AP-1 Family Research Articles

Dissecting the role of transcription factor AP2-M in Babesia asexual replication.

Babesia spp. are obligate intracellular parasites that invade host cells to complete their asexual development and transmission. Here, we identified a transcription factor AP2-M (BXIN_0799) in Babesia sp. Xinjiang (Bxj), a member of the Apicomplexan AP2 family, which regulates gene expression related to red blood cell (RBC) invasion and cell cycle progression. Our genome-wide analysis of (Cut-Tag) data shows that AP2-M specifically recognized DNA motifs in the promoters of target genes. AP2-M target genes included other AP2 gene family members and epigenetic markers, which could modulate gene expression involved in RBC invasion, merozoite morphology, and cell cycle phases, as indicated by RNA sequencing, proteomics, and single-cell RNA sequencing (scRNA-seq) data from an ap2-m gene disrupted strain (AP2-M (-)). We conclude that AP2-M appeared to contribute to the process of red blood cell invasion, maintain merozoite morphology, and cell cycle progression through GS and MS phases.

Dynamic chromatin accessibility and transcriptome changes following PDGF-BB treatment of bone-marrow derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) are multipotent stem cells that are under investigation for use in clinical trials because they are capable of self-renewal and differentiating into different cell types under defined conditions. Nonetheless, the therapeutic effects of MSCs have been constrained by low engraftment rates, cell fusion, and cell survival. Various strategies have been explored to improve the therapeutic efficacy of MSCs, with platelet-derived growth factor (PDGF)-BB emerging as a promising candidate. To enhance our comprehension of the impact of PDGF-BB on the gene expression profile and chromosomal accessibility of MSCs, RNA-sequencing and analysis of chromatin accessibility profiles were conducted on three human primary MSCs in culture, both with and without stimulation by PDGF-BB.ResultsIntegrative analysis of gene expression and chromatin accessibility demonstrated that PDGF-BB treatment modified the chromatin accessibility landscape, marking regions for activation or repression through the AP-1 family transcription factors TEAD, CEBP, and RUNX2. These changes in AP-1 transcription factor expression, in turn, led to cell proliferation and differentiation potential towards osteoblasts, adipocytes, or chondrocytes. The degree of MSC differentiation varies among cells isolated from different donors. The presence of an enrichment of exosome-related genes is also noted among all the differentially expressed genes.ConclusionsIn conclusion, the observed changes in AP-1 transcription factor expression not only induced cellular proliferation and differentiation, but also revealed variations in the degree of MSC differentiation based on donor-specific differences. Moreover, the enrichment of exosome-related genes among differentially expressed genes suggests a potential significant role for PDGF-BB in facilitating intercellular communication.

Integrated Transcriptional and Metabolomic Analysis of Factors Influencing Root Tuber Enlargement during Early Sweet Potato Development.

Sweet potato (Ipomoea batatas (L.) Lam.) is widely cultivated as an important food crop. However, the molecular regulatory mechanisms affecting root tuber development are not well understood. The aim of this study was to systematically reveal the regulatory network of sweet potato root enlargement through transcriptomic and metabolomic analysis in different early stages of sweet potato root development, combined with phenotypic and anatomical observations. Using RNA-seq, we found that the differential genes of the S1 vs. S2, S3 vs. S4, and S4 vs. S5 comparison groups were enriched in the phenylpropane biosynthesis pathway during five developmental stages and identified 67 differentially expressed transcription factors, including AP2, NAC, bHLH, MYB, and C2H2 families. Based on the metabolome, K-means cluster analysis showed that lipids, organic acids, organic oxides, and other substances accumulated differentially in different growth stages. Transcriptome, metabolome, and prophetypic data indicate that the S3-S4 stage is the key stage of root development of sweet potato. Weighted gene co-expression network analysis (WGCNA) showed that transcriptome differential genes were mainly enriched in fructose and mannose metabolism, pentose phosphate, selenium compound metabolism, glycolysis/gluconogenesis, carbon metabolism, and other pathways. The metabolites of different metabolites are mainly concentrated in amino sugar and nucleotide sugar metabolism, flavonoid biosynthesis, alkaloid biosynthesis, pantothenic acid, and coenzyme A biosynthesis. Based on WGCNA analysis of gene-metabolite correlation, 44 differential genes and 31 differential metabolites with high correlation were identified. This study revealed key gene and metabolite changes in early development of sweet potato root tuber and pointed out potential regulatory networks, providing new insights into sweet potato root tuber development and valuable reference for future genetic improvement.

