Abstract 2446 Evaluation of DNA-binding preferences of human SIX family transcription factors using DAP-seq

Transcription factors (TFs) are proteins that bind to specific DNA sequences known as cis-regulatory elements (CREs). These proteins interact with the genome to regulate gene expression, making them essential for virtually every cellular process. There are 1,639 TFs currently identified in humans, accounting for nearly 8% of the gene content. This is important to consider since both mutations in TFs and TF-binding sites (TFBS) have been associated with multiple diseases. Therefore, progress towards understanding how TFs mediate binding with the genome will progress our understanding of gene regulation and its implications on health and disease. The sine oculis homeobox (SIX) family of TFs are known to be regulators of developmental processes during embryogenesis. Additionally, several human congenital conditions have been connected to genetic mutations in human SIX genes, including Branchio-oto-renal (BOR) Syndrome and Holoprosencephaly. This study aims to elucidate the DNA-protein interactions of the human SIX family of TFs. To achieve this goal, we will use the high-throughput DNA-binding assay known as DNA Affinity Purification Sequencing (DAP-seq). This method depicts the local sequence context through genome-wide-binding site maps. The first step we took to carry out our protocol was to create a human genomic DNA (gDNA) library. PCR amplification of the gDNA library was also done to remove secondary DNA modifications, creating an ampDAP-seq library. The second step involved the expression of the SIX TF proteins through a cell-free system. SIX2 and SIX4 were expressed successfully, and an Electrophoretic Mobility Shift Assay (EMSA) showed that these SIX proteins were functional by forming complexes with DNA probes. Our future steps involve the DNA affinity purification of the DNA-protein complexes. Subsequently, DNA elution, PCR enrichment and pooling will be performed. Next, DNA sequencing will be carried out by next-generation sequencing on the Illumina platform, then the files of the sequencing reads will be aligned to a reference genome using a standard short read mapping software. DAP-seq data will be analyzed by standard peak calling using the mapped read files, identifying peaks that indicate TFBS. The mapped reads and peaks will be examined in a genome browser and motif characterization programs will be used to identify binding sequences of the TFs. Finally, by taking advantage of the DAP-seq method, we anticipate that our findings will advance the field of genomics and contribute to the broader efforts in deciphering the intricacies of transcriptional regulation and their effects on human health.