig muscle growth and fat deposition and further provides the new molecular markers for understanding the complex biological mechanisms of pig muscle growth and fat deposition.

An increasing number of studies have shown that long intergenic non-coding RNAs (lincRNAs) are a very important class of non-coding RNAs that plays a vital role in many biological processes. Adipose tissue is an important place for storing energy, but few studies on lincRNAs were related to pig subcutaneous fat development. Here, we used published RNA-seq data from subcutaneous adipose tissue of Italian Large White pigs and identified 252 putative lincRNAs, wherein 34 were unannotated. These lincRNAs had relatively shorter length, lower number of exons, and lower expression level compared with protein-coding transcripts. Gene ontology and pathway analysis indicated that the adjacent genes of lincRNAs were involved in lipid metabolism. In addition, differentially expressed lincRNAs (DELs) between low and high backfat thickness pigs were identified. Through the detection of quantitative trait locus (QTL), DELs were mainly located in QTLs related to adipose development. Based on the expression correlation of DEL genes and their differentially expressed potential target genes, we constructed a co-expression network and a potential pathway of DEL's effect on lipid metabolism. Our study identified and analyzed lincRNAs in subcutaneous adipose tissue, and results suggested that lincRNAs may be involved in the regulation of subcutaneous fat development. Our findings provided new insights into the biological function of porcine lincRNAs.

Long intergenic non-coding RNAs (lincRNAs) have been considered to play a key regulatory role in various biological processes. An increasing number of studies have utilized transcriptome analysis to obtain lincRNAs with functions related to cancer, but lincRNAs affecting growth rates in weaned piglets are rarely described. Although lincRNAs have been systematically identified in various mouse tissues and cell lines, studies of lincRNA in pigs remain rare. Therefore, identifying and characterizing novel lincRNAs affecting the growth performance of weaned piglets is of great importance. Here, we reconstructed 101,988 lincRNA transcripts and identified 1,078 lincRNAs in two groups of longissimus dorsi muscle (LDM) and subcutaneous fat (SF) based on published RNA-seq datasets. These lincRNAs exhibit typical characteristics, such as shorter lengths and lower expression relative to protein-encoding genes. Gene ontology analysis revealed that some lincRNAs could be involved in weaned piglet related processes, such as insulin resistance and the AMPK signaling pathway. We also compared the positional relationship between differentially expressed lincRNAs (DELs) and quantitative trait loci (QTL) and found that some of DELs may play an important role in piglet growth and development. Our work details part of the lincRNAs that may affect the growth performance of weaned piglets and promotes future studies of lincRNAs for molecular-assisted development in weaned piglets.