7657 Single Cell RNAseq And Omics Revealed Novel Role Of Liver Group 2 Innate Lymphocytes In Regulation Of Hepatic Gluconeogenesis

Abstract Disclosure: M. Fujimoto: None. T. Tanaka: None. Background: The liver is an important organ that maintains homeostasis of glucose metabolism in the body through gluconeogenesis. It is considered that gluconeogenesis in hepatocytes is particularly active around the portal vein. However, since excessive gluconeogenesis aggravates the pathogenesis of diabetes, it is necessary to elucidate the regulatory mechanism of gluconeogenesis. Although several studies revealed that group 2 innate lymphocytes (ILC2) contribute to blood glucose maintenance in adipose and pancreatic tissues, the contribution of liver ILC2s to glucose metabolism is unknown. This study aims to elucidate the contribution of liver ILC2s to glucose metabolism and its molecular mechanism. Methods: We administered IL-33, an endogenous ILC2 activator, to BALB/c and NSG mice, and measured fasting blood glucose levels and blood glucose elevation by gluconeogenic substrate pyruvate, and blood glucose levels in liver ILC2s transplanted NSG mice. Molecular mechanisms were analyzed by single cell-RNAseq (scRNA-seq) of liver ILC2s, lung ILC2s and hepatocytes, GATA3 transcription complex, ATAC-seq, GATA3-ChIP-seq, and Metabolomics of primary hepatocytes. Results: IL-33 strongly induced IL-13 production by liver ILC2s, lowered fasting blood glucose, and suppressed pyruvate-induced glucose elevation; these effects were not observed in NSG mice lacking immune cells including ILC2s. A hypoglycemic effect of IL-33 was observed in liver ILC2-transplanted NSG mice, but not in mice transplanted with IL-13-deficient ILC2s, suggesting the effect is IL-13-dependent. scRNA-seq results showed that liver ILC2s showed higher IL-13 expression than lung ILC2s, and IL-33 treatment suppressed the expression of gluconeogenic enzymes in the G6pc-high hepatocyte population. The interaction between liver ILC2s and G6pc-high hepatocytes via IL-13/IL-13 receptors was also demonstrated; co-culture of ILC2s with primary cultured hepatocytes resulted in decreased expression of hepatocyte gluconeogenic enzymes and accumulation of multiple gluconeogenic substrates. In addition, IL-13 neutralizing antibody treatment attenuated the effect of the decreased expression of gluconeogenic enzymes in hepatocytes. Transcription factor GATA3 complex analysis and GATA3-ChIPseq suggested binding of GATA3 of the AP-1 family (JunB) in liver ILC2s. Overexpression, knockdown, and scRNA-seq results suggest that AP1 may act in a GATA3-repressive manner in liver ILC2s. Discussion The functional characteristics of liver ILC2s, hepatocyte heterogeneity and subcluster were demonstrated by scRNA-seq, suggesting that liver ILC2s may directly suppress hepatocyte gluconeogenesis via IL-13. This may play a novel role in the homeostasis of hepatic glucose metabolism. Presentation: 6/1/2024

Integrated transcriptomic and miRNA-seq analysis of Pak-choi provides insight into the molecular mechanisms involved in the preservation of postharvest quality by white LED light irradiation

Low-intensity white light-emitting diode (LED) irradiation effectively delays postharvest senescence in pak-choi at 20 °C and 90 % relative humidity. In this study, RNA-seq and miRNA-seq analyses were conducted on samples collected at 0 d (initial sample) and 5 d (CK and LED-treated samples) to evaluate the regulatory effects of white LED irradiation on miRNA. The resulting data revealed a total of 23 differentially expressed miRNAs (DEmiRNAs) in the CK/LED group on 5 d of storage. LED irradiation induced the expression of AP2 family genes and the biosynthesis of aliphatic glucosinolates by modulating the level of bra-miR172 family miRNAs. LED irradiation also stimulated the expression of genes associated with the photosynthetic pathway in pak-choi and regulated biosynthesis of ascorbic acid and resistance-associated transcription factors by modulating the level of novel miRNAs. Furthermore, LED irradiation suppressed cellulase and polygalacturonase activity, and maintained the pectin content. These findings demonstrate the ability of white LED light irradiation to significantly delay the postharvest senescence process in pak-choi by modulating miRNA expression.

Activating mutations remodel the chromatin accessibility landscape to drive distinct regulatory networks in KMT2A-rearranged acute leukemia.

