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.

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.

BackgroundWilliams 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).ResultsTo 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.ConclusionsGiven 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.

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.

Ubiquitin-specific peptidase 15 (USP15) is a deubiquitinating enzyme implicated in critical cellular and oncogenic processes. We report that USP15 mRNA and protein are overexpressed in human acute myeloid leukemia (AML) as compared to normal hematopoietic progenitor cells. This high expression of USP15 in AML correlates with KEAP1 protein and suppression of NRF2. Knockdown or deletion of USP15 in human and mouse AML models significantly impairs leukemic progenitor function and viability and de-represses an antioxidant response through the KEAP1-NRF2 axis. Inhibition of USP15 and subsequent activation of NRF2 leads to redox perturbations in AML cells, coincident with impaired leukemic cell function. In contrast, USP15 is dispensable for human and mouse normal hematopoietic cells in vitro and in vivo. A preclinical small-molecule inhibitor of USP15 induced the KEAP1-NRF2 axis and impaired AML cell function, suggesting that targeting USP15 catalytic function can suppress AML. Based on these findings, we report that USP15 drives AML cell function, in part, by suppressing a critical oxidative stress sensor mechanism and permitting an aberrant redox state. Furthermore, we postulate that inhibition of USP15 activity with small molecule inhibitors will selectively impair leukemic progenitor cells by re-engaging homeostatic redox responses while sparing normal hematopoiesis.