Induction Of Gene Transcription Research Articles

Aβ-targeting synNotch Receptor for Alzheimer's Disease: Expanding Applications to Extracellular Protein Aggregates.

The synthetic Notch receptor (synNotch) system is a versatile platform that induces gene transcription in response to extracellular signals. However, its application has been largely confined to membrane-bound targets due to specific activation requirements. Whether synNotch can also target extracellular protein aggregates, such as amyloid beta (Aβ) in Alzheimer's disease (AD), is unclear. To address this, we engineered an Aβ-targeting synNotch receptor controlling the production of chimeric human-mouse versions of Lecanemab (Leqembi®) or Aducanumab (Aduhelm®), both FDA-approved antibodies for AD. We demonstrate that NIH 3T3 cells expressing this synNotch system detect and respond to extracellular Aβ aggregates by synthesizing and secreting Aducanumab or Lecanemab. These findings broaden the potential applications of synNotch, extending its targets beyond membrane-bound proteins to extracellular protein aggregates, providing obvious benefits to research in this scientific arena.

HEXIM1 is correlated with Alzheimer's disease pathology and regulates immediate early gene dynamics in neurons.

ABSTRACTImpaired memory formation and recall is a distinguishing feature of Alzheimer’s disease, and memory requires de novo gene transcription in neurons. Rapid and robust transcription of many genes is facilitated by the formation of a poised basal state, in which RNA polymerase II (RNAP2) has initiated transcription, but is paused just downstream of the gene promoter. Neuronal depolarization releases the paused RNAP2 to complete the synthesis of messenger RNA (mRNA) transcripts. Paused RNAP2 release is controlled by positive transcription elongation factor b (P-TEFb), which is sequestered into a larger inactive complex containing Hexamethylene bisacetamide inducible protein 1 (HEXIM1) under basal conditions. In this work, we find that neuronal expression ofHEXIM1mRNA is highly correlated with human Alzheimer’s disease pathologies. Furthermore, P-TEFb regulation by HEXIM1 has a significant impact on the rapid induction of neuronal gene transcription, particularly in response to repeated depolarization. These data indicate that HEXIM1/P-TEFb has an important role in inducible gene transcription in neurons, and for setting and resetting the poised state that allows for the robust activation of genes necessary for synaptic plasticity.GRAPHICAL ABSTRACT

Identification of immunity-related genes distinctly regulated by Manduca sexta Spӓtzle-1/2 and Escherichia coli peptidoglycan

The immune system of Manduca sexta has been well studied to understand molecular mechanisms of insect antimicrobial responses. While evidence supports the existence of major immune signaling pathways in this species, it is unclear how induced production of defense proteins is specifically regulated by the Toll and Imd pathways. Our previous studies suggested that diaminopimelic acid-type peptidoglycans (DAP-PG) from Gram-negative and some Gram-positive bacteria, more than Lys-type peptidoglycans (Lys-PG) from other Gram-positive bacteria, triggers both pathways through membrane-bound receptors orthologous to Drosophila Toll and PGRP-LC. In this study, we produced M. sexta proSpätzle-1 and proSpätzle-2 in Sf9 cells, identified their processing enzymes, and used prophenoloxidase activating protease-3 to activate the cytokine precursors. After Spätzle-1 and -2 were isolated from the reaction mixtures, we separately injected the purified cytokines into larval hemocoel to induce gene transcription in fat body through the Toll pathway solely. On the other hand, we treated a M. sexta cell line with E. coli DAP-PG to only induce the Imd pathway and target gene expression. RNA-Seq analysis of the fat body and cultured cells collected at 0, 6, and 24 h after treatment indicated that expression of diapausin-4, -10, -12, -13, cecropin-2, -4, -5, attacin-5, -11, and lebocin D is up-regulated predominantly via Toll signaling, whereas transcription of cecropin-6, gloverin, lysozyme-1, and gallerimycin-2 is mostly induced by DAP-PG via Imd signaling. Other antimicrobial peptides are expressed in response to both pathways. Transcripts of most Toll-specific genes (e.g., lebocin D) peaked at 6 h, contrasting the gradual increase and plateauing of drosomycin mRNA level at 24−48 h in Drosophila. We also used T (oll)-I (md) ratios to estimate relative contributions of the two pathways to transcriptional regulation of other components of the immune system. The differences in pathway specificity and time course of transcriptional regulation call for further investigations in M. sexta and other insects.

