Ubiquitous Transcription Factor Research Articles

YY1 knockout in pro-B cells impairs lineage commitment, enabling unusual hematopoietic lineage plasticity.

During B-cell development, cells progress through multiple developmental stages, with the pro-B-cell stage defining commitment to the B-cell lineage. YY1 is a ubiquitous transcription factor that is capable of both activation and repression functions. We found here that knockout of YY1 at the pro-B-cell stage eliminates B lineage commitment. YY1 knockout pro-B cells can generate T lineage cells in vitro using the OP9-DL4 feeder system and in vivo after injection into sublethally irradiated Rag1-/- mice. These T lineage-like cells lose their B lineage transcript profile and gain a T-cell lineage profile. Single-cell RNA-seq experiments showed that as YY1 knockout pro-B cells transition into T lineage cells in vitro, various cell clusters adopt transcript profiles representing a multiplicity of hematopoietic lineages, indicating unusual lineage plasticity. In addition, YY1 KO pro-B cells in vivo can give rise to other hematopoietic lineages in vivo. Evaluation of RNA-seq, scRNA-seq, ChIP-seq, and scATAC-seq data indicates that YY1 controls numerous chromatin-modifying proteins leading to increased accessibility of alternative lineage genes in YY1 knockout pro-B cells. Given the ubiquitous nature of YY1 and its dual activation and repression functions, YY1 may regulate commitment in multiple cell lineages.

CREB activates the MafA promoter through proximal E-boxes and a CCAAT motif in pancreatic β-cells.

MafA is a key transcriptional regulator of pancreatic islet β-cell function. Its target genes include those encoding preproinsulin and the glucose transporter Glut2 (Slc2a2); thus, MafA function is essential for glucose-stimulated insulin secretion. Expression levels of MafA are reduced in β-cells of diabetic mouse models and human subjects, suggesting that β-cell dysfunction associated with type 2 diabetes is attributable to the loss of MafA. On the other hand, MafA is transcriptionally upregulated by incretin hormones through activation of CREB and its co-activator CRTC2. β-cell-specific expression of MafA relies on a distal enhancer element. However, the precise mechanism by which CREB-CRTC2 regulates the enhancer and proximal promoter regions of MafA remains unclear. In this report, we analyzed previously published ChIP-seq data and found that CREB and NeuroD1, a β-cell-enriched transactivator, bound to both the promoter and enhancer regions of human MAFA. A series of reporter assays revealed that CREB activated the enhancer through a conserved cAMP-responsive element (CRE) but stimulated MAFA promoter activity even when the putative CRE was deleted. Two E-box elements and a CCAAT motif, which bind NeuroD1 and ubiquitous NF-Y transcription factors, respectively, were necessary for transcriptional activation of the MAFA promoter by CREB. Genome-wide analysis of CREB-bound loci in β-cells revealed that they were enriched with CCAAT motifs. Furthermore, promoter analysis of the Isl1 gene encoding a β-cell-enriched transcription factor revealed that a CRE-like element and two CCAAT motifs, but not the E-box, were necessary for activation by CREB. These results provide clues to elucidate the detailed mechanism by which CREB regulates MafA as well as β-cell-specific genes.

Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation.

Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome may also play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutation and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and significantly change the chromosome organization at domain scales. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulator (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (>200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.

Sex-Dependent Regulation of Mucin Gene Transcription and Goblet Cell Secretion Machinery Following Intra-Airway IL-13 in Mice with Conditional Loss of Club Cell Creb1