Activating FLT3 and RAS mutations commonly occur in leukemia with KMT2A-gene rearrangements (KMT2A-r). However, how these mutations cooperate with the KMT2A-r to remodel the epigenetic landscape is unknown. Using a retroviral acute myeloid leukemia (AML) mouse model driven by KMT2A::MLLT3, we show that FLT3 ITD , FLT3 N676K , and NRAS G12D remodeled the chromatin accessibility landscape and associated transcriptional networks. Although the activating mutations shared a common core of chromatin changes, each mutation exhibits unique profiles with most opened peaks associating with enhancers in intronic or intergenic regions. Specifically, FLT3 N676K and NRAS G12D rewired similar chromatin and transcriptional networks, distinct from those mediated by FLT3 ITD . Motif analysis uncovered a role for the AP-1 family of transcription factors in KMT2A::MLLT3 leukemia with FLT3 N676K and NRAS G12D , whereas Runx1 and Stat5a/Stat5b were active in the presence of FLT3 ITD . Furthermore, transcriptional programs linked to immune cell regulation were activated in KMT2A-r AML expressing NRAS G12D or FLT3 N676K , and the expression of NKG2D-ligands on KMT2A-r cells rendered them sensitive to CAR T cell-mediated killing. Human KMT2A-r AML cells could be pharmacologically sensitized to NKG2D-CAR T cells by treatment with the histone deacetylase inhibitor LBH589 (panobinostat) which caused upregulation of NKG2D-ligand levels. Co-treatment with LBH589 and NKG2D-CAR T cells enabled robust AML cell killing, and the strongest effect was observed for cells expressing NRAS G12D . Finally, the results were validated and extended to acute leukemia in infancy. Combined, activating mutations induced mutation-specific changes in the epigenetic landscape, leading to changes in transcriptional programs orchestrated by specific transcription factor networks.

Two-polarized roles of transcription factor FOSB in lung cancer progression and prognosis: dependent on p53 status

BackgroundActivator protein-1 (AP-1) represents a transcription factor family that has garnered growing attention for its extensive involvement in tumor biology. However, the roles of the AP-1 family in the evolution of lung cancer remain poorly characterized. FBJ Murine Osteosarcoma Viral Oncogene Homolog B (FOSB), a classic AP-1 family member, was previously reported to play bewilderingly two-polarized roles in non-small cell lung cancer (NSCLC) as an enigmatic double-edged sword, for which the reasons and significance warrant further elucidation.Methods and ResultsBased on the bioinformatics analysis of a large NSCLC cohort from the TCGA database, our current work found the well-known tumor suppressor gene TP53 served as a key code to decipher the two sides of FOSB – its expression indicated a positive prognosis in NSCLC patients harboring wild-type TP53 while a negative one in those harboring mutant TP53. By constructing a panel of syngeneically derived NSCLC cells expressing p53 in different statuses, the radically opposite prognostic effects of FOSB expression in NSCLC population were validated, with the TP53-R248Q mutation site emerging as particularly meaningful. Transcriptome sequencing showed that FOSB overexpression elicited diversifying transcriptomic landscapes across NSCLC cells with varying genetic backgrounds of TP53 and, combined with the validation by RT-qPCR, PREX1 (TP53-Null), IGFBP5 (TP53-WT), AKR1C3, and ALDH3A1 (TP53-R248Q) were respectively identified as p53-dependent transcriptional targets of FOSB. Subsequently, the heterogenous impacts of FOSB on the tumor biology in NSCLC cells via the above selective transcriptional targets were confirmed in vitro and in vivo. Mechanistic investigations revealed that wild-type or mutant p53 might guide FOSB to recognize and bind to distinct promoter sequences via protein-protein interactions to transcriptionally activate specific target genes, thereby creating disparate influences on the progression and prognosis in NSCLC.ConclusionsFOSB expression holds promise as a novel prognostic biomarker for NSCLC in combination with a given genetic background of TP53, and the unique interactions between FOSB and p53 may serve as underlying intervention targets for NSCLC.

