Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 188 genes then get expressed (with activation by ABI3/VP1 factors), implying a hand-off from a repressed but open state to an active state. This scenario matches the concept of poised chromatin: the chromatin is open (perhaps to allow rapid activation potential) but transcription is held in check by repressor proteins. The VAL1/VAL2 example has been documented in Arabidopsis and our findings suggest a similar mechanism could be at play in maize seeds (Yuan et al., 2020). Another example of gene silencing comes from distant OCRs that might work like silencers. We found several open regions in the genome, located between genes, that could be silencer elements. These spots were near genes that are usually turned off, such as imprinted genes or known negative regulators. Some of these regions were open in one tissue, like the maternal layer or aleurone, but closed in another, like the embryo or starchy endosperm. This matched the activity of nearby genes. In the tissue where the OCR was open, the linked gene was turned off. One idea is that these open regions might attract tissue-specific repressors. These could be MYB or homeodomain proteins that help shut down gene expression in one part of the seed. Here’s one example. A gene that codes for a transcription factor needed for germination was active in the embryo but silent in the endosperm. We found an open DNA region upstream of this gene, but only in the endosperm. That region contained repeat sequences, which might be bound by a repressor. This suggests the endosperm might open that area just to bring in a silencing complex. That way, it keeps the germination gene turned off until the right time. It is also worth mentioning that some genes displayed discordant chromatin–expression patterns due to possibly post-transcriptional regulation. For instance, a gene might have an open promoter and even produce transcripts, but those transcripts are not accumulating (perhaps due to RNA instability or translation being blocked). In such cases, our RNA-seq might show low steady-state mRNA despite an accessible promoter. However, these scenarios are harder to identify without further data (like nascent transcription assays or proteomics) and constituted a minority in our observations. Overall, the majority of OCRs in maize seeds act as conventional positive regulatory elements (promoters or enhancers facilitating gene expression), but a subset seem to mark regions of active chromatin that nonetheless correspond to repressed genes. The existence of these cases highlights the complexity of epigenetic regulation: an open chromatin state is generally permissive but must be interpreted in the context of bound factors. Accessible chromatin can serve as a platform for either activation or repression, depending on the proteins recruited to that site. Our data provide candidates for such regulatory phenomena in maize seed development, such as VAL-like repressor targets and potential silencer elements. Recognizing these will be important for a complete understanding of the chromatin code governing seed development – it is not a simple on/off switch, but a combinatorial platform where open DNA may need further cues to drive or block transcription. Importantly, these findings caution that strategies to modulate gene expression by targeting open chromatin (for crop improvement purposes) must account for the possible presence of repressive complexes at those sites. 4 Key Transcription Factors and Regulatory Network Construction 4.1 Cis-element discovery and transcription factor footprinting We performed de novo motif enrichment analysis on the sequences of our OCRs to uncover over-represented DNA sequence motifs, which likely correspond to binding sites of relevant TF families. This analysis yielded a rich collection of motifs, many of which could be matched to known plant cis-elements. Strikingly, the highest enrichment scores were observed for motifs belonging to a few major TF families, consistent with those families’ known roles in seed development. MIKC MADS-box motifs were among the top hits: a specific 8-bp CC[A/T]6GG sequence (the CArG-box motif) appeared frequently in accessible regions that open during early endosperm and embryo differentiation. This motif is recognized by MADS-domain transcription factors, such as AGAMOUS-like proteins. In fact, our motif resembled the binding site of Arabidopsis SEPALLATA (SEP) and SHORT VEGETATIVE PHASE (SVP) MADS factors, which are known to play roles not only in floral development but also in embryogenesis and endosperm development. The enrichment of the CArG motif in cluster III OCRs (those open at 4–6 DAP) suggests that MADS-box TFs are active in early seed development. Indeed, maize homologs of AGL15/18 (such as ZmMADS47) have been implicated in embryonic tissue development, and our data support that they bind accessible sites during those stages (Galli et al., 2018).
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