AP-1 Family Research Articles

A novel transcription factor, ScAIL1, modulates plant defense responses by targeting DELLA and regulating gibberellin and jasmonic acid signaling in sugarcane.

DELLA proteins are important repressors of gibberellin signaling, regulating plant development and defense responses through crosstalk with various phytohormones. Sugarcane ScGAI encodes a DELLA protein that regulates culm development. However, it is unclear which transcription factors mediate the transcription of ScGAI. Here, we identified two different ScGAI promoter sequences that cooperatively regulate ScGAI transcription. We also identified a nuclear-localized AP2 family transcription factor, ScAIL1, which inhibits the transcription of ScGAI by directly binding to two ScGAI promoters. ScAIL1 was expressed in all sugarcane tissues tested and was induced by gibberellin and various stressors, including NaCl, polyethylene glycol, and pathogenic fungi and bacteria. Overexpression of ScAIL1 in rice significantly improved resistance to bacterial blight and rice blast, while reducing growth and development. In addition, several genes associated with stress responses were significantly up-regulated in transgenic rice overexpressing ScAIL1. Endogenous phytohormone content and expression analysis further revealed that ScAIL1-overexpressing lines improved resistance to bacterial blight and rice blast instead of promoting growth, and that this response was associated with increased jasmonic acid synthesis and gibberellin inactivation. These results provide molecular evidence that the role of ScAIL1 in the plant defense response is related to jasmonic acid and gibberellin signaling.

Fra-1 induces apoptosis and neuroinflammation by targeting S100A8 to modulate TLR4 pathways in spinal cord ischemia/reperfusion injury.

Spinal cord ischemia/reperfusion injury (SCII) is a severe complication driven by apoptosis and neuroinflammation. An increase in the expression of c-Fos, a member of the AP-1 family, is known as a neuronal activation marker in SCII. The AP-1 family is composed of Jun, Fos, and is associated with the regulation of cytokines expression and apoptosis. Fra-1 is a member of the Fos family, however, the contribution of Fra-1 to SCII is still unclear. In our study, Fra-1 was highly upregulated especially in neurons and microglia and promoted apoptosis by changing the expression of Bax/Bcl-2 after SCII. Furthermore, we found that Fra-1 directly regulated the transcription expression of S100A8. We demonstrated that knockdown of Fra-1 alleviated S100A8 mediated neuronal apoptosis and inflammatory factor release, thus improved motor function after SCII. Interestingly, we showed that administration of TAK-242, the TLR4 inhibitor, to the ischemia/reperfusion (I/R) injury induced rats suppressed the activation of the ERK and NF-κB pathways, and further reduced Fra-1 expression. In conclusion, we found that Fra-1-targeted S100A8 was expressed the upstream of Fra-1, and the Fra-1/S100A8 interaction formed a feedback loop in the signaling pathways activated by SCII.

The chromatin remodeling protein ATRX positively regulates IRF3-dependent type I interferon production and interferon-induced gene expression.

The chromatin remodeling protein alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a component of promyelocytic leukemia nuclear bodies (PML-NBs) and thereby mediates intrinsic immunity against several viruses including human cytomegalovirus (HCMV). As a consequence, viruses have evolved different mechanisms to antagonize ATRX, such as displacement from PML-NBs or degradation. Here, we show that depletion of ATRX results in an overall impaired antiviral state by decreasing transcription and subsequent secretion of type I IFNs, which is followed by reduced expression of interferon-stimulated genes (ISGs). ATRX interacts with the transcription factor interferon regulatory factor 3 (IRF3) and associates with the IFN-β promoter to facilitate transcription. Furthermore, whole transcriptome sequencing revealed that ATRX is required for efficient IFN-induced expression of a distinct set of ISGs. Mechanistically, we found that ATRX positively modulates chromatin accessibility specifically upon IFN signaling, thereby affecting promoter regions with recognition motifs for AP-1 family transcription factors. In summary, our study uncovers a novel co-activating function of the chromatin remodeling factor ATRX in innate immunity that regulates chromatin accessibility and subsequent transcription of interferons and ISGs. Consequently, ATRX antagonization by viral proteins and ATRX mutations in tumors represent important strategies to broadly compromise both intrinsic and innate immune responses.

