Precise transcriptional regulation is necessary for processes from the stress response to the specification of cellular identity. Alterations in transcription are found in human diseases including cancers, neurological disorders, and developmental diseases. Thus, understanding core mechanisms of transcriptional control is essential to understanding both basic cellular processes and the pathogenesis of many human diseases. We seek to understand how transcriptional initiation is regulated through the use of genetic and genomic tools in budding yeast and mammalian cells.
Epigenomic technology development
Genome-wide mapping of protein binding sites has become a staple in many areas of modern molecular biology. This is most commonly done by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). However, recent studies have indicated that caution may be warranted when using currently available ChIP-based methods. Crosslinking ChIP-seq (X-ChIP-seq) methods rely on formaldehyde to preserve protein-DNA interactions. However, formaldehyde preferentially generates protein-protein crosslinks and thus may cause epitope masking and capture of transient mass action-driven interactions with chromatin. Recent studies have also described the existence of "hyper-ChIPable" regions, wherein multiple unrelated proteins including nuclear-localized GFP and a Golgi protein can be localized to actively transcribed regions. To circumvent these issues, we adapted chromatin endogenous cleavage (ChEC), originally developed by Ulrich Laemmli, to a high-throughput sequencing readout (ChEC-seq). In ChEC, microccocal nuclease (MNase) is genetically fused to a protein of interest to target calcium-dependent cleavage to specific genomic loci in vivo. We found that ChEC-seq not only provides high spatial resolution of transcription factor (TF) binding sites in yeast, but provided kinetic information about TF association with the genome.
Transcriptional regulation by Mediator
Mediator is a conserved, essential transcriptional coactivator complex thought to be generally required for transcription by RNA Polymerase II. We have used ChEC-seq to map Mediator binding to the yeast genome, revealing binding exclusively to UASs under normal growth conditions as well as an uncoupling of transcriptional output from Mediator levels at the UAS. In addition, we found a mutual dependency in chromatin recruitment between Mediator and the TFIID coactivator complex, a component of the transcription pre-initiation complex (PIC) that is one of the first factors to binding during PIC assembly. We are now exploring the functions of Mediator in recruiting other components of the PIC to understand its role in promoting transcription initiation.
Functions of CTCF and its paralog BORIS
CCCTC-binding factor (CTCF) is well known as a Mediator of chromatin architecture via its binding to insulator elements. CTCF has a paralog, Brother of the Regulator of Imprinted Sites (BORIS) that is normally expressed only in germ cells but is aberrantly expressed in cell lines representing a variety of cancers. 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.