ABSTRACT
The growing epidemic of obesity and metabolic diseases calls for a better understanding of adipocyte biology. The regulation of transcription in adipocytes is particularly important, as it is a target for several therapeutic approaches. Transcriptional outcomes are influenced by both histone modifications and transcription factor binding. Although the epigenetic states and binding sites of several important transcription factors have been profiled in the mouse 3T3-L1 cell line, such data are lacking in human adipocytes. In this study, we identified H3K56 acetylation sites in human adipocytes derived from mesenchymal stem cells. H3K56 is acetylated by CBP and p300, and deacetylated by SIRT1, all are proteins with important roles in diabetes and insulin signaling. We found that while almost half of the genome shows signs of H3K56 acetylation, the highest level of H3K56 acetylation is associated with transcription factors and proteins in the adipokine signaling and Type II Diabetes pathways. In order to discover the transcription factors that recruit acetyltransferases and deacetylases to sites of H3K56 acetylation, we analyzed DNA sequences near H3K56 acetylated regions and found that the E2F recognition sequence was enriched. Using chromatin immunoprecipitation followed by high-throughput sequencing, we confirmed that genes bound by E2F4, as well as those by HSF-1 and C/EBPα, have higher than expected levels of H3K56 acetylation, and that the transcription factor binding sites and acetylation sites are often adjacent but rarely overlap. We also discovered a significant difference between bound targets of C/EBPα in 3T3-L1 and human adipocytes, highlighting the need to construct species-specific epigenetic and transcription factor binding site maps. This is the first genome-wide profile of H3K56 acetylation, E2F4, C/EBPα and HSF-1 binding in human adipocytes, and will serve as an important resource for better understanding adipocyte transcriptional regulation.
Subject(s)
Adipocytes/metabolism , Genome, Human/genetics , Histones/metabolism , Transcription Factors/metabolism , 3T3-L1 Cells , Acetylation , Adipocytes/cytology , Animals , Base Sequence , Binding Sites/genetics , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , CREB-Binding Protein/genetics , CREB-Binding Protein/metabolism , Cell Differentiation/genetics , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/metabolism , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , Gene Expression Profiling , Heat Shock Transcription Factors , Humans , Lysine/metabolism , Mesoderm/cytology , Mesoderm/metabolism , Mice , Sirtuin 1/genetics , Sirtuin 1/metabolism , Transcription Factors/geneticsABSTRACT
INTRODUCTION: CD44 is a cell adhesion molecule believed to play a critical role in T cell and monocyte infiltration in the inflammatory process. The reduction of CD44 expression or its ability to properly interact with its key ligand, hyaluronic acid (HA), inhibits migration and subsequent activation of cells within sites of inflammation. CD44-deficient mice exhibit decreased disease in a mouse arthritis model. METHODS: Accordingly, we developed PF-03475952, a fully human IgG2 anti-CD44 monoclonal antibody (mAb). RESULTS: Binding of PF-03475952 to CD44 inhibits binding of HA and induces loss of CD44 from the cell surface. PF-03475952 also passed a series of safety pharmacology assays designed to assess the risk of the mAb to bind Fc gamma receptors, stimulate cytokine release from human whole blood, and stimulate cytokine release from peripheral blood mononuclear cells (PBMC) using plate-bound antibodies. The latter assay was designed specifically to evaluate the risk of cytokine storm that had been observed with TGN1412 (immunostimulatory CD28 superagonist mAb). PF-003475952 exhibits high-affinity binding to both human and cynomolgus monkey CD44, but does not cross-react with rodent CD44. Thus, a rat anti-mouse CD44 mAb was used to demonstrate a dose-dependent decrease of disease in mouse collagen-induced arthritis. Importantly, efficacy was correlated with >50% loss of cell surface CD44 on circulating cells. Loss of CD44 expression on CD3+ lymphocytes was monitored following a single dose of PF-03475952 in cynomolgus monkeys as a pharmacodynamic marker. The recovery of CD44 expression was found to be dose-dependent. PF-03475952 doses of 1, 10, and 100 mg/kg reduced CD44 expression below 50% for 218, 373, and >504 hours, respectively. CONCLUSION: Targeting of CD44 is a unique mechanism of action in the treatment of inflammatory diseases and is expected to reduce joint damage induced by inflammatory mediators, resulting in disease modification in inflammatory diseases such as rheumatoid arthritis.