Nonpruritic hypopigmented macules on an immunocompromised patient

Nonpruritic hypopigmented macules on an immunocompromised patient

Integrative genome‐wide chromatin accessibility and transcriptome profiling of diffuse large B‐cell lymphoma

Integrative genome‐wide chromatin accessibility and transcriptome profiling of diffuse large B‐cell lymphoma

In vitro single molecule and bulk phase studies reveal the AP-1 transcription factor cFos binds to DNA without its partner cJun

The AP-1 transcription factor family crucially regulates progression of the cell cycle, as well as playing roles in proliferation, differentiation, and the stress response. The two best described AP-1 family members, cFos and cJun, are known to dimerize to form a functional AP-1 heterodimer that binds to a consensus response element sequence. Although cJun can also homodimerize and bind to DNA, the canonical view is that cFos cannot bind DNA without heterodimerizing with cJun. Here, we show that cFos can actually bind to DNA in the absence of cJun in vitro. Using dual color single molecule imaging of cFos alone, we directly visualize binding to and movement on DNA. Of all these DNA-bound proteins, detailed analysis suggested 30 to 46% were homodimers. Furthermore, we constructed fluorescent protein fusions of cFos and cJun for Förster resonance energy transfer experiments. These constructs indicated complete dimerization of cJun, but although cFos could dimerize, its extent was reduced. Finally, to provide orthogonal confirmation of cFos binding to DNA, we performed bulk-phase circular dichroism experiments that showed clear structural changes in DNA; these were found to be specific to the AP-1 consensus sequence. Taken together, our results clearly show cFos can interact with DNA both as monomers and dimers independently of its archetypal partner, cJun.

Microbiota, not host origin drives ex vivo intestinal epithelial responses

ABSTRACT Microbial dysbiosis is an established finding in patients with inflammatory bowel disease (IBD), but host-microbial interactions are poorly understood. We aimed to unravel the effect of microbiota exposure on intestinal epithelial cells. Confluent Transwell® organoid monolayers of eight UC patients and eight non-IBD controls were co-cultured for six hours with microbiota (3x108 cells) of UC patients or a healthy volunteer (HV), in the presence or absence of an inflammatory cytokine mix. Transepithelial electrical resistance (TEER), fluorescein isothiocyanate (FITC) dextran measurements, and RNA sequencing were performed on epithelial cells, and 16S rRNA sequencing on microbiota samples before and after co-culture. Transcriptomic response following microbiota exposure was not different between epithelial cells from UC patients or non-IBD controls. Following UC microbiota exposure, but not HV microbiota, a strong decrease in epithelial barrier integrity was observed in both UC and HV epithelial cells by TEER and FITC dextran measurements. Exposure of inflamed epithelium to UC microbiota induced transcriptomic stress pathways including activation of EGR1, MAPK and JAK/STAT signaling, as well as AP-1 family and FOSL transcripts. Stress responses after HV microbiota stimulation were milder. We conclude that not the epithelial cell origin (UC versus non-IBD) but the microbial donor drives transcriptomic responses, as exposure to UC microbiota was sufficient to induce stress responses in all epithelial cells. Further research on therapies to restore the microbial balance, to remove the constant trigger of dysbiosis, is required.

Modulation of viral replication, apoptosis and antiviral response by induction and mutual regulation of EGR and AP-1 family genes during coronavirus infection

ABSTRACT Coronaviruses have evolved a variety of strategies to exploit normal cellular processes and signalling pathways for their efficient reproduction in a generally hostile cellular environment. One immediate-early response gene (IEG) family, the AP-1 gene family, was previously shown to be activated by coronavirus infection. In this study, we report that another IEG family, the EGR family, is also activated in cells infected with four different coronaviruses in three genera, i.e. gammacoronavirus infectious bronchitis virus (IBV), alphacoronaviruses porcine epidemic diarrhoea virus (PEDV) and human coronavirus-229E (HCoV-229E), and betacoronavirus HCoV-OC43. Knockdown of EGR1 reduced the expression of cJUN and cFOS, and knockdown of cJUN and/or cFOS reduced the expression of EGR1, demonstrating that these two IEG families may be cross-activated and mutual regulated. Furthermore, ERK1/2 was identified as an upstream kinase, and JNK and p38 as inhibitors of EGR1 activation in coronavirus-infected cells. However, upregulation of EGR family genes, in particular EGR1, appears to play a differential role in regulating viral replication, apoptosis and antiviral response. EGR1 was shown to play a limited role in regulation of coronavirus replication, and an anti-apoptotic role in cells infected with IBV or PEDV, but not in cells infected with HCoV-229E. Upregulation of EGR1 may also play a differential role in the regulation of antiviral response against different coronaviruses. This study reveals a novel regulatory network shared by different coronaviruses in the immediate-early response of host cells to infection.

