Shechter Lab > Projects

Histone chaperones and their regulation by glutamylation

Chaperone proteins bind to histones and escort them through the cell. In the nucleus, they remove and deposit histones during transcription, replication, and repair of the DNA. We study how chaperones bind to histones both to stabilize them and to deposit histones into nucleosomes. We propose that the major clue to chaperone molecular mechanisms lies in their only conserved feature: their acidic-stretches embedded in intrinsically disordered regions (IDRs). In this "Fly Fishing for Histones" model (here), the IDRs are baited to catch and release histone proteins. Our studies use novel structural and biological approaches to target these chaperone IDRs and their post-translational modifications, particularly the unusual glutamylation of the acidic IDRs. Our studies, ongoing in both Xenopus laevis cell-free extracts and in AML cancer cells, are exploring the critical role of chaperone glutamylation in regulation of these essential biological processes.

PRMTs and Arginine Methylation

PRMTs1-9 (Protein Arginine Methyltransferases) catalyze the post-translational methylation of protein arginines, including in histones, RNA-binding proteins, and splicing factors. They are critical for early development; they are also outstanding candidates for cancer chemotherapy because misregulation of their activity contributes to proliferative and invasive cellular phenotypes. We study how PRMTs select their substrates, how their activity is regulated, and what arginine methylation does in the cell to regulate biological function. We are currently focused on the major enzymes PRMT1 and PRMT5, their lung cancer relevance, transcriptional and splicing regulation, and the "reading" of methylarginine by effector proteins such as WDR5 (left and here). We use biochemistry, enzymology, structural biology, mass spectrometry, cell culture, RNA-Seq and PRO-Seq transcriptomics, and ChIP-Seq epigenomics to unravel the complex biological phenomena regulated by PRMTs and arginine methylation.

There are three types of arginine methylation: monomethyl (Rme1 or MMA); asymmetric dimethyl (Rme2a or ADMA); and symmetric dimethyl (Rme2s or SDMA)

Cancer biology

Cancer cells use many strategies to overcome growth and differentiation controls leading to the variety of disease presentations. In addition to mutation of oncogenes and tumor suppressors, it is now clear that simply alteration in gene expression, RNA splicing, and protein production is a frequent occurrence in human cancers. In particular, regulators of chromatin and histones and splicing factors are commonly misregulated in cancer cells.

We are specifically interested in the role of PRMTs, histones, and splicing factor methylation in lung cancers. Because their activity contributes to proliferative and invasive phenotypes, protein methyltransferases are outstanding candidates for cancer chemotherapy. In many lung cancer patients, overexpression of the protein arginine methyltransferases PRMT1, PRMT4/CARM1, and PRMT5 are associated with poor overall prognoses. Furthermore, at least five Phase I clinical trials–including in non-small cell lung cancer–are currently underway for drugs inhibiting these enzymes. However, as these enzymes have hundreds of similar substrate proteins, their mechanisms of promoting lung cancer tumorigenesis and metastasis are unclear. Lack of detailed mechanistic understanding of both of cellular and molecular roles of PRMTs in lung cancer is detrimental to effective future use of PRMT inhibitors in the clinic. Our goal is to determine how and why drugging PRMTs could be effective lung cancer chemotherapies.

We also focus on the role of histone chaperones in leukemia cells. Mutations in the histone chaperone Nucleophosmin/NPM1 (annotated as NPM1c) are found in up to 35% of adult patients with acute myeloid leukemia (AML) (1). However, the mechanisms by which the NPM1c mutation transforms hematopoietic cells are still poorly understood. We are studying mechanisms regulating NPM1 and other chaperones to understand how they function to alter cancer cell gene expression.

Xenopus laevis biology

Cell-free extracts of Xenopus laevis (African clawed frog) are our classic model system, providing a robust biochemical approach for testing our hypotheses in quasi-in vivo fashion. These extracts allow us to probe chromatin and other activities in the absence of on-going transcription and to identify critical post-translational modifications. Extracts recapitulate all critical cellular and nuclear functions, but in a test tube. These experiments are unique and important to understanding how PRMTs and histone chaperones function.

Additionally, we are interested in the biology of gametogenesis and egg laying. Using PET and CT scanning, we are conducting studies into consequences of hormones on oocyte maturation.