Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.

Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.

Characterization of a SILAC method for proteomic analysis of primary rat microglia.

Microglia play important and dynamic roles in mediating a variety of physiological and pathological processes during the development, normal function and degeneration of the central nervous system. Application of SILAC-based proteomic analysis would greatly facilitate the identification of cellular pathways regulating the multifaceted phenotypes of microglia. We and others have successfully SILAC-labeled immortalized murine microglial cell lines in previous studies. In this study, we report the development and evaluation of a SILAC-labeled primary rat microglia model. Although the isotope labeling scheme for primary microglia is drastically different from that of immortalized cell lines, our de novo and uninterrupted primary culture labeling protocol (DUP-SILAC) resulted in sufficient incorporation of SILAC labels for mass spectrometry-based proteomic profiling. In addition, label incorporation did not alter their morphology and response to endotoxin stimulation. Proteomic analysis of the endotoxin-stimulated SILAC-labeled primary microglia identified expected as well as potentially novel activation markers and pro-inflammatory pathways that could be quantified in a more physiologically relevant cellular model system compared to immortalized cell lines. The establishment of primary microglia SILAC model will further expand our capacity for global scale proteomic profiling of pathways under various physiological and pathological conditions. Proteomic MS data are available via ProteomeXchange with identifier PXD002759.

Loss of WSTF results in spontaneous fluctuations of heterochromatin formation and resolution, combined with substantial changes to gene expression.

BACKGROUND: Williams syndrome transcription factor (WSTF) is a multifaceted protein that is involved in several nuclear processes, including replication, transcription, and the DNA damage response. WSTF participates in a chromatin-remodeling complex with the ISWI ATPase, SNF2H, and is thought to contribute to the maintenance of heterochromatin, including at the human inactive X chromosome (Xi). WSTF is encoded by BAZ1B, and is one of twenty-eight genes that are hemizygously deleted in the genetic disorder Williams-Beuren syndrome (WBS). RESULTS: To explore the function of WSTF, we performed zinc finger nuclease-assisted targeting of the BAZ1B gene and isolated several independent knockout clones in human cells. Our results show that, while heterochromatin at the Xi is unaltered, new inappropriate areas of heterochromatin spontaneously form and resolve throughout the nucleus, appearing as large DAPI-dense staining blocks, defined by histone H3 lysine-9 trimethylation and association of the proteins heterochromatin protein 1 and structural maintenance of chromosomes flexible hinge domain containing 1. In three independent mutants, the expression of a large number of genes were impacted, both up and down, by WSTF loss. CONCLUSIONS: Given the inappropriate appearance of regions of heterochromatin in BAZ1B knockout cells, it is evident that WSTF performs a critical role in maintaining chromatin and transcriptional states, a property that is likely compromised by WSTF haploinsufficiency in WBS patients.

Site-specific identification and validation of hepatic histone nitration in vivo: Implications for alcohol-induced liver injury.

Oxidative and nitrative stress have been implicated in the molecular mechanisms underlying a variety of biological processes and disease states including cancer, aging, cardiovascular disease, neurological disorders, diabetes, and alcohol-induced liver injury. One marker of nitrative stress is the formation of 3-nitrotyrosine, or protein tyrosine nitration (PTN), which has been observed during inflammation and tissue injury; however, the role of PTN in the progression or possibly the pathogenesis of disease is still unclear. We show in a model of alcohol-induced liver injury that an increase in PTN occurs in hepatocyte nuclei within the liver of wild-type male C57BL/6J mice following chronic ethanol exposure (28 days). High-resolution mass spectrometric analysis of isolated hepatic nuclei revealed several novel sites of tyrosine nitration on histone proteins. Histone nitration sites were validated by tandem mass spectrometry (MS/MS) analysis of representative synthetic nitropeptides equivalent in sequence to the respective nitrotyrosine sites identified in vivo. We further investigated the potential structural impact of the novel histone H3 Tyr41 (H3Y41) nitration site identified using molecular dynamics (MD) simulations. MD simulations of the nitrated and non-nitrated forms of histone H3Y41 showed significant structural changes at the DNA interface upon H3Y41 nitration. The results from this study suggest that, in addition to other known post-translational modifications that occur on histone proteins (e.g., acetylation and methylation), PTN could induce chromatin structural changes, possibly affecting gene transcription processes associated with the development of alcohol-induced liver injury.

TNFAIP3 Plays a Role in Aging of the Hematopoietic System.

Hematopoietic stem and progenitor cells (HSPC) experience a functional decline in response to chronic inflammation or aging. Haploinsufficiency of A20, or TNFAIP3, an innate immune regulator, is associated with a variety of autoimmune, inflammatory, and hematologic malignancies. Based on a prior analysis of epigenomic and transcriptomic changes during normal human aging, we find that the expression of A20 is significantly reduced in aged HSPC as compared to young HSPC. Here, we show that the partial reduction of A20 expression in young HSPC results in characteristic features of aging. Specifically, heterozygous deletion of A20 in hematopoietic cells resulted in expansion of the HSPC pool, reduced HSPC fitness, and myeloid-biased hematopoiesis. These findings suggest that altered expression of A20 in HSPC contributes to an aging-like phenotype, and that there may be a common underlying mechanism for diminished HSPC function between inflammatory states and aging.

The WSTF-ISWI chromatin remodeling complex transiently associates with the human inactive X chromosome during late S-phase prior to BRCA1 and γ-H2AX.

Replicating the genome prior to each somatic cell division not only requires precise duplication of the genetic information, but also accurately reestablishing the epigenetic signatures that instruct how the genetic material is to be interpreted in the daughter cells. The mammalian inactive X chromosome (Xi), which is faithfully inherited in a silent state in each daughter cell, provides an excellent model of epigenetic regulation. While much is known about the early stages of X chromosome inactivation, much less is understood with regards to retaining the Xi chromatin through somatic cell division. Here we report that the WSTF-ISWI chromatin remodeling complex (WICH) associates with the Xi during late S-phase as the Xi DNA is replicated. Elevated levels of WICH at the Xi is restricted to late S-phase and appears before BRCA1 and γ-H2A.X. The sequential appearance of WICH and BRCA1/γ-H2A.X implicate each as performing important but distinct roles in the maturation and maintenance of heterochromatin at the Xi.