The Buck lab integrates experimental and computational approaches to determine the rules dictating transcription factor (TF) targeting in a Eukaryotic genome and apply our findings to human clinical samples. To address this question we us model systems (mice and yeast) and human cell culture. Specifically, we investigate TF binding selection in response to environmental stress, characterize the chromatin mediated mechanisms directing TF target selection, determine how developmental signals reshape the epigenetic landscape during cellular development, and develop bioinformatics tools to analyze and interpret next-generation chromatin datasets. The lab uses state of the art genomic techniques including ChIP-seq, FAIRE-seq, and MNase-seq and is composed of both a molecular and computational lab. Currently the lab is focusing on the following projects:

Determining how the Tup1 co-repressor is targeted and how it regulates chromatin structure in budding yeast

Up-regulation of Transducin-like Enhancer of Split (TLE) proteins is associated with astrocytoma, meningioma, pituitary adenoma, synovial sarcoma, and lung adenocarcinoma. TLE1, the human homolog of fly Groucho and yeast Tup1, represses transcription by recruiting chromatin remodeling proteins which establishes repressive chromatin architecture, and is involved in several signal-transduction cascades, such as Notch, Wingless/Wnt, and DPP/BMP. The budding yeast homolog Tup1 has been a model for studying similar repressor complexes in multicellular eukaryotes. Tup1-Ssn6 does not bind DNA directly, but is directed to individual promoters by one or more DNA-binding proteins, referred to as Tup1 recruiters. The goal of this project is to determine how Tup1 identifies its sites across the genome and how it regulates chromatin structure at it targets.

Hanlon, S.E., Rizzo, J.M., Tatomer, D.C, Lieb, JD, and Buck, M.J. (2011). The stress response factors Yap6, Cin5, Phd1, and Skn7 direct targeting of the conserved co-repressor Tup1-Ssn6 in S. cerevisiae. PLOS One, 6(4): e19060.

Rizzo, J.M., Mieczkowski, P.A., Buck, M.J. (2011). Tup1 stabilizes promoter nucleosome positioning and occupancy at transcriptionally plastic genes. Nucleic Acids Research. 2011 Jul 23. Epub ahead of print

Nucleosome inhibition of transcription factor binding

Chromatin structure and nucleosome positioning has been postulated as the most important factor directing TFs to their appropriate binding sites. The goal of this project is to determine the principals and characteristics of nucleosome inhibition of TF binding. TF binding sites, located within DNA that is tightly wrapped within a nucleosome are typically inaccessible. Nucleosome inhibition of TF binding depends on at least seven factors: the type of TF and its concentration, nucleosome occupancy, histone tail modifications, binding site affinity, binding site location and site rotational setting within a nucleosome. Because these variables are not independent of each other, they need to be explored simultaneously or controlled stringently when studying regulation of TF targeting. However, this is highly impractical and requires thousands of experiments. To overcome this limitation, a unique approach combining yeast genetics with next-generation sequencing is applied, which allows us to study the relationships between the above factors and TF binding.

Buck, M.J., and Lieb, J.D. (2006). A chromatin-mediated mechanism for specification of conditional transcription factor targets.  Nature Genetics Dec; 38(12): 1446-51.

Identification of Epigenetic Biomarkers

Epigenetic alterations have been associated with cancer-specific expression differences in development of human tumors. The ability to recognize and detect the progression of epigenetic events occurring during tumorigenesis is critical to developing strategies for therapeutic intervention. Key epigenetic alterations, leading to silencing or activation, are associated with changes in nucleosome occupancy. We use a straightforward, reproducible, genomic approach for measuring chromatin accessibility Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) combined with next generation sequencing (FAIRE-seq). FAIRE isolates nucleosome-depleted genomic regions, which are the functionally active regions, and these regions represent ideal locations to identify chromosomal aberrations or SNP's associated with tumor formation.

Identification of shared chromatin architectures

Currently the only way to characterize chromatin architecture is to have an accurately mapped functional element in the genome. Functional elements include genes for protein and non-coding RNAs, and regulatory sequences that direct essential functions such as gene expression, DNA replication, and chromosome inheritance. With an accurately mapped functional element, chromatin structural data is aligned by the genomic coordinates and an average profile is created.To determine the chromatin architecture at unknown or at inaccurately mapped functional elements we are developing chromatin alignment algorithms and applying them to genome wide chromatin datasets.

Lai, W., and Buck, M.J. (2010) ArchAlign: Coordinate-free alignment of chromatin datasets reveals novel architectures.  Genome Biology Dec 23; 11(12):R126.