In order to contribute to the development of scientists working on problems involving gene dysregulation, such as rare diseases, I wish to mentor a postdoctoral fellow as part of my recently funded SC1 grant project. I wish to recruit a fellow with expertise in cell culture,and other general molecular biology laboratory skills, such as transfections, protein expression and purification, etc. The postdoctoral fellow will assist the PI in the execution of all procedures. Moreover, this fellow will also interact/train with the collaborators, students and technician involved.
The long-term goal of the research project, which is titled "Mechanisms of TWIST bHLH Transcription Factors Binding to Functional Target Regions" is to determine the molecular mechanisms by which TWIST proteins decode genomic information and how genetic variation modulates TWIST2-genome interactions that impact craniofacial development. Two specific aims are proposed in this developmental proposal.
In Specific Aim 1, we will determine the binding affinities of TWIST proteins and selected TWIST mutant proteins as well as assess structural properties of the purified proteins and DNA-protein complexes. We will assay wild type and mutant proteins found in SS, BSS, AMS, SCS and SwCS patients by biolayer interferometry, electrophoretic mobility shift assays and structural studies via spectrophotometric methods such as circular dichroism and other methods for 3D structure determination. These experiments will help us to better understand the pathogenic mechanisms of mutant TWIST proteins as well as contribute to the development of methods to express and characterize transcription factors.
In Specific Aim 2, we will determine the DNA-sequence specificity of TWIST1/2 complexes (as homodimers or heterodimers with E12 as partner). We will use in vivo (ChIP) and in vitro (SELEX) DNA binding assays combined with DNA sequencing to determine the DNA-binding specificity of these complexes and the role that specific histone modifications (both activating and inactivating marks) and chromatin structure (using ATAC-Seq) have. Bioinformatic analyses will be performed in order to interpret changes in gene targets between wild-type and mutant proteins and determine the TWIST2 binding site sequences used to regulate gene expression of target genes.