Ultrasonic treatment can improve maize seed germination and abiotic stress resistance

Constant-frequency ultrasonic treatment helped to improve seed germination. However, variable-frequency ultrasonic treatment on maize seed germination were rarely reported. In this study, maize seeds were exposed to 20–40 kHz ultrasonic for 40 s. The germination percentage and radicle length of maize seeds increased by 10.4% and 230.5%. Ultrasonic treatment also significantly increased the acid protease, α-amylase, and β-amylase contents by 96.4%, 73.8%, and 49.1%, respectively. Transcriptome analysis showed that 11,475 differentially expressed genes (DEGs) were found in the ultrasonic treatment and control groups, including 5,695 upregulated and 5,780 downregulated. Metabolic pathways and transcription factors (TFs) were significantly enriched among DEGs after ultrasonic treatment. This included metabolism and genetic information processing, that is, ribosome, proteasome, and pyruvate metabolism, sesquiterpenoid, triterpenoid, and phenylpropanoid biosynthesis, and oxidative phosphorylation, as well as transcription factors in the NAC, MYB, bHLH, WRKY, AP2, bZIP, and ARF families. Variable-frequency ultrasonic treatment increased auxin, gibberellin, and salicylic acid by 5.5%, 37.3%, and 28.9%, respectively. Abscisic acid significantly decreased by 33.2%. The related DEGs were upregulated and downregulated to varying degrees. Seed germination under the abiotic stress conditions of salt stress (NaCl solution), drought (PEG solution), and waterlogging (water-saturated sand bed) under ultrasonic treatment were promoted, radicle length was significantly increased by 30.2%, 30.5%, and 27.3%, respectively; and germination percentage by 14.8%, 20.1%, and 21.6%, respectively. These findings provide new insight into the mechanisms through ultrasonic to promote maize seed germination.

Super-enhancer-driven IRF2BP2 enhances ALK activity and promotes neuroblastoma cell proliferation.

Super-enhancers (SEs) typically govern the expression of critical oncogenes and play a fundamental role in the initiation and progression of cancer. Focusing on genes that are abnormally regulated by SE in cancer may be a new strategy for understanding pathogenesis. In the context of this investigation, we have identified a previously unreported SE-driven gene IRF2BP2 in neuroblastoma (NB). The expression and prognostic value of IRF2BP2 were detected in public databases and clinical samples. The effect of IRF2BP2 on NB cell growth and apoptosis was evaluated through in vivo and in vitro functional loss experiments. The molecular mechanism of IRF2BP2 was investigated by the study of chromatin regulatory regions and transcriptome sequencing. The sustained high expression of IRF2BP2 results from the activation of a novel SE established by NB master transcription factors MYCN, MEIS2, and HAND2, and they form a new complex that regulates the gene network associated with the proliferation of NB cell populations. We also observed a significant enrichment of the AP-1 family at the binding sites of IRF2BP2. Remarkably, within NB cells, AP-1 plays a pivotal role in shaping the chromatin accessibility landscape, thereby exposing the binding site for IRF2BP2. This orchestrated action enables AP-1 and IRF2BP2 to collaboratively stimulate the expression of the NB susceptibility gene ALK, thereby upholding the highly proliferative phenotype characteristic of NB. Our findings indicate that SE-driven IRF2BP2 can bind to AP-1 to maintain the survival of tumor cells via regulating chromatin accessibility of the NB susceptibility gene ALK.

Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus.

Systemic lupus erythematosus (SLE) is prototypical autoimmune disease driven by pathological T cell-B cell interactions1,2. Expansion of T follicular helper (TFH) and T peripheral helper (TPH) cells, two T cell populations that provide help to B cells, is a prominent feature of SLE3,4. Human TFH and TPH cells characteristically produce high levels of the B cell chemoattractant CXCL13 (refs. 5,6), yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4+ T cell phenotypes in patients with SLE, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4+ T cells. Transcriptomic, epigenetic and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ TPH/TFH cell differentiation and promote an IL-22+ phenotype. Type I interferon, a pathogenic driver of SLE7, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ TPH/TFH cells on a polarization axis opposite from T helper 22(TH22) cells and reveal AHR, JUN and interferon as key regulators of these divergent T cell states.

Engineered uterine primary myometrial cells with high-mobility group AT-hook 2 overexpression display a leiomyoma-like transcriptional and epigenomic phenotype

