Functional characterization of cis-regulatory variation underlying trait diversity in maize

• Applicant: Thomas Hartwig, Ph.D.
• Subject Area: Plant Genetics and Genomics
• Term: from 2021 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 458854361

• Final Report Year: 2025

Final Report Abstract

Transcription factors (TF) play pivotal roles in patterning the spatial and temporal expression of genes, thus allowing for complex developmental trajectories and rapid responses to changing environments. Accordingly, a central goal of genetics has long been identifying the sequences bound by TFs and understanding how variation at these regulatory sites effects gene expression differences. Historically, identification of cis-acting regulatory elements such as TF- binding sites has been arduous, done one TF at a time with ChIP-seq and with relatively low resolution. Genomic approaches have sought to approximate these efforts, identifying open chromatin genome-wide, but at the cost of resolution and interpretability. In this project, we established a high-throughput TF footprinting assay and demonstrated how haplotype-specific quantification of TF footprints in their native chromatin context enabled us to construct a firstgeneration plant pancistrome. Our approach, focusing on hybrids of well-studied inbred lines, allowed haplotype-specific resolution of TF-binding sites in cis, circumventing biological, technical, and trans-factor differences that otherwise complicate the quantification of cis-element occupancy. We identified ~210,000 variable-occupied cis-elements across a diverse set of maize lines, which constitutes the largest collection of genetic/epigenetic variants at cis-elements in plants to date. This collection of variants coincided with numerous loci known to govern variation in flowering time, plant architecture, and organ size. In many cases, we demonstrated direct correlations between allele-specific cis-element occupancy and transcript abundance. Importantly, we observed that variation at TF-binding sites explains the majority of the heritability for a remarkable ~70% of the almost 150 phenotypes studied. We also demonstrated that high-resolution TF footprinting can be effective in identifying thousands of confident candidate loci for crop improvement associated with the response to agricultural important traits, specifically heat and drought stress. Lastly, we demonstrated that cis-elements identified through TF footprinting can improve deep learning model predictions of cis-regulatory elements, or help validate their predictions at an unprecedented scale and precession.

Publications

• The native cistrome and sequence motif families of the maize ear. PLOS Genetics, 17(8), e1009689.
  Savadel, Savannah D.; Hartwig, Thomas; Turpin, Zachary M.; Vera, Daniel L.; Lung, Pei-Yau; Sui, Xin; Blank, Max; Frommer, Wolf B.; Dennis, Jonathan H.; Zhang, Jinfeng & Bass, Hank W.
  
• Mapping responsive genomic elements to heat stress in a maize diversity panel. Genome Biology, 23(1).
  Liang, Zhikai; Myers, Zachary A.; Petrella, Dominic; Engelhorn, Julia; Hartwig, Thomas & Springer, Nathan M.
  
• Conserved and variable heat stress responses of the Heat Shock Factor transcription factor family in maize and Setaria viridis. Plant Direct, 7(4).
  Myers, Zachary A.; Wootan, Clair M.; Liang, Zhikai; Zhou, Peng; Engelhorn, Julia; Hartwig, Thomas & Nathan, Springer M.
  
• Genetic variation at transcription factor binding sites largely explains phenotypic heritability in maize. openRxiv.
  Engelhorn, Julia; Snodgrass, Samantha J.; Kok, Amelie; Seetharam, Arun S.; Schneider, Michael; Kiwit, Tatjana; Singh, Ayush; Banf, Michael; Khaipho-Burch, Merritt; Runcie, Daniel E.; Sanchez-Camargo, Victor A.; Torres-Rodriguez, J. Vladimir; Sun, Guangchao; Stam, Maike; Fiorani, Fabio; Beier, Sebastian; Schnable, James C.; Bass, Hank W.; Hufford, Matthew B. ... & Hartwig, Thomas
  
• Hybrid allele-specific ChIP-seq analysis identifies variation in brassinosteroid-responsive transcription factor binding linked to traits in maize. Genome Biology, 24(1).
  Hartwig, Thomas; Banf, Michael; Prietsch, Gisele Passaia; Zhu, Jia-Ying; Mora-Ramírez, Isabel; Schippers, Jos H. M.; Snodgrass, Samantha J.; Seetharam, Arun S.; Huettel, Bruno; Kolkman, Judith M.; Yang, Jinliang; Engelhorn, Julia & Wang, Zhi-Yong
  
• MINI‐AC : inference of plant gene regulatory networks using bulk or single‐cell accessible chromatin profiles. The Plant Journal, 117(1), 280-301.
  Manosalva Pérez, Nicolás; Ferrari, Camilla; Engelhorn, Julia; Depuydt, Thomas; Nelissen, Hilde; Hartwig, Thomas & Vandepoele, Klaas
  
• Genetic variation at transcription factor binding sites largely explains phenotypic heritability in maize. Nature Genetics, 57(9), 2313-2322.
  Engelhorn, Julia; Snodgrass, Samantha J.; Kok, Amelie; Seetharam, Arun S.; Schneider, Michael; Kiwit, Tatjana; Singh, Ayush; Banf, Michael; Doan, Duong Thi Hai; Khaipho-Burch, Merritt; Runcie, Daniel E.; Sánchez-Camargo, Victor A.; Bader, Rechien; Vladimir Torres-Rodriguez, J.; Sun, Guangchao; Stam, Maike; Fiorani, Fabio; Beier, Sebastian; Schnable, James C. ... & Hartwig, Thomas
  
• Modelling genetic variation effects in plant gene regulatory networks using transfer learning on genomic and transcription factor binding data. Springer Science and Business Media LLC.
  Peleke, Fritz; Zumkeller, Simon; Hartwig, Thomas; Engelhorn, Julia; Schmitt, Armin & Szymański, Jędrzej