Gene Transcriptional Profiles of Individuals with HbS Trait Are Different from Individuals without HbS Trait

Background: Although hemoglobin S (HbS) trait has historically been considered a relatively benign condition, emerging data support it is a risk factor for several clinical manifestations such as thrombosis, exertional-related complications, renal medullary cell carcinoma, chronic kidney disease, and osteonecrosis. Data also support HbS trait mice have an intermediary behavioral pain phenotype between control and sickle cell disease (SCD) mice. Despite these emerging data regarding the clinical phenotype associated with HbS trait, little is known about the biology of this condition. To investigate the biology of HbS trait, we analyzed gene expression profiles in healthy individuals with HbS trait and compared these to individuals without HbS trait and individuals with SCD. We hypothesized that those with HbS trait will have gene expression patterns that are different than individuals without HbS trait and those with SCD. Methods: Plasma samples were obtained from individuals with SCD and healthy Black individuals during baseline health. Systematic HbS trait testing was completed for all healthy controls. Plasma-based transcription analyses were conducted. Briefly, gene transcription was assessed in healthy blood donor peripheral blood mononuclear cells (PMBCs) after being co-cultured with participant plasma. PMBCs served as sensors to plasma born factors that induced gene transcription which was then measured with an Affymetrix array. Gene transcription profiles were analyzed to identify differentially expressed transcripts between individuals with SCD at baseline health and healthy controls with and without HbS trait that exhibited a fold change of |Log2R|>0.263, ANOVA p<0.05. Further, differentially expressed transcripts were also assessed between those with and without HbS trait using the same fold change cutoff. Mean gene expression heat maps of these differentially expressed transcripts were created using Genesis for three groups: individuals with SCD, individuals with HbS trait, and individuals without HbS trait. An annotated heat map of differentially expressed genes between those with and without HbS trait was also created. Results: Plasma-induced mean gene expression signatures were obtained from 87 individuals with SCD at baseline health and 52 healthy Black controls; 30 (58%) had documented HbS trait and 22 (42%) did not. Mean age of SCD patients during baseline health was 14.5 (SD 14.2) years, 51.7% were female, and 70.1% had HbSS/HbSβ 0thal. Mean age of Black controls with HbS trait was 28.4 (SD 12.8) years, and 80.6% were female and mean age of those without HbS trait was 20.5 (SD 18.2) years and 70.8% were female. Figure 1 shows the mean plasma-induced gene expression profiles for individuals with SCD at baseline health and healthy Black controls with and without HbS trait. Data show controls with HbS trait have intermediary gene expression patterns between those without HbS trait and those with SCD. We found 43 transcripts were significantly differentially expressed between those with and without HbS trait. Figure 2 displays the annotated heat map of these 43 differentially expressed transcripts. Conclusions: Healthy individuals with HbS trait have transcriptional signatures that are intermediary between those without HbS trait and those with SCD. Further, individuals with and without HbS trait have differentially expressed transcripts suggesting different biological processes in HbS trait may play a role in the pathophysiology of clinical complications in individuals with HbS trait. These findings also underscore the need to account for HbS trait status of healthy controls when including them as a comparison group for individuals with SCD in biological investigations. Further work is needed to understand the biological meaning behind these findings.

Role and molecular regulatory mechanisms of Hippo signaling pathway in Caenorhabditis elegans and mammalian cell models of Alzheimer’s disease

Although there is increasing evidence for the involvement of Hippo signaling in Alzheimer’s disease (AD), the detailed functions and regulatory mechanisms are not fully understood, given the diverse biological effects of this pathway. In the present work, we used Caenorhabditis elegans and mammalian cell models to investigate changes in the Hippo signaling pathway in response to Aβ and the downstream effects on AD development. Aβ1–42 production in the AD models decreased phosphorylation of the upstream CST-1/WTS-1 kinase cascade and promoted an interaction between LIN-10 and YAP-1, leading to the nuclear translocation of YAP-1 and inducing gene transcription in conjunction with the transcription factor EGL-44. The YAP-1/EGL-44 complex suppressed the autophagy-lysosome pathway by modulating mTOR signaling, which enhanced Aβ1–42 accumulation and promoted AD progression. These results demonstrate for the first time that crosstalk between Hippo and mTOR signaling contributes to AD development by enhancing Aβ production, resulting in inhibition of Hippo signaling and autophagy-lysosome pathway and Aβ accumulation, suggesting potential therapeutic targets for the treatment or prevention of AD.

