MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v01/MSV000082363/primaryOK2000000Aaron Johnsonhttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=e64ba6c81e8640fa87351dd56cebb790aaron.m.johnson@ucdenver.eduMassIVE38UNIMOD:188 - "13C(6) Silac label."UNIMOD:259 - "13C(6) 15N(2) Silac label."UNIMOD:1 - "Acetylation."UNIMOD:28 - "Pyro-glu from Q."UNIMOD:267 - "13C(6) 15N(4) Silac label."UNIMOD:35 - "Oxidation or Hydroxylation."UNIMOD:6 - "Iodoacetic acid derivative."ProteomicsQ ExactiveSaccharomyces Cerevisiae (ncbitaxon:4932)University of Colorado DenverDNA packaged into chromatin is the core structure of the human genome. Nearly all eukaryotic genome regulation must interface with this genomic structure, and modification of the chromatin can influence molecular mechanisms that regulate the underlying DNA. Many processes are governed by regulated stepwise assembly mechanisms that build complex machinery on chromatin to license a specific activity such as transcription. Transcriptional activators drive the initial steps of gene expression, regulated in part by chromatin. Here we describe tools to study the stepwise assembly of protein complexes on chromatin in a highly controlled manner using reconstituted human chromatin platforms and quantitative proteomic profiling. We profile the early steps in transcriptional activation and highlight the potential for understanding the multiple ways chromatin can influence transcriptional regulation. We also describe modifications of this approach to study the activity of a long noncoding RNA to act as a dynamic scaffold for proteins to be recruited to chromatin. This approach has the potential to provide a more comprehensive understanding of important macromolecular complex assembly that occurs on the human genome. The reconstituted nature of the chromatin substrate offers a tunable system that can be trapped at specific substeps to understand how chromatin interfaces with genome regulation machinery.SILAC-MS Profiling of Reconstituted Human Chromatin Platforms for the Study of Transcription and RNA Regulation.Balas Maggie M MM, Porman Allison M AM, Hansen Kirk C KC, Johnson Aaron M AMnuclear chromatin, Regulations, study, ribonucleic acid, Acid, cytoplasmic chromatin, RNA, Ribonucleic, human being, Man (Taxonomy), Social Controls., ribose nucleic acid, Formal Social Control, Non Polyadenylated RNA, Modern, ribonucleic acids, Non-Polyadenylated, Control, RNS, Ribonucleic Acid, Controls, human, Human, Non-Polyadenylated RNA, Social, Homo sapiens, Social Control, bacterial transcription, yeast nucleic acid, Modern Man, Ribonukleinsaeure, chromosome scaffold, Gene Products, pentosenucleic acids, Ribonucleic acids, regulation, transcription from bacterial-type RNA polymerase promoter, Regulation, Man, Non Polyadenylated, Formal Social Controls, Chromatins, humans, RNA Gene Productsnuclear chromatin, Regulations, study, ribonucleic acid, Acid, cytoplasmic chromatin, RNA, Ribonucleic, human being, Man (Taxonomy), Social Controls., ribose nucleic acid, Formal Social Control, Non Polyadenylated RNA, Modern, ribonucleic acids, Non-Polyadenylated, Control, RNS, Ribonucleic Acid, Controls, human, Human, Non-Polyadenylated RNA, Social, Homo sapiens, Social Control, bacterial transcription, yeast nucleic acid, Modern Man, Ribonukleinsaeure, chromosome scaffold, Gene Products, pentosenucleic acids, Ribonucleic acids, regulation, transcription from bacterial-type RNA polymerase