IL-13 is an important effector molecule in allergic asthma, converting secretoglobin-positive club cells into mucin-secreting goblet cells and promoting mucin hypersecretion. IL-13-mediated conversion of club cells to goblet cells requires decreased gene expression of forkhead box A2 ( FOXA2) and increased gene expression of SAM pointed domain containing ETS transcription factor ( SPDEF). In addition, IL-13-mediated mucin hypersecretion may include modulation of purinergic and muscarinic receptors that control basal and stimulated mucus secretion, respectively. We recently found that the cAMP response element-binding protein (CREB), a ubiquitous transcription factor, directly binds to FOXA2 and modulates mucus secretion mechanisms in mice. Here we tested the hypothesis that conditional loss of club cell Creb1 in murine airways modulates the pro-mucin effects of IL-13. Loss of club cell Creb1 augmented IL-13-mediated increases in gene expression of the major gel-forming secreted mucins Muc5ac and Muc5b in male murine airways, with no effect on IL-13-mediated increases in Muc5ac and Muc5b in female murine airways. Examination of mucin secretion mechanisms revealed that IL-13 decreased muscarinic 3 receptor ( M3R) mRNA expression in male murine airways, whereas loss of club cell Creb1 prevented this, resulting in a significant treatment by genotype interaction. Interestingly, in female airways, a main effect of genotype was observed, with loss of club cell Creb1 reducing M3R mRNA expression in the airway. Examination of the purinergic receptor P2Y ( P2ry2) mRNA revealed a main treatment effect for IL-13 to increase P2ry2 expression in male murine airways with no impact of loss of club cell Creb1. In the females, IL-13 increased P2ry2 mRNA in both genotypes, with loss of club cell Creb1 significantly blunting the effect of IL-13 on P2ry2 mRNA. Lastly, we examined goblet cell density and mucin secretion mechanisms using Alcian Blue-PAS staining post in vivo stimulation with a muscarinic agent. We found no major effect of treatment or genotype in the density of goblet cells containing acidic mucins in either male or female airways. In contrast, both males and females showed IL-13-mediated increases in the density of goblet cells containing neutral mucins that was not impacted by genotype. Our preliminary findings suggest that loss of club cell Creb1 decreases molecular mucin secretion mechanisms in male and female airways, and increases Muc5ac and Muc5b mRNA in male airways, though a corresponding genotype-dependent increase in goblet cell density post cholinergic stimulation was not observed. Thus, we conclude that loss of club cell Creb1 causes changes to molecules important for IL-13-mediated mucin hypersecretion, without apparent functional benefit or detriment to the murine airway. This work was supported by the National Institutes of Health (OD026582, HL152101), the Cystic Fibrosis Foundation (REZNIKO20I0, REZNIKO19I0), the Katie Rose Foundation (AWD08203), and the University of Florida McKnight Career Accelerator Award. Angelina Bonilla was funded by the National institutes of Health (R25GM115298). Pedro Trevizan Bau receives the University of Florida McKnight Brain Institute Gator NeuroScholar Fellowship. 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.

Abstract 3787: Important role of GPX1 and NF-kb signaling pathway in human gastric cancer including cell proliferation and invasion

Abstract Background: Glutathione peroxidases(GPXs) are member of an antioxidant enzyme family. Their role is to eliminate the reactive oxygen species(ROS). Many available studies confirm that over expression of GPXs can promote cancer cell proliferation and invasion. Among the 8 GPXs, Glutathione peroxidase-1 (GPX1) is the most plentiful. Overexpression of GPX1 has been noted to promote invasion, migration, cisplatin resistance and proliferation in breast, colorectal and lung cancer. NF-kB is a ubiquitous nuclear transcription factor in the human body. that key role in cell proliferation, apoptosis and tumor progression. In reliable studies, inhibition of NF-kB expression has been shown to reduce the malignancy of Esophageal squamous cell carcinoma. Accordingly, the purpose of this study is to identify the GPX1 and NF-kB signaling pathway and their correlation with cancer cell proliferation and invasion in vitro using gastric cancer cell. Methods: In our study, we used cell culture, Northern blotting, cDNA microarray analysis, western blotting, RT-PCR, Zymography, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, GPX1 knock-down with short hairpin RNS(shRNA), Standard two-chamber invasion assay, Chromatin immunoprecipitation assay. Results: First, we confirmed that the expression level of GPX1 was up-regulated by HGF(hepatocyte growth factor) in gastric cancer cells. The cells were treated with NF-kB inhibitor, Pyrrolidine dithiocarbamate(PDTC), and analyzed by Western blotting to identify the associated pathway of HGF-induced GPX1 with NF-kB. The HGF-induced GPX1 protein level was down regulated by PDTC. The role of GPX1 associated with NF-kB was determined by knock down GPX1 expression using GPX1-sh RNA. The GPX1-sh RNA treated cells showedvdown regulation of NF-kB and urokinase Plasminogen activator(uPA) also. To fine the upper regulator of GPX1 signaling pathway, we treated AKT inhibitor(LY294002) ant then analyzed by Western blotting to identify associated pathway of HGF-induced GPX1 with AKT. When AKT pathway was inhibited, GPX1 and NF-kB were down-regulated in gastric cancer cell. HGF-mediated cell proliferation and invasion in vitro was decreased in GPX1 knock down cell. We identified the putative binding site of GPX1 promotor containing NF-kB binding site and confirmed the function by CHIP assay. Conclusions: This study aimed to investigate relationship between GPX1 and NF-kB in gastric cancer cells. Results showed that HGF induced GPX1 expression through the NF-kB and AKT pathway and GPX1 may play a significant role in cell proliferation and invasion in gastric cancer. Therefore, GPX1 can be a potential therapeutic target of gastric cancer for the treatment. Citation Format: Byeong Il Jang, Ji Yoon Jung, Sung Ae Koh, Kyung Hee Lee. Important role of GPX1 and NF-kb signaling pathway in human gastric cancer including cell proliferation and invasion [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 3787.