GATA zinc finger protein p66β promotes breast cancer cell migration by acting as a co-activator of Snail

The transcriptional repressor Snail induces EMT during embryonic development and tumor metastasis. Growing evidence indicates that Snail functions as a trans-activator to induce gene expression; however, the underlying mechanism remains elusive. Here, we report that Snail cooperates with GATA zinc finger protein p66β to transactivate genes in breast cancer cells. Biologically, depletion of p66β reduces cell migration and lung metastasis in BALB/c mice. Mechanistically, Snail interacts with p66β and cooperatively induces gene transcription. Notably, a group of genes induced by Snail harbor conserved G-rich cis-elements (5′-GGGAGG-3′, designated as G-box) in their proximal promoter regions. Snail directly binds to G-box via its zinc fingers and transactivates the G-box-containing promoters. p66β enhances Snail binding affinity to G-box, whereas depletion of p66β results in a decreased binding affinity of Snail to the endogenous promoters and concomitantly reduces the transcription of Snail-induced genes. Taken together, these data demonstrated that p66β is critical for Snail-mediated cell migration by acting as a co-activator of Snail to induce genes containing G-box elements in the promoters.

Preclinical development of RAG1-40-31L: A novel small activating RNA-lipid conjugate targeting tumor suppressor gene p21 for treatment of non-muscle invasive bladder cancer.

e16620 Background: Loss-of-function of tumor suppressor genes are the dominant driving force in tumorigenesis. Restoration in function and/or expression of these genes holds tremendous therapeutic potential in cancer treatment. Bladder cancer is the 9th leading cause of cancer-related deaths in the United States in which ~75% of all newly diagnosed cases are non-muscle invasive bladder cancer (NMIBC). Standard-of-care includes intravesical installation of Bacillus Calmette-Guerin following tumor resection with an expected failure rate of ~50% within 6 months for patients with high-grade tumors. RNA activation (RNAa) is a mechanism in which short RNA duplexes termed small activating RNA (saRNA) induce gene transcription by targeting non-coding regulatory sequence ( i.e., promoters) embedded in chromatin. Thus, saRNA-induced overexpression of p21WAF1/CIP1 (p21) – a tumor suppressor of several anti-growth pathways – may hold therapeutic potential in bladder cancer. Through conjugation of a novel lipid ( i.e., C5X5), we have created a p21-targeting saRNA drug candidate referred to as RAG01-40-31L for delivery to bladder cancer cells in vivo. The aim of this work was to characterize the intravesical administration of RAG01-40-31L on p21 induction and its anti-tumor potential in NMBIC models. Methods: In vitro expression analyses were performed to investigate p21 induction via saRNA in KU7 and T24 bladder cancer cell lines by RT-qPCR and immunoblot analysis. Cell cycle analysis and tumorigenicity assays examined impact of RAG1-40-31L on cancer cell growth. Orthotopic human bladder tumors were established in female BALB/c nude mice using the recombinant cell line KU7 engineered to overexpress luciferase reporter. Tumor growth was monitored in live animals via luciferase bioluminescence using an in vivo imaging system. Plasma and tissue were harvested for drug exposure and expression analytics. Results: In vitro analyses indicated RAG1-40-31L activated p21 expression by approximately 6-fold and inhibited bladder cancer cell growth ( i.e., cell cycle arrest, apoptosis, and senescence) in a dose-dependent manner with low nanomolar potency. Intravesical administration of RAG1-40-31L (3 times per week for 2 weeks) inhibited growth of orthotopic tumors by 65% in mouse bladder models. Single-dose pharmacokinetics demonstrated a half-life (t1/2) of ~114 h with minimal systemic exposure (~0.05% of that of bladder tissue). Conclusions: These data constitute a preclinical proof-of-concept for saRNA as a novel p21-targeting modality in the treatment of cancer. The C5X5 lipid conjugate appears to be a viable approach to enhance oligonucleotide delivery and activity in bladder cancer. Preclinical safety assessment studies to enable clinical trials of RAG01-40-31L are currently underway.

JAK/STAT Signaling and Cervical Cancer: From the Cell Surface to the Nucleus.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway constitutes a rapid signaling module from the cell surface to the nucleus, and activates different cellular responses, such as proliferation, survival, migration, invasion, and inflammation. When the JAK/STAT pathway is altered, it contributes to cancer progression and metastasis. STAT proteins play a central role in developing cervical cancer, and inhibiting the JAK/STAT signaling may be necessary to induce tumor cell death. Several cancers show continuous activation of different STATs, including cervical cancer. The constitutive activation of STAT proteins is associated with a poor prognosis and overall survival. The human papillomavirus (HPV) oncoproteins E6 and E7 play an essential role in cervical cancer progression, and they activate the JAK/STAT pathway and other signals that induce proliferation, survival, and migration of cancer cells. Moreover, there is a crosstalk between the JAK/STAT signaling cascade with other signaling pathways, where a plethora of different proteins activate to induce gene transcription and cell responses that contribute to tumor growth. Therefore, inhibition of the JAK/STAT pathway shows promise as a new target in cancer treatment. In this review, we discuss the role of the JAK/STAT pathway components and the role of the HPV oncoproteins associated with cellular malignancy through the JAK/STAT proteins and other signaling pathways to induce tumor growth.