promoter, Regulation, Man, Non Polyadenylated, Formal Social Controls, Chromatins, humans, RNA Gene ProductsTranscription Activation, Non Protein Coding, Activity, 2410041A17Rik, ectodin, ACTG, ACTE, protein, fs(1)M34, sci, csp2, Act5, Social Controls, l(1)G0420, DmelCG12051, SUB, Noncoding RNA, protein polypeptide chains, Readability, Genetic Induction, DmelCG12298, GRP1, Grp1, HEL-176, HOW, How, CG4601, SCRAMBLED, protein aggregate, Fs(3)Hor, Formal Social Controls, KIF20A, Chromatins, l(3)j5D5, 24B, Transcriptional, A, C, DmelCG2684, cyt5C, reference sample, DHO, Genomes, Non-Protein-Coding RNA, long, PTPSTEP, stru, CG18572, USAG-1, proteins, l(3)S053606, NTef2, CG10293, Social, l(3)j5B5, Non Coding, act 42A, l(2)k08110, chromosome scaffold, Ac5C, CG4027, ACT, Act, GPH, 0610006G05Rik, nuclear chromatin, 0904/17, l(1)G0330, Actin/BAP47, CTE-II, AACT, STRUBBELIG, CTE-IIa, act, Ach1, hBACH, ACH1, LACH1, Fs(3)Sz11, act42A, GIG25, Induction, Su(b), Alpha-1-antichymotrypsin His-Pro-less, Non-Coding RNA, SZ1, AFFX-Dros-ACTIN_M_r_at, GIG24, 3.1.3.48, DFNA26, actin, LACH, Non-Peptide-Coding, GAT, DFNA20, Transactivation, DmelCG4027, Step, CG11628, l(1)G0117, Control, Actin, Striatum-enriched protein-tyrosine phosphatase, Nontranslated, whole genome, Controls, Non-Protein-Coding, beta-actin/Bap47, early, l(1)G0486, l(1)G0245, actin5C, l(1)G0009, STEP, Sostl, ACTL3, Horka, Lach1, Wise, CG2684, Fs(3)Horka, anon-EST:Liang-2.39, Regulation, BACH, l(1)Ab, Regulations, AT1G11140, Act5c, cytoplasmic chromatin, Trans Activation, l(1)G0010, GRP1/cytohesin 1, Nontranslated RNA, npcRNA, P62, number, ACT5C, Gene, CG12051, Serpin A3, protein-containing complex, Bach, presence, CG12298, CG11633, cytohesin/GRP1, l(1)G0025, polypeptide chain, Gene Products, Cell growth-inhibiting gene 24|25 protein, DmF2, lod, study, Transcription, act5C, anatomical systems, Genetic, l(3)s2612, Activation, Non-Coding, BRWS2, mei-1794, l(2)SH2 0323, fs(1)829, Act42a, anon-EST:fe2D2, Neural-specific protein-tyrosine phosphatase, 1700027G07Rik, DmelCG18572, DmelCG10293, SRF9, T11, Act-5C, Cte-II, Controlled, l(1)G0177, 42A, Controlling, RNA, CPS, Social Controls., Formal Social Control, Non Peptide Coding, protein complex, clone 2.39, Proteins, dJ393D12.2, stepk, qkr, l(3)S090417, STRUBBELIG-RECEPTOR FAMILY 9, count in organism, Non-Peptide-Coding RNA, CAD, native protein, natural protein, Social Control, beta-actin, KH93F, Protein, M32055, Act42, Genetic Trans-Activation, ACTA3, l(2)SH0323, Untranslated RNA, who, CYH1, l(1)G0079, PYR1, VSCM, Dub, Gene Activation, act 5C, INSDC_qualifier:other, Who/How, Understanding, Lds, DmelCG11628, Protein Gene Products, Gene Proteins, qkr[93F], DRORUD, Noncoding, regulation, BAP47, ACTSG, T19D16.8, General activity, Bap47, SCM, Actin5C, Trans-Activationprojections, Transcription Activation, Non Protein Coding, Activity, 2410041A17Rik, ACTG, ACTE, protein, fs(1)M34, sci, csp2, Act5, Social Controls, l(1)G0420, DmelCG12051, Noncoding RNA, protein polypeptide chains, Readability, Genetic Induction, macromolecular complex assembly, HEL-176, HOW, How, CG4601, protein aggregate, Fs(3)Hor, Formal Social Controls, Chromatins, l(3)j5D5, 24B, Transcriptional, A, C, thymus nucleic acid, DmelCG2684, cyt5C, Gene Expressions, Man (Taxonomy), reference sample, Genomes, DHO, Non-Protein-Coding RNA, long, stru, CG18572, proteins, l(3)S053606, NTef2, CG10293, Social, l(3)j5B5, Human Genomes, Non Coding, act 42A, papilla, chromosome