Nuclear Factor AP2X-4 Governs the Expression of Cell Cycle- and Life Stage-Regulated Genes and is Critical for Toxoplasma Growth.

ABSTRACTToxoplasma gondii is a ubiquitous pathogen infecting one third of the world’s population and diverse animals. It has a complex life cycle alternating among different developmental stages, which contributes to its transmission and pathogenesis. The parasite has a sophisticated gene regulation network that enables timely expression of genes at designated stages. However, little is known about the underlying regulatory mechanisms. Here, we identified an AP2 family transcription factor named TgAP2X-4, which was crucial for parasite growth during the acute infection stage. TgAP2X-4 deletion leads to reduced expression of many genes that are normally upregulated during the M phase of the cell cycle. These include genes that encode rhoptry neck proteins that are key for parasite invasion. As a result, the Δap2X-4 mutant displayed significantly decreased efficiency of host cell invasion. Transcriptomic analyses suggested that TgAP2X-4 also regulates a large group of genes that are typically induced during chronic infection, such as BAG1 and LDH2. Given the diverse impacts on gene expression, TgAP2X-4 inactivation results in severely impaired parasite growth, as well as drastic attenuation of parasite virulence and complete inability to form chronic infection. Therefore, TgAP2X-4 represents a candidate for antitoxoplasmic drug and vaccine designs.IMPORTANCE Toxoplasma gondii has a complicated gene regulation network that allows “just in time” expression of genes to cope with the physiological needs at each stage during the complex life cycle. However, how such regulation is achieved is largely unknown. Here, we identified a transcription factor named TgAP2X-4 that is critical for the growth and life cycle progression of the parasite. Detailed analyses found that TgAP2X-4 regulated the expression of many cell cycle-regulated genes, including a subset of rhoptry genes that were essential for the parasites to enter host cells. It also regulated the expression of many genes involved in the development of chronic infection. Because of the diverse impacts on gene expression, TgAP2X-4 inactivation caused reduced parasite growth in vitro and attenuated virulence in vivo. Therefore, it is a potential target for drug or vaccine designs against Toxoplasma infections.

Abstract 2822: Enhanced effector activity of mediator CDK8 kinase module deficient CAR-T Cells

Abstract Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes that limit T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were components of the CDK8 kinase module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. CDK8 kinase module deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. CDK8 kinase module deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation most notably involving transcription factors that play a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in CDK8 kinase module deficient CAR-T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK8 kinase module as a target for enhancing the potency of anti-tumor T cell responses. Citation Format: Katherine A. Freitas, Julia A. Belk, Elena Sotillo, Bence Daniel, Katalin Sandor, Dorota Klysz, Vandon T. Duong, Peng Xu, Meena Malipatlolla, Evan W. Weber, Robbie G. Majzner, Howard Y. Chang, Ansuman T. Satpathy, Crystal Mackall. Enhanced effector activity of mediator CDK8 kinase module deficient CAR-T Cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2822.

Glypican-3-specific CAR-NKT cells overexpressing BATF3 mediate potent antitumor activity against hepatocellular carcinoma.

