Phf15 - a novel transcriptional repressor regulating inflammation in a mouse microglial cell line

Aim: Excessive microglial inflammation has emerged as a key player in mediating the effects of aging and neurodegeneration on brain dysfunction. Thus, there is great interest in discovering transcriptional repressors that can control this process. We aimed to examine whether Phf15 - one of the top differentially expressed genes in microglia during aging in humans - could regulate transcription of proinflammatory mediators in microglia.Methods: Real-time quantitative PCR was used to assess Phf15 mRNA expression in mouse brain during aging. Loss-of-function [short hairpin RNA (shRNA) -mediated knockdown (KD) and CRISPR/Cas9-mediated knockout (KO) of Phf15] and gain-of-function [retroviral overexpression (OE) of murine Phf15 cDNA] studies in a murine microglial cell line (SIM-A9) followed by immune activation with lipopolysaccharide were used to determine the effect of Phf15 on proinflammatory factor (Tnfα, IL-1β, and Nos2) mRNA expression. RNA sequencing was used to determine global transcriptional changes after Phf15 knockout under basal conditions and after lipopolysaccharide stimulation.Results:Phf15 expression increases in mouse brain during aging, similar to humans. KD, KO, and OE studies determined that Phf15 represses mRNA expression levels of proinflammatory mediators such as Tnfα, IL-1β, and Nos2. Global transcriptional changes after Phf15 KO showed that Phf15 specifically represses genes related to the antiviral (type I interferon) response and cytokine production in microglia.Conclusion: We provide the first evidence that Phf15 is an important transcriptional repressor of microglial inflammation, regulating the antiviral response and proinflammatory cytokine production. Importantly, Phf15 regulates both basal and signal-dependent activation and controls the magnitude and duration of the microglial inflammatory response

Versatile Histochemical Approach to Detection of Hydrogen Peroxide in Cells and Tissues Based on Puromycin Staining

Hydrogen peroxide (H₂O₂) is a central reactive oxygen species (ROS) that contributes to diseases from obesity to cancer to neurodegeneration but is also emerging as an important signaling molecule. We now report a versatile histochemical approach for detection of H₂O₂ that can be employed across a broad range of cell and tissue specimens in both healthy and disease states. We have developed a first-generation H₂O₂-responsive analogue named Peroxymycin-1, which is based on the classic cell-staining molecule puromycin and enables covalent staining of biological samples and retains its signal after fixation. H₂O₂-mediated boronate cleavage uncages the puromycin aminonucleoside, which leaves a permanent and dose-dependent mark on treated biological specimens that can be detected with high sensitivity and precision through a standard immunofluorescence assay. Peroxymycin-1 is selective and sensitive enough to image both exogenous and endogenous changes in cellular H₂O₂ levels and can be exploited to profile resting H₂O₂ levels across a panel of cell lines to distinguish metastatic, invasive cancer cells from less invasive cancer and nontumorigenic counterparts, based on correlations with ROS status. Moreover, we establish that Peroxymycin-1 is an effective histochemical probe for in vivo H₂O₂ analysis, as shown through identification of aberrant elevations in H₂O₂ levels in liver tissues in a murine model of nonalcoholic fatty liver disease, thus demonstrating the potential of this approach for studying disease states and progression associated with H₂O₂. This work provides design principles that should enable development of a broader range of histochemical probes for biological use that operate via activity-based sensing

The Proximal J Kappa Germline-Transcript Promoter Facilitates Receptor Editing through Control of Ordered Recombination

<div><p>V(D)J recombination creates antibody light chain diversity by joining a Vκ gene segment with one of four Jκ segments. Two Jκ germline-transcript (GT) promoters control Vκ-Jκ joining, but the mechanisms that govern Jκ choice are unclear. Here, we show in gene-targeted mice that the proximal GT promoter helps targeting rearrangements to Jκ1 by preventing premature DNA breaks at Jκ2. Consequently, cells lacking the proximal GT promoter show a biased utilization of downstream Jκ segments, resulting in a diminished potential for receptor editing. Surprisingly, the proximal—in contrast to the distal—GT promoter is transcriptionally inactive prior to Igκ recombination, indicating that its role in Jκ choice is independent of classical promoter function. Removal of the proximal GT promoter increases H3K4me3 levels at Jκ segments, suggesting that this promoter could act as a suppressor of recombination by limiting chromatin accessibility to RAG. Our findings identify the first <i>cis</i>-element critical for Jκ choice and demonstrate that ordered Igκ recombination facilitates receptor editing.</p></div

The proximal Jκ GT promoter controls Jκ choice.

<p><b>A)</b> LM-PCR detects total DNA breaks at Jκ gene segments in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Linker ligated genomic DNA was first amplified with several Jκ-specific forward primers (FP) and a linker-specific reverse primer (LP) and then hybridized with Jκ RSS probes. Results are representative of at least two independent experiments. <b>B)</b> LM-PCR detects premature DNA breaks at Jκ2, Jκ4, and Jκ5 in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Linker ligated genomic DNA was first amplified with several Jκ-specific forward primers (FP) and a linker-specific reverse primer (LP) and then hybridized with Jκ RSS probes. Results are representative of at least two independent experiments. <b>C)</b> VJ coding joint PCR detects individual Jκ segments in completed VκJκ joints in B cells from bone marrow or spleen of wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Genomic DNA was first amplified with a degenerate Vκ-specific forward primer and a reverse primer (MAR35) that binds downstream of Jκ5 and then hybridized with a probe (5’MAR35) that binds downstream of Jκ5 but upstream of the reverse primer. Results are representative of at least two independent experiments. </p

The proximal Jκ GT promoter is inactive prior to Igκ recombination.

