MATRIN3 deficiency triggers autoinflammation via cGAS-STING activation.

Interferon-stimulated genes (ISGs) comprise a program of immune effectors important for host immune defense. When uncontrolled, ISGs play a central role in interferonopathies and other inflammatory diseases. The mechanisms responsible for turning on ISGs are not completely known. By investigating MATRIN3 (MATR3), a nuclear RNA-binding protein mutated in familial ALS, we found that perturbing MATR3 results in elevated expression of ISGs. Using an integrative approach, we elucidate a pathway that leads to activation of cGAS-STING. This outlines a plausible mechanism for pathogenesis in a subset of ALS, and suggests new diagnostic and therapeutic approaches for this fatal disease.

Binary outcomes of enhancer activity underlie stable random monoallelic expression.

Mitotically stable random monoallelic gene expression (RME) is documented for a small percentage of autosomal genes. We developed an in vivo genetic model to study the role of enhancers in RME using high-resolution single-cell analysis of natural killer (NK) cell receptor gene expression and enhancer deletions in the mouse germline. Enhancers of the RME NK receptor genes were accessible and enriched in H3K27ac on silent and active alleles alike in cells sorted according to allelic expression status, suggesting enhancer activation and gene expression status can be decoupled. In genes with multiple enhancers, enhancer deletion reduced gene expression frequency, in one instance converting the universally expressed gene encoding NKG2D into an RME gene, recapitulating all aspects of natural RME including mitotic stability of both the active and silent states. The results support the binary model of enhancer action, and suggest that RME is a consequence of general properties of gene regulation by enhancers rather than an RME-specific epigenetic program. Therefore, many and perhaps all genes may be subject to some degree of RME. Surprisingly, this was borne out by analysis of several genes that define different major hematopoietic lineages, that were previously thought to be universally expressed within those lineages: the genes encoding NKG2D, CD45, CD8α, and Thy-1. We propose that intrinsically probabilistic gene allele regulation is a general property of enhancer-controlled gene expression, with previously documented RME representing an extreme on a broad continuum.

Identification of Small Molecule Inhibitors of a Mir155 Transcriptional Reporter in Th17 Cells.

MicroRNA miR-155 is an important regulatory molecule in the immune system and is highly expressed and functional in Th17 cells, a subset of CD4+ T helper cells which are key players in autoimmune diseases. Small molecules that can modulate miR-155 may potentially provide new therapeutic avenues to inhibit Th17 cell-mediated autoimmune diseases. Here, we present a novel high-throughput screening assay using primary T cells from genetically engineered Mir155 reporter mice, and its use to screen libraries of small molecules to identify novel modulators of Th17 cell function. We have discovered a chemical series of (E)-1-(phenylsulfonyl)-2-styryl-1H-benzo[d] imidazoles as novel down-regulators of Mir155 reporter and cytokine expression in Th17 cells. In addition, we found that FDA approved antiparasitic agents belonging to the 'azole' family also down-regulate Mir155 reporter and cytokine expression in Th17 cells, and thus could potentially be repurposed to treat Th17-driven immunopathologies.

Posttranscriptional regulation of human endogenous retroviruses by RNA-binding motif protein 4, RBM4.

The human genome encodes for over 1,500 RNA-binding proteins (RBPs), which coordinate regulatory events on RNA transcripts. Most studies of RBPs have concentrated on their action on host protein-encoding mRNAs, which constitute a minority of the transcriptome. A widely neglected subset of our transcriptome derives from integrated retroviral elements, termed endogenous retroviruses (ERVs), that comprise ∼8% of the human genome. Some ERVs have been shown to be transcribed under physiological and pathological conditions, suggesting that sophisticated regulatory mechanisms to coordinate and prevent their ectopic expression exist. However, it is unknown how broadly RBPs and ERV transcripts directly interact to provide a posttranscriptional layer of regulation. Here, we implemented a computational pipeline to determine the correlation of expression between individual RBPs and ERVs from single-cell or bulk RNA-sequencing data. One of our top candidates for an RBP negatively regulating ERV expression was RNA-binding motif protein 4 (RBM4). We used photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation to demonstrate that RBM4 indeed bound ERV transcripts at CGG consensus elements. Loss of RBM4 resulted in an elevated transcript level of bound ERVs of the HERV-K and -H families, as well as increased expression of HERV-K envelope protein. We pinpointed RBM4 regulation of HERV-K to a CGG-containing element that is conserved in the LTRs of HERV-K-10, -K-11, and -K-20, and validated the functionality of this site using reporter assays. In summary, we systematically identified RBPs that may regulate ERV function and demonstrate a role for RBM4 in controlling ERV expression.