e14521 Background: Hepatocellular carcinoma (HCC), the fourth leading cause of cancer-related deaths worldwide, is amenable to immunotherapies that target glypican-3 (GPC3), a protein expressed by over 75% of HCCs but not by healthy tissues. The invariant natural killer T cells (NKTs) prevent HCC tumorogenesis through interactions with the gut microbiome in mice, and the presence of tumor-infiltrating NKTs is associated with improved clinical outcomes. We previously tested GPC3-specific chimeric antigen receptors (CARs) and selected a receptor with both CD28 and 41BB costimulatory endodomains based on its ability to mediate superior antitumor activity of transduced NKTs in vivo. Co-expression of interleukin-15 (IL15), a cytokine that supports NKT expansion and persistence, with the chosen GPC3-CAR further enhanced this activity. GPC3-CAR NKTs co-expressing IL15 (15.GPC3-CAR) overexpressed the Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF3), a member of the AP-1 family. Since AP-1 family proteins are master regulators of cell survival and proliferation, we hypothesized that overexpressing BATF3 in GPC3-CAR NKTs (BATF3.GPC3-CAR) will lead to superior expansion, persistence, and antitumor activity compared to 15.GPC3-CAR NKTs. Methods: We used RNAseq and flow cytometry to define the transcriptomic and phenotypic characteristics, respectively, of BATF3-overexpressing NKTs at baseline and after tumor cell killing in co-culture. We also used co-culture assays to evaluate the ability of BATF3- or IL15-expressing NKTs to proliferate, produce effector cytokines, and kill tumor cells after multiple rounds of exposure. Finally, we evaluated the ability of BATF3.GPC3-CAR and IL15.GPC3-CAR NKTs to expand, persist, and target tumors in an HCC xenograft murine model. Results: BATF3 overexpression in NKTs induced significant differences in gene expression profiles compared to a GFP control at baseline (DEGs up: 20; down: 26, p < 0.01) and after tumor cell killing (DEGs up: 19; down: 32; p < 0.01; fold change > 2 or < 0.5, respectively) corresponding to enrichment of gene sets related to metabolic fitness. Importantly, CD62L+ NKTs, which have enhanced antitumor properties, were enriched in BATF3-expressing NKTs (27.6% vs 8.2%, p < 0.05, versus GFP control). Further, BATF3 significantly enhanced NKT proliferation (area under the curve of absolute NKT cell counts: 5x1011 vs 1.1x1011, p = 0.001; fold expansion: 6.12x104 vs 1.2x104, p = 0.001, versus IL15 NKTs). Finally, BATF3.GPC3-CAR NKTs induced long-term tumor control and significantly improved survival of HCC xenograft-bearing mice compared to 15.GPC3-CAR NKTs (p = 0.0002, Mantel-Cox analysis). Conclusions: BATF3.GPC3-CAR NKTs induce robust antitumor activity in pre-clinical models of HCC. These results provide a foundation for evaluating BATF3.GPC3-CAR NKTs in patients with relapsed/refractory HCC.

Single-Cell RNA and ATAC Sequencing Reveal Hemodialysis-Related Immune Dysregulation of Circulating Immune Cell Subpopulations.

BackgroundAn increased risk of infection, malignancy, and cardiovascular diseases in maintenance hemodialysis patients is associated with hemodialysis-related immunity disturbances. Although defects in T-lymphocyte-dependent immune responses and preactivation of antigen-presenting cells have been documented in hemodialysis patients, the effects of long-term hemodialysis on the transcriptional program and chromosomal accessibility of circulating immune cell subpopulations remain poorly defined.MethodsWe integrated single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to characterize the transcriptome profiles of peripheral mononuclear cells (PBMCs) from healthy controls and maintenance hemodialysis patients. Validation of differentially expressed genes in CD4+ T cells and monocytes were performed by magnetic bead separation and quantitative real-time PCR.ResultsWe identified 16 and 15 PBMC subgroups in scRNA-seq and scATAC-seq datasets, respectively. Hemodialysis significantly suppressed the expression levels of T cell receptor (TCR) genes in CD4+ T cell subsets (e.g., TRAV4, CD45, CD3G, CD3D, CD3E) and major histocompatibility complex II (MHC-II) pathway-related genes in monocytes (HLA-DRB1, HLA-DQA2, HLA-DQA1, HLA-DPB1). Downstream pathways of TCR signaling, including PI3K-Akt-mTOR, MAPK, TNF, and NF-κB pathways, were also inhibited in CD4+ T cell subpopulations during the hemodialysis procedure. Hemodialysis altered cellular communication patterns between PBMC subgroups, particularly TGF-TGFBR, HVEM-BTLA, and IL16-CD4 signalings between CD4+ T cells and monocytes. Additionally, we found that hemodialysis inhibited the expression of AP-1 family transcription factors (JUN, JUND, FOS, FOSB) by interfering with the chromatin accessibility profile.ConclusionsOur study provides a valuable framework for future investigations of hemodialysis-related immune dysregulation and identifies potential therapeutic targets for reconstituting the circulating immune system in maintenance hemodialysis patients.

