Shaping the Innate Immune Response Through Post-Transcriptional Regulation of Gene Expression Mediated by RNA-Binding Proteins

Innate immunity is the frontline of defense against infections and tissue damage. It is a fast and semi-specific response involving a myriad of processes essential for protecting the organism. These reactions promote the clearance of danger by activating, among others, an inflammatory response, the complement cascade and by recruiting the adaptive immunity. Any disequilibrium in this functional balance can lead to either inflammation-mediated tissue damage or defense inefficiency. A dynamic and coordinated gene expression program lies at the heart of the innate immune response. This expression program varies depending on the cell-type and the specific danger signal encountered by the cell and involves multiple layers of regulation. While these are achieved mainly via transcriptional control of gene expression, numerous post-transcriptional regulatory pathways involving RNA-binding proteins (RBPs) and other effectors play a critical role in its fine-tuning. Alternative splicing, translational control and mRNA stability have been shown to be tightly regulated during the innate immune response and participate in modulating gene expression in a global or gene specific manner. More recently, microRNAs assisting RBPs and post-transcriptional modification of RNA bases are also emerging as essential players of the innate immune process. In this review, we highlight the numerous roles played by specific RNA-binding effectors in mediating post-transcriptional control of gene expression to shape innate immunity.

Induced reprogramming of adult murine cardiomyocytes to pluripotency in vivo

Partial cell reprogramming has been demonstrated in certain mouse tissues by in situ overexpression of Oct3/4, Klf4, Sox2 and cMyc (OKSM) transcription factors, and can trigger rejuvenation and/or augment regeneration of aged or injured tissues. In vivo reprogramming of adult mouse cardiomyocytes has been elusive, but success could overcome the lack of endogenous cardiomyocyte turnover that contributes to the poor resolution of heart disease. Here, we exploited cell type-specific Cre recombination and conditional, doxycycline-inducible, control of gene expression to generate cardiomyocyte-specific, inducible, reprogrammable mice. Eighteen days of doxycycline-induced OKSM expression in this model established a gene expression program characteristic of the pluripotent state and triggered the generation of teratomas of confirmed cardiomyocyte origin. These findings confirm that OKSM reprograms adult mouse cardiomyocytes to pluripotency and will enable studies of the contribution of reprogrammed cardiomyocytes to cardiac regeneration.

Predicting the Ultimate Axial Capacity of Uniaxially Loaded CFST Columns Using Multiphysics Artificial Intelligence

The object of this research is concrete-filled steel tubes (CFST). The article aimed to develop a prediction Multiphysics model for the circular CFST column by using the Artificial Neural Network (ANN), the Adaptive Neuro-Fuzzy Inference System (ANFIS) and the Gene Expression Program (GEP). The database for this study contains 1667 datapoints in which 702 are short CFST columns and 965 are long CFST columns. The input parameters are the geometric dimensions of the structural elements of the column and the mechanical properties of materials. The target parameters are the bearing capacity of columns, which determines their life cycle. A Multiphysics model was developed, and various statistical checks were applied using the three artificial intelligence techniques mentioned above. Parametric and sensitivity analyses were also performed on both short and long GEP models. The overall performance of the GEP model was better than the ANN and ANFIS models, and the prediction values of the GEP model were near actual values. The PI of the predicted Nst by GEP, ANN and ANFIS for training are 0.0416, 0.1423, and 0.1016, respectively, and for Nlg these values are 0.1169, 0.2990 and 0.1542, respectively. Corresponding OF values are 0.2300, 0.1200, and 0.090 for Nst, and 0.1000, 0.2700, and 0.1500 for Nlg. The superiority of the GEP method to the other techniques can be seen from the fact that the GEP technique provides suitable connections based on practical experimental work and does not rely on prior solutions. It is concluded that the GEP model can be used to predict the bearing capacity of circular CFST columns to avoid any laborious and time-consuming experimental work. It is also recommended that further research should be performed on the data to develop a prediction equation using other techniques such as Random Forest Regression and Multi Expression Program.