To determine if engineered HMGA2 overexpressing uterine primary myometrial cells recapitulate the transcriptional and epigenomic features of HMGA2-subtype leiomyomas. Isolated primary, "normal" myometrial cells from 3 patients were engineered to overexpress HMGA2 to determine how HMGA2 establishes transcriptomic and epigenomic features of HMGA2-overexpressing leiomyoma. Academic research laboratory PATIENTS: Primary myometrial cells were isolated from normal myometrium obtained from three patients undergoing hysterectomy. Not applicable MAIN OUTCOME MEASURES: Determined genome-wide transcriptomic and epigenomic features of engineered HMGA2-overexpressing uterine primary myometrial cells. Engineered HMGA2-V5-overexpressing primary myometrial cells approximated the HMGA2 expression level observed in HMGA2-overexpression subtype leiomyoma. HMGA2-V5 expression resulted in differential expression of 1612 genes (FDR < 0.05) which were found to be enriched in pathways associated with leiomyoma formation, including extracellular matrix organization. Comparative gene expression analysis between HMGA2-V5 engineered primary cells and HMGA2-overexpression subtype leiomyoma revealed significant overlap of differentially expressed genes. Mechanistically, HMGA2-V5 overexpression resulted in 41,323 regions with differential H3K27ac deposition (FDR < 0.05) and 205,605 regions of altered chromatin accessibility (FDR < 0.05). Transcription factor binding site analysis implicated the AP-1 family of transcription factors. • What clinical problem is addressed by these studies? About 10-15% of the uterine leiomyoma cases exhibit increased expression of HMGA2; our study addresses the primary role of HMGA2 overexpression in the pathogenesis of leiomyoma. • What are the key findings? Ectopic expression of HMGA2 in primary myometrial cells transforms transcriptional and epigenomic machinery in these cells which to some extent resemble features of leiomyoma. • How do these findings apply to human fertility or the reproductive process? Our study provides a comprehensive analysis into the changes occurring in epigenome of myometrial cells when HMGA2 is overexpressed. Because epigenome can be targeted with drugs, therefore understanding alterations in epigenome provides new avenues for treating leiomyoma which affects reproductive age women.

A Transcriptomic Analysis Sheds Light on the Molecular Regulation of Wood Formation in Populus trichocarpa during Drought Stress

Wood is an abundant and essential renewable resource whose production is threatened in some parts of the world by drought. A better understanding of the molecular mechanisms underlying wood formation during drought is critical to maintaining wood production under increasingly adverse environmental conditions. In this study, we investigated wood formation in black cottonwood (Populus trichocarpa) during drought stress. The morphological changes during drought stress in P. trichocarpa included the wilting and drooping of leaves, stem water loss, and a reduction in whole plant biomass. The water embolism rate indicated that the water transport in stems was blocked under drought conditions. An anatomical analysis of the xylem and cambium revealed that drought stress changed the structure of vessel cells, increased lignin accumulation, and decreased the cambium cell layers. We subsequently identified 12,438 and 9156 differentially expressed genes from stem xylem and cambium tissues under well-watered and drought conditions, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that these genes were mainly involved in hormone signal transduction and amino sugar and nucleotide sugar metabolism. To further explore the molecular mechanism of wood formation in response to drought, we analyzed the expression patterns of the genes involved in lignin, cellulose, and hemicellulose biosynthesis in xylem and the genes involved in cambial activity in the cambium. To better understand the regulatory networks governing xylem development and cambium activity in response to drought, we analyzed the MYB (138), AP2 (130), bHLH (89), and NAC (81) transcription factor families to shed light on the interactions between the TFs in these families and the genes they regulate. Identifying the key genes that regulate wood formation in P. trichocarpa during drought provides a genetic foundation for further research on the molecular regulatory networks and physiology underpinning wood formation during drought stress.

Catecholamine treatment induces reversible heart injury and cardiomyocyte gene expression

BackgroundCatecholamines are commonly used as therapeutic drugs in intensive care medicine to maintain sufficient organ perfusion during shock. However, excessive or sustained adrenergic activation drives detrimental cardiac remodeling and may lead to heart failure. Whether catecholamine treatment in absence of heart failure causes persistent cardiac injury, is uncertain. In this experimental study, we assessed the course of cardiac remodeling and recovery during and after prolonged catecholamine treatment and investigated the molecular mechanisms involved.ResultsC57BL/6N wild-type mice were assigned to 14 days catecholamine treatment with isoprenaline and phenylephrine (IsoPE), treatment with IsoPE and subsequent recovery, or healthy control groups. IsoPE improved left ventricular contractility but caused substantial cardiac fibrosis and hypertrophy. However, after discontinuation of catecholamine treatment, these alterations were largely reversible. To uncover the molecular mechanisms involved, we performed RNA sequencing from isolated cardiomyocyte nuclei. IsoPE treatment resulted in a transient upregulation of genes related to extracellular matrix formation and transforming growth factor signaling. While components of adrenergic receptor signaling were downregulated during catecholamine treatment, we observed an upregulation of endothelin-1 and its receptors in cardiomyocytes, indicating crosstalk between both signaling pathways. To follow this finding, we treated mice with endothelin-1. Compared to IsoPE, treatment with endothelin-1 induced minor but longer lasting changes in cardiomyocyte gene expression. DNA methylation-guided analysis of enhancer regions identified immediate early transcription factors such as AP-1 family members Jun and Fos as key drivers of pathological gene expression following catecholamine treatment.ConclusionsThe results from this study show that prolonged catecholamine exposure induces adverse cardiac remodeling and gene expression before the onset of left ventricular dysfunction which has implications for clinical practice. The observed changes depend on the type of stimulus and are largely reversible after discontinuation of catecholamine treatment. Crosstalk with endothelin signaling and the downstream transcription factors identified in this study provide new opportunities for more targeted therapeutic approaches that may help to separate desired from undesired effects of catecholamine treatment.

