Direct Transcriptional Activation Research Articles

The aryl hydrocarbon receptor and FOS mediate cytotoxicity induced by Acinetobacter baumannii

Acinetobacter baumannii is a pathogenic and multidrug-resistant Gram-negative bacterium that causes severe nosocomial infections. To better understand the mechanism of pathogenesis, we compare the proteomes of uninfected and infected human cells, revealing that transcription factor FOS is the host protein most strongly induced by A. baumannii infection. Pharmacological inhibition of FOS reduces the cytotoxicity of A. baumannii in cell-based models, and similar results are also observed in a mouse infection model. A. baumannii outer membrane vesicles (OMVs) are shown to activate the aryl hydrocarbon receptor (AHR) of host cells by inducing the host enzyme tryptophan-2,3-dioxygenase (TDO), producing the ligand kynurenine, which binds AHR. Following ligand binding, AHR is a direct transcriptional activator of the FOS gene. We propose that A. baumannii infection impacts the host tryptophan metabolism and promotes AHR- and FOS-mediated cytotoxicity of infected cells.

EGR2 is an epigenomic regulator of phagocytosis and antifungal immunity in alveolar macrophages.

Alveolar macrophages (AMs) act as gatekeepers of the lung's immune responses, serving essential roles in recognizing and eliminating pathogens. The transcription factor (TF) early growth response 2 (EGR2) has been recently described as required for mature AMs in mice; however, its mechanisms of action have not been explored. Here, we identified EGR2 as an epigenomic regulator and likely direct proximal transcriptional activator in AMs using epigenomic approaches (RNA sequencing, ATAC sequencing, and CUT&RUN). The predicted direct proximal targets of EGR2 included a subset of AM identity genes and ones related to pathogen recognition, phagosome maturation, and adhesion, such as Clec7a, Atp6v0d2, Itgb2, Rhoc, and Tmsb10. We provided evidence that EGR2 deficiency led to impaired zymosan internalization and reduced the capacity to respond to Aspergillus fumigatus. Mechanistically, the lack of EGR2 altered the transcriptional response, secreted cytokines (i.e., CXCL11), and inflammation-resolving lipid mediators (i.e., RvE1) of AMs during in vivo zymosan-induced inflammation, which manifested in impaired resolution. Our findings demonstrated that EGR2 is a key proximal transcriptional activator and epigenomic bookmark in AMs responsible for select, distinct components of cell identity and a protective transcriptional and epigenomic program against fungi.

Variations in the Antivirulence Effects of Fatty Acids and Virstatin against Vibrio cholerae Strains.

The expression of two major virulence factors of Vibrio cholerae, cholera toxin (CT) and toxin coregulated pilus (TCP), is induced by environmental stimuli through a cascade of interactions among regulatory proteins known as the ToxR regulon when the bacteria reach the human small intestine. ToxT is produced via the ToxR regulon and acts as the direct transcriptional activator of CT (ctxAB), TCP (tcp gene cluster), and other virulence genes. Unsaturated fatty acids (UFAs) and several smallmolecule inhibitors of ToxT have been developed as antivirulence agents against V. cholerae. This study reports the inhibitory effects of fatty acids and virstatin (a small-molecule inhibitor of ToxT) on the transcriptional activation functions of ToxT in isogenic derivatives of V. cholerae strains containing various toxT alleles. The fatty acids and virstatin had discrete effects depending on the ToxT allele (different by 2 amino acids), V. cholerae strain, and culture conditions, indicating that V. cholerae strains could overcome the effects of UFAs and small-molecule inhibitors by acquiring point mutations in toxT. Our results suggest that small-molecule inhibitors should be examined thoroughly against various V. cholerae strains and toxT alleles during development.