Identification of Small Molecule Enhancers of Immunotherapy for Melanoma.

Small molecule based targeted therapies for the treatment of metastatic melanoma hold promise but responses are often not durable, and tumors frequently relapse. Response to adoptive cell transfer (ACT)-based immunotherapy in melanoma patients are durable but patients develop resistance primarily due to loss of antigen expression. The combination of small molecules that sustain T cell effector function with ACT could lead to long lasting responses. Here, we have developed a novel co-culture cell-based high throughput assay system to identify compounds that could potentially synergize or enhance ACT-based immunotherapy of melanoma. A BRAFV600E mutant melanoma cell line, SB-3123p which is resistant to Pmel-1-directed ACT due to low gp100 expression levels was used to develop a homogenous time resolve fluorescence (HTRF), screening assay. This high throughput screening assay quantitates IFNγ released upon recognition of the SB-3123p melanoma cells by Pmel-1 CD8+ T-cells. A focused collection of approximately 500 small molecules targeting a broad range of cellular mechanisms was screened, and four active compounds that increased melanoma antigen expression leading to enhanced IFNγ production were identified and their in vitro activity was validated. These four compounds may provide a basis for enhanced immune recognition and design of novel therapeutic approaches for patients with BRAF mutant melanoma resistant to ACT due to antigen downregulation.

Anxiolytic Drug FGIN-1-27 Ameliorates Autoimmunity by Metabolic Reprogramming of Pathogenic Th17 Cells.

Th17 cells are critical drivers of autoimmune diseases and immunopathology. There is an unmet need to develop therapies targeting pathogenic Th17 cells for the treatment of autoimmune disorders. Here, we report that anxiolytic FGIN-1-27 inhibits differentiation and pathogenicity of Th17 cells in vitro and in vivo using the experimental autoimmune encephalomyelitis (EAE) model of Th17 cell-driven pathology. Remarkably, we found that the effects of FGIN-1-27 were independent of translocator protein (TSPO), the reported target for this small molecule, and instead were driven by a metabolic switch in Th17 cells that led to the induction of the amino acid starvation response and altered cellular fatty acid composition. Our findings suggest that the small molecule FGIN-1-27 can be re-purposed to relieve autoimmunity by metabolic reprogramming of pathogenic Th17 cells.

RNA-binding protein IGF2BP1 maintains leukemia stem cell properties by regulating HOXB4, MYB, and ALDH1A1.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an oncofetal protein expressed in various cancers including leukemia. In this study, we assessed the role of IGF2BP1 in orchestrating leukemia stem cell properties. Tumor-initiating potential, sensitivity to chemotherapeutic agents, and expression of cancer stem cell markers were assessed in a panel of myeloid, B-, and T-cell leukemia cell lines using gain- and loss-of-function systems, cross-linking immunoprecipitation (CLIP), and photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) techniques. Here, we report that genetic or chemical inhibition of IGF2BP1 decreases leukemia cells' tumorigenicity, promotes myeloid differentiation, increases leukemia cell death, and sensitizes leukemia cells to chemotherapeutic drugs. IGF2BP1 affects proliferation and tumorigenic potential of leukemia cells through critical regulators of self-renewal HOXB4 and MYB and through regulation of expression of the aldehyde dehydrogenase, ALDH1A1. Our data indicate that IGF2BP1 maintains leukemia stem cell properties by regulating multiple pathways of stemness through transcriptional and metabolic factors.

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3.

Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.

A forward genetic screen reveals novel independent regulators of ULBP1, an activating ligand for natural killer cells.

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.