Direct interaction between the PRDM3 and PRDM16 tumor suppressors and the NuRD chromatin remodeling complex.

Aberrant isoform expression of chromatin-associated proteins can induce epigenetic programs related to disease. The MDS1 and EVI1 complex locus (MECOM) encodes PRDM3, a protein with an N-terminal PR-SET domain, as well as a shorter isoform, EVI1, lacking the N-terminus containing the PR-SET domain (ΔPR). Imbalanced expression of MECOM isoforms is observed in multiple malignancies, implicating EVI1 as an oncogene, while PRDM3 has been suggested to function as a tumor suppressor through an unknown mechanism. To elucidate functional characteristics of these N-terminal residues, we compared the protein interactomes of the full-length and ΔPR isoforms of PRDM3 and its closely related paralog, PRDM16. Unlike the ΔPR isoforms, both full-length isoforms exhibited a significantly enriched association with components of the NuRD chromatin remodeling complex, especially RBBP4. Typically, RBBP4 facilitates chromatin association of the NuRD complex by binding to histone H3 tails. We show that RBBP4 binds to the N-terminal amino acid residues of PRDM3 and PRDM16, with a dissociation constant of 3.0 µM, as measured by isothermal titration calorimetry. Furthermore, high-resolution X-ray crystal structures of PRDM3 and PRDM16 N-terminal peptides in complex with RBBP4 revealed binding to RBBP4 within the conserved histone H3-binding groove. These data support a mechanism of isoform-specific interaction of PRDM3 and PRDM16 with the NuRD chromatin remodeling complex.

5-Hydroxymethylcytosine preferentially targets genes upregulated in isocitrate dehydrogenase 1 mutant high-grade glioma.

Gliomas demonstrate epigenetic dysregulation exemplified by the Glioma CpG Island Methylator Phenotype (G-CIMP) seen in IDH1 mutant tumors. 5-Hydroxymethylcytosine (5hmC) is implicated in glioma pathogenesis; however, its role in IDH1 mutant gliomas is incompletely understood. To characterize 5hmC in IDH1 mutant gliomas further, we examine 5hmC in a cohort of IDH1 mutant and wild-type high-grade gliomas (HGG) using a quantitative locus-specific approach. Regions demonstrating high 5hmC abundance and differentially hydroxymethylated regions (DHMR) enrich for enhancers implicated in glioma pathogenesis. Among these regions, IDH1 mutant tumors possess greater 5hmC compared to wild type. 5hmC contributes to overall methylation status of G-CIMP genes. 5hmC targeting gene body regions correlates significantly with increased gene expression. In particular, a strong correlation between increased 5hmC and increased gene expression is identified for genes highly expressed in the IDH1 mutant cohort. Overall, locus-specific gain of 5hmC targeting regulatory regions and associated with overexpressed genes suggests a significant role for 5hmC in IDH1 mutant HGG.

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.