The MLL3/4 complexes and MiDAC act antagonistically as genome-wide regulators of H4K20ac to control a specific gene expression program

The mitotic deacetylase complex MiDAC has recently been shown to play a vital physiological role in embryonic development and neurite outgrowth. However, how MiDAC functionally intersects with other chromatin-modifying regulators is poorly understood. Here, we describe a physical interaction between the histone H3K27 demethylase UTX, a complex-specific subunit of the enhancer-associated MLL3/4 complexes, and MiDAC. We demonstrate that UTX bridges the association of the MLL3/4 complexes and MiDAC by interacting with ELMSAN1, a scaffolding subunit of MiDAC. Our data shows that MiDAC constitutes a negative genome-wide regulator of H4K20ac, an activity which is counteracted by the MLL3/4 complexes. MiDAC and the MLL3/4 complexes co-localize at many genomic regions, that are enriched for H4K20ac and the enhancer marks H3K4me1, H3K4me2 and H3K27ac. We find that MiDAC antagonizes the recruitment of the MLL3/4 complexes to negatively regulate H4K20ac, H3K4me2 and H3K27ac resulting in transcriptional attenuation of associated genes. In summary, our findings provide a paradigm how the opposing roles of chromatin-modifying components, such as MiDAC and the MLL3/4 complexes, balance the transcriptional output of specific gene expression programs.

TBX2 controls a proproliferative gene expression program in melanoma

Senescence shapes embryonic development, plays a key role in aging, and is a critical barrier to cancer initiation, yet how senescence is regulated remains incompletely understood. TBX2 is an antisenescence T-box family transcription repressor implicated in embryonic development and cancer. However, the repertoire of TBX2 target genes, its cooperating partners, and how TBX2 promotes proliferation and senescence bypass are poorly understood. Here, using melanoma as a model, we show that TBX2 lies downstream from PI3K signaling and that TBX2 binds and is required for expression of E2F1, a key antisenescence cell cycle regulator. Remarkably, TBX2 binding in vivo is associated with CACGTG E-boxes, present in genes down-regulated by TBX2 depletion, more frequently than the consensus T-element DNA binding motif that is restricted to Tbx2 repressed genes. TBX2 is revealed to interact with a wide range of transcription factors and cofactors, including key components of the BCOR/PRC1.1 complex that are recruited by TBX2 to the E2F1 locus. Our results provide key insights into how PI3K signaling modulates TBX2 function in cancer to drive proliferation.

674 IL-27 signaling drives a type 1 interferon-like gene expression program of immunoregulatory pathways associated with cancer progression