Structural and dynamical aspect of DNA motif sequence specific binding of AP-1 transcription factor.

Activator protein-1 (AP-1) comprises one of the largest and most evolutionary conserved families of ubiquitous eukaryotic transcription factors that act as a pioneer factor. Diversity in DNA binding interaction of AP-1 through a conserved basic-zipper (bZIP) domain directs in-depth understanding of how AP-1 achieves its DNA binding selectivity and consequently gene regulation specificity. Here, we address the structural and dynamical aspects of the DNA target recognition process of AP-1 using microsecond-long atomistic simulations based on the structure of the human AP-1 FosB/JunD bZIP-DNA complex. Our results show the unique role of DNA shape features in selective base specific interactions, characteristic ion population, and solvation properties of DNA grooves to form the motif sequence specific AP-1-DNA complex. The TpG step at the two terminals of the AP-1 site plays an important role in the structural adjustment of DNA by modifying the helical twist in the AP-1 bound state. We addressed the role of intrinsic motion of the bZIP domain in terms of opening and closing gripper motions of DNA binding helices, in target site recognition and binding of AP-1 factors. Our observations suggest that binding to the cognate motif in DNA is mainly accompanied with the precise adjustment of closing gripper motion of DNA binding helices of the bZIP domain.

Dynamic modulation of the non-canonical NF-κB signaling pathway for HIV shock and kill.

HIV cure still remains an elusive target. The "Shock and Kill" strategy which aims to reactivate HIV from latently infected cells and subsequently kill them through virally induced apoptosis or immune mediated clearance, is the subject of widespread investigation. NF-κB is a ubiquitous transcription factor which serves as a point of confluence for a number of intracellular signaling pathways and is also a crucial regulator of HIV transcription. Due to its relatively lower side effect profile and proven role in HIV transcription, the non-canonical NF-κB pathway has emerged as an attractive target for HIV reactivation, as a first step towards eradication. A comprehensive review examining this pathway in the setting of HIV and its potential utility to cure efforts is currently lacking. This review aims to summarize non-canonical NF-κB signaling and the importance of this pathway in HIV shock-and-kill efforts.

The transcription factor NF-YA is crucial for neural progenitor maintenance during brain development

In contrast to stage-specific transcription factors, the role of ubiquitous transcription factors in neuronal development remains a matter of scrutiny. Here, we demonstrated that a ubiquitous factor NF-Y is essential for neural progenitor maintenance during brain morphogenesis. Deletion of the NF-YA subunit in neural progenitors by using nestin-cre transgene in mice resulted in significant abnormalities in brain morphology, including a thinner cerebral cortex and loss of striatum during embryogenesis. Detailed analyses revealed a progressive decline in multiple neural progenitors in the cerebral cortex and ganglionic eminences, accompanied by induced apoptotic cell death and reduced cell proliferation. In neural progenitors, the NF-YA short isoform lacking exon 3 is dominant and co-expressed with cell cycle genes. ChIP-seq analysis from the cortex during early corticogenesis revealed preferential binding of NF-Y to the cell cycle genes, some of which were confirmed to be downregulated following NF-YA deletion. Notably, the NF-YA short isoform disappears and is replaced by its long isoform during neuronal differentiation. Forced expression of the NF-YA long isoform in neural progenitors resulted in a significant decline in neuronal count, possibly due to the suppression of cell proliferation. Collectively, we elucidated a critical role of the NF-YA short isoform in maintaining neural progenitors, possibly by regulating cell proliferation and apoptosis. Moreover, we identified an isoform switch in NF-YA within the neuronal lineage in vivo, which may explain the stage-specific role of NF-Y during neuronal development.