FOS gene associated immune infiltration signature in perivascular adipose tissues of abdominal aortic aneurysm

Abdominal aortic aneurysms (AAA) are pathological dilations in local aortic wall. The inflammatory infiltrates of the perivascular adipose tissue (PAT) surrounding AAAs were associated with AAAs and have been shown to contribute vascular pathology. However, the mechanism by which PAT inflammation contributes to vascular pathology in AAA remains to be clarified. This study aimed to explore the association between immune cell infiltration and key gene expression profile in PAT of AAA. For that, a gene expression dataset of human dilated perivascular adipose tissue (dPAT), non-dilated perivascular adipose tissue (ndPAT), subcutaneous abdominal fat (SAF) and omental-visceral fat (OVF) samples, as well as another microarray dataset of the abdominal perivascular adipose tissue in peripheral artery disease patients were downloaded from GEO database for analysis in this study. The CIBERSORT algorithm, weighted gene co-expression network analysis (WGCNA) and LASSO algorithm were used for the identification of immune infiltration, immune-related genes and the development of diagnostic signature. Our data discovered a significant higher proportion of activated mast cells and follicular helper T (Tfh) cells in dPAT than ndPAT, OVT and SAF samples. Moreover, AP-1 family members (FOS, FOSB, ATF3, JUN and JUNB) were found to compose the hub genes of purple module in WGCNA. Among them, FOS gene acts as a higher efficient marker to discriminate dPAT from ndPAT, OVT and SAF in AAA. Meanwhile, the expression profiles of the AP-1 family members are all significantly positive correlated with activated mast cell, plasma cell and Tfh cell infiltration in dPAT of AAA. Therefore, in the PAT surrounding AAA, the signature of inflammatory infiltration might be represented by a FOS-dominated cell network consist of activated mast cell, plasma cell and Tfh cell. Given the complicated etiology of AAA, our results are likely to shed new light on the pathophysiologic mechanism of AAA influenced by the local dPAT.

Role of RNA Binding Protein with Multiple Splicing (RBPMS) Variants in Cell Proliferation, Invasion and Angiogenesis in Ovarian Cancer

Worldwide, the number of cancer‐related deaths continues to increase due to the ability of cancer cells to become chemotherapy‐resistant and metastasize. For women with ovarian cancer, a staggering 70% will become resistant to the front‐line therapy, cisplatin. RNA binding protein with multiple splicing (RBPMS) is a member of family proteins that bind to the nascent RNA transcripts regulate their processing, which includes splicing, transport, localization and stability. Evidence indicates that RBPMS interact with c‐Fos, a member of the AP‐1 family that dimerizes with c‐Jun to stimulate AP‐1 transcriptional activity. Furthermore, RBPMS interacted with transcription factor Smad3, which bind to c‐Jun increasing AP‐1 transcriptional activity. Three major RBPMS isoforms have been described, RBPMS‐A (21.8 kDa), RBPMS‐B (22.4 kDa) and RBPMS‐C (24.2 kDa). Reports have shown that each RBPMS isoform interacts with proteins and/or RNAs responsible for cell growth, proliferation, and tumor progression. Previous reports of our laboratory have found that RBPMS expression decrease in cisplatin resistance compared with cisplatin sensitive cells. However, the specific RBPMS isoform responsible of this observation has not been elucidated and less is known about its role on invasiveness ability and angiogenesis capacity. We overexpressed each RBPMS variants (A, B and C) in the cisplatin resistant ovarian cancer cells, A2780CP20, and performed cell viability, cell proliferation and invasion assays. Significant reduction in the number of colonies and their area were observed in both isoform A and isoform C clones, compared with isoform B and control empty vector clones. However, clones of isoform A were more sensitive to cisplatin treatment in comparation with isoform B and C clones. In vivo experiments showed a significant reduction in tumor growth between RBPMS A and C when compared with the controls. Tumor tissues were subjected to immunohistochemical analysis for proliferation (Ki67) and angiogenesis (CD31). Elevated signals of both antibodies were detected in tissues from control groups when compared with RBPMS A and C overexpressing tumor tissues. These studies along with the RNAseq suggest that RBPMS, regulate transcripts involved in cell proliferation and invasion capacity of ovarian cancer cells. Moreover, RBPMS A and C represses uncontrolled proliferation, invasiveness, and angiogenesis in ovarian cancer cells. However, only the isoform A contributed to the cisplatin sensitivity of ovarian cancer cells. Future efforts on this project will be focused on determining the RBPMS isoform that represses AP‐1 (c‐Fos and c‐Jun) dependent gene regulation in cisplatin sensitive ovarian cancer cells.