Epigenetic Memory of Intestinal Epithelial Cells in Inflammatory Bowel Disease

Background: The pathogenesis of human Inflammatory Bowel Disease (IBD) is a multifactorial process including interaction between immune, epithelial, and environmental factors. Growing evidence suggests that epithelial cell defects play major role in the pathogenesis of IBD. Epigenetic memory involves lasting, cell-type specific modifications to chromatin at multiple scales, which impacts transcriptional programs required for normal cell functions. A major gap in knowledge are the mechanisms involved in establishing and maintaining memory in cell types and diseases of the intestine. The objective of this study was to determine if intestinal epithelial cells (IECs) retain a memory of altered chromatin architectures and resultant regulation following inflammation. The hypothesis was that the IECs would show lasting altered chromatin structure landscapes (epigenetic memory) in response to inflammation. Methods: We have developed a multicellular model system of IBD using adult stem cell derived organoids from humans with IBD, from regions of the colon with and without active inflammation. We performed ATAC-seq, bulk RNA-seq and single cell RNA-seq on these organoids derived from the uninflamed and inflamed tissues of IBD patients, specifically Ulcerative Colitis (UC). Results: Regions of increased chromatin accessibility are enriched in organoids derived from areas of inflammation in UC patients compared to organoids from patient-matched, uninflamed regions. These open chromatin regions are associated with proinflammatory genes, with modest transcriptional alterations, pointing to an epigenetic inflammatory memory. Motif analysis for accessibly peaks in UC showed an enrichment of the AP-1 family of transcription factors and chromatin remodelers (SMARCA4). This data is consistent with other studies of inflammatory memory from other epithelial cell types, but we show this for the first time in IECs in the context of IBD. Conclusions: This study presents a novel line of investigation and development of model systems to examine epigenetic memory of IECs in human IBD. We expect that our studies will identify new chromatin-based mechanisms of IEC function that are impacted by inflammation. This research was funded by American Gastroenterological Association Research Scholar Award and KL2 Scholar Career Development Award (KL2 TR002379, National Center for Advancing Translational Science). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elucidating the changes in the heterogeneity and function of radiation-induced cardiac macrophages using single-cell RNA sequencing.

A mouse model of irradiation (IR)-induced heart injury was established to investigate the early changes in cardiac function after radiation and the role of cardiac macrophages in this process. Cardiac function was evaluated by heart-to-tibia ratio, lung-to-heart ratio and echocardiography. Immunofluorescence staining and flow cytometry analysis were used to evaluate the changes of macrophages in the heart. Immune cells from heart tissues were sorted by magnetic beads for single-cell RNA sequencing, and the subsets of macrophages were identified and analyzed. Trajectory analysis was used to explore the differentiation relationship of each macrophage subset. The differentially expressed genes (DEGs) were compared, and the related enriched pathways were identified. Single-cell regulatory network inference and clustering (SCENIC) analysis was performed to identify the potential transcription factors (TFs) which participated in this process. Cardiac function temporarily decreased on Day 7 and returned to normal level on Day 35, accompanied by macrophages decreased and increased respectively. Then, we identified 7 clusters of macrophages by single-cell RNA sequencing and found two kinds of stage specific macrophages: senescence-associated macrophage (Cdkn1ahighC5ar1high) on Day 7 and interferon-associated macrophage (Ccr2highIsg15high) on Day 35. Moreover, we observed cardiac macrophages polarized over these two-time points based on M1/M2 and CCR2/major histocompatibility complex II (MHCII) expression. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses suggested that macrophages on Day 7 were characterized by an inflammatory senescent phenotype with enhanced chemotaxis and inflammatory factors, while macrophages on Day 35 showed enhanced phagocytosis with reduced inflammation, which was associated with interferon-related pathways. SCENIC analysis showed AP-1 family members were associated with IR-induced macrophages changes. We are the first study to characterize the diversity, features, and evolution of macrophages during the early stages in an IR-induced cardiac injury animal model.