BackgroundInterleukin (IL)-27 is a heterodimeric immunoregulatory cytokine that signals through the JAK/STAT pathway to increase the expression of coinhibitory receptors on immune cells (e.g. PD-L1, TIM-3, LAG-3) and dampen inflammatory cytokine production. Blockade of IL-27 leads to antitumor activity in preclinical mouse models of lung metastases. A Phase 1 trial of SRF388 (NCT04374877), a first-in-class anti–IL-27 antibody, has demonstrated monotherapy antitumor activity in a patient with non-small cell lung cancer (NSCLC).1 The current study aimed to characterize the immunoregulatory impact of IL-27 signaling by gene expression profiling.MethodsGene expression changes induced by IL-27 were examined in activated human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs), and the IL-27RA–expressing lung cancer cell line NCI-H2228 by microarray or single cell RNA-sequencing. The resulting IL-27 signature genes were interrogated by Gene Set Enrichment Analysis (GSEA) using publicly available datasets, including single cell RNA-seq analysis of the tumor microenvironment, from patients with NSCLC.2ResultsIL-27 induced a robust gene expression program in human immune cells that included several inhibitory receptors and canonical interferon regulated genes such as guanylate-binding proteins and interferon regulatory factors. GSEA and interferon signature analysis showed a striking overlap with those genes regulated by interferon-beta, a cytokine known to drive immune suppression associated with chronic viral infection and that is used therapeutically for controlling inflammation associated with the autoimmune disease multiple sclerosis. Moreover, interferon regulated pathways have recently emerged as a mechanism of resistance to immune checkpoint blockade in cancer. Exploration of the IL-27 gene signature in published datasets showed enrichment in macrophage populations associated with progressive disease in patients with NSCLC. While many of the properties of IL-27–mediated immune regulation have focused on hematopoietic cells, IL-27RA is also expressed on tumor cells from NSCLC patients with progressive disease as well as lung cancer cell lines in which IL-27 can upregulate PD-L1, IDO1, and other canonical interferon regulated genes.ConclusionsThese studies elucidate the transcriptional networks that are engaged after IL-27 signaling in immune and cancer cells and highlight the parallels with interferon-associated immune regulation. Blockade of IL-27 provides a novel therapeutic strategy to alleviate a gene transcriptional program implicated in immune suppression and checkpoint resistance.ReferencesPatnaik A, Morgensztern D, Mantia C, et al. Results of a phase 1 study of SRF388, a first-in-human, first-in-class, high-affinity anti-IL-27 antibody in advanced solid tumors. J Clin Oncol 2021;39:2551–2551.Maynard A, McCoach CE, Rotow JK, et al. Therapy-induced evolution of human lung cancer revealed by single-cell RNA sequencing. Cell 2020;182:1232–1251.

BMP7 Increases UCP1-Dependent and Independent Thermogenesis with a Unique Gene Expression Program in Human Neck Area Derived Adipocytes

White adipocytes contribute to energy storage, accumulating lipid droplets, whereas brown and beige adipocytes mainly function in dissipating energy as heat primarily via the action of uncoupling protein 1 (UCP1). Bone morphogenic protein 7 (BMP7) was shown to drive brown adipocyte differentiation in murine interscapular adipose tissue. Here, we performed global RNA-sequencing and functional assays on adipocytes obtained from subcutaneous (SC) and deep-neck (DN) depots of human neck and differentiated with or without BMP7. We found that BMP7 did not influence differentiation but upregulated browning markers, including UCP1 mRNA and protein in SC and DN derived adipocytes. BMP7 also enhanced mitochondrial DNA content, levels of oxidative phosphorylation complex subunits, along with PGC1α and p-CREB upregulation, and fragmentation of mitochondria. Furthermore, both UCP1-dependent proton leak and UCP1-independent, creatine-driven substrate cycle coupled thermogenesis were augmented upon BMP7 addition. The gene expression analysis also shed light on the possible role of genes unrelated to thermogenesis thus far, including ACAN, CRYAB, and ID1, which were among the highest upregulated ones by BMP7 treatment in both types of adipocytes. Together, our study shows that BMP7 strongly upregulates thermogenesis in human neck area derived adipocytes, along with genes, which might have a supporting role in energy expenditure.