TFEB safeguards trophoblast syncytialization in humans and mice

Nutrient sensing and adaptation in the placenta are essential for pregnancy viability and proper fetal growth. Our recent study demonstrated that the placenta adapts to nutrient insufficiency through mechanistic target of rapamycin (mTOR) inhibition-mediated trophoblast differentiation toward syncytiotrophoblasts (STBs), a highly specialized multinucleated trophoblast subtype mediating extensive maternal-fetal interactions. However, the underlying mechanism remains elusive. Here, we unravel the indispensable role of the mTORC1 downstream transcriptional factor TFEB in STB formation both in vitro and in vivo. TFEB deficiency significantly impaired STB differentiation in human trophoblasts and placenta organoids. Consistently, systemic or trophoblast-specific deletion of Tfeb compromised STB formation and placental vascular construction, leading to severe embryonic lethality. Mechanistically, TFEB conferred direct transcriptional activation of the fusogen ERVFRD-1 in human trophoblasts and thereby promoted STB formation, independent of its canonical function as a master regulator of the autophagy-lysosomal pathway. Moreover, we demonstrated that TFEB directed the trophoblast syncytialization response driven by mTOR complex 1 (mTORC1) signaling. TFEB expression positively correlated with the reinforced trophoblast syncytialization in human fetal growth-restricted placentas exhibiting suppressed mTORC1 activity. Our findings substantiate that the TFEB-fusogen axis ensures proper STB formation during placenta development and under nutrient stress, shedding light on TFEB as a mechanistic link between nutrient-sensing machinery and trophoblast differentiation.

HOXC6-mediated transcriptional activation ofENO2 promotes oral squamous cell carcinoma progression through the Warburg effect.

Oral squamous cell carcinoma (OSCC) requires an in-depth exploration of its molecular mechanisms. The Warburg effect, along with the oncogenesenolase 2(ENO2) andhomeobox C6 (HOXC6), plays a central role in cancer. However, the specific interaction between ENO2 and HOXC6 in driving the Warburg effect and OSCC progression remains poorly understood. Through differential gene expression analysis in head and neck squamous cell carcinomas using Gene Expression Profiling Interactive Analysis, we identified upregulated ENO2 in OSCC. Silencing ENO2 in OSCC cells revealed its involvement in migration, invasion, and aerobic glycolysis of OSCC cells. Further exploration of ENO2's regulatory network identified HOXC6 as a potential transcriptional regulator. Subsequently, HOXC6 was silenced in OSCC cells, and expressions of ENO2 were assessed to validate its relationship with ENO2. Chromatin Immunoprecipitation and luciferase assays were utilized to investigate the direct transcriptional activation of ENO2 by HOXC6. A rescue assay co-overexpressing ENO2 and silencing HOXC6 in OSCC cells affirmed HOXC6's role in ENO2-associated glycolysis. High ENO2 expression in OSCC was validated through quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry analyses, which correlated with poor patient survival. Functional assays demonstrated that ENO2 silencing inhibited glycolysis and attenuated the aggressiveness of OSCC cells. In vivo studies confirmed the oncogenic role of ENO2 in OSCC growth. Notably, HOXC6 exhibited a positive correlation with ENO2 expression in clinical samples. Mechanistically, HOXC6 was identified as a direct transcriptional activator of ENO2, orchestrating the Warburg effect in OSCC cells. This study reveals the intricate link between HOXC6-mediated ENO2 transcriptional activation and the Warburg effect in OSCC, offering a potential therapeutic target for treating OSCC patients.

FOXF1 promotes tumor vessel normalization and prevents lung cancer progression through FZD4

Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/β-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/β-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/β-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.

σE of Streptomyces coelicolor can function both as a direct activator or repressor of transcription

σ factors are considered as positive regulators of gene expression. Here we reveal the opposite, inhibitory role of these proteins. We used a combination of molecular biology methods and computational modeling to analyze the regulatory activity of the extracytoplasmic σE factor from Streptomyces coelicolor. The direct activator/repressor function of σE was then explored by experimental analysis of selected promoter regions in vivo. Additionally, the σE interactome was defined. Taken together, the results characterize σE, its regulation, regulon, and suggest its direct inhibitory function (as a repressor) in gene expression, a phenomenon that may be common also to other σ factors and organisms.

Pirin Inhibits FAS-Mediated Apoptosis to Support Colorectal Cancer Survival.

