Biology subjects

Huang, S.-s. C.

Publications and source records attributed to Huang, S.-s. C..

8 recordsLinked to original sources

bioRxiv2025

Transcription Factor (TF) validation using Dam-IT simultaneously captures genome-wide TF-DNA binding, direct gene regulation, and chromatin accessibility in plant cells

Transcription Factors (TFs) govern vast networks of gene regulation. However, TF-DNA binding and TF-gene regulation datasets are typically measured separately due to experimental constraints, making it challenging to disentangle true biological relationships from batch effects. To fill this gap, we developed DamID-seq Incorporating Transcriptomics (Dam-IT), which simultaneously captures TF-DNA binding, direct TF-gene regulation, and chromatin accessibility in the same batch of cells. Dam-IT uses a transient cell-based TF-target validation system that is scalable and flexible to many experimental designs. As proof of concept, we used Dam-IT to reveal that bZIP1 directly regulates genes by binding to DNA regions of relatively low chromatin accessibility, supporting a "Hit-and-Run" mechanism of transcription.

systems biology↗

bioRxiv2025

CircRNA hypomethylation in the human amygdala implicates FKBP5 in alcohol use disorder

Genome- and phenome-wide association studies implicate the RNA demethylase FTO in alcohol use disorder (AUD), yet the RNA methylation landscape in AUD remains poorly characterized. Analyzing postmortem human basolateral amygdala (BLA) tissue, a key brain region in AUD-related behaviors, we found extensive m6A hypomethylation uniquely affecting circular RNAs (circRNAs). Notably, FKBP5-hosted circRNAs (circFKBP5s) exhibited pronounced hypomethylation correlating with elevated expression of FKBP5 mRNA isoforms. These findings were replicated in an animal model of alcohol dependence. Predictive analyses suggest that circFKBP5s influence genes involved in neurodevelopmental processes and neuronal identity. These findings uncover a novel aspect of AUD neurobiology linked to circRNA methylation.

molecular biology↗

bioRxiv2024

Diversification and conservation of DNA binding specificities of SPL family of transcription factors

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/612952v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1154c42org.highwire.dtl.DTLVardef@1419f5borg.highwire.dtl.DTLVardef@1454a7aorg.highwire.dtl.DTLVardef@1cbd5f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG SQUAMOSA Promoter-Binding Protein-Like (SPL) transcription factors are key regulators of plant development and stress responses. Here, we present a comprehensive DNA affinity purification sequencing (DAP-seq) analysis of 14 of the 16 SPL members in Arabidopsis thaliana, revealing two major classes based on distinct DNA-binding motifs. Gene Ontology enrichment of target genes indicated both shared and specialized functions among AtSPLs, including roles in hormone signaling, metal ion homeostasis, and developmental transitions. Comparative analysis of closely related paralogs, AtSPL9 and AtSPL15, uncovered divergence in their genomic binding locations and target gene regulation, particularly in auxin and abscisic acid pathways. These differences were supported by motif analysis and protoplast reporter assays. Extending our study across species, we analyzed DAP-seq data from maize (Zea mays) and wheat (Triticum aestivum) SPL homologs, identifying conserved target genes but species-specific DNA motifs. Notably, wheat SPLs preferentially bind to longer, palindromic motifs distinct from the GTAC core motif in Arabidopsis. Using AlphaFold3 structure modeling, we show that these motif differences may arise from enhanced protein-protein interactions that stabilize dimeric binding. Our results highlight how SPL family members diversify through DNA-binding evolution and suggest that transcription factor dimerization contributes to motif complexity and regulatory specialization across plant genomes.

plant biology↗

bioRxiv2024

Transcription factor binding site divergence across maize inbred lines drives transcriptional and phenotypic variation

Regulatory elements are important constituents of plant genomes that have shaped ancient and modern crops. Their identification, function, and diversity in crop genomes however are poorly characterized, thus limiting our ability to harness their power for further agricultural advances using induced or natural variation. Here, we use DNA affinity purification-sequencing (DAP-seq) to map transcription factor (TF) binding events for 200 maize TFs belonging to 30 distinct families and heterodimer pairs in two distinct inbred lines historically used for maize hybrid plant production, providing empirical binding site annotation for 5.3% of the maize genome. TF binding site comparison in B73 and Mo17 inbreds reveals widespread differences, driven largely by structural variation, that correlate with gene expression changes. TF binding site presence-absence variation helps clarify complex QTL such as vgt1, an important determinant of maize flowering time, and DICE, a distal enhancer involved in herbivore resistance. Modification of TF binding regions via CRISPR-Cas9 mediated editing alters target gene expression and phenotype. Our functional catalog of maize TF binding events enables collective and comparative TF binding analysis, and highlights its value for agricultural improvement.

plant biology↗

bioRxiv2023

Transcription Factor Dynamics in Cross-Regulation of Plant Hormone Signaling Pathways

