Abstract
Arrest of cell differentiation is one of the leading causes of leukemia and other cancers. Induction of cell differentiation using pharmaceutical agents has been clinically attempted for the treatment of these cancers. Epigenetic regulation may be one of the underlying molecular mechanisms controlling cell proliferation or differentiation. Here, we report on the use of proteomics-based differential protein expression analysis in conjunction with quantification of histone modifications to decipher the interconnections among epigenetic modifications, their modifying enzymes or mediators, and changes in the associated pathways/networks that occur during cell differentiation. During phorbol-12-myristate 13-acetate-induced differentiation of U937 cells, fatty acid synthesis and its metabolic processing, the clathrin-coated pit endocytosis pathway, and the ubiquitin/26 S proteasome degradation pathways were up-regulated. In addition, global histone H3/H4 acetylation and H2B ubiquitination were down-regulated concomitantly with impaired chromatin remodeling machinery, RNA polymerase II complexes, and DNA replication. Differential protein expression analysis established the networks linking histone hypoacetylation to the down-regulated expression/activity of p300 and linking histone H2B ubiquitination to the RNA polymerase II-associated FACT-RTF1-PAF1 complex. Collectively, our approach has provided an unprecedentedly systemic set of insights into the role of epigenetic regulation in leukemia cell differentiation.
Highlights
Both histone acetylation and H2B ubiquitination are associated with gene activation as defined by the “histone code” and “cross-talk” theories [1]
We report on the use of proteomics-based differential protein expression analysis in conjunction with quantification of histone modifications to decipher the interconnections among epigenetic modifications, their modifying enzymes or mediators, and changes in the associated pathways/networks that occur during cell differentiation
Our results demonstrated that hypoacetylation of H3/H4 and hypoubiquitination of H2B, which is regulated by an array of pathways, including fatty acid synthesis and metabolism, protein degradation, chromatin remodeling, and DNA replication, is associated with cell differentiation
Summary
Cell Culture and Protein Extraction—U937 cells were cultured in RPMI 1640 (Mediatech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum (FBS; PAA Laboratories Inc.), 1 mM L- glutamine (Mediatech, Inc.), and 200 units/ml penicillin and 200 g/ml streptomycin (Lonza, Walkersville, MD) at a density of 2 ϫ 105 cells/ml in a humid incubator with 5% CO2 at 37 °C. One- and Two-dimensional RPLC MS—Peptides were separated by online RPLC using an Eksigent nanoLC Ultra 1D Plus and nanoLC AS-2 Autosampler (Eksigent Technologies) coupled online with the Thermo Scientific LTQ Orbitrap Velos mass spectrometer This hybrid instrument features a dual pressure ion trap configuration along with the OrbitrapTM detector and a higher energy collision induced dissociation (HCD) collision cell [20, 21]. Histone Acetyltransferase Activity Assay—Whole cell extracts containing equal amounts of proteins (500 g) from U937 and HL60 cells and their PMA-induced differentiated counterparts were incubated for 30 min with 3 g of human recombinant histone H3 in the presence of 14C-acetyl-CoA. The protein G-agarose beads were incubated for 30 min with 50 l of HAT assay mixture (10 l of 5ϫ HAT assay buffer, 20 l of D.I. water, 10 l of biotin-conjugated H4 peptides, and 10 l of 100 M acetyl-CoA containing 0.5 Ci/l 14C-acetyl-CoA). Analysis of Global DNA Cytosine Methylation—Global cytosine methylation was measured by GC/MS as previously described [23]
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