scaffold, Ac5C, CG4027, ACT, Act, Double-Stranded DNA, deoxyribonucleic acids, DNAn, transcription from bacterial-type RNA polymerase promoter, nuclear chromatin, 0904/17, l(1)G0330, Actin/BAP47, CTE-II, Modern, AACT, lamina, CTE-IIa, flanges, act, Ach1, hBACH, ACH1, Double-Stranded, LACH1, Fs(3)Sz11, act42A, (Deoxyribonucleotide)n+m, GIG25, chaperone activity, Induction, Su(b), Alpha-1-antichymotrypsin His-Pro-less, Non-Coding RNA, SZ1, AFFX-Dros-ACTIN_M_r_at, GIG24, shelf, DFNA26, actin, desoxyribose nucleic acid, LACH, Non-Peptide-Coding, GAT, DFNA20, Transactivation, DmelCG4027, Drives, shelves, l(1)G0117, Control, protein complex formation, Actin, whole genome, Nontranslated, Controls, Non-Protein-Coding, projection, ridge, beta-actin/Bap47, human, early, l(1)G0486, l(1)G0245, actin5C, l(1)G0009, ACTL3, Horka, Lach1, bacterial transcription, ds DNA, cellular protein complex assembly, CG2684, Fs(3)Horka, anon-EST:Liang-2.39, DNA, Regulation, BACH, humans, l(1)Ab, Regulations, Act5c, cytoplasmic chromatin, Trans Activation, l(1)G0010, human being, DNS, Nontranslated RNA, npcRNA, (Deoxyribonucleotide)n, conformation, lamellae, P62, number, ACT5C, Gene, CG12051, Serpin A3, protein-containing complex, Bach, process of organ, presence, Deoxyribonucleic acids, Human, lamella, l(1)G0025, Deoxyribonucleic Acid, polypeptide chain, Homo sapiens, Gene Products, Cell growth-inhibiting gene 24|25 protein, DmF2, Man, lod, study, Transcription, act5C, anatomical systems, Genetic, l(3)s2612, Activation, Non-Coding, BRWS2, Double Stranded, Deoxyribonucleic acid, ridges, Expressions, fs(1)829, Act42a, anon-EST:fe2D2, 1700027G07Rik, DmelCG18572, cellular protein-containing complex assembly, DmelCG10293, (Deoxyribonucleotide)m, Expression, T11, laminae, relational structural quality, Act-5C, Cte-II, Controlled, l(1)G0177, 42A, Controlling, RNA, CPS, Social Controls., Formal Social Control, Non Peptide Coding, protein complex, clone 2.39, DNAn+1, Proteins, dJ393D12.2, qkr, l(3)S090417, count in organism, Non-Peptide-Coding RNA, CAD, native protein, Human Genome, natural protein, Social Control, beta-actin, KH93F, Protein, M32055, core, Act42, Genetic Trans-Activation, ds-DNA, ACTA3, Untranslated RNA, protein complex assembly, macromolecule complex assembly, flange, organ process, who, l(1)G0079, cellular macromolecule complex assembly, PYR1, VSCM, Gene Activation, act 5C, INSDC_qualifier:other, Who/How, Understanding, Lds, Protein Gene Products, processes, Gene Proteins, Modern Man, Desoxyribonukleinsaeure, qkr[93F], DRORUD, Noncoding, regulation, BAP47, ACTSG, General activity, Bap47, Actin5C, Trans-Activation0PXD009790trueHuman chromatin platforms for the study of transcription and RNA regulationHere we describe tools to study the step-wise assembly of protein complexes on chromatin in a highly-controlled manner using reconstituted chromatin platforms and quantitative proteomic profiling. We profile the early steps in transcriptional activation and highlight the potential for understanding the multiple ways chromatin can influence transcriptional regulation. We also describe modifications of this approach to study the activity of a long noncoding RNA to act as a dynamic scaffold for proteins to be recruited to chromatin. The reconstituted nature of the chromatin substrate offers a tune-able system, able to be trapped at specific sub-steps, to understand how chromatin interfaces with genome regulation machinery.Tue May 15 21:11:00 BST 2018MSV00008236330192551