Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 184 stage. This suggests a tight coupling of promoter chromatin state with developmental gene activation. Besides promoters, a large fraction of OCRs (~50% or more) resided in intergenic or intronic regions, often far from gene transcription start sites. Many of these distal OCRs likely represent enhancer elements or other distal CREs that regulate gene expression through long-range interactions. We found numerous examples where an intergenic OCR could be assigned (by proximity or through known QTL) to a particular gene and developmental process. A notable case is the distal open region upstream of the UNBRANCHED3 (UB3) gene in maize inflorescences: this region, known as KRN4, is an enhancer that influences UB3 expression and hence kernel row number (Du et al., 2020). In our seed dataset, the orthologous region corresponding to KRN4 showed accessible chromatin specifically in inflorescence tissues but not in developing seeds, consistent with its specialized role in ear development (and validating our method’s tissue-specific resolution). Conversely, we identified distal OCRs within the seed that could function as enhancers controlling seed-specific genes. Many such OCRs were found near genes involved in hormone biosynthesis/signaling, storage accumulation, or developmental transitions, suggesting the presence of enhancers coordinating these gene networks. Interestingly, some intergenic OCRs were located within transposable element (TE) sequences or flanking them. It has been noted that TEs contribute to regulatory innovation in maize by donating sequences that can act as enhancers. We observed stage-specific accessibility at certain MITE and LTR retrotransposon sequences in the seed genome, hinting that they might serve as developmental enhancers or chromatin boundary elements (Bubb et al., 2024). Indeed, transposon-derived enhancers have played roles in maize evolution (e.g., Hopscotch at tb1) and likely also in development. Globally, while the absolute number of promoter OCRs changed somewhat over time (tracking the number of expressed genes), the proportion of OCRs in promoters vs. distal regions remained relatively stable across stages. However, the specific identity of accessible intergenic regions changed markedly: early in development one set of enhancers is open (e.g. those for embryo patterning genes), whereas later, a different set of enhancers (e.g. for storage product genes) becomes accessible. These observations underscore that dynamic enhancer usage is a feature of seed development. Our findings align with prior single-cell ATAC-seq results in maize, which showed that roughly one-third of detected OCRs are cell-type or stage specific – reflecting enhancers that turn on or off depending on developmental context. Overall, the developmental stage of the seed can be “read out” in terms of its chromatin accessibility profile: certain promoter and enhancer regions are hallmarks of specific stages, reinforcing the idea that chromatin accessibility changes are an integral part of developmental gene regulation. 2.3 Genome-wide visualization of chromatin accessibility dynamics To understand the changes in chromatin accessibility during the growth of maize seeds, we examined the open chromatin regions (OCRs) across the entire genome. We tracked which regions were open or closed at different stages. One of our methods was to display the ATAC-seq signal trajectories of different chromosomal regions at different time points. This made it easier for us to identify regions where chromatin accessibility increased or decreased over time. For example, we used a genome browser to examine a 2 Mb region on chromosome 1. In this region, some peaks remained strong throughout all stages. These stable peaks were mostly near the constitutively active housekeeping genes. However, we also found some peaks that only appeared at certain time points. In a region containing a group of storage protein genes, the signal was almost non-existent three days after pollination (DAP3). But by 8 to 12 days after pollination, we saw clear and strong peaks. This was in good agreement with the time when these genes began to be active in the endosperm. Therefore, by observing the ATAC-seq signals over time, we can see when certain regions of the genome become more open, which usually matches the activation time of nearby genes (Zheng et al., 2025). We also created heatmaps to display the ATAC-seq read levels around all open chromatin regions (OCRs). The samples were arranged by developmental stage to facilitate the observation of accessibility changes over time. Some regions were open early but closed later. Others remained closed initially and only opened later. Some regions remained open throughout. These different patterns were clearly shown in the heatmaps. Next, we classified the OCRs into different types. We called them "early open", "late open", and "always open". Early open OCRs were usually close to genes related to cell division and tissue growth. These genes were typically active
RkJQdWJsaXNoZXIy MjQ4ODYzNA==