Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.

ATF2 orchestrates macrophage differentiation and activation to promote antibacterial responses.

The differentiation and activation of macrophages are critical regulatory programs that are central to host inflammation and pathogen defense. However, the transcriptional regulatory pathways involved in these programs are not well understood. Herein, we demonstrate that the activity and expression of the transcription factor ATF2 is precisely regulated during primary human monocyte-to-macrophage differentiation and that its activation is linked to M1 polarization and antibacterial responses. Genetic perturbation experiments demonstrated that deletion of ATF2 (THP-ΔATF2) resulted in irregular and abnormal macrophage morphology, whereas macrophages overexpressing ATF2 (THP-ATF2) developed round and pancake-like morphology, resembling classically activated (M1) macrophages. Mechanistically, we show that ATF2 binds to the core promoter of PPM1A, a phosphatase that regulates monocyte-to-macrophage differentiation, to regulate its expression. Functionally, overexpression of ATF2 sensitized macrophages to M1 polarization, resulting in increased production of major histocompatibility complex class II, IL-1ß, and IP-10; improved phagocytic capacity; and enhanced control of the intracellular pathogen Mycobacterium tuberculosis. Gene expression profiling revealed that overexpression of ATF2 reprogramed macrophages to promote antibacterial pathways enriched in chemokine signaling, metabolism, and antigen presentation. Consistent with pathways analysis, metabolic profiling revealed that genetic overexpression or stimuli-induced activation of ATF2 alters the metabolic capacity of macrophages and primes these cells for glycolytic metabolism during M1 polarization or bacterial infection. Our findings reveal that ATF2 plays a central role during macrophage differentiation and M1 polarization to enhance the functional capacities of macrophages.

Foxo3a tempers excessive glutaminolysis in activated T cells to prevent fatal gut inflammation in the murine IL-10-/- model of colitis.

Mutations in susceptibility alleles correlate with gut-inflammatory diseases, such as Crohn's disease; however, this does not often impact the disease progression indicating the existence of compensatory genes. We show that a reduction in Foxo3a expression in IL-10-deficient mice results in a spontaneous and aggressive Crohn's- like disease with 100% penetrance, which is rescued by deletion of myeloid cells, T cells and inhibition of mTORC1. In Foxo3a-/- IL-10-/- mice, there is poor cell death of myeloid cells in the gut, leading to increased accumulation of myeloid and T cells in the gut. Myeloid cells express high levels of inflammatory cytokines, and regulatory T cells are dysfunctional despite increased abundance. Foxo3a signaling represses the transcription of glutaminase (GLS/GLS2) to prevent over-consumption of glutamine by activated T cells and its conversion to glutamate that contributes to the TCA cycle and mTORC1 activation. Finally, we show that Foxo3a restricts the abundance of colitogenic microbiota in IL-10-deficient mice. Thus, by suppressing glutaminolysis in activated T cells Foxo3a mediates a critical checkpoint that prevents the development of fulminant gut inflammatory disease.

Chromatin and transcription factor profiling in rare stem cell populations using CUT&Tag.

Muscle stem cells (MuSCs) are a rare stem cell population that provides myofibers with a remarkable capacity to regenerate after tissue injury. Here, we have adapted the Cleavage Under Target and Tagmentation technology to the mapping of the chromatin landscape and transcription factor binding in 50,000 activated MuSCs isolated from injured mouse hindlimb muscles. We have applied this same approach to human CD34+ hematopoietic stem and progenitor cells. This protocol could be adapted to any rare stem cell population. For complete details on the use and execution of this protocol, please refer to Robinson et al. (2021).

Insights into interplay between rexinoid signaling and myogenic regulatory factor-associated chromatin state in myogenic differentiation.

While skeletal myogenesis is tightly coordinated by myogenic regulatory factors including MyoD and myogenin, chromatin modifications have emerged as vital mechanisms of myogenic regulation. We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the specification and differentiation of skeletal muscle lineage. Here, we examine the genome-wide impact of rexinoids on myogenic differentiation through integral RNA-seq and ChIP-seq analyses. We found that bexarotene promotes myoblast differentiation through the coordination of exit from the cell cycle and the activation of muscle-related genes. We uncovered a new mechanism of rexinoid action which is mediated by the nuclear receptor and largely reconciled through a direct regulation of MyoD gene expression. In addition, we determined a rexinoid-responsive residue-specific histone acetylation at a distinct chromatin state associated to MyoD and myogenin. Thus, we provide novel molecular insights into the interplay between RXR signaling and chromatin states pertinent to myogenic programs in early myoblast differentiation.

