We are an interdisciplinary team that studies the basic biochemical and biophysical processes that drive the regulation of chromatin structure. As part of this pursuit, we are developing new and innovative in vitro systems, “omic” technologies and computational approaches to answer several questions:
To this end, we are actively engaged in two major areas of research:
DNA methylation in mammals occurs primarily at cytosine residues of CpG dinucleotides. The faithful mitotic inheritance of DNA methylation patterns is essential for mammalian development and has been well studied for its role in the stable repression of transposons and endogenous retroviruses, gene silencing, genomic imprinting and X-chromosome inactivation. DNA methylation represses transcription directly by inhibiting the retention of sequence-specific DNA-binding transcription factors and indirectly through the recruitment of co-repressor complexes. DNA methylation patterns are established by the de novo methyltransferases DNMT3A and DNMT3B and are epigenetically inherited through somatic cell divisions by the replication-coupled maintenance DNA methyltransferase DNMT1. DNA methylation is erased passively with cell division in the absence of DNMT activity and actively by the TET family of DNA hydroxylases.
The protein UHRF1 has emerged in recent years as a key regulator of DNA methylation inheritance. Mice lacking either DNMT1 or UHRF1 show similar phenotypes of global DNA hypomethylation (Figure 1A) and early embryonic death. We and others have shown that the DNA methylation regulatory function of UHRF1 depends on its ability to bind chromatin (Rothbart et al 2012 Nat Struc Mol Biol; Rothbart et al 2013 Genes Dev), where it facilitates histone H3K18 and H3K23 mono-ubiquitination, a docking site for DNMT1 (Harrison et al 2016 eLife). Our studies show that the UHRF1 reader and writer domains function in a coordinated manner to engage the histone and DNA components of chromatin, to direct its ubiquitin ligase activity towards H3K18 and H3K23, and to regulate the epigenetic inheritance of DNA methylation (Vaughan et al 2018 Nuc Acids Res; Vaughan et al 2018 Proc Natl Acad Sci USA) (Figure 1B-C).
Notably, our findings assign a key biological function to hemi-methylated DNA as a member of an emerging class of non-protein ligand activators of E3 ligases, and they define the relationship between the enzymatic, histone and DNA-binding activities of UHRF1 as a multidomain epigenetic regulator of DNA methylation inheritance. Our studies present the first demonstration of an E3 ubiquitin ligase being activated by DNA binding and are the first to show that an epigenetic modification controls E3 ligase function. Using UHRF1 as a model system, we are working to define the underlying principles of multivalent engagement and allosteric regulation of enzymatic activity on chromatin, and we are applying these conceptual advances to the study of other biologically interesting and disease-relevant chromatin regulators.
The appreciation of UHRF1 as a key regulator of DNA methylation inheritance suggests UHRF1 antagonism as an innovative therapeutic strategy to target aberrant DNA methylation patterning, which is a hallmark of human cancers. The role of UHRF1 in tumorigenesis and the therapeutic potential of UHRF1 inhibition have now become major translational foci of the lab. This translational potential also drives our continuing basic studies of UHRF1 function and mechanism.
Histone post-translational modifications (PTMs) and their combinations function in two ways:
We contributed to the development of peptide microarray technology to query the activities of known and putative histone PTM regulators (Rothbart et al 2012 Methods Enzymol; Cornett et al 2016 Methods Enzymol) (Figure 2). We have used this platform extensively to characterize the reader, writer and eraser activities of these regulators and the behavior of antibodies that recognize histones and their PTMs (Rothbart et al 2015 Mol Cell; Shah et al 2018 Mol Cell). Because histone PTM antibodies are key reagents for immunoblotting, immunostaining and chromatin immunoprecipitation (ChIP) experiments, we continually profile the behavior of the most used and cited commercial histone PTM antibodies and compile this information into a public database (www.histoneantibodies.com). This interactive web resource is a valuable tool for the research community that routinely uses these antibodies in a wide range of applications.
From large-scale cancer exome sequencing studies, we now appreciate that chromatin regulators (including writers, erasers and readers of histone PTMs) are among the most frequently amplified and mutated genes in human cancers. This has sparked tremendous interest from the cancer and chromatin biology communities and has led to the development of small-molecule inhibitors that are in various phases of preclinical and clinical development. Defining the specificities of histone (and non-histone) PTM writers, erasers and readers is therefore critical for understanding the mechanisms of action of emerging epigenetic drugs, for rationalizing potential combination therapies and for identifying reliable biomarkers of clinical activity. To this end, we are developing new functional proteomics platforms that query the activities methyllysine writers (Cornett et al 2018 Science Adv) (Figure 3), erasers and readers. We consider these screening efforts analogous to genome and epigenome roadmap initiatives, where cartography and public data dissemination provide a foundation for later molecular studies. Datasets and bioinformatics tools generated from these studies are being used to facilitate comprehensive mapping of methylation sites throughout the human proteome, to promote the discovery of previously unknown substrates and interaction networks, and to identify optimal substrates for drug screening and structural studies.