The long-term goal of our group is to understand how a single transcription factor can regulate vastly different sets of genes depending on the cell type and to identify and study processes that influence the expression level of individual genes.
We study transcriptional regulation using the glucocorticoid receptor (GR), a member of the steroid hormone receptor family. Upon hormone stimulation, GR binds to specific DNA sequences to regulate the expression of target genes. Although GR is expressed throughout the body, the genes regulated and the genomic loci bound by GR show little overlap between cell-types. Current efforts are aimed to investigate the role of sequence motifs and chromatin in cell-type-specific genomic binding and transcriptional regulation. Further, we study signals involved in fine-tuning expression levels of individual target genes, specifically the role of DNA as a ligand that allosterically modulates the activity of GR.
Focus 1: Role of genetic and epigenetic landscape in guiding GR to specific genomic loci
Binding-site motifs of eukaryotic transcription factors typically only have a handful of constrained nucleotide positions. Hence, these sequences are ubiquitously found in the genome whereas binding only occurs at a small subset of these putative genomic binding sites. Consequently, the binding site motif in itself provides insufficient information and a combination of inputs needs to be integrated to specify where transcription factors bind (Figure 3). Our major goal is to identify genetic (DNA sequence elements) and epigenetic (e.g. histone modifications) inputs that specify where GR binds genomically.
We map the genomic loci of GR binding using ChIP-seq in cell types derived from different tissues including a cell line with a well-characterized epigenome (IMR90: data includes DNA methylation, DNAse-I sensitivity, >20 histone modifications, RNA-seq, www.roadmapepigenomics.org.). To identify genetic and epigenetic features that correlate with genomic binding we employ bioinformatical approaches including linear modeling. The relevance of the identified features is probed in cell lines and animal models including zebrafish. For example by assaying how perturbation of these signals (e.g. knockdown of genes responsible for depositing certain histone modifications) alters the binding profile of GR. Similarly, transcription reporter constructs allow us to test the role of identified DNA sequence motifs in determining where GR binds.
We have identified genetic and epigenetic features that correlate with GR binding. Interestingly, many of these sequence signals are cell type specific and functional studies using transcriptional reporters indicate that they play a critical role in directing cell-type specific transcriptional regulation by GR.
We also find sequence motifs and epigenetic features that are depleted at sites of GR binding making these features candidates to prevent the binding of GR. Studies using zinc finger nucleases to create isogenic cell lines with genomically integrated reporters and experiments in zebrafish showed that these depleted motifs interfere with GR binding and with GR-dependent activation of transcription (>90% reduction in activation).