Double DAP-seq uncovered synergistic DNA binding of interacting bZIP transcription factors

Abstract

Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo- versus heterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis and show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4 cis-elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.

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

“…These interactions have the potential to alter binding site specificity and thus expand the repertoire of TF binding sites beyond those achieved by single TFs. However, the impact of protein interactions on DNA binding in plants has only been investigated for a few families at the genome-wide level, despite its importance and implications for crop improvement ( Lai et al 2020 ) ( Li et al 2023 ). For example, breeding-based selection for certain traits often acts upon quantitative changes in TF expression levels ( Alonge et al 2020 ) ( Aguirre et al 2023 ); for dosage sensitive TFs whose interactions with partners influences DNA binding, such selections would be best informed by knowledge of protein partners and how those protein interactions affect DNA binding.…”

“…Among DNA-binding TF families, several employ homodimeric and/or heterodimeric DNA binding domains (DBDs), in which a functional DBD is formed by the dimerization of two identical or distinct monomers. To explore further how combinatorial heterodimeric interactions among TF family members impact DNA binding specificity we employed doubleDAP-seq, a modified version of DAP-seq for the analysis of heterodimeric complexes ( Li et al 2023 ) ( Supplemental figure 5a ). We focused on the large maize BHLH family which has 175 members comprising twelve main groups based on structural similarities to Arabidopsis BHLHs ( Heim et al 2003 ).…”

“…DoubleDAP-seq experiments were carried out by simultaneously expressing pIX-HALO-TFs and pIX-SBPtag clones ( Li et al 2023 ) in a 50 ul TNT rabbit reticulocyte reaction containing 1 ug of each plasmid in a 2 hour incubation at 30 degrees C. Subsequently 1 ug of adapter-ligated library was diluted in wash buffer and added to the reticulocyte reaction together with 10 ul of MagneHALO beads for a final volume 100 ul. Samples were rotated for 2 hours at room temperature, washed eight to ten times in wash buffer with the use of a magnet.…”

Recent advances in exploring transcriptional regulatory landscape of crops

“…Nonetheless, DAP-seq method does not fully capture chromatin state information or the cofactors of TFs that can influence TF-DNA binding in vivo . To solve these issues, modified methods such as sequential DAP-seq (seq-DAP-seq) and double DAP-seq (dDAP-seq) techniques have been developed ( Lai et al., 2020 ; Li et al., 2023a ). In seq-DAP-seq, a sequential purification based on multiple tags was used.…”

Classification and Regression Trees to predict Transcription Factor Combinatorial Interaction in scRNA-seq data

“…For instance LEAFY (LFY), being a major regulator of flower development, changes its binding site preference under the cooperative action of UNUSUAL FLORAL ORGANS (UFO) (Rieu et al, 2023). Another recent example shows that bZIP transcription factors are in combination binding to variable Cis-Regulatory-Elements (CREs) (Li et al, 2023). From a regulatory perspective it has also been demonstrated that genome wide transcriptomic response triggered by Nitrogen provision is also the result of a combinatorial action of TFs (Brooks et al, 2019).…”