Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks

Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin

p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins

Identifying a potential substrate of Plasmodium Falciparum cell cycle regulatory Kinase PFPK5

Malaria remains a global health problem, despite over a century of efforts towards control and prevention. It is responsible for over 2 million deaths a year. Plasmodium falciparum, the protozoan parasite that causes malaria, presents quite an unexplored field of study, significant both for the purposes of understanding the complex life cycle of the parasite, and for identifying novel and unique targets for anti-malarial therapy. Cyclin-dependent kinases. (CDK.s) play a number of crucial roles in the progression of the cell cycle such as regulating the onset of DNA replication and entry into mitosis. Plasmodium falciparum protein kinase 5, PfPK5, manifests characteristics of eukaryotic CDKs. It is a serine/threonine kinase, has 60% amino acid identity to eukaryotic cyclin-dependent kinase cdc2, and shares the mechanism of activation with CDKs. To establish if PfPK5 indeed is the major cell cycle regulatory kinase, as well as to expand our knowledge about the signaling networks of the parasite, it is necessary to identify proteins that interact with the kinase, such as its putative substrates. Currently, only one Plasmodium falciparum protein is known to interact with PfPK5 - its cyclin partner, Pfcycl. Identifying substrates of PfPKS is a particularly important research endeavor since it would provide insight into the yet unknown downstream signaling pathways of PfPK5. It is likely that pathways unique to Plasmodium falciparum will be found, which may be specifically targeted for anti-malaria therapy. A potential substrate of Plasmodium falciparum cell cycle regulatory kinase PfPK5 has been identified. The new protein, which we call SPOK, was identified by screening a phage display cDNA library. Since SPOK is a large protein of approximately 140kDa, a domain containing a tandem CDK/cdc2 phosphorylation motif of SPEK (single amino acid code, S/TPXK/R) was expressed in E.coli. Our results show that this domain of SPOK is indeed phosphorylated in vitro by PfPK5. This raises the possibility that SPOK could be an in vivo substrate of PfPK5 and may play a role in regulating the cell cycle of the parasite

The MBT Repeats of L3MBTL1 Link SET8-mediated p53 Methylation at Lysine 382 to Target Gene Repression*

The p53 tumor suppressor protein is regulated by multiple post-translational modifications, including lysine methylation. We previously found that monomethylation of p53 at lysine 382 (p53K382me1) by the protein lysine methyltransferase (PKMT) SET8/PR-Set7 represses p53 transactivation of target genes. However, the molecular mechanism linking p53K382 monomethylation to repression is not known. Here we show in biochemical and crystallographic studies the preferential recognition of p53K382me1 by the triple malignant brain tumor (MBT) repeats of the chromatin compaction factor L3MBTL1. We demonstrate that SET8-mediated methylation of p53 at Lys-382 promotes the interaction between L3MBTL1 and p53 in cells, and the chromatin occupancy of L3MBTL1 at p53 target promoters. In the absence of DNA damage, L3MBTL1 interacts with p53K382me1 and p53-target genes are repressed, whereas depletion of L3MBTL1 results in a p53-dependent increase in p21 and PUMA transcript levels. Activation of p53 by DNA damage is coupled to a decrease in p53K382me1 levels, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from p53-target promoters. Together, we identify L3MBTL1 as the second known methyl-p53 effector protein, and provide a molecular explanation for the mechanism by which p53K382me1 is transduced to regulate p53 activity