Cross-regulation between hormone signaling pathways is indispensable for plant growth and development. However, the molecular mechanisms by which multiple hormones interact and co-ordinate activity need to be understood. Here, we generated a cross-regulation network explaining how hormone signals are integrated from multiple pathways in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. To do so we comprehensively characterized transcription factor activity during plant hormone responses and reconstructed dynamic transcriptional regulatory models for six hormones; abscisic acid, brassinosteroid, ethylene, jasmonic acid, salicylic acid and strigolactone/karrikin. These models incorporated target data for hundreds of transcription factors and thousands of protein-protein interactions. Each hormone recruited different combinations of transcription factors, a subset of which were shared between hormones. Hub target genes existed within hormone transcriptional networks, exhibiting transcription factor activity themselves. In addition, a group of MITOGEN-ACTIVATED PROTEIN KINASES (MPKs) were identified as potential key points of cross-regulation between multiple hormones. Accordingly, the loss of function of one of these (MPK6) disrupted the global proteome, phosphoproteome and transcriptome during hormone responses. Lastly, we determined that all hormones drive substantial alternative splicing that has distinct effects on the transcriptome compared with differential gene expression, acting in early hormone responses. These results provide a comprehensive understanding of the common features of plant transcriptional regulatory pathways and how cross-regulation between hormones acts upon gene expression.

plant biology↗

bioRxiv2022

Uncovering the "ZIP code" for bZIP dimers reveals novel motifs, regulatory rules and one billion years of cis-element evolution

Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. For example, dimerization properties of the basic leucine zipper (bZIP) family play a critical role in regulating the unique biological functions in all eukaryotes. However, the molecular mechanism underlying the binding sequence and functional specificity of homo- versus heterodimers remains elusive. To fill this gap, we developed a double DNA Affinity Purification sequencing (dDAP-seq) technique that maps heterodimer DNA binding sites in an endogenous genome context. Our genome-wide binding profiles of twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis revealed that heterodimerization significantly expands the DNA binding preferences of bZIP TFs. Analysis of the heterodimer target genes in stress response and development suggest heterodimerization gives rise to regulatory responses that are distinct from the homodimers. In addition to the classic ACGT elements recognized by plant bZIPs, we found that the C/S1 heterodimers bound to motifs that might share an origin with the GCN4 cis-elements in yeast that diverged from plants more than one billion years ago. Importantly, heterodimer binding specificities can be distinguished by their relative preference for ACGT motifs versus GCN4-related motifs. More broadly, our study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.

molecular biology↗

bioRxiv2021

The grape MYB24 mediates the coordination of light-induced terpene and flavonol accumulation in response to berry anthocyanin sunscreen depletion

The presence of naturally-occurring color mutants in plants has permitted the identification of many regulatory genes implicated in the synthesis of discrete metabolic compounds, mostly anthocyanins and carotenoids. Conversely, transcription factors that coordinate more than one specialized metabolic pathway seem challenging to screen from a forward genetics perspective. We explored the relationship between different branches of the phenylpropanoid and isoprenoid pathways while examining an infrequent berry skin color variegation in grapevine. Red and white berry skin sections were compared at the genetic, transcriptomic and metabolomic levels showing that, as in most cultivated white grape varieties, the uncolored skin section convened the non-functional alleles of the anthocyanin regulators MYBA1 and MYBA2, explaining the lack of pigments. In contrast, light-responsive flavonols and monoterpenes increased in anthocyanin-depleted areas. We disclosed an enrichment of the flavonol, terpene and carotenoid pathways among up-regulated genes from white-skin sections, accompanied by increased expressions of flavonol regulators and the still uncharacterized MYB24 gene. We used DAP-seq to examine the in vitro binding of affinity-purified MYB24 protein to genomic DNA and demonstrated its binding in the promoter regions of terpene (22) and carotenoid (6) genes, in addition to more than 30 photosynthesis and light-response genes, including the flavonol-regulator HY5 homologue (HYH). We confirmed the activation of TPS35 and HYH promoter:luciferase reporters in the presence of MYB24 and the grape bHLH MYC2, all of which correlate in their higher expression in white skin variegated sections. The integration of several datasets allowed to define a list of high confidence targets, suggesting MYB24 as a modulator of light responses including the synthesis of flavonoids (flavonols) and isoprenoids (terpenes, and putatively carotenoids). The correspondence between MYB24 and monoterpenes in all conditions surveyed implies that this regulatory network is broadly triggered towards berry ripening, and that the absence of anthocyanin sunscreens accelerates its activation most likely in a dose-dependent manner due to increased radiation exposure.

plant biology↗

bioRxiv2021

Orchestration of the stilbene synthase gene family and their regulators by subgroup 2 MYB genes

The stilbenoid pathway is responsible for the production of resveratrol and its derivatives in grapevine. A few transcription factors (TFs) have been previously identified as regulators of this pathway but the extent of this control is yet to be fully understood. Here we demonstrate how DNA affinity purification sequencing (DAP-Seq) allows for genome-wide TF binding site interrogation in a non-model species. We obtained 5,190 and 4,443 binding events assigned to 4,041 and 3,626 genes for MYB14 and MYB15, respectively (around 40% of peaks being located within -10kb of transcription start sites). DAP-Seq of MYB14 and MYB15 was combined with aggregate gene centred co-expression networks built from more than 1,400 transcriptomic datasets from leaves, fruits and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15 and MYB13, a third uncharacterised member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS ) targets, these three regulators bind to 30 out of 47 STS family genes. Moreover all three MYBs bind to several PAL, C4H and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and novel resveratrol-modifying gene candidates amongst which ROMT2 -3 were validated enzymatically. A high proportion of DAP-Seq bound genes was induced in the activated transcriptomes of transient MYB15 -overexpressing stilbenoid-producing grapevine leaves, validating our methodological approach for identifying gene regulatory networks of specialised metabolism. Overall, MYB genes from Subgroup 2 appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production.

plant biology↗