Regulation of Hspb7 by MEF2 and AP-1: implications for Hspb7 in muscle atrophy.

Mycocyte enhancer factor 2 (MEF2) and activator protein 1 (AP-1) transcription complexes have been individually implicated in myogenesis, but their genetic interaction has not previously been addressed. Using MEF2A, c-Jun and Fra-1 chromatin immunoprecipitation sequencing (ChIP-seq) data and predicted AP-1 consensus motifs, we identified putative common MEF2 and AP-1 target genes, several of which are implicated in regulating the actin cytoskeleton. Because muscle atrophy results in remodelling or degradation of the actin cytoskeleton, we characterized the expression of putative MEF2 and AP-1 target genes (Dstn, Flnc, Hspb7, Lmod3 and Plekhh2) under atrophic conditions using dexamethasone (Dex) treatment in skeletal myoblasts. Heat shock protein b7 (Hspb7) was induced by Dex treatment and further analyses revealed that loss of MEF2A using siRNA prevented Dex-regulated induction of Hspb7. Conversely, ectopic Fra-2 or c-Jun expression reduced Dex-mediated upregulation of Hspb7 whereas AP-1 depletion enhanced Hspb7 expression. In vivo, expression of Hspb7 and other autophagy-related genes was upregulated in response to atrophic conditions in mice. Manipulation of Hspb7 levels in mice also impacted gross muscle mass. Collectively, these data indicate that MEF2 and AP-1 confer antagonistic regulation of Hspb7 gene expression in skeletal muscle, with implications for autophagy and muscle atrophy.

Inhibition of ROS and upregulation of inflammatory cytokines by FoxO3a promotes survival against Salmonella typhimurium.

Virulent intracellular pathogens, such as the Salmonella species, engage numerous virulence factors to subvert host defence mechanisms to induce a chronic infection that leads to typhoid or exacerbation of other chronic inflammatory conditions. Here we show the role of the forkhead transcription factor FoxO3a during infection of mice with Salmonella typhimurium (ST). Although FoxO3a signalling does not affect the development of CD8(+) T cell responses to ST, FoxO3a has an important protective role, particularly during the chronic stage of infection, by limiting the persistence of oxidative stress. Furthermore, FoxO3a signalling regulates ERK signalling in macrophages, which results in the maintenance of a proinflammatory state. FoxO3a signalling does not affect cell proliferation or cell death. Thus, these results reveal mechanisms by which FoxO3a promotes host survival during infection with chronic, virulent intracellular bacteria.

BRG1 interacts with GLI2 and binds Mef2c gene in a hedgehog signalling dependent manner during in vitro cardiomyogenesis.

BACKGROUND: The Hedgehog (HH) signalling pathway regulates cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (mES) cell model. To further assess the transcriptional role of HH signalling during cardiomyogenesis in stem cells, we studied the effects of overexpressing GLI2, a primary transducer of the HH signalling pathway, in mES cells. RESULTS: Stable GLI2 overexpression resulted in an enhancement of cardiac progenitor-enriched genes, Mef2c, Nkx2-5, and Tbx5 during mES cell differentiation. In contrast, pharmacological blockade of the HH pathway in mES cells resulted in lower expression of these genes. Mass spectrometric analysis identified the chromatin remodelling factor BRG1 as a protein which co-immunoprecipitates with GLI2 in differentiating mES cells. We then determined that BRG1 is recruited to a GLI2-specific Mef2c gene element in a HH signalling-dependent manner during cardiomyogenesis in P19 EC cells, a mES cell model. CONCLUSIONS: Thus, we propose a mechanism where HH/GLI2 regulates the expression of Mef2c by recruiting BRG1 to the Mef2c gene, most probably via chromatin remodelling, to ultimately regulate in vitro cardiomyogenesis.

Opposing regulation of Sox2 by cell-cycle effectors E2f3a and E2f3b in neural stem cells.