Combined Analysis of the Metabolome and Transcriptome to Explore Heat Stress Responses and Adaptation Mechanisms in Celery (Apium graveolens L.).

Celery is an important leafy vegetable that can grow during the cool season and does not tolerate high temperatures. Heat stress is widely acknowledged as one of the main abiotic stresses affecting the growth and yield of celery. The morphological and physiological indices of celery were investigated in the present study to explore the physiological mechanisms in response to high temperatures. Results showed that the antioxidant enzyme activity, proline, relative conductivity, and malondialdehyde were increased, while chlorophyll and the water content of leaves decreased under high-temperature conditions. Short-term heat treatment increased the stomatal conductance to cool off the leaves by transpiration; however, long-term heat treatment led to stomatal closure to prevent leaf dehydration. In addition, high temperature caused a disordered arrangement of palisade tissue and a loose arrangement of spongy tissue in celery leaves. Combined metabolomic and transcriptomic analyses were further used to reveal the regulatory mechanisms in response to heat stress at the molecular level in celery. A total of 1003 differential metabolites were identified and significantly enriched in amino acid metabolism and the tricarboxilic acid (TCA) cycle. Transcriptome sequencing detected 24,264 different genes, including multiple transcription factor families such as HSF, WRKY, MYB, AP2, bZIP, and bHLH family members that were significantly upregulated in response to heat stress, suggesting that these genes were involved in the response to heat stress. In addition, transcriptional and metabolic pathway analyses showed that heat stress inhibited the glycolysis pathway and delayed the TCA cycle but increased the expression of most amino acid synthesis pathways such as proline, arginine, and serine, consistent with the results of physiological indicators. qRT-PCR further showed that the expression pattern was similar to the expression abundance in the transcriptome. The important metabolites and genes in celery that significantly contributed to the response to high temperatures were identified in the present study, which provided the theoretical basis for breeding heat-resistant celery.

MYOCD is Required for Cardiomyocyte-like Cells Induction from Human Urine Cells and Fibroblasts Through Remodeling Chromatin.

Despite direct reprogramming of human cardiac fibroblasts into induced cardiomyocytes (iCM) holds great potential for heart regeneration, the mechanisms are poorly understood. Whether other human somatic cells could be reprogrammed into cardiomyocytes is also unknown. Here, we report human urine cells (hUCs) could be converted into CM-like cells from different donors and the related chromatin accessibility dynamics (CAD) by assay for transposase accessible chromatin(ATAC)-seq. hUCs transduced by MEF2C, TBX5, MESP1 and MYOCD but without GATA4 expressed multiple cardiac specific genes, exhibited Ca2+ oscillation potential and sarcomeric structures, and contracted synchronously in coculture with mouse CM. Additionally, we found that MYOCD is required for both closing and opening critical loci, mainly by hindering the opening of loci enriched with motifs for the TEAD and AP1 family and promoting the closing of loci enriched with ETS motifs. These changes differ partially from CAD observed during iCM induction from human fibroblasts. Collectively, our study offers one practical platform for iCM generation and insights into mechanisms for iCM fate determination.

Aryl hydrocarbon receptor–targeted therapy for CD4+ T cell–mediated idiopathic pneumonia syndrome in mice