Multiple lineage-specific epigenetic landscapes at the antigen receptor loci.

Antigen receptors (AgRs) expressed on B and T cells provide the adaptive immune system with ability to detect numerous foreign antigens. Epigenetic features of B cell receptor (BCR) and T cell receptor (TCR) genes were previously studied in lymphocytes, but little is known about their epigenetic features in other cells. Here, we explored histone modifications and transcription markers at the BCR and TCR loci in lymphocytes (pro-B, DP T cells, and mature CD4+ T cells), compared to embryonic stem (ES) cells and neurons. In B cells, the BCR loci exhibited active histone modifications and transcriptional markers indicative of active loci. Similar results were observed at the TCR loci in T cells. All loci were largely inactive in neurons. Surprisingly, in ES cells all AgR loci displayed a high degree of active histone modifications and markers of active transcription. Locations of these active histone modifications in ES cells were largely distinct from those in pro-B cells, and co-localized at numerous binding locations for transcription factors Oct4, Sox2, and Nanog. ES and pro-B cells also showed distinct binding patterns for the ubiquitous transcription factor YY1 and chromatin remodeler Brg1. On the contrary, there were many overlapping CCCTC-binding factor (CTCF) binding patterns when comparing ES cells, pro-B cells, and neurons. Our study identifies epigenetic features in ES cells and lymphocytes that may be related to ES cell pluripotency and lymphocyte tissue-specific activation at the AgR loci.

The Role of Skeletal Muscle Nrf2 on Cardiac Function in Mice

Introduction: Excessive oxidant stress has been shown to damage cardiac tissue and result in impaired cardiac function. Animals with chronic heart failure (CHF) exhibit lower levels of antioxidant enzymes in many tissues including the heart, brain, and skeletal muscle (SkM). Nrf2 is a ubiquitous master transcription factor that regulates antioxidant proteins during enhanced oxidative stress. Earlier studies from this laboratory showed that exercise training was cardioprotective following ischemia reperfusion injury in mice. This protective effect was abrogated in mice in which the Nrf2 gene was deleted selectively in skeletal muscle. Given that skeletal muscle is approximately 40% of body weight and in light of recent findings on skeletal muscle derived extracellular vesicles, we sought to determine if up and downregulation of Nrf2 in skeletal muscle altered cardiac function. We hypothesized that skeletal muscle knockout of Nrf2 would impair cardiac function and the response to the beta 1 agonist dobutamine. Materials and Methods: Experiments were carried out in 37 males iMS-Keap1flox/flox and iMS-Nrf-2flox/flox (i.e. SkM specific) mice. Gene deletion was conditional upon the administration of the estrogen receptor agonist tamoxifen at 3 months of age; floxed mice given vehicle instead of tamoxifen were considered WT. The mice were assigned to 4 groups: Keap1 knockout (KO) (10), Keap1 WT (8), Nrf-2 KO (11), and Nrf-2 WT (8). Results: Keap1 deletion resulted in an increase in skeletal muscle hemoxygenase 1 (HO1) (p = 0.0286) while Nrf2 deletion resulted in a reduction in HO1 (p = 0.0286). However, there were no changes in the level of HO1 or other antioxidant proteins in the heart. Cardiac function was assessed with high frequency echocardiography (Vevo 3100). Systolic and diastolic function were analyzed. Up and downregulation of skeletal muscle Nrf2 did not evoke any change in either systolic or diastolic function. Conclusions: Previous studies have shown that Nrf2 upregulation salvaged cardiac function in mice subjected to either myocardial infarction or thoracic aortic constriction. Therefore, it may be that our lack of an effect of SkM specific Nrf2 manipulation is due to this study being carried out in normal animals and not in a disease setting such as CHF. In future work, this effect will be examined in mice with cardiac dysfunction. Supported by P0162222 and SURF from the APS This is the full abstract presented at the American Physiology Summit 2023 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.