The Menin-MLL1 interaction is a molecular dependency in NUP98-rearranged AML

Translocations involving the NUP98 gene produce NUP98-fusion proteins and are associated with a poor prognosis in acute myeloid leukemia (AML). MLL1 is a molecular dependency in NUP98-fusion leukemia, and therefore we investigated the efficacy of therapeutic blockade of the Menin-MLL1 interaction in NUP98-fusion leukemia models. Using mouse leukemia cell lines driven by NUP98-HOXA9 and NUP98-JARID1A fusion oncoproteins, we demonstrate that NUP98-fusion driven leukemia is sensitive to the Menin-MLL1 inhibitor VTP50469, with an IC50 similar to what we have previously reported for MLL-rearranged and NPM1c leukemia cells. Menin-MLL1 inhibition upregulates markers of differentiation such as CD11b and downregulates expression of pro-leukemogenic transcription factors such as Meis1 in NUP98-fusion transformed leukemia cells. We demonstrate that MLL1 and the NUP98 fusion protein itself are evicted from chromatin at a critical set of genes that are essential for maintenance of the malignant phenotype. In addition to these in vitro studies, we established patient-derived xenograft (PDX) models of NUP98-fusion driven AML to test the in vivo efficacy of Menin-MLL1 inhibition. Treatment with VTP50469 significantly prolongs survival of mice engrafted with NUP98-NSD1 and NUP98-JARID1A leukemias. Gene expression analysis revealed that Menin-MLL1 inhibition simultaneously suppresses a pro-leukemogenic gene expression program, including downregulation of the HOXA cluster, and upregulates tissue-specific markers of differentiation. These preclinical results suggest that Menin-MLL1 inhibition may represent a rational, targeted therapy for patients with NUP98-rearranged leukemias.

BMP7 increases UCP1-dependent and independent thermogenesis with a unique gene expression program in human neck area derived adipocytes

White adipocytes contribute to energy storage accumulating lipid droplets, whereas brown and beige adipocytes mainly function in dissipating energy as heat primarily via the action of uncoupling protein 1 (UCP1). Bone morphogenic protein 7 (BMP7) has been shown to drive brown adipocyte differentiation in mice. In this study, we have performed global RNA-sequencing and functional assays on adipocytes obtained from subcutaneous (SC) and deep-neck (DN) depots of human neck, and differentiated with or without BMP7. We found that BMP7 did not influence differentiation but upregulated browning markers, including UCP1 mRNA and protein. BMP7 also enhanced mitochondrial DNA content, fragmentation, and levels of oxidative phosphorylation complex subunits along with PGC1α and p-CREB upregulation. Furthermore, both UCP1-dependent proton leak and UCP1-independent, creatine driven substrate cycle coupled thermogenesis were augmented upon BMP7 treatment in SC and DN derived adipocytes. The gene expression analysis shed light also on possible role of genes unrelated to thermogenesis so far, including ACAN, CRYAB, and ID1, which were amongst the highest upregulated ones by BMP7 treatment in both types of adipocytes. Together, our study shows that BMP7 strongly upregulates thermogenesis in human neck area derived adipocytes, along with genes, which might have a supporting role in energy expenditure.

Defining the AHR regulated transcriptome in NK cells reveal gene expression program relevant to development and function

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular processes in cancer and immunity, including innate immune cell development and effector function. However, the transcriptional repertoire through which AHR mediates these effects remains largely unexplored. To elucidate the transcriptional elements directly regulated by AHR in NK cells, we performed RNA- and ChIP-sequencing on NK cells exposed to AHR agonist or antagonist. We show that mature peripheral blood NK cells lack AHR, but its expression is induced by Stat3 during IL-21-driven activation and proliferation, coincident with increased NCAM1 (CD56) expression resulting in a CD56bright phenotype. Compared to control conditions, NK cells expanded in the presence of the AHR antagonist, StemRegenin-1, were unaffected in proliferation or cytotoxicity, had no increase in NCAM1 transcription and maintained the CD56dim phenotype. However, it showed altered expression of 1,004 genes including those strongly associated with signaling pathways. In contrast, NK cells expanded in the presence of the AHR agonist, kynurenine, showed decreased cytotoxicity and altered expression of 97 genes including those strongly associated with oxidative stress and cellular metabolism. By overlaying these differentially expressed genes with AHR chromatin binding we identified 160 genes directly regulated by AHR, including hallmark AHR targets AHRR and CYP1B1, and known regulators of phenotype, development, metabolism, and function such as NCAM1, KIT, NQO1, and TXN. In summary, we define the AHR transcriptome in NK cells, propose a model of AHR and Stat3 coregulation, and identify potential pathways that may be targeted to overcome AHR-mediated immune suppression.