Resistance to immunotherapy in colorectal cancer (CRC) is associated with obstruction of FAS (Apo-1 or CD95)-dependent apoptosis, a hallmark of cancer. Here it is demonstrated that the upregulation of pirin (PIR) protein in colon cancers promotes tumorigenesis. Knockout or inhibition of PIR dramatically increases FAS expression, FAS-dependent apoptosis and attenuates colorectal tumor formation in mice. Specifically, NFκB2 is a direct transcriptional activator of FAS and robustly suppressed by PIR in dual mechanisms. One is the disruption of NFκB2 complex (p52-RELB) association with FAS promoter, the other is the inhibition of NIK-mediated NFκB2 activation and nuclear translocation, leading to the inability of active NFκB2 complex toward the transcription of FAS. Furthermore, PIR interacts with FAS and recruits it in cytosol, preventing its membrane translocation and assembling. Importantly, knockdown or knockout of PIR dramatically sensitizes cells to FAS mAb- or active CD8+ T cells-triggered cell death. Taken together, a PIR-NIK-NFκB2-FAS survival pathway is established, which plays a key role in supporting CRC survival.

The transcription factor ETV1 regulates expression of the atrial specific potassium channel TASK-1 and modulates cardiac action potentials

Abstract Background In recent years, the two-pore domain potassium channel TASK-1 has been identified to play an important role in the pathophysiology of atrial fibrillation (AF). In cardiac tissue, TASK-1 is almost exclusively expressed in the atria and significantly upregulated in AF patients. Moreover, it has been characterized as a key player in action potential (AP) shortening observed during AF, making it a promising target for antiarrhythmic therapy. However, the underlying transcriptional mechanisms responsible for TASK-1 upregulation during AF are still unclear. A few years ago, the transcription factor ETV1 has been described to induce atrial remodeling and arrhythmia. Similar to TASK-1, ETV1 is predominantly expressed in the atria and upregulated in AF patients. Therefore, investigating possible interactions between ETV1 and TASK-1 is crucial to further understand atrial remodeling and develop new antiarrhythmic strategies. Purpose The purpose of this study was to investigate the effects of ETV1 regulation on TASK-1 expression. For that, electrophysiological effects of ETV1 modulation on TASK-1 currents and action potential formation were studied on HL-1 cardiomyocytes. Methods ETV1 modulation was achieved either through application of the pharmacological ETV1-inhibitor BRD32048 (1 µM) or transfection of HL-1 cells with a specific ETV1-siRNA. Electrophysiological effects of ETV1 inhibition were assessed using two-electrode voltage clamp experiments on Xenopus laevis oocytes heterologously expressing TASK-1 as well as whole-cell patch clamp measurements on HL-1 cells after pharmacological- and siRNA-mediated ETV1 inhibition. Chromatin immunoprecipitation combined with real-time qPCR (ChIP-qPCR) and ATAC-seq experiments were performed to identify the epigenetic interaction between ETV1 and the KCNK3 gene encoding TASK-1. Results TASK-1 currents were markedly reduced in Xenopus laevis oocytes after inhibition of ETV1. Furthermore, both pharmacological inhibition and siRNA-mediated knockdown of ETV1 caused a significant decrease of TASK-1 currents in HL-1 cells by 35.8% and 39.3%, respectively. These effects were associated with a statistically significant prolongation of the action potential duration at 90% repolarization (APD90) by 28.5% after functional inhibition and 24.9% after siRNA-knockdown. This correlated with a significant decrease of TASK-1 expression on mRNA and protein level. In addition, ATAC-seq data and ChIP-qPCR revealed an enrichment of ETV1 binding within accessible regulatory elements at the KCNK3 gene locus. Conclusions ETV1-knockdown shows strong inhibitory effects on TASK-1 while ChIP-qPCR and ATAC-seq data suggest that ETV1 acts as a direct transcriptional activator of KCNK3. The finding that ETV1 is involved in the regulation of TASK-1 improves our understanding of the pathomechanisms underlying AF and will hopefully help to advance TASK-1-based AF therapy.