Endothelial Nrf2 modulates blood pressure and vasoconstrictor responses in conscious mice

Introduction: Excessive oxidant stress has been shown to mediate a variety of cardiovascular pathological conditions, including hypertension. The level of reactive oxygen species (ROS) in tissues is regulated by the balance between the production of ROS and the ability to scavenge superoxide and hydrogen peroxide by a myriad of antioxidant enzymes and small molecules. One of the most important regulators of antioxidant activity is the ubiquitous transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 binds to antioxidant response elements of multiple genes following its release from its inhibitory protein Keap1 and part of the E3 ubiquitin ligase complex. In this study we hypothesized that endothelial Nrf2 is a critical factor in blood pressure regulation following vasoconstrictor stimuli. Methods: Transgenic mice were created using the endothelial Tie2 promoter expressing Cre recombinase (Tie2-Cre) crossed with either Nrf2 floxed or Keap1 floxed mice so that Nrf2 would be deleted or upregulated respectively, and selectively in the endothelium. Cre negative mice were used as wild type (WT) controls. Mice were instrumented with radiotelemetry transducers to chronically record arterial blood pressure and heart rate in the conscious state. Blood pressure and heart rate were recorded for 24 hours once per week. After baseline recordings, animals were challenged with an osmotic minipump infusion of Angiotensin II (Ang II) (600ng/Kg/min) for 7 days or L-nitroarginine methyl ester (L-NAME) (100 mg/kg/d in drinking water). Results: Baseline mean arterial pressure (MAP) was 101.6 ± 2.5 mm Hg (mean±SEM; n=7) in WT mice (daytime =95.4±1.5; nighttime =106.1±1.0). In mice with endothelial Nrf2 deletion baseline MAP was significantly higher(p<0.01) during nighttime (112.1±1.1). In addition, the response to Ang II was significantly augmented (p < 0.001) in Nrf2 KO mice compared to WT mice (148.9±5.3 vs 116.8±6.7 mm Hg). Both day and nighttime values were higher in Nrf2 KO mice. Furthermore, Nrf2 KO mice exhibited a delayed return to baseline upon pump emptying. In contrast, in one Keap1 KO mouse there was a response to Ang II that was similar to WT (92.1 mm Hg baseline to 116.3 mmHg post Ang II). In Nrf2 deleted mice, the hypertensive response to L-NAME was no different than that of WT mice. Conclusion: In summary, these preliminary data suggest that endothelial Nrf2 plays an important role in modulation of blood pressure at rest and during oxidative stress induced by Ang II. The L-NAME data suggests that upregulation of Nrf2 increases the bioavailability of nitric oxide to blunt Ang II mediated vasoconstriction. Supported by NHLBI P01-62222 This is the full abstract presented at the American Physiology Summit 2023 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.

Proteomic basis of mortality resilience mediated by FOXO3 longevity genotype

FOXO3 is a ubiquitous transcription factor expressed in response to cellular stress caused by nutrient deprivation, inflammatory cytokines, reactive oxygen species, radiation, hypoxia, and other factors. We showed previously that the association of inherited FOXO3 variants with longevity was the result of partial protection against mortality risk posed by aging-related life-long stressors, particularly cardiometabolic disease. We then referred to the longevity-associated genotypes as conferring “mortality resilience.” Serum proteins whose levels change with aging and are associated with mortality risk may be considered as “stress proteins.” They may serve as indirect measures of life-long stress. Our aims were to (1) identify stress proteins that increase with aging and are associated with an increased risk of mortality, and (2) to determine if FOXO3 longevity/resilience genotype dampens the expected increase in mortality risk they pose. A total of 4500 serum protein aptamers were quantified using the Somalogic SomaScan proteomics platform in the current study of 975 men aged 71–83 years. Stress proteins associated with mortality were identified. We then used age-adjusted multivariable Cox models to investigate the interaction of stress protein with FOXO3 longevity-associated rs12212067 genotypes. For all the analyses, the p values were corrected for multiple comparisons by false discovery rate. This led to the identification of 44 stress proteins influencing the association of FOXO3 genotype with reduced mortality. Biological pathways were identified for these proteins. Our results suggest that the FOXO3 resilience genotype functions by reducing mortality in pathways related to innate immunity, bone morphogenetic protein signaling, leukocyte migration, and growth factor response.

Genome-wide identification of the B3 gene family in soybean and the response to melatonin under cold stress.

