Journal: Nucleic Acids Research
Article Title: Allele-specific quantitative proteomics unravels molecular mechanisms modulated by cis-regulatory PPARG locus variation
Figure Lengend Snippet: Discovery of allele-specific binding proteins at cis -regulatory variants. ( A ) Workflow: (1) cis-regulatory variant prediction at disease associated variants ( PPARG ) in high LD ( r 2 ≥ 0.7 ( 6 )) by integrating bioinformatics phylogenetic TFBS module complexity analysis and regulatory chromatin marks; (2) protein–DNA binding assessed by Cy5 labeled oligonucleotides matching the risk and nonrisk allele, respectively, in electrophoretic mobility shift assay (EMSA); (3) protein enrichment with biotin (bio) labeled oligonucleotides on streptavidin-beads (str) and elution of native protein complexes with increasing concentration of NaCl; (4) protein–DNA binding in eluted fractions; (5) protein identification and quantification by LC–MS/MS and subsequent label-free quantitative analysis; and (6) molecular mechanisms, experimental and genetics verification of significant allele-specific binding transcription factors and related coregulators. (B–D) Bioinformatics and public domain epigenomic marks of regulatory regions infer the cis -regulatory variant rs7647481 at the PPARG locus (related to Supplementary Figure S1 ). ( B ) PMCA analysis of cross-species TFBS pattern conservation predicted six indicated candidate cis -regulatory SNPs at complex regions ( 6 ) (red) out of 23 noncoding proxy SNPs ( r 2 ≥ 0.7 ( 6 )) at the type 2 diabetes (T2D) associated PPARG locus (tagSNP rs1801282). ( C ) Overlap of six variants identified in (B) with H3K27ac (histone H3-lysine 27 acetylation), H3K4me1 and H3K4me2 (histone H3–lysine 4 mono- and di-methylation) histone modification regions at the PPARG locus during adipogenic differentiation of primary human adipocyte stem cells ( 36 ), GSE21366, genomic coordinates are given conform to hg19. ( D ) Localization of cis- regulatory (red) and non cis -regulatory (grey) variants subjected to workflow (A2–6) relative to transcriptional start site of the PPARG1–3 mRNA isoforms. rs7647481 overlapping with both, day 3 and day 9, tested late stage of adipogenesis histone modification regions (Figure 1C ) and with adipocyte DNase-seq regions (see Supplementary Figure S1 ). * rs4684847 previously identified as specifically overlapping with homeobox TFBS ( 6 ). Blue boxes = coding exons, dashed white boxes = untranslated exons, blue lines = introns, black arrows = promoters.
Article Snippet: According to the manufacturer΄s instructions, streptavidin coupled magnetic beads (Dynabeads M-280, Invitrogen, Darmstadt, Germany) were washed and collected using Bind & Wash buffer (Dynabeads M-280, Invitrogen, Darmstadt, Germany) and Magnetic particle separator (Magna-Sep™, Invitrogen, Darmstadt, Germany), discarding the supernatant.
Techniques: Binding Assay, Variant Assay, Labeling, Electrophoretic Mobility Shift Assay, Protein Enrichment, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Methylation, Modification