Genome-wide identification and comprehensive analysis heat shock transcription factor (Hsf) members in asparagus (Asparagus officinalis) at the seeding stage under abiotic stresses

Heat shock transcription factors (Hsf) are pivotal as essential transcription factors. They function as direct transcriptional activators of genes regulated by thermal stress and are closely associated with various abiotic stresses. Asparagus (Asparagus officinalis) is a vegetable of considerable economic and nutritional significance, abundant in essential vitamins, minerals, and dietary fiber. Nevertheless, asparagus is sensitive to environmental stresses, and specific abiotic stresses harm its yield and quality. In this context, Hsf members have been discerned through the reference genome, and a comprehensive analysis encompassing physical and chemical attributes, evolutionary aspects, motifs, gene structure, cis-acting elements, collinearity, and expression patterns under abiotic stresses has been conducted. The findings identified 18 members, categorized into five distinct subgroups. Members within each subgroup exhibited analogous motifs, gene structures, and cis-acting elements. Collinearity analysis unveiled a noteworthy pattern, revealing that Hsf members within asparagus shared one, two, and three pairs with counterparts in Arabidopsis, Oryza sativa, and Glycine max, respectively.Furthermore, members displayed tissue-specific expression during the seedling stage, with roots emerging as viable target tissue. Notably, the expression levels of certain members underwent modification under the influence of abiotic stresses. This study establishes a foundational framework for understanding Hsf members and offers valuable insights into the potential application of molecular breeding in the context of asparagus cultivation.

SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.

OsTOC1 plays dual roles in the regulation of plant circadian clock by functioning as a direct transcription activator or repressor

Plant clock function relies on precise timing of gene expression through complex regulatory networks consisting of activators and repressors at the core of oscillators. Although TIMING OF CAB EXPRESSION 1 (TOC1) has been recognized as a repressor involved in shaping oscillations and regulating clock-driven processes, its potential to directly activate gene expression remains unclear. In this study, we find that OsTOC1 primarily acts as a transcriptional repressor for core clock components, including OsLHY and OsGI. Here, we show that OsTOC1 possesses the ability to directly activate the expression of circadian target genes. Through binding to the promoters of OsTGAL3a/b, transient activation of OsTOC1 induces the expression of OsTGAL3a/b, indicating its role as an activator contributing to pathogen resistance. Moreover, TOC1 participates in regulating multiple yield-related traits in rice. These findings suggest that TOC1's function as a transcriptional repressor is not inherent, providing flexibility to circadian regulations, particularly in outputs.

Lysine Methyltransferase SMYD1 Regulates Myogenesis via skNAC Methylation.

The SMYD family is a unique class of lysine methyltransferases (KMTases) whose catalytic SET domain is split by a MYND domain. Among these, Smyd1 was identified as a heart- and skeletal muscle-specific KMTase and is essential for cardiogenesis and skeletal muscle development. SMYD1 has been characterized as a histone methyltransferase (HMTase). Here we demonstrated that SMYD1 methylates is the Skeletal muscle-specific splice variant of the Nascent polypeptide-Associated Complex (skNAC) transcription factor. SMYD1-mediated methylation of skNAC targets K1975 within the carboxy-terminus region of skNAC. Catalysis requires physical interaction of SMYD1 and skNAC via the conserved MYND domain of SMYD1 and the PXLXP motif of skNAC. Our data indicated that skNAC methylation is required for the direct transcriptional activation of myoglobin (Mb), a heart- and skeletal muscle-specific hemoprotein that facilitates oxygen transport. Our study revealed that the skNAC, as a methylation target of SMYD1, illuminates the molecular mechanism by which SMYD1 cooperates with skNAC to regulate transcriptional activation of genes crucial for muscle functions and implicates the MYND domain of the SMYD-family KMTases as an adaptor to target substrates for methylation.

The role of FUT8‐catalyzed core fucosylation in Alzheimer’s amyloid‐β oligomer‐induced activation of human microglia