Melatonin is a multipotent molecule that exists widely in animals and plants and plays an active regulatory role in abiotic stresses. The B3 superfamily is a ubiquitous transcription factor with a B3 functional domain in plants, which can respond temporally to abiotic stresses by activating defense compounds and plant hormones. Despite the fact that the B3 genes have been studied in a variety of plants, their role in soybean is still unknown. The regulation of melatonin on cold resistance of soybean and the response of B3 genes to cold stress were investigated by measuring biochemical indexes of soybean. Meanwhile, the genome-wide identification of B3 gene family was conducted in soybean, and B3 genes were analyzed based on phylogeny, motifs, gene structure, collinearity, and cis-regulatory elements analysis. We found that cold stress-induced oxidative stress in soybean by producing excessive reactive oxygen species. However, exogenous melatonin treatment could increase the content of endogenous melatonin and other hormones, including IAA and ABA, and enhance the antioxidative system, such as POD activity, CAT activity, and GSH/GSSG, to scavenge ROS. Furthermore, the present study first revealed that melatonin could alleviate the response of soybean to cold stress by inducing the expression of B3 genes. In addition, we first identified 145 B3 genes in soybean that were unevenly distributed on 20 chromosomes. The B3 gene family was divided into 4 subgroups based on the phylogeny tree constructed with protein sequence and a variety of plant hormones and stress response cis-elements were discovered in the promoter region of the B3 genes, indicating that the B3 genes were involved in several aspects of the soybean stress response. Transcriptome analysis and results of qRT-PCR revealed that most GmB3 genes could be induced by cold, the expression of which was also regulated by melatonin. We also found that B3 genes responded to cold stress in plants by interacting with other transcription factors. We found that melatonin regulates the response of soybean to cold stress by regulating the expression of the transcription factor B3 gene, and we identified 145 B3 genes in soybean. These findings further elucidate the potential role of the B3 gene family in soybean to resist low-temperature stress and provide valuable information for soybean functional genomics study.

The human glucocorticoid receptor.

Glucocorticoids are members of steroid hormones that are biosynthesized in the intermediate cellular zone of the adrenal cortex (zona fasciculata) and released into the peripheral blood as final products of the hypothalamic-pituitary-adrenal (HPA) axis, as well as under the control of the circadian biologic system. These molecules regulate every physiologic function of the organism as they bind to an almost ubiquitous hormone-activated transcription factor, the glucocorticoid receptor (GR), which influences the rate of transcription of a huge number of target genes amounting to up to 20% of the mammalian genome. The evolving progress of cellular, molecular and computational-structural biology and the implication of epigenetics in every-day clinical practice have enabled us a deeper and ever-increasing understanding of how target tissues respond to natural and synthetic glucocorticoids. In this chapter, we summarize the current knowledge on the structure, expression, function and signaling of the human glucocorticoid receptor in normal and pathologic conditions.

In silico discovery of blood cell macromolecular associations

BackgroundPhysical molecular interactions are the basis of intracellular signalling and gene regulatory networks, and comprehensive, accessible databases are needed for their discovery. Highly correlated transcripts may reflect important functional associations, but identification of such associations from primary data are cumbersome. We have constructed and adapted a user-friendly web application to discover and identify putative macromolecular associations in human peripheral blood based on significant correlations at the transcriptional level.MethodsThe blood transcriptome was characterized by quantification of 17,328 RNA species, including 341 mature microRNAs in 105 clinically well-characterized postmenopausal women. Intercorrelation of detected transcripts signal levels generated a matrix with > 150 million correlations recognizing the human blood RNA interactome. The correlations with calculated adjusted p-values were made easily accessible by a novel web application.ResultsWe found that significant transcript correlations within the giant matrix reflect experimentally documented interactions involving select ubiquitous blood relevant transcription factors (CREB1, GATA1, and the glucocorticoid receptor (GR, NR3C1)). Their responsive genes recapitulated up to 91% of these as significant correlations, and were replicated in an independent cohort of 1204 individual blood samples from the Framingham Heart Study. Furthermore, experimentally documented mRNAs/miRNA associations were also reproduced in the matrix, and their predicted functional co-expression described. The blood transcript web application is available at http://app.uio.no/med/klinmed/correlation-browser/blood/index.php and works on all commonly used internet browsers.ConclusionsUsing in silico analyses and a novel web application, we found that correlated blood transcripts across 105 postmenopausal women reflected experimentally proven molecular associations. Furthermore, the associations were reproduced in a much larger and more heterogeneous cohort and should therefore be generally representative. The web application lends itself to be a useful hypothesis generating tool for identification of regulatory mechanisms in complex biological data sets.

Interrelation between α-Cardiac Actin Treadmilling and Myocardin-Related Transcription Factor-A Nuclear Shuttling in Cardiomyocytes.