AbstractWe previously demonstrated that interferon γ (IFN-γ) derived from donor T cells co-opts the indoleamine 2,3-dioxygenase 1 (IDO1) → aryl hydrocarbon receptor (AHR) axis to suppress idiopathic pneumonia syndrome (IPS). Here we report that the dysregulated expression of AP-1 family genes in Ahr−/− lung epithelial cells exacerbated IPS in allogeneic bone marrow transplantation settings. AHR repressed transcription of Jund by preventing STAT1 from binding to its promoter. As a consequence, decreased interleukin-6 impaired the differentiation of CD4+ T cells toward Th17 cells. IFN-γ– and IDO1-independent induction of Ahr expression indicated that the AHR agonist might be a better therapeutic target for IPS than the IDO1 activator. We developed a novel synthetic AHR agonist (referred to here as PB502) that potently inhibits Jund expression. PB502 was highly effective at inducing AHR activation and ameliorating IPS. Notably, PB502 was by far superior to the endogenous AHR ligand, L-kynurenine, in promoting the differentiation of both mouse and human FoxP3+ regulatory CD4+ T cells. Our results suggest that the IDO1-AHR axis in lung epithelial cells is associated with IPS repression. A specific AHR agonist may exhibit therapeutic activity against inflammatory and autoimmune diseases by promoting regulatory T-cell differentiation.

Integrated Chromatin Accessibility and Transcriptome Landscapes of 5-Fluorouracil-Resistant Colon Cancer Cells.

Background: 5-Fluorouracil (5-FU) is one of the most effective and widely used chemotherapeutic drugs in the treatment of colon cancer, yet chemoresistance is a common feature of colon cancer treatment, resulting in poor prognosis and short survival. Dynamic reprogramming of chromatin accessibility is crucial for proper regulation of gene transcription associated with cancer drug resistance by providing the gene regulatory machinery with rapid access to the open genomic DNA. Methods: Here, we explored the global chromatin accessibility and transcription changes by the assay for transposase-accessible chromatin using sequencing (ATAC-seq) in combination with transcriptome sequencing of both parental and 5-FU-resistant HCT15 cells, followed by integrative analysis to better understand the regulatory network underlying 5-FU resistance in colon cancer cells. Results: A total of 3,175 differentially expressed mRNAs (DEGs), lncRNAs (DELs), and miRNAs (DEMs) related to 5-FU resistance were identified, including significantly upregulated IL33, H19, and miR-17-5p; the downregulated AKR1B10, LINC01012, and miR-125b-5p; and chromatin modifiers such as INO80C, HDAC6, and KDM5A. The construction of the ceRNA regulatory network revealed that H19, HOXA11-AS, and NEAT1 might function as ceRNAs associated with 5-FU resistance in HCT15 cells. Moreover, 9,868 differentially accessible regions (DARs) were obtained, which were positively (r = 0.58) correlated with their nearest DEGs and DELs. The upregulated genes related to 4,937 hyper-accessible regions were significantly enriched in signaling pathways of MAPK, FOX, and WNT, while the 4,931 hypo-accessible regions were considered to be involved in declined biosynthesis of amino acids and nucleotide sugars, signaling pathways of Notch, and HIF-1. Analyses of the DAR sequences revealed that besides the AP-1 family, the TF motifs of FOX and KLF family members were highly enriched in hyper- and hypo-accessible regions, respectively. Finally, we obtained several critical TFs and their potential targets associated with DARs and 5-FU resistance, including FOXA1 and KLF3. Conclusion: These data provided clear insights and valuable resources for an improved understanding of the non-genetic landscape of 5-FU-resistant colon cancer cells based on chromatin accessibility and transcript levels, which allowed for genome-wide detection of TF binding sites, potential cis-regulatory elements and therapeutic targets.

Genome-wide identification and expression profiling analysis of maize AP2/ERF superfamily genes reveal essential roles in abiotic stress tolerance

