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A4727 - Chromatin Immunoprecipitation (ChIP) Sequencing Identifies a Potential Role of SP-R210 in Regulating Cellular Metabolism and Trafficking Genes in Macrophages
Author Block: E. Yau1, Y. Chen1, A. Salzberg2, Y. I. Kawasawa3, C. Song1, Z. C. Chroneos1; 1Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA, United States, 2Institute of Personalized Medicine, Pennsylvania State University College of Medicine, Hershey, PA, United States, 3Pharmacology, and Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA, United States.
Rationale: Alveolar macrophages, the resident innate immune cells in the lung, are often the first immune cells to come into contact with airborne pathogens such as influenza. Previous studies have identified that a surfactant protein A (SP-A) receptor, SP-R210, also known as Myosin 18a (Myo18a), regulates innate immune functions of macrophages. This receptor can be alternatively spliced into two predominant protein isoforms: a long form containing a cytosolic PDZ domain (SP-R210L), and a shorter form without the PDZ domain (SP-R210S). These isoforms of SP-R210 differentially regulate macrophage function, especially with SP-R210L playing an anti-inflammatory role. Unexpectedly, we found that both SP-R210 isoforms translocate to the nucleus. This finding, along with published data showing differences in cytokine expression between SP-R210L knockout (where SP-R210S is dominant) and wild type (WT) cells (where both isoforms exist), has led us to hypothesize that SP-R210 moves to the nucleus to modulate macrophage gene expression. To address this hypothesis, we used ChIP and sequencing (ChIP-seq) analysis to investigate SP-R210:DNA interactions in the macrophage genome.
Methods: We performed ChIP-seq of WT and SP-R210L knockout macrophage cell lines to identify regions of the chromosome differentially bound by the two isoforms of SP-R210. We utilized antibodies against SP-R210, PU.1, and NFκB to pull down the respective protein-bound DNA fragments in both WT and SP-R210L knockouts. The DNA fragments were sequenced via Illumina next-generation sequencing and aligned to the mouse reference genome. DNA regions with differential antibody binding were called using the bdgdiff function of the MACS2 (v. 2.1.0) package and annotated using the ChIPpeakAnno v.3.8.9 R package.
Results: Genes we identified with significant association to SP-R210 common to WT and SP-R210L-deficient macrophages were Phosphoglycerate kinase 1 (Pgk1); Camk2b, a calcium/calmodulin serine/threonine kinase; and Ykt6, a synaptobrevin homologue that acts as a v-SNARE receptor. Dpy19l1, a transmembrane tryptophan mannosyltransferase, was identified in WT but not the SP-R210L-deficient macrophages. Furthermore, the Pgk1 was enriched in PU.1- and NFκB-associated immunoprecipitates as well.
Conclusions: Our study identified a potential role of nuclear SP-R210 isoforms in cellular metabolism and trafficking functions in macrophages. The identification of Pgk1 and Ykt6 in both cell types suggests a role of SP-R210S in macrophage metabolism and vesicle trafficking, respectively. The enrichment of Dpy19l1 only in WT cells suggests a selective role of SP-R210L in modulating secretory proteins in macrophages.