Myocardin-related transcription factors (MRTFs) play a central role in the regulation of actin expression and cytoskeletal dynamics that are controlled by Rho GTPases. SRF is a ubiquitous transcription factor strongly expressed in muscular tissues. The depletion of SRF in the adult mouse heart leads to severe dilated cardiomyopathy associated with the down-regulation of target genes encoding sarcomeric proteins including α-cardiac actin. The regulatory triad, composed of SRF, its cofactor MRTFA and actin, plays a major role in the coordination of the nuclear transcriptional response to adapt actin filament dynamics associated with changes in cell shape, and contractile and migratory activities. Most of the knowledge on the regulation of the SRF–MRTF–Actin axis has been obtained in non-muscle cells with α-actin and smooth muscle cells with α-smooth actin. Here, we visualized for the first time by a time-lapse video, the nucleocytoplasmic shuttling of MRTFA induced by serum or pro-hypertrophic agonists such as angiotensin II, phenylephrine and endothelin-1, using an MRTFA-GFP adenovirus in cultures of neonatal rat cardiomyocytes. We showed that an inhibitor of the RhoA/ROCK signaling pathway leads to an α-cardiac actin polymerization disruption and inhibition of MRTFA nucleocytoplasmic shuttling. Moreover, inhibition of the PI3K/Akt signaling pathway also prevents the entry of MRTFA into the nuclei. Our findings point out a central role of the SRF–MRTFA–actin axis in cardiac remodeling.

Abstract 1165: Therapeutic targeting of glucocorticoid receptor in ETS rearranged cancer

Abstract The Nuclear receptor Glucocorticoid receptor (GR) acts as a ubiquitous hormone-dependent transcription factor, which regulates many cellular functions. Here, we discovered that some ETS (E-twenty-six) family transcription factors form physical complexes with GR. Two members of the family, FLI1 and ERG exhibited stronger binding with the ligand-activated GR. GR-FLI1 interaction enhance the transcriptional activity of DNA-bound GR. Antagonizing GR or lowering cortisol levels in Ewing sarcoma (ES) animal models, a pediatric bone malignancy driven by EWS-FLI1 fusion, robustly retarded tumor growth, as well as inhibited bone to lung metastasis. These findings prompted us to identify prognostic, GR-regulated gene signature in ES. Because the genomic binding sites of GR in ES are still unknown, using chromatin immunoprecipitation followed by high-throughput DNA sequencing in A673 cells we found previously known and new target genes of GR. By utilizing the protein-fragment complementation assay (PCA), we discovered that GR physically interacts with additional ETS factors. Disruption of this complex is enabled by clinically approved GR antagonist RU486 or newly developed selective GR modulators (i.e., SGRMs without AR or PR cross-reactivity). ETS proteins has been strongly associated with tumor progression and metastasis in several cancers. Taken together, we hypothesised that these mechanisms are also relevant for other ETS rearranged cancers. Results of co-immunoprecipitation analysis confirm the interaction of GR and ERG/ETV4 in ETS-positive prostate cancer cells. In addition, migration and proliferation were reduced when we knocked-down or antagonized GR. It is important noting that the pharmacology of GR is very well developed and some GR-targeting drugs are among the most prescribed medicines. These discoveries will open the way to clinical tests and eventual application of anti- hormone therapy in the discipline of pediatric sarcomas and ETS-rearranged cancers. Citation Format: Arunachalam Sekar, Nishanth Belugali Nataraj, Yosef Yarden. Therapeutic targeting of glucocorticoid receptor in ETS rearranged cancer [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 1165.

Krüppel-like factor 6 (KLF6) requires its amino terminal domain to promote villous trophoblast cell fusion.