Abstract 3940: Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8

Abstract Breast cancer is a highly complex multifactorial disease, which can be driven by the aberrant regulation of different signaling pathways including receptor tyrosine kinase (RTK) signaling. Notably, while RTKs have been classically described as transmembrane receptors, they can also translocate to the nucleus, even though the functional importance of this process remains largely unexplored for most RTKs. Here, we report a novel role for the nuclear form of the RTK human epidermal growth factor receptor 3 (HER3) in driving primary breast cancer growth and metastasis. Firstly, using different patient-derived organoids (PDOs) established from metastatic breast cancer patients, we demonstrated the robust translocation of the activated phosphorylated HER3 receptor (pHER3) from the plasma membrane to the nucleus in response to its ligand Neuregulin 1 (NRG1). Interestingly, the nuclear translocation was observed not only in the HER2-positive breast cancer subtype but also in luminal and triple-negative breast cancer-derived cells. In order to decipher the functional role of nuclear HER3 (nHER3), we identified and mutated its nuclear localization signal, resulting in decreased nuclear translocation of the receptor while its membrane form remained intact. This reduction in nHER3 remarkably impeded primary tumor growth and metastatic spread in different patient-derived xenograft (PDX) models. Conversely, selective overexpression of HER3 in the nucleus led to increased primary tumor growth and metastatic burden in vivo. In order to decipher the mechanism of action of nHER3, we performed co-immunoprecipitation followed by global mass spectrometric analysis to identify the protein binding partners of the receptor in the nucleus. Specifically, nHER3 was found to interact with different transcription factors such as the AP-2 family of transcription factors and with major chromatin remodeling complexes like the nucleosome remodeling and deacetylase (NuRD) complex, implicating its role in transcriptional regulation. Furthermore, gene expression analysis of PDOs expressing the wild-type or the mutated form of HER3 revealed the modulation of several key growth regulators such as early growth response 3 (EGR3) by nHER3. The nHER3-EGR3 axis further controlled downstream effectors like the immune chemoattractants CXCL1 and CXCL8. Accordingly, PDX from breast cancer cells overexpressing nHER3 showed significantly increased immune infiltration, suggesting a possible role of nHER3 in regulating the tumor microenvironment. Altogether, we report here a novel non-canonical role of the nuclear form of HER3 receptor in driving breast tumorigenesis, with key implications for our understanding and targeting of RTK signaling in breast cancer. Citation Format: Tasneem Cheytan, Roberto Würth, Nina Hahnen, Elisa Donato, Corinna Klein, Rebecca Weber, Daniele Colombo, Jeroen Krijgsveld, Martin Sprick, Andreas Trumpp. Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3940.

Expansion of memory Vδ2 T cells following SARS-CoV-2 vaccination revealed by temporal single-cell transcriptomics

γδ T cells provide rapid cellular immunity against pathogens. Here, we conducted matched single-cell RNA-sequencing and γδ-TCR-sequencing to delineate the molecular changes in γδ T cells during a longitudinal study following mRNA SARS-CoV-2 vaccination. While the first dose of vaccine primes Vδ2 T cells, it is the second administration that significantly boosts their immune response. Specifically, the second vaccination uncovers memory features of Vδ2 T cells, shaped by the induction of AP-1 family transcription factors and characterized by a convergent central memory signature, clonal expansion, and an enhanced effector potential. This temporally distinct effector response of Vδ2 T cells was also confirmed in vitro upon stimulation with SARS-CoV-2 spike-peptides. Indeed, the second challenge triggers a significantly higher production of IFNγ by Vδ2 T cells. Collectively, our findings suggest that mRNA SARS-CoV-2 vaccination might benefit from the establishment of long-lasting central memory Vδ2 T cells to confer protection against SARS-CoV-2 infection.