BackgroundAs one of the largest transcription factor families in plants, the APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily is involved in various biological processes and plays significant roles in plant growth, development and responses to various stresses. Although identification and characterization of AP2/ERF superfamily genes have been accomplished in many plant species, very little is known regarding the structure and function of AP2/ERF genes in maize.ResultsIn this study, a total of 214 genes encoding ZmAP2/ERF proteins with complete AP2/ERF domain were eventually identified according to the AGPv4 version of the maize B73 genome. Based on the number of AP2/ERF domain and similarities of amino acid sequences among AP2/ERF proteins from Arabidopsis, rice and maize, all 214 putative ZmAP2/ERF proteins were categorized into three distinct families, including the AP2 family (44), the ERF family (166) and the RAV family (4), respectively. Among them, the ERF family was further subdivided into two diverse subfamilies, including the DREB and ERF subfamilies with 61 and 105 members, respectively. Further, based on phylogenetic analysis, the members of DREB and ERF subfamilies were subdivided into four (Group I-IV) and eight (Group V-XII) groups, respectively. The characteristics of exon-intron structure of these putative ZmAP2/ERF genes and conserved protein motifs of their encoded ZmAP2/ERF proteins were also presented respectively, which was in accordance with the results of group classification. Promoter analysis suggested that ZmAP2/ERF genes shared many stress- and hormone-related cis-regulatory elements. Gene duplication and synteny analysis revealed that tandem or segmental duplication and purifying selection might play significant roles in evolution and functional differentiation of AP2/ERF superfamily genes among three various gramineous species (maize, rice and sorghum). Using RNA-seq data, transcriptome analysis indicated that the majority of ZmAP2/ERF genes displayed differential expression patterns at different developmental stages of maize. In addition, the following analyses of co-expression network among ZmAP2/ERF genes and protein protein interaction between ZmAP2 and ZmERF proteins further enabled us to understand the regulatory relationship among members of the AP2/ERF superfamily in maize. Furthermore, by quantitative real-time PCR analysis, twenty-seven selected ZmAP2/ERF genes were further confirmed to respond to three different abiotic stresses, suggesting their potential roles in various abiotic stress responses. Collectively, these results revealed that these ZmAP2/ERF genes play essential roles in abiotic stress tolerance.ConclusionsTaken together, the present study will serve to present an important theoretical basis for further exploring the function and regulatory mechanism of ZmAP2/ERF genes in the growth, development, and adaptation to abiotic stresses in maize.

P004 Microbiota, not host origin drives ex vivo epithelial response in ulcerative colitis patients and non-IBD controls

Abstract Background Host-microbial interactions in inflammatory bowel disease (IBD) are poorly understood. Furthermore, patients with IBD have microbial dysbiosis and their epithelial cells exhibit intrinsic defects. We aimed to unravel the effect of exposure to microbiota on epithelial cells from both patients with ulcerative colitis (UC) and non-IBD controls. Methods Confluent Transwell® organoid-derived monolayers of 8 UC patients and 8 non-IBD controls were co-cultured for 6 hours with microbiota (3.108 cells) derived from active UC patients (n=3, endoscopic Mayo score ≥2) or healthy volunteer (HV, n=1, selected on high microbial cell count and presence of selected phyla1). For this purpose, fresh faecal samples were filtered and frozen in 0.9% NaCl. Inflammation was re-induced with 100 ng/ml TNF-α, 20 ng/ml IL-1β, and 1 µg/ml flagellin in confluent Transwell® cultures, 24 hours prior to microbiota co-culture. Transepithelial electrical resistance (TEER), 4 kDa fluorescein isothiocyanate (FITC) dextran (2 mg/ml) measurements, and RNA sequencing by Truseq were performed on epithelial cells, and 16S rRNA sequencing on microbiota samples before and after co-culture. Results The transcriptomic response of epithelial cells to microbiota was clearly influenced by the type of microbiota stimulation (no, UC or HV microbiota). However, this response was not influenced by the origin of the epithelial cells (UC or non-IBD controls) (Figure 1A). TEER and FITC dextran measurements showed a strong decrease in epithelial integrity (in both UC and non-IBD epithelium) following stimulation with UC microbiota, in contrast to HV microbiota (Figure 1B). In UC epithelial cells, stimulation with microbiota from UC patients induced a stress induced phenotype including activation of EGR1 signalling, AP-1 family, FOSL genes, MAPK and JNK signalling (Figure 2). Activation of stress pathways (MAPK family cascades) and key markers (e.g. FOSB, FOSL1, MYC, EGR1, ATF3, NOTCH1) was lower after HV microbiota stimulation (Figure 3–4). Expression levels of key markers were UC microbiota specific and similar in epithelial cells of UC and non-IBD controls (Figure 4). Conclusion Not the host (UC vs non-IBD epithelial cells) but the microbial donor (UC vs HV) is driving the transcriptomic response. Epithelial cells from UC patients did not show an increased sensitivity towards microbiota stimulation compared to non-IBD epithelial cells. In contrast, exposure of epithelial cells to UC microbiota was sufficient to induce a strong stress response and barrier disruption. Further research on therapies to restore the microbial balance, to remove the constant trigger of dysbiosed microbiota, is required.