Villous cytotrophoblast (vCTB) cells fuse to generate and maintain the syncytiotrophoblast layer required for placental development and function. Krüppel-like factor 6 (KLF6) is a ubiquitous transcription factor with an N-terminal acidic transactivation domain and a C-terminal zinc finger DNA-binding domain. KLF6 is highly expressed in placenta, and it is required for proper placental development. We have demonstrated that KLF6 is necessary for cell fusion in human primary vCTBs, and in the BeWo cell line. Full length KLF6 or a mutant lacking its N-terminal domain were expressed in BeWo cells or in primary vCTB cells isolated from human term placentas. Cell fusion, gene and protein expression, and cell proliferation were analyzed. Moreover, Raman spectroscopy and atomic force microscopy (AFM) were used to identify biochemical, topography, and elasticity cellular modifications. The increase in KLF6, but not the expression of its deleted mutant, is sufficient to trigger cell fusion and to raise the expression of β-hCG, syncytin-1, the chaperone protein 78 regulated by glucose (GRP78), the ATP Binding Cassette Subfamily G Member 2 (ABCG2), and Galectin-1 (Gal-1), all molecules involved in vCTB differentiation. Raman and AFM analysis revealed that KLF6 reduces NADH level and increases cell Young's modulus. KLF6-induced differentiation correlates with p21 upregulation and decreased cell proliferation. Remarkable, p21 silencing reduces cell fusion triggered by KLF6 and the KLF6 mutant impairs syncytialization and decreases syncytin-1 and β-hCG expression. KLF6 induces syncytialization through a mechanism that involves its regulatory transcriptional domain in a p21-dependent manner.

A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

Inflammation is a physiological process whose deregulation causes some diseases including cancer. Nuclear Factor kB (NF-kB) is a family of ubiquitous and inducible transcription factors, in which the p65/p50 heterodimer is the most abundant complex, that play critical roles mainly in inflammation. Glucocorticoid Receptor (GR) is a ligand-activated transcription factor and acts as an anti-inflammatory agent and immunosuppressant. Thus, NF-kB and GR are physiological antagonists in the inflammation process. Here we show that in mice and humans there is a spliced variant of p65, named p65 iso5, which binds the corticosteroid hormone dexamethasone amplifying the effect of the glucocorticoid receptor and is expressed in the liver of patients with hepatic cirrhosis and hepatocellular carcinoma (HCC). Furthermore, we have quantified the gene expression level of p65 and p65 iso5 in the PBMC of patients affected by SARS-CoV-2 disease. The results showed that in these patients the p65 and p65 iso5 mRNA levels are higher than in healthy subjects. The ability of p65 iso5 to bind dexamethasone and the regulation of the glucocorticoid (GC) response in the opposite way of the wild type improves our knowledge and understanding of the anti-inflammatory response and identifies it as a new therapeutic target to control inflammation and related diseases.

Abstract 9172: Feasibility and Efficacy of Targeted Cardiac NF-kB Inhibition in Experimental Diastolic Dysfunction Using Drug-Delivery Technology: A Proof-of-Concept Study

Introduction: Elastin-like polypeptides (ELP) are novel drug delivery vectors that can be fused to therapeutic agents and cell penetrating peptides (CPPs), slowing their plasma clearance and increasing uptake by target tissues. We developed a CPP-ELP fusion to a specific inhibitor of the p50 subunit of NF-kB (SynB1-ELP-p50i). We developed a swine model of chronic kidney disease (CKD) that also fosters hypertension, cardiac remodeling, and diastolic dysfunction, and observed that cardiac abnormalities associate with increased cardiac expression of NF-kB, a ubiquitous transcription factor that modulates cardiac inflammation, fibrosis, and vascular remodeling. Hypothesis: We aimed to define the feasibility and efficacy of intra-coronary (IC) SynB1-ELP-p50i administration to test whether a targeted inhibition of NF-kB can improve cardiac abnormalities in CKD. Methods: We first performed a biodistribution study (BDS) in 3 pigs, 4 hours after IC injection of fluorescently labeled SynB1-ELP-p50i (10 mg/kg). Then, CKD was induced in 6 pigs (bilateral renovascular disease and dyslipidemia), treated with a single IC SynB1-ELP-p50i or placebo at 6 weeks and observed for 8 more weeks. Cardiac morphology and function were quantified before/after treatment by echocardiography. After completion of the studies, pigs were euthanized and hearts excised for ex vivo studies. Results: Using IVIS technology, the BDS showed significant retention of SynB1-ELP-p50i in the heart. In the efficacy study, a single IC administration of IC SynB1-ELP-p50i reduced cardiac NF-kB expression, improved cardiac relaxation, and remodeling compared to untreated CKD (Figure). Conclusions: We show for the first time the feasibility of a targeted inhibition of cardiac NF-kB using ELP technology in a translational model of CKD and diastolic dysfunction. Our study may set the stage for a new therapeutic strategy using unique drug-delivery technology never tested before to protect the heart.