Uncovering the multi-layer cis-regulatory landscape of rice via integrative nascent RNA analysis

Background: Efforts to characterize regulatory elements in plant genomes traditionally rely on evolutionary conservation and chromatin accessibility. Recently, intergenic bi-directional nascent transcript has emerged as a putative hallmark of active enhancers. Here, we integrate these approaches to better define the cis-regulatory landscape of the rice genome. Results: In juvenile leaf tissues of the Azucena rice variety, we analyze conserved noncoding sequences, intergenic bi-directional transcripts, and regions of open chromatin. These three features highlight distinct classes of regulatory targets, each exhibiting complexity and regulatory roles. Conserved noncoding sequences are associated with more complex regulatory interactions, while regions marked by chromatin accessibility or bi-directional nascent transcription tend to promote more stable regulatory activity. Some transcribed regulatory sites harbor elements linked to transposable element silencing, whereas others correlate with increased expression of nearby genes, pointing to candidate transcribed regulatory elements. We further identified molecular interactions between genic regions and intergenic transcribed regulatory elements using 3-dimensional chromatin contact data, we identify physical interactions between transcribed intergenic regions and genic regions. These interactions often co-localize with expression quantitative trait loci and coincide with increased transcription, further supporting a regulatory role. Conclusions: Our integrative analysis reveals multiple distinct classes of regulatory elements in the rice genome, with overlapping but non-identical targets and signatures. Many candidate elements share features consistent with transcriptional enhancement, though the specific criteria for defining active enhancers in plants require further characterization. These findings underscore the importance of using complementary genomic signals to discover and characterize functionally diverse regulatory elements in plant genomes.