Session Title: Genetics, Genomics and Proteomics II
Session Type: Abstract Submissions (ACR)
Background/Purpose There is an imbalance of inflammatory M1 vs. anti-inflammatory M2 macrophages (MΦs) in rheumatoid arthritis (RA). The epigenetic codes underlying this M1 dominating pathogenesis in RA have not been well established. Our studies have demonstrated that terminal fucosylation, adding a fucose to the glycan termini by fucosyltransferases is a hallmark of M1 MΦs. A terminal fucosylation inhibitor, 2-Deoxy-D-galactose (2-D-gal), precluded the differentiation of M1 MΦ and skewed them to an M2 phenotype, resulting in suppression of collage II-induced arthritis. The purpose of the study is to investigate how terminal fucosylation regulates MΦ plasticity by modulating the epigenetic landscape.
Methods Mouse bone marrow derived M1 and M2 MΦs were differentiated by GM-CSF and M-CSF respectively. Ulex Europaeus Agglutinin I [UEA 1, recognizing terminal Fucα(1-2)Gal] pull down coupled to liquid chromatography mass spectrometry (LCMS) was performed to identify the fucosylated proteins in M1 MΦs. Histone H1 was purified from mouse M1 MΦs by using high salt/acid extraction and SDS-PAGE. N-linked glycans on histone H1 was released by in gel PNGase F digestion, followed by permethylation and analyzed by MALDI/TOF-MS and NSI-FT-MS. MS/MS and MS3rd were further carried out to sequence the fucose moieties. Chromatin immunoprecipitations (ChIPs) were performed using anti-H3K4me1 and H3K27ac, followed by next generation sequencing (illumina HiSeq 2500). ChIP-Seq data were aligned to build version mm9 of mouse genome using Bowtie, and regions of enrichment were identified by the MACS peak-finding algorithm. A p value threshold of enrichment of 1×10-9was used.
Results Decreased chromatin condensation was observed in M1 compared to M2 macrophages by chromatin staining, which is reversed by 2-D-gal. This suggested that histone H1, a chromatin packing regulator, might be the fucosylated target. Indeed, MS analysis revealed that H1 (Q value 117.5), but not H2A, H2B, H3 and H4 (Q value 15.2, 6.1, 5.1 and 0), is the key protein that is highly fucosylated in M1 MΦs. Terminal fucose moieties and 23 distinct N-glycans were further identified on histone H1 from mouse M1 MΦs by MS analysis. ChIP-seq revealed that active enhancers, characterized by the co-enrichment of H3K4me1 and H3K27ac, exhibited a significantly higher activity at the M1 signature gene loci (including Irf5, Tnf, Il12, Ifng, and H2 loci) in M1 compared to M2 MΦs. Importantly, inhibition of H1 fucosylation in M1 MΦs by 2-D-gal dramatically reduced the enhancer activity at these loci to a level that is comparable to or even lower than that of M2 macrophages; On the other hand, 2-D-gal promoted the enhance activity at the M2 signature gene loci, including Arg1, in M1 MΦs. Gene transcription was verified by illumina WG-6 gene array.
Conclusion Histone H1 fucosylation is a novel chromatin mark. It regulates macrophage subset plasticity and determines their identities by dynamically interacting with histone H3 and modulating the enhancer activity at the M1and M2 signature gene loci. These processes can be reversed by the fucosylation inhibitor, which is a potential biologic agent that acts at the epigenetic level to reshape the inflammatory MΦs and reestablish immune homeostasis in RA.
D. M. Spalding,
J. A. Mobley,
S. L. Bridges Jr.,
H. C. Hsu,
J. D. Mountz,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-novel-epigenetic-mark-histone-h1-fucosylation-orchestrates-macrophage-differentiation-and-plasticity-by-remodeling-the-enhancer-landscape-in-rheumatoid-arthritis/