AbstractBackgroundA previous RNA‐seq study of human Alzheimer’s disease (AD) brain samples revealed significant upregulation of FUT8 coding for α1‐6 fucosyltransferase in parahippocampal gyrus, suggesting a role of FUT8 and the core fucosylation it catalyzes in early AD. Despite the well‐recognized roles of core fucosylation in immune regulation, its significance in microglial function, especially in neurodegenerative conditions such as AD, has rarely been studied.MethodHuman microglia derived from induced pluripotent stem cells (iMG) were used as the model system. To simulate microglia in the early stage of AD, we stimulated iMG derived from a subject of APOE3/3 genotype with amyloid‐β oligomer (AβO, 3 μM). The degree of microglia activation was determined by cytokine induction and MAPK phosphorylation. Lectins were used in flow cytometry, cytochemical staining, and lectin blotting to determine the changes in core fucosylation. Fucosylation was also examined by MS/MS‐based N‐glycan analysis. FUT8 expression in brain or cell samples was determined by quantitative PCR and Western blotting. FUT8 inhibition was induced by siRNA‐mediated gene silencing. The transcriptional regulation of FUT8 was investigated by ChIP‐PCR using p53 antibodies.ResultHuman iMG responded to AβO with a pattern of pro‐inflammatory activation within 24 hours. Using two lectins specifically recognizing α1,6 core fucose, AAL and LCA, in flow cytometry and fluorescence staining, we showed that such an activation was associated with increased core fucosylation. This observation will be further verified by N‐glycomic analysis. Inhibition of fucosylation by two inhibitors, 2FF and 6‐Alk‐Fuc, as well as FUT8 knockdown by specific siRNAs suppressed AβO‐induced iMG activation. AβO‐activated iMG also showed increased protein levels of FUT8 and p53. Because p53 was previously shown to be a direct transcriptional activator of FUT8 in tumor cells, we will test whether p53 is responsible for FUT8 upregulation by ChIP‐PCR. The in vivo relevance of our results is supported by qPCR results showing increased levels of FUT8 transcript in human temporal cortex, 5xFAD mouse cortex, and microglia isolated from 5xFAD mice.ConclusionOur data suggests that FUT8‐catalyzed α1‐6 core fucosylation is required for AβO‐induced microglia activation and may play a role in the pathogenesis of AD.

Lung endothelial cells regulate pulmonary fibrosis through FOXF1/R-Ras signaling

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.

Dietary L-tryptophan determines the number of colonic regulatory T-cells and susceptibility to colitis via GPR15

Abstract Environmental factors play a prominent role in the onset of immunological disorders such as ulcerative colitis, but the mechanisms are unclear. Diet is a major environmental factor, mostly known to influence the gut microbiota, which in turn affects the host immune system. Here, we demonstrated the microbiota-independent effect of the ubiquitous dietary component, L-tryptophan (L-Trp) on mucosal immune responses. We discovered that the amount of ingested L-Trp determines the number of colonic CD4 +T-cells through the aryl hydrocarbon receptor (AhR), which triggers the direct transcriptional activation of the colon T-cell homing receptor, GPR15. However, the default pathway connecting L-Trp, AhR, GPR15, and T-cells is microbiota-independent and involves host IDO1 and IDO2 enzymes, selectively increasing GPR15 +Tregs. Consequently, two weeks of L-Trp supplementation nearly doubled the colonic GPR15 +Tregs and significantly decreased the future risk of colitis. Our extensive, in vitro screening of AhR ligands suggests that AhR ligand-specific variations in target gene expression may account for the Treg-selective response to L-Trp. We also identified benzo[a]pyrene, a compound enriched in cigarette smoke and grilled meat, as an AHR agonist that strongly promotes GPR15+FOXP3+ Treg generation. Thus, our findings uncover mechanisms by which routine dietary and behavioral practices have a significant impact on colonic Treg responses, suggesting a potential non- invasive therapeutic approach for colitis. Supported by grant from NIH (R01AI141787, R21AI142318, T32 AI134646, 5P30CA056036-22) Supported by grant from CCFA (Career Development Award 329388)

Developmental growth plate cartilage formation suppressed by artificial light at night via inhibiting BMAL1-driven collagen hydroxylation.

Exposure to artificial light at night (LAN) can induce obesity, depressive disorder and osteoporosis, but the pernicious effects of excessive LAN exposure on tissue structure are poorly understood. Here, we demonstrated that artificial LAN can impair developmental growth plate cartilage extracellular matrix (ECM) formation and cause endoplasmic reticulum (ER) dilation, which in turn compromises bone formation. Excessive LAN exposure induces downregulation of the core circadian clock protein BMAL1, which leads to collagen accumulation in the ER. Further investigations suggest that BMAL1 is the direct transcriptional activator of prolyl 4-hydroxylase subunit alpha 1 (P4ha1) in chondrocytes, which orchestrates collagen prolyl hydroxylation and secretion. BMAL1 downregulation induced by LAN markedly inhibits proline hydroxylation and transport of collagen from ER to golgi, thereby inducing ER stress in chondrocytes. Restoration of BMAL1/P4HA1 signaling can effectively rescue the dysregulation of cartilage formation within the developmental growth plate induced by artificial LAN exposure. In summary, our investigations suggested that LAN is a significant risk factor in bone growth and development, and a proposed novel strategy targeting enhancement of BMAL1-mediated collagen hydroxylation could be a potential therapeutic approach to facilitate bone growth.

