Design, Synthesis, and Validation - Genome Scale Optimization of Energy Flux through Compartmentalized Metabolic Networks in a Model Photosynthetic Eukaryotic Microbe - Genome-wide promoter characterization using DAP-Seq

Sep 15, 2026

Uploaded by Asher

Final report for Award Number DE-SC0018344 documenting a genome-scale approach to optimize energy flux in a compartmentalized metabolic network in a model photosynthetic eukaryotic microbe, alongside genome-wide characterization of transcription factor binding sites. DAP-Seq–derived catalogs for P. tricornutum (58 TFs) identify diel-cycle–linked motif enrichment, with bZIP-associated promoters enriched in early light-phase modules and HSF-associated promoters enriched in dark-phase modules. Motif density correlates with expression intensity during light-to-dark transitions. Results further highlight evolutionary conservation of core eukaryotic TF motifs and define the nitrate-sensitive HNS_A 13 nt DNA motif linked to nitrate-responsive gene regulation.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages15

Reading Time38 min

AccessFree to download

Reporting Summary

Award and Project Information

Genome-wide TFBS Characterization with DAP-Seq

Diel (Light:Dark) Regulation and Motif-Enrichment Results

Nitrogen Response and the HNS_A Motif

Reporting Period: Final Report Award Number: DE-SC0018344 Principal Investigator: Allen, Andrew Institution: J. Craig Venter Institute, Rockville, MD Title: Design, Synthesis, and Validation: Genome Scale Optimization of Energy Flux through Compartmentalized Metabolic Networks in a Model Photosynthetic Eukaryotic Microbe PI(s) Allen & Dupont (JCVI) Characterization of promoter architecture genome-wide using DAP-Seq Knowledge ofTFBS found within endogenous promoters can guide design of synthetic or refactored promoters with optimized or scalable expression characteristics. However, for diatoms, only fragmented knowledge of the regulatory significance of specific TFBS exists for a few transcription factors (TFs) and virtually nothing is known about the significance of native promoter architectures. A mechanistic understanding of how promoters modulate expression under various specific conditions, such as the light:dark (LD) transition, will expand the catalog of available parts that could be used in rational design of synthetic chromosomes. Using a genome-wide catalog of TFBS for P. tricornutum (58 TFs) constructed from DNA-affinity purification sequencing data Figure 1. Identification of transcription factor binding sites (TFBS) and enrichment in the promoters of gene coexpression modules. A) Heatmap shows motif enrichment in promoters of coexpression modules over diel cycles (modules from Smith et al., ref (14). B) Average gene expression (transcript, standardized) for selected strong diel modules. C) Positive correlation between bZIP motif density and light phase expression, and HSF motif density and dark phase expression. D) Motifs (from DAP-Seq data) of the most highly enriched light (bZIP) and dark (HSF) regulators, E) A motif (HNS_A) predicted from the promoters of a highly nitrate sensitive gene co-expression module correlates with intensity of nitrate sensitivity, F) Comparison of conserved diatom and animal Hox binding motifs. (DAP-Seq, we screened for motif enrichment in the promoters of genes that were co-expressed over diel cycles from a previously published RNA-seq dataset. Significant enrichment of several bZIP TFBS was observed in the promoters of genes maximally expressed early in the light phase, while enrichment of various HSF TFBS was observed in genes expressed in the dark phase, implicating these two major classes ofTFs (which have been evolutionarily expanded in diatoms) in the regulation of gene programs used in LD transitions (Fig. 1A,B) . Further, a positive relationship between the density of these motifs and the intensity of expression observed in response to the shift from light to dark was also observed (Fig. 1C), suggesting that transcriptional activity could be modulated by diel shifts by engineering higher densities of bZIP or HSF motifs into synthetic promoters (Fig. 1D) . In addition to bZIPs and HSFs, diatoms have homologs of eukaryotic TFs often associated with roles in function and development of multicellular organisms, like CCAAT-enhancer-binding proteins or Hox TFs. DAP-seq reveals that despite evolutionary distances, these P. tricornutum core eukaryotic TFs bind identical motifs to distant mammalian orthologs from humans and mice (Fig. 1F) . Though the roles of these TFs are completely unknown, this evolutionary conservation suggests they have an essential core function in regulating diatom gene expression that must be elucidated to fully harness the ability to rationally redesign and synthetically optimize promoters. Further, an understanding that proximal promoter/enhancer-mediated gene expression is achieved not only by the binding affinity ofTFs to TFBS, but by the spacing, orientation, and order of TFBS is currently emerging. These principles or “grammar”of promoters/enhancers must also be dissected in order to inform design principles leading to elements that can yield predictable and precise control over engineered metabolic pathways or cellular processes

FAQs

What method was used to characterize genome-wide promoter architecture in P. tricornutum?

DAP-Seq was used to construct a genome-wide catalog of transcription factor binding sites (58 TFs) and assess motif enrichment in endogenous promoters.

How do bZIP and HSF motifs relate to diel-cycle gene expression?

bZIP TFBS enrichment is observed in promoters of genes maximally expressed early in the light phase, while HSF TFBS enrichment is observed in genes expressed in the dark phase, linking these TF classes to LD transition regulation.

What is the role of the HNS_A motif in nitrate-responsive gene regulation?

A 13 nt DNA motif (HNS_A) enriched in promoters of highly nitrate sensitive genes is associated with nitrate availability, and motif comparisons suggest it may be assembled from multiple TFBS with different input-signal controls.

Sep 15, 2026

Uploaded by Asher