<p><b>A)</b> Flow cytometry detects expression of κGFP (proximal GT promoter reporter; top) and κhCD4 (distal GT promoter reporter; bottom) in RAG-deficient developing B cells carrying either no transgene, a B1-8^wt HC transgene, or a B1-8^wt HC transgene plus a κHEL transgene. Pro-B and pre-B cells are gated B220⁺ IgM⁻, immature (imm) B cells are gated B220⁺ IgM⁺ IgD⁻, transitional (trans) B cells are gated B220⁺ IgM⁺ IgD^low, and mature (mat) B cells are gated B220⁺ IgM⁺ IgD^high. Grey shaded histograms show cells from a C57Bl/6 control mouse. Results are representative of at least two independent experiments. <b>B)</b> Flow cytometry detects expression of κGFP (top) and κhCD4 (bottom) in non-editing (B1-8^wtHC/κHEL/RAG−/−) or receptor-editing (B1-8^lowHC/κHEL/RAG−/−) B cells (gated B220+ IgM−). Grey shaded histograms show cells from a C57Bl/6 control mouse. Results are representative of at least two independent experiments. <b>C)</b> Northern blotting of Jκ GTs in an Abelson virus-transformed pre-B cell line treated with either STI-571 (STI, which mimics pre-BCR signaling) or the TLR4 ligand LPS. mRNA was hybridized with a Cκ-specific probe (top) that recognizes mature Igκ transcripts, distal GTs, and proximal GTs, the latter of which can be identified by their smaller size. Additionally, the blot was hybridized with a probe specific for distal GTs (middle). Beta-tubulin transcripts (bottom) served as a loading control. Results are representative of two independent experiments. </p

Removal of the proximal Jκ GT promoter increases H3K4me3 levels in the Jκ region and upregulates distal GT promoter activity.

<p><b>A)</b> ChIP analysis of H3K4me3 levels in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Immunoprecipitated genomic DNA was analyzed by qPCR. Specific enrichment was calculated with the formula 2^{Ct(Input)-Ct(IP)}. Results are representative of two independent experiments. Error bars represent standard deviations of triplicate qPCR assays. <b>B)</b> RT-qPCR analysis of distal GT promoter activity in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Jκ GT specific amplification was normalized to HPRT. Locations of forward (FP) and reverse (RP) primers are indicated above the diagram (D, distal GT promoter). Results are representative of two independent experiments. Error bars represent standard deviations of triplicate qPCR assays. </p

Mitohormesis reprogrammes macrophage metabolism to enforce tolerance.

Macrophages generate mitochondrial reactive oxygen species and mitochondrial reactive electrophilic species as antimicrobials during Toll-like receptor (TLR)-dependent inflammatory responses. Whether mitochondrial stress caused by these molecules impacts macrophage function is unknown. Here, we demonstrate that both pharmacologically driven and lipopolysaccharide (LPS)-driven mitochondrial stress in macrophages triggers a stress response called mitohormesis. LPS-driven mitohormetic stress adaptations occur as macrophages transition from an LPS-responsive to LPS-tolerant state wherein stimulus-induced pro-inflammatory gene transcription is impaired, suggesting tolerance is a product of mitohormesis. Indeed, like LPS, hydroxyoestrogen-triggered mitohormesis suppresses mitochondrial oxidative metabolism and acetyl-CoA production needed for histone acetylation and pro-inflammatory gene transcription, and is sufficient to enforce an LPS-tolerant state. Thus, mitochondrial reactive oxygen species and mitochondrial reactive electrophilic species are TLR-dependent signalling molecules that trigger mitohormesis as a negative feedback mechanism to restrain inflammation via tolerance. Moreover, bypassing TLR signalling and pharmacologically triggering mitohormesis represents a new anti-inflammatory strategy that co-opts this stress response to impair epigenetic support of pro-inflammatory gene transcription by mitochondria

Actomyosin-Mediated Tension Orchestrates Uncoupled Respiration in Adipose Tissues

The activation of brown/beige adipose tissue (BAT) metabolism and the induction of uncoupling protein 1 (UCP1) expression are essential for BAT-based strategies to improve metabolic homeostasis. Here, we demonstrate that BAT utilizes actomyosin machinery to generate tensional responses following adrenergic stimulation, similar to muscle tissues. The activation of actomyosin mechanics is critical for the acute induction of oxidative metabolism and uncoupled respiration in UCP1+ adipocytes. Moreover, we show that actomyosin-mediated elasticity regulates the thermogenic capacity of adipocytes via the mechanosensitive transcriptional co-activators YAP and TAZ, which are indispensable for normal BAT function. These biomechanical signaling mechanisms may inform future strategies to promote&nbsp;the expansion and activation of brown/beige adipocytes