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

The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells.The linker histone H1s “beads-on-a-string” structure aids chromatin folding into highly compacted 30 nm chromatin fibers (1). Previous studies demonstrated that histone H1s are differentially expressed and incorporated into chromatin during embryonic stem cell differentiation and reprogramming to pluripotency (2). More than being accumulated after differentiation, the three histone H1 isoforms, H1.3, H1.4, and H1.5, are required for embryonic stem cell differentiation as demonstrated by in vivo H1.3/H1.4/H1.5 triple null experiments (3). Histone H1 null cells exhibit altered nucleosome architecture (4) which may cause epigenetic reprogramming (2), specific changes in gene regulation including repression of pluripotency gene Oct4 expression (3, 5), and cell growth (6, 7). In human blood or bone marrow, hematopoietic stem cells give rise to two major pluripotent progenitor cell lineages, myeloid and lymphoid progenitors, from which are derived mature blood cells including erythrocytes, megakaryocytes, and cells of the myeloid and lymphoid lineages. However, epigenetic regulation or reprogramming in this complex differentiation system has not yet been fully understood. As a follow up to our proteomics studies on epigenetic networks in U937 cell differentiation (8), we have performed proteomics studies on primary human monocyte differentiation. In this report, using proteomics and bioinformatics tools in lieu of microarray analysis of gene expression, we describe the presence of unique protein expression profiles, specifically the linker histones, in monocyte differentiation into macrophages and dendritic cells.Differentiation of monocytes from primary leukemia cell lines or from human peripheral blood mononuclear cells into macrophages or macrophage-like cells using different differentiating reagents has been frequently used as a mimic model for understanding the process of innate and adaptive immune responses to inflammatory stimuli, viral infection, and environmental cues. Either phorbol myristate acetate (PMA)¹ or granulocyte-macrophage colony-stimulating factor (GMCSF) has normally been used for differentiation of monocytes, though the former is generally for differentiation of primary monocytic cell lines, while the latter for differentiation of human blood monocytes (9–11). In our experiments, CD14+ monocytes were treated with PMA, PMA + ionomycin, GMCSF, or GMCSF + IL4. After treatment, monocyte differentiation into macrophages or dendritic cells was monitored by mass spectrometry and bioinformatics analyses. We report here that monocytic cell lineages can be distinguished based on protein expression profiles, specifically, histone H1.4 and H1.5 expression patterns. We identified H3K9-methyl/S10-phos/K14-acetyl tri-modification forms in the monocyte and macrophages but not in dendritic cells. In addition, histone H4 K16 acetylation was low in monocytes and macrophages but significantly higher in dendritic cells. Our findings suggest a switch from H3 tri-modification and linker histone expression to histone H4 K16 acetylation occurs during the monocyte-to-dendritic cell transition.