Psi promotes Drosophila wing growth via direct transcriptional activation of cell cycle targets and repression of growth inhibitors.

The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Our previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Here, through the first genome-wide binding and expression profiles obtained for a FUBP family protein, we demonstrate that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFβ signalling). We further demonstrate tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.

HNF1β-associated cyst development and electrolyte disturbances are not explained by BAIAP2L2 expression.

Mutations or deletions in transcription factor hepatocyte nuclear factor 1 homeobox β (HNF1β) cause renal cysts and/or malformation, maturity-onset diabetes of the young and electrolyte disturbances. Here, we applied a comprehensive bioinformatic approach on ChIP-seq, RNA-seq, and gene expression array studies to identify novel transcriptional targets of HNF1β explaining the kidney phenotype of HNF1β patients. We identified BAR/IMD Domain Containing Adaptor Protein 2 Like 2 (BAIAP2L2), as a novel transcriptional target of HNF1β and validated direct transcriptional activation of the BAIAP2L2 promoter by a reporter luciferase assay. Using mass spectrometry analysis, we show that BAIAP2L2 binds to other members of the I-BAR domain-containing family: BAIAP2 and BAIAP2L1. Subsequently, the role of BAIAP2L2 in maintaining epithelial cell integrity in the kidney was assessed using Baiap2l2 knockout cell and mouse models. Kidney epithelial cells lacking functional BAIAP2L2 displayed normal F-actin distribution at cell-cell contacts and formed polarized three-dimensional spheroids with a lumen. In vivo, Baiap2l2 knockout mice displayed normal kidney and colon tissue morphology and serum and urine electrolyte concentrations were not affected. Altogether, our study is the first to characterize the function of BAIAP2L2 in the kidney in vivo and we report that mice lacking BAIAP2L2 exhibit normal electrolyte homeostasis and tissue morphology under physiological conditions.

Abstract B009: A SOX9-B7x axis safeguards dedifferentiated tumor cells from immunosurveillance to enable DCIS progression

Abstract Accumulating evidence has demonstrated a central role of dedifferentiation in carcinogenesis. In basal-like breast cancer mouse models, upregulation of SOX9 drives dedifferentiation of luminal progenitors to cells with luminal-basal hybrid features resembling embryonic multipotent mammary stem cells. Additionally, genetic inactivation of SOX9 inhibits dedifferentiation and blocks the progression of in situ tumors to malignant carcinoma. Whether the host immune system can recognize these dedifferentiated cells and how they eventually evade immunosurveillance are unclear. Here, we showed that SOX9 was necessary for limiting infiltrating T lymphocytes to protect dedifferentiated cells from immune surveillance in premalignant lesions of basal-like breast cancer. SOX9 deletion in spontaneous BLBC tumor models caused massive T lymphocyte infiltration and elimination of dedifferentiated cells. Antibody-mediated T cell depletion rescued the accumulation of dedifferentiated tumor cells in SOX9-deficient DCIS and its progression to invasive tumors. Mechanistically, through both stimulation of the STAT3 pathway and direct transcriptional activation, SOX9 upregulated the expression of the immune checkpoint B7x, which mediated the action of SOX9 in immunosuppression. In advanced tumors, B7x blockade reactivated antitumor immunity and effectively inhibited tumor growth. Furthermore, B7x depletion overcame the resistance to anti-PD-L1 immunotherapy. Collectively, these findings uncovered a dedifferentiation-associated immune evasion mechanism and demonstrated the therapeutic potential of targeting the SOX9-B7x pathway to eradicate dedifferentiated tumor cells for intercepting breast cancer progression. Citation Format: Yu Liu, Peter John, John R Christin, Maja Oktay, XingXing Zang, Wenjun Guo. A SOX9-B7x axis safeguards dedifferentiated tumor cells from immunosurveillance to enable DCIS progression [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B009.