Chromatin profiling reveals TFAP4 as a critical transcriptional regulator of bovine satellite cell differentiation

BackgroundSatellite cells are myogenic precursor cells in adult skeletal muscle and play a crucial role in skeletal muscle regeneration, maintenance, and growth. Like embryonic myoblasts, satellite cells have the ability to proliferate, differentiate, and fuse to form multinucleated myofibers. In this study, we aimed to identify additional transcription factors that control gene expression during bovine satellite cell proliferation and differentiation.ResultsUsing chromatin immunoprecipitation followed by sequencing, we identified 56,973 and 54,470 genomic regions marked with both the histone modifications H3K4me1 and H3K27ac, which were considered active enhancers, and 50,956 and 59,174 genomic regions marked with H3K27me3, which were considered repressed enhancers, in proliferating and differentiating bovine satellite cells, respectively. In addition, we identified 1,216 and 1,171 super-enhancers in proliferating and differentiating bovine satellite cells, respectively. Analyzing these enhancers showed that in proliferating bovine satellite cells, active enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation whereas repressed enhancers were associated with genes essential for myoblast differentiation, and that in differentiating satellite cells, active enhancers were associated with genes essential for myoblast differentiation or muscle contraction whereas repressed enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation. Active enhancers in proliferating bovine satellite cells were enriched with binding sites for many transcription factors such as MYF5 and the AP-1 family transcription factors; active enhancers in differentiating bovine satellite cells were enriched with binding sites for many transcription factors such as MYOG and TFAP4; and repressed enhancers in both proliferating and differentiating bovine satellite cells were enriched with binding sites for NF-kB, ZEB-1, and several other transcription factors. The role of TFAP4 in satellite cell or myoblast differentiation was previously unknown, and through gene knockdown and overexpression, we experimentally validated a critical role for TFAP4 in the differentiation and fusion of bovine satellite cells into myofibers.ConclusionsSatellite cell proliferation and differentiation are controlled by many transcription factors such as AP-1, TFAP4, NF-kB, and ZEB-1 whose roles in these processes were previously unknown in addition to those transcription factors such as MYF5 and MYOG whose roles in these processes are widely known.

Activator protein transcription factors coordinate human IL-33 expression from noncanonical promoters in chronic airway disease.

IL-33 is a cytokine central to type 2 immune pathology in chronic airway disease. This cytokine is abundantly expressed in the respiratory epithelium and increased in disease, but how expression is regulated is undefined. Here we show that increased IL33 expression occurs from multiple noncanonical promoters in human chronic obstructive pulmonary disease (COPD), and it facilitates production of alternatively spliced isoforms in airway cells. We found that phorbol 12-myristate 13-acetate (PMA) can activate IL33 promoters through protein kinase C in primary airway cells and lines. Transcription factor (TF) binding arrays combined with RNA interference identified activator protein (AP) TFs as regulators of baseline and induced IL33 promoter activity. ATAC-Seq and ChIP-PCR identified chromatin accessibility and differential TF binding as additional control points for transcription from noncanonical promoters. In support of a role for these TFs in COPD pathogenesis, we found that AP-2 (TFAP2A, TFAP2C) and AP-1 (FOS and JUN) family members are upregulated in human COPD specimens. This study implicates integrative and pioneer TFs in regulating IL33 promoters and alternative splicing in human airway basal cells. Our work reveals a potentially novel approach for targeting IL-33 in development of therapeutics for COPD.

AP-2α/AP-2β Transcription Factors Are Key Regulators of Epidermal Homeostasis

AP-2 transcription factors regulate ectodermal development, but their roles in epidermal homeostasis in adult skin are unknown. We find that AP-2α is the predominant AP-2 family member in adult epidermis, followed by AP-2β. Through inactivation of AP-2α, AP-2β, or both in keratinocytes, we assessed the effects of a gradient of epidermal AP-2 activity on skin function. We find that (i) loss of AP-2β in keratinocytes is compensated for by AP-2α, (ii) loss of AP-2α impairs terminal keratinocyte differentiation and hair morphogenesis, and (iii) the combined loss of AP-2α/AP-2β results in more severe skin and hair abnormalities. Keratinocyte differentiation defects precede progressive neutrophilic skin inflammation. Inducible inactivation of AP-2α/AP-2β in the adult phenocopies these manifestations. Transcriptomic analyses of epidermis lacking AP-2α or AP-2α/AP-2β in keratinocytes demonstrate a terminal keratinocyte differentiation defect with upregulation of alarmin keratins and of several immune pathway regulators. Moreover, our analyses suggest a key role of reduced AP-2α-dependent gene expression of CXCL14 and the keratin 15 gene K15 as an early pathogenic event toward the manifestation of skin inflammation. Thus, AP-2α and AP-2β are critical regulators of epidermal homeostasis in adult skin.