The mechanisms through which cell-cycle control and cell-fate decisions are coordinated in proliferating stem cell populations are largely unknown. Here, we show that E2f3 isoforms, which control cell-cycle progression in cooperation with the retinoblastoma protein (pRb), have critical effects during developmental and adult neurogenesis. Loss of either E2f3 isoform disrupts Sox2 gene regulation and the balance between precursor maintenance and differentiation in the developing cortex. Both isoforms target the Sox2 locus to maintain baseline levels of Sox2 expression but antagonistically regulate Sox2 levels to instruct fate choices. E2f3-mediated regulation of Sox2 and precursor cell fate extends to the adult brain, where E2f3a loss results in defects in hippocampal neurogenesis and memory formation. Our results demonstrate a mechanism by which E2f3a and E2f3b differentially regulate Sox2 dosage in neural precursors, a finding that may have broad implications for the regulation of diverse stem cell populations.

The Rb/E2F pathway modulates neurogenesis through direct regulation of the Dlx1/Dlx2 bigene cluster.

During brain morphogenesis, the mechanisms through which the cell cycle machinery integrates with differentiation signals remain elusive. Here we show that the Rb/E2F pathway regulates key aspects of differentiation and migration through direct control of the Dlx1 and Dlx2 homeodomain proteins, required for interneuron specification. Rb deficiency results in a dramatic reduction of Dlx1 and Dlx2 gene expression manifested by loss of interneuron subtypes and severe migration defects in the mouse brain. The Rb/E2F pathway modulates Dlx1/Dlx2 regulation through direct interaction with a Dlx forebrain-specific enhancer, I12b, and the Dlx1/Dlx2 proximal promoter regions, through repressor E2F sites both in vitro and in vivo. In the absence of Rb, we demonstrate that repressor E2Fs inhibit Dlx transcription at the Dlx1/Dlx2 promoters and Dlx1/2-I12b enhancer to suppress differentiation. Our findings support a model whereby the cell cycle machinery not only controls cell division but also modulates neuronal differentiation and migration through direct regulation of the Dlx1/Dlx2 bigene cluster during embryonic development.

Traf7, a MyoD1 transcriptional target, regulates nuclear factor-κB activity during myogenesis.

We have identified the E3 ligase Traf7 as a direct MyoD1 target and show that cell cycle exit-an early event in muscle differentiation-is linked to decreased Traf7 expression. Depletion of Traf7 accelerates myogenesis, in part through downregulation of nuclear factor-κB (NF-κB) activity. We used a proteomic screen to identify NEMO, the NF-κB essential modulator, as a Traf7-interacting protein. Finally, we show that ubiquitylation of NF-κB essential modulator is regulated exclusively by Traf7 activity in myoblasts. Our results suggest a new mechanism by which MyoD1 function is coupled to NF-κB activity through Traf7, regulating the balance between cell cycle progression and differentiation during myogenesis.

Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms.

Polycomb group proteins are critical to maintaining gene repression established during Drosophila development. Part of this group forms the PRC2 complex containing Ez that catalyzes di- and trimethylation of histone H3 lysine 27 (H3K37me2/3), marks repressive to transcription. We report that the mammalian homologs Ezh1 and Ezh2 form similar PRC2 complexes but exhibit contrasting repressive roles. While PRC2-Ezh2 catalyzes H3K27me2/3 and its knockdown affects global H3K27me2/3 levels, PRC2-Ezh1 performs this function weakly. In accordance, Ezh1 knockdown was ineffectual on global H3K27me2/3 levels. Instead, PRC2-Ezh1 directly and robustly represses transcription from chromatinized templates and compacts chromatin in the absence of the methyltransferase cofactor SAM, as evidenced by electron microscopy. Ezh1 targets a subset of Ezh2 genes, yet Ezh1 is more abundant in nonproliferative adult organs while Ezh2 expression is tightly associated with proliferation, as evidenced when analyzing aging mouse kidney. These results might reflect subfunctionalization of a PcG protein during evolution.

Retinoblastoma tumor suppressor protein-dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit.

The retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes. Remarkably, we also uncover a function for the pRb-related proteins p107 and p130 as enforcers of a G2/M phase checkpoint that prevents progression into mitosis in cells that have lost pRb. We further demonstrate that pRb effects permanent cell cycle exit in part by maintaining trimethylation of histone H3 lysine 27 (H3K27) on cell cycle genes. H3K27 trimethylation silences other genes, including Cyclin D1, in a pRb-independent but polycomb-dependent manner. Thus, our data distinguish two distinct chromatin-based regulatory mechanisms that lead to terminal differentiation.

ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene.

In the present study, we show that E2Fs (E2 promoter-binding factors) regulate the expression of ASK-1 (apoptosis signal-regulating kinase 1), which encodes a mitogen-activated protein kinase kinase kinase, also known as MAP3K5. Its mRNA expression is cell-cycle-regulated in human T98G cells released from serum starvation. Moreover, overexpression and RNA interference experiments support the requirement of endogenous E2F/DP (E2F dimerization partner) activity for ASK-1 expression. Characterization of the human ASK-1 promoter demonstrates that the -95/+11 region is critical for E2F-mediated up-regulation. Chromatin immunoprecipitation assays show that E2F1-E2F4 are bound in vivo to the ASK-1 promoter in cycling cells, probably through a non-consensus E2F-binding site located 12 bp upstream of the transcription start site. Mutation of this site completely abolishes the ASK-1 promoter response to E2Fs as well as the E2F1 binding in electrophoretic mobility-shift experiments. Our results indicate that E2Fs modulate the expression of ASK-1 and suggest that some of the cellular functions of ASK-1 may be under the control of E2F transcription factors. Moreover, the up-regulation of ASK-1 may also favour the p53-independent E2F1 apoptotic activity.

A common set of gene regulatory networks links metabolism and growth inhibition.

Using genome-wide analysis of transcription factor occupancy, we investigated the mechanisms underlying three mammalian growth arrest pathways that require the pRB tumor suppressor family. We found that p130 and E2F4 cooperatively repress a common set of genes under each growth arrest condition and showed that growth arrest is achieved through repression of a core set of genes involved not only in cell cycle control but also mitochondrial biogenesis and metabolism. Motif-finding algorithms predicted the existence of nuclear respiratory factor-1 (NRF1) binding sites in E2F target promoters, and genome-wide factor binding analysis confirmed our predictions. We showed that NRF1, a factor known to regulate expression of genes involved in mitochondrial function, is a coregulator of a large number of E2F target genes. Our studies provide insights into E2F regulatory circuitry, suggest how factor occupancy can predict the expression signature of a given target gene, and reveal pathways deregulated in human tumors.

Overexpression of a dominant negative type II bone morphogenetic protein receptor inhibits the growth of human breast cancer cells.

Bone morphogenetic proteins (BMPs) exert cell type-specific effects on cell proliferation. To clarify the role of the BMP pathway in human breast cancer cells, we used a dominant negative strategy with a truncated human type II BMP receptor (DN-BMPRII; amino acid 1-172) fused to the NH2 terminus of enhanced green fluorescent protein. Transient overexpression of DN-BMPRII interfered with BMP-2-induced Smad1 transcriptional activity and caused cells to accumulate in G1. Stable cell lines that constitutively overexpressed DN-BMPRII were resistant to BMP-2-induced Smad1 phosphorylation and proliferated much more slowly than control stable cell lines. These results suggest that BMPs interacting with type II BMP receptors contribute to the proliferation and/or survival of human breast cancer cells.

Regulation of the human cyclin-dependent kinase inhibitor p18INK4c by the transcription factors E2F1 and Sp1.

The p18(INK4c) cyclin-dependent kinase inhibitor is an important regulator of cell cycle progression and cellular differentiation. We and others found that overexpressed E2F proteins up-regulate p18 expression. To better understand this phenomenon, we performed a functional analysis of the human p18 promoter. Deletion studies revealed that the E2F-responsive elements of the promoter are located within 131 bp upstream of the transcription start site. This region contains putative Sp1- and E2F-binding sites. Mutational inactivation of these elements revealed that the Sp1 sites were important for the basal activity of the promoter but could also mediate the effects of E2F1 on the p18 promoter. Moreover, we found that E2F1 and Sp1 can synergistically enhance the activity of the proximal p18 promoter. Gel shift analyses using p18 promoter-derived probes led to the identification of several multiprotein complexes that were found to contain different combinations of E2F proteins and/or Sp1. Recombinant E2F1 was also capable of binding to the E2F-binding sites. Chromatin immunoprecipitation experiments demonstrated that E2F1 and E2F4 associate with the p18 promoter in unperturbed cells. Based on these findings, we conclude that E2F proteins and Sp1 play an important role in the control of p18 expression.