Project description:In eukaryotes, many transcription factors (TF) usually form dimers or oligomers with itself or other TFs to regulate gene expression. To study how homo- and heterodimerization of TFs affect DNA binding specificity, we developed a double DNA Affinity Purification sequencing (double DAP-seq; dDAP-seq) technique that maps heterodimer binding sites in endogenous genome context and applied it to elucidate the binding profiles of homo- and heterodimers of the Group C and S1 basic leucine zipper (bZIP) transcription factors in Arabidopsis. Genome-wide binding profiles of twenty pairs of bZIP C/S1 heterodimers and bZIP S1 homodimers revealed that heterodimerization significantly expands the DNA binding preferences of homodimers, creating unique binding sites and target gene functions. In addition to the classical ACGT elements recognized by plant bZIPs, we found the Group C/S1 dimers bind to sequence motifs that might share an origin with the yeast bZIP GCN4. Further analysis of heterodimer-specific binding sequences uncovered two types of motif recognition patterns that mediate heterodimer specificity. Our study shed light on the functions and mechanisms of TF dimerization and demonstrated the potential of dDAP-seq in deciphering the complexity of these interactions within or across TF families.
Project description:We assessed how the effects of transcription factor binding sites (TFBSs) on the activity of a cis-regulatory sequence (CRS) depend on the local sequence context. We constructed a library of synthetic CRSs composed of TFBSs enriched in mouse photoreceptors and assayed them by massively parallel reporter assay in live mouse retina. We used the resulting data to train neural network-based models that predict CRS activity from sequence.
Project description:Development of the human malaria parasite, Plasmodium falciparum is regulated by a limited number of sequence-specific transcription factors (TFs). However, the mechanisms by which these TFs recognize genome-wide binding sites to regulate target genes is still largely unknown. To address TF target specificity, we investigated the binding of two TF subsets that either bind CACACA or GTGCAC and further characterized PfAP2-G and PfAP2-EXP which bind unique DNA motifs (GTAC and TGCATGCA). We interrogated the impact of DNA sequence context and the chromatin landscape on P. falciparum TF binding using high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post-translational modifications, and chromatin accessibility. We determined that DNA sequence context does not greatly impact binding site selection for CACACA-binding TFs, while chromatin accessibility, epigenetic patterns, co-factor recruitment, and dimerization contribute to differential binding. In contrast, GTGCAC-binding TFs prefer different sequence contexts, DNA shape profiles, and chromatin dynamics. Finally, we find that TFs that preferentially bind divergent DNA motifs may bind overlapping genomic regions in vivo due to low-affinity binding to other sequence motifs. Our results demonstrate that TF binding site selection relies on a combination of DNA sequence and chromatin features, thereby contributing to the complexity of P. falciparum gene regulatory mechanisms.
Project description:We identified the DNA binding sites of DPY-27 in C. elegans. DPY-27 was C-terminally tagged with GFP and the expression pattern was examined through fluorescent microscopy. The binding sites were determined using chromatin immunoprecipitation with anti-GFP antibody followed by illumina high-throughput sequencing (ChIP-seq). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Identification of DPY-27 binding sites at embryonic stage
Project description:We identified the DNA binding sites of MAB-5 in C. elegans. MAB-5 was C-terminally tagged with GFP and the expression pattern was examined through fluorescent microscopy. The binding sites were determined using chromatin immunoprecipitation with anti-GFP antibody followed by illumina high-throughput sequencing (ChIP-seq). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Identification of MAB-5 binding sites at L3 stage