Discovery and Mechanism of Small Molecule Inhibitors Selective for the Chromatin-Binding Domains of Oncogenic UHRF1.

Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist.

Molecular investigation of the tandem Tudor domain and plant homeodomain histone binding domains of the epigenetic regulator UHRF2.

Ubiquitin-like containing PHD and ring finger (UHRF)1 and UHRF2 are multidomain epigenetic proteins that play a critical role in bridging crosstalk between histone modifications and DNA methylation. Both proteins contain two histone reader domains, called tandem Tudor domain (TTD) and plant homeodomain (PHD), which read the modification status on histone H3 to regulate DNA methylation and gene expression. To shed light on the mechanism of histone binding by UHRF2, we have undergone a detailed molecular investigation with the TTD, PHD and TTD-PHD domains and compared the binding activity to its UHRF1 counterpart.

Catalysis by protein acetyltransferase Gcn5.

Gcn5 serves as the defining member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of proteins that display a common structural fold and catalytic mechanism involving the transfer of the acyl-group, primarily acetyl-, from CoA to an acceptor nucleophile. In the case of Gcn5, the target is the ε-amino group of lysine primarily on histones. Over the years, studies on Gcn5 structure-function have often formed the basis by which we understand the complex activities and regulation of the entire protein acetyltransferase family.

High-throughput strategy to identify inhibitors of histone-binding domains.

Many epigenetic proteins recognize the posttranslational modification state of chromatin through their histone-binding domains and thereby recruit nuclear complexes to specific loci within the genome. A number of these domains have been implicated in cancer and other diseases through aberrant binding of chromatin; therefore, identifying small molecules that disrupt histone binding could be a powerful mechanism for disease therapy. We have developed a high-throughput assay for the detection of histone peptide-domain interactions utilizing AlphaScreen technology.

Autoacetylation of the histone acetyltransferase Rtt109.

Rtt109 is a yeast histone acetyltransferase (HAT) that associates with histone chaperones Asf1 and Vps75 to acetylate H3K56, H3K9, and H3K27 and is important in DNA replication and maintaining genomic integrity. Recently, mass spectrometry and structural studies of Rtt109 have shown that active site residue Lys-290 is acetylated. However, the functional role of this modification and how the acetyl group is added to Lys-290 was unclear.

KAT(ching) metabolism by the tail: insight into the links between lysine acetyltransferases and metabolism.

Post-translational modifications of histones elicit structural and functional changes within chromatin that regulate various epigenetic processes. Epigenetic mechanisms rely on enzymes whose activities are driven by coenzymes and metabolites from intermediary metabolism. Lysine acetyltransferases (KATs) catalyze the transfer of acetyl groups from acetyl-CoA to epsilon amino groups.

Catalytic activation of histone acetyltransferase Rtt109 by a histone chaperone.

Most histone acetyltransferases (HATs) function as multisubunit complexes in which accessory proteins regulate substrate specificity and catalytic efficiency. Rtt109 is a particularly interesting example of a HAT whose specificity and catalytic activity require association with either of two histone chaperones, Vps75 or Asf1. Here, we utilize biochemical, structural, and genetic analyses to provide the detailed molecular mechanism for activation of a HAT (Rtt109) by its activating subunit Vps75.

Kinetic mechanism of the Rtt109-Vps75 histone acetyltransferase-chaperone complex.

Rtt109 is a histone acetyltransferase (HAT) involved in promoting genomic stability, DNA repair, and transcriptional regulation. Rtt109 associates with the NAP1 family histone chaperone Vps75 and stimulates histone acetylation. Here we explore the mechanism of histone acetylation and report a detailed kinetic investigation of the Rtt109-Vps75 complex.

Where in the cell is SIRT3?--functional localization of an NAD+-dependent protein deacetylase.

Sirtuins are NAD+-dependent enzymes that have been implicated in a wide range of cellular processes, including pathways that affect diabetes, cancer, lifespan and Parkinson's disease. To understand their cellular function in these age-related diseases, identification of sirtuin targets and their subcellular localization is paramount. SIRT3 (sirtuin 3), a human homologue of Sir2 (silent information regulator 2), has been genetically linked to lifespan in the elderly.

Catalytic mechanism of a MYST family histone acetyltransferase.

Distinct catalytic mechanisms have been proposed for the Gcn5 and MYST histone acetyltransferase (HAT) families. Gcn5-like HATs utilize an ordered sequential mechanism involving direct nucleophilic attack of the N-epsilon-lysine on the enzyme-bound acetyl-CoA. Recently, MYST enzymes were reported to employ a ping-pong route of catalysis via an acetyl-cysteine intermediate.