Conference Program - ABRF 2011 - Association of Biomolecular ...

(S4-2) Chromatin Dynamics in Melanoma: A Role forMacroH2AA. Kapoor 1,2 , M. Goldberg 1,2 , L. Cumberland 1,2 *,K. Ratnakumar 1,2 *, M. Segura 4,6 , P. Emanuel 2,3 ,S. Menendez 4,6 , C. Vardabasso 1,2 , G. LeRoy 7 , C. Vidal 2,3 †,D. Polsky 4,5,6 , I. Osman 5,6 , B. Garcia 7 , E. Hernando 4,6 ,E. Bernstein 1,21Department of Oncological Sciences, Mount Sinai Schoolof Medicine, New York, NY, United States; 2 Departmentof Dermatology, Mount Sinai School of Medicine, NewYork, NY, United States; 3 Department of Pathology, MountSinai School of Medicine, New York, NY, United States;4Department of Pathology, New York University LangoneMedical Center, New York, NY, United States; 5 Departmentof Dermatology, New York University Langone MedicalCenter, New York, NY, United States; 6 InterdisciplinaryMelanoma Cooperative Group, New York UniversityLangone Medical Center, New York, NY, United States;7Department of Molecular Biology, Princeton University,Schultz Laboratory, Princeton, NJ, United States; †PresentAddress: Department of Dermatology, Saint Louis UniversitySchool of Medicine, St. Louis, MO, United States; *Theseauthors contributed equally to this work.Cancer is a disease consisting of both genetic and epigenetic changes.Although increasing evidence demonstrates that tumour progressionentails chromatin-mediated changes such as DNA methylation, therole of histone variants in cancer initiation and progression currentlyremains unclear. Histone variants replace conventional histones withinthe nucleosome and confer unique biological functions to chromatin.Using well characterized, paired series of murine and human melanomacells lines, we probed the epigenetic profile of melanoma. Analysisof histones from both series using multiplexed quantitative massspectrometryrevealed changes in several histone posttranslationalmodifications and histone variants. The loss of mH2A isoforms,histone variants generally associated with condensed chromatin andfine-tuning of developmental gene expression programs is positivelycorrelated with increasing malignant phenotype of melanoma cellsin culture and human tissue samples. Knockdown of mH2A isoformsin melanoma cells of low malignancy results in significantly increasedproliferation and migration in vitro and growth and metastasis invivo. Restored expression of mH2A isoforms rescues these malignantphenotypes in vitro and in vivo. We demonstrate that the tumourpromotingfunction of mH2A loss is mediated, at least in part, throughdirect transcriptional upregulation of CDK8. Suppression of CDK8, acolorectal cancer oncogene inhibits proliferation of melanoma cells,and knockdown of CDK8 in cells depleted of mH2A suppresses theproliferative advantage induced by mH2A loss. Moreover, a significantinverse correlation between mH2A and CDK8 expression levels existsin melanoma patient samples. Taken together, our results demonstratethat mH2A is a critical component of chromatin that suppresses thedevelopment of malignant melanoma, a highly intractable cutaneousneoplasm.(S4-3) Using Protein Domain Microarrays to Read theHistone CodeM.T. BedfordMD Anderson Cancer Center, Department of MolecularCarcinogenesis, Smithville, TX, United StatesFor cells to survive, differentiate, and grow, information has to betransferred from the cell surface to the nucleus. This process is referredto as signal transduction. A hallmark of cancer is the deregulation ofsignal transduction pathways. Signaling events in eukaryotic cells involvethe assembly and disassembly of large protein-protein complexes.These diverse associations are mediated through interactions ofa limited number of modular signaling units or protein-domains.Protein interactions involving domains are often regulated by posttranslationalmodification (PTM – like phosphorylation, methylationand acetylation) of the smaller protein motif within the ligand. Wehave developed a chip-size protein microarray that harbors a displayof over 300 modular protein-interacting domains including SH2, SH3,PDZ, FHA, 14-3-3, WW, Chromo, Tudor, PHD and MBT domains. Inthe emerging proteomic era, it is becoming easier to identify proteinsusing tryptic digestion followed by mass spectrometric approaches.These same methods also detect sites of posttranslational modificationon proteins. Many of these posttranslational modifications likelygenerate docking sites for protein modules. We have developedprotein-domain microarray technology to help identify proteins thatcan interact with motifs that are either methylated or phosphorylated.This high-throughput approach facilitates the rapid identificationof protein-protein interactions in vitro. Further in vivo studies areneeded to confirm that these interactions do indeed occur in biologicalsystems. Protein domains are cloned into a GST expression vector, andrecombinant protein is produced in bacteria. These fusion proteinsare then arrayed onto nitrocellulose coated glass slides using a robot.These slides are probed with biotinylated peptides that are preconjugatedto streptavidin-Cy3. The peptides used in this experimentare synthesized as 15 mers, and both the modified and unmodifiedforms of the peptides are tested on the array. In this manner, we canidentify novel methyl- and phospho-dependent interactions. We havebuilt three types of arrays: (1) A phospho-tyrosine reader harbors 70SH2 domains and 5 PTB domains (total = 75 domains). (2) A phosphothreonine/serinereader that harbors 7 14-3-3 domains, 5 FHAdomains, 15 BRCT domains and a WW domain (total = 28 domains).(3) An epigenetic reading array that harbors methyl and acetyl readers.This array is composed of 50 tudor domains, 22 bromo domains, 36PHD domains, 17 MBT domains, 11 WD40 domains, 9 SANT domains,28 chromo domains, 15 PWWP domains, 5 BRK domains, 5 CWdomains, and 9 Ank repeats (total = 207 domains). More and moreposttranslational modifications are being discovered on proteins. Theroles of many of these methylation and phosphorylation events oftenremain obscure. This approach provides an easy way for a researcherto identify potential binding partners for their favorite proteins.These arrays thus offer researchers tools to get at “mechanism”. Onceinvestigators know that they are working with a clearly functional PTM,they can proceed with confidence to generate modification specificantibodies and interrogate the signaling pathway that is engaged bythe identified PTM-driven protein-protein interaction.Scientific SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 39