Mass Spectrometric Studies on Epigenetic Interaction Networks in Cell Differentiation

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.Both histone acetylation and H2B ubiquitination are associated with gene activation as defined by the "histone code" and "cross-talk" theories (1). As demonstrated previously in Berk's laboratory (2), small interfering RNA (siRNA)-mediated dual knockdown of CBP 5 and p300 resulted in specific histone H3Lys-18 hypoacetylation in vivo in both IMR90 and HeLa cells, indicating that p300/CBP, possibly together with their closely associated protein PCAF, are the major histone acetyltransferases required for maintaining global H3 Lys-18 acetylation.On the other hand, the putative histone H2B ubiquitination enzyme, UBCH6 (also known as UBE2E1), physically interacts with RNF20/40 (homologs of Bre1, the E3 ligase in yeast) and PAF1 to form a trimeric complex, which colocalizes with RNA polymerase II at transcriptionally active genes (3). RTF1, which has a function similar to Rad6 (mediates ubiquitination at lysine 123 of H2B in yeast) and is a component of the PAF1/RNA polymerase II complex, cooperatively interacts with RNF20/40, and FACT (facilitates chromatin transcription) to regulate H2B ubiquitination (4 -8).

Quantitative Proteomics Reveals a Role for Epigenetic Reprogramming During Human Monocyte Differentiation

“…This difference suggested that PMA differentiation protocols might not be as effective on primary cells as they are on cell lines such as U937 cells. The expression of H1.4/H1.5 was significantly increased in monoblast and premonocyte U937 cells after 72 h treatment with PMA (8). However, H1.4/H1.5 expression was markedly decreased in macrophages and dendritic cells differentiated from monocytes, suggesting that expression of these proteins might be a dynamic process (supplemental Fig.…”

“…In parallel, as previously reported, the promonocyte/monoblast U937 cells were also cultured in RPMI 1640 media. U937 cells were differentiated with PMA for 72 h to monocytes/macrophages (8) (Fig. 1A).…”

Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry

“…Several proteins involved in H2B ubiquitination and deubiquitination were identified and quantified. In this specific study, down-regulation of H2B ubiquitination has been validated using targeted LC-MS/MS by multiple reaction monitoring (MRM) [70]. Recently, SUMOylation [71], propionylation/butyrylation [72], and O-linked N-acetylglucosamine (O-GlcNAc) [73] were also discovered as variable modifications on histone proteins.…”

Proteome Analysis of Coinfection of Epithelial Cells with Filifactor alocis and Porphyromonas gingivalis Shows Modulation of Pathogen and Host Regulatory Pathways

“…Protein samples were analyzed using a Thermo Scientific LTQ Orbitrap Velos mass spectrometer and the data processed as previously reported (45). The four-part protocol used for the MS and tandem MS (MS/MS) analyses was carried out, and the data collection was achieved using Xcalibur software (Thermo Electron) followed by screening performed with Bioworks 3.1.…”