- Postdoctoral Fellow, Fred Hutchinson Cancer Research Center, 2011-2015
- Ph.D., Case Western Reserve University, 2011
Affiliated Assistant Professor, Biology
Affiliated Assistant Professor, Biology
Myers Hall 230
Proper transcriptional regulation is essential for the establishment and maintenance of cellular identity. Consistent with its central role in cellular function, transcription is dysregulated in numerous human disorders, including cancer. We use genetic and genomic approaches to study the control of initiation, a critical step in transcription where numerous regulatory inputs are integrated. As many basic transcriptional mechanisms are conserved throughout evolution, we use the budding yeast Saccharomyces cerevisiae as a model system.
We use genome-wide methodologies to study transcriptional regulation. We develoed chromatin endogenous cleavage followed by high-throughput sequencing (ChEC-seq) as a method to map the genome-wide binding of proteins with high spatiotemporal resolution. ChEC-seq involves fusion of micrococcal nuclease (MNase), a calcium-dependent exo-/endonuclease to a chromatin-associated protein of interest. Upon addition of exogenous calcium to cells, MNase cleaves DNA in proximity to binding sites for the factor of interest. Total DNA is then purified and small fragments are sequenced to reveal genomic loci bound by the fusion protein. ChEC-seq has numerous advantages relative to ChIP-based methods: it avoids artifacts associated with crosslinking, sonication, and poor antibody quality, is controlled by calcium, and provides high spatial resolution. We leveraged the inducible nature of MNase to characterize two classes of transcription factor binding sites in budding yeast. We found that sites displaying rapid cleavage displayed robust matches to known DNA motifs, while sites that were cleaved more slowly had no such sequences. We are currently testing ChEC-seq with additional classes of chromatin-binding proteins in budding yeast and are developing the method in metazoan systems.
We are particularly interested in the functions of the Mediator complex, a conserved, essential coactivator generally required for transcription by RNA Polymerase II (RNAPII). Studies of Mediator function in budding yeast have been complicated by conflicting data regarding its genome-wide localization as determined by ChIP-seq. We used ChEC-seq to map the binding of Mediator to the budding yeast genome and confirmed that, under normal growth conditions, it is confined to upstream activating sequences (UASs). We also found that its binding to UASs is generally uncoupled from transcriptional output and that its binding to chromatin is cooperative with that of TFIID, another coactivator complex that is part of the RNAPII pre-initiation complex (PIC). We are using a combination of budding yeast genetics and ChEC-seq to furthur elucidate the role of Mediator in PIC recruitment.
In addition to addressing questions related to basic transcription mechanisms, we are interested in the function of CCCTC-binding factor (CTCF), well known for its function in chromatin insulation. While CTCF is ubiquitously expressed, it has a paralog, brother of the regulator of imprinted sites (BORIS), that is expressed in germ and cancer cells and is comparatively poorly understood. Recent work has demonstrated the existence of clustered CTCF target sites (2xCTSes) that are occupied by CTCF homodimers in BORIS-negative cells but are occupied by CTCF/BORIS heterodimers or BORIS homodimers in BORIS-positive cells. In contrast to single CTCF target sites (1xCTSes), which are though to be involved in chromatin insulation, 2xCTSes are associated with active gene regulatory elements. We are investigating the mechanisms by which CTCF and BORIS influence transcription and chromatin architecture through these two classes of sites and are also exploring novel functions for CTCF and BORIS through mapping their protein-protein interaction landscapes.
Tourigny JP, Saleh MM, Schumacher K, Devys D, Zentner GE (2018). Mediator is essential for small nuclear and nucleolar RNA transcription in yeast. Mol Cell Biol 38(24):e00296-18.
Bruzzone MJ, Grünberg S, Kubik S, Zentner GE, Shore D (2018). Distinct patterns of histone acetyltransferase and Mediator deployment at yeast protein-coding genes. Genes Dev 32(17-18):1252-1265.
Drury DW, Dapper AL, Siniard DJ, Zentner GE, Wade MJ (2017). CRISPR/Cas9 gene drives in genetically variable and nonrandomly mating wild populations. Sci Adv 3(5):e1601910.
Grünberg S, Henikoff S, Hahn S, Zentner GE (2016). Mediator binding to UASs is broadly uncoupled from transcription and cooperative with TFIID recruitment to promoters. EMBO J 35(22):2435-2446.
Zentner GE, Kasinathan S, Xin B, Rohs R, Henikoff S (2015). ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo. Nat Commun 6:8733.