Developmental expression of CREB1 and NFATC2 in pig embryos.

The CREB1 gene encodes the cAMP response element binding protein 1 (CREB1), a leucine zipper transcription factor that regulates cellular gene expression in response to elevated levels of intracellular cAMP. When activated by phosphorylation, CREB1 binds to the cAMP response element (CRE) of the promoters of its target genes. CREB1 is an essential component in many physiological processes, and its function is correlated to neurodevelopment, plasticity and cell survival, and learning and memory. The NFATC2 gene codes for the nuclear factor of activated T-cells 2 protein. The NFATC2 protein is a DNA-binding protein that functions as an inducer of gene transcription during immune response. The aim of the present study was to examine the developmental expression of porcine CREB1 and NFACT2 transcripts. The expression of CREB1 and NFACT2 mRNA was examined by quantitative real-time RT-PCR. For the CREB1 transcript, we found significant reduction in transcript levels in the brain stem and basal ganglia during porcine embryo development, determined from day 60 to day 115 of gestation. In contrast, a significant increase in CREB1 mRNA was detected in the lungs during embryo development. No significant changes in the NFATC2 transcript were detected in porcine brain tissue during embryo development. Differential CREB1 mRNA expression was found in pig brain tissues during embryo development.

An observational human study investigating the effect of anabolic androgenic steroid use on the transcriptome of skeletal muscle and whole blood using RNA-Seq

BackgroundThe effects of Anabolic Androgenic Steroids (AAS) are largely illustrated through Androgen Receptor induced gene transcription, yet RNA-Seq has yet to be conducted on human whole blood and skeletal muscle. Investigating the transcriptional signature of AAS in blood may aid AAS detection and in muscle further understanding of AAS induced hypertrophy.MethodsMales aged 20–42 were recruited and sampled once: sedentary controls (C), resistance trained lifters (RT) and resistance trained current AAS users (RT-AS) who ceased exposure ≤ 2 or ≥ 10 weeks prior to sampling. RT-AS were sampled twice as Returning Participants (RP) if AAS usage ceased for ≥ 18 weeks. RNA was extracted from whole blood and trapezius muscle samples. RNA libraries were sequenced twice, for validation purposes, on the DNBSEQ-G400RS with either standard or CoolMPS PE100 reagents following MGI protocols. Genes were considered differentially expressed with FDR < 0.05 and a 1.2- fold change.ResultsCross-comparison of both standard reagent whole blood (N = 55: C = 7, RT = 20, RT-AS ≤ 2 = 14, RT-AS ≥ 10 = 10, RP = 4; N = 46: C = 6, RT = 17, RT-AS ≤ 2 = 12, RT-AS ≥ 10 = 8, RP = 3) sequencing datasets, showed that no genes or gene sets/pathways were differentially expressed between time points for RP or between group comparisons of RT-AS ≤ 2 vs. C, RT, or RT-AS ≥ 10. Cross-comparison of both muscle (N = 51, C = 5, RT = 17, RT-AS ≤ 2 = 15, RT-AS ≥ 10 = 11, RP = 3) sequencing (one standard & one CoolMPS reagent) datasets, showed one gene, CHRDL1, which has atrophying potential, was upregulated in RP visit two. In both muscle sequencing datasets, nine differentially expressed genes, overlapped with RT-AS ≤ 2 vs. RT and RT-AS ≤ 2 vs. C, but were not differentially expressed with RT vs. C, possibly suggesting they are from acute doping alone. No genes seemed to be differentially expressed in muscle after the long-term cessation of AAS, whereas a previous study found long term proteomic changes.ConclusionA whole blood transcriptional signature of AAS doping was not identified. However, RNA-Seq of muscle has identified numerous differentially expressed genes with known impacts on hypertrophic processes that may further our understanding on AAS induced hypertrophy. Differences in training regimens in participant groupings may have influenced results. Future studies should focus on longitudinal sampling pre, during and post-AAS exposure to better control for confounding variables.

The RNA-binding protein Mex3B regulates airway remodeling through modulation of TGFBR3 mRNA stability in chronic rhinosinusitis.

Abstract Objective: To explore the pathogenic function of Mex3 RNA binding family member B (Mex3b) in chronic rhinosinusitis(CRS). Methods: The expression of Mex3b, TGF-β receptor III (TGFBR3), TGF-β2, p-Smad2 were studied by quantitative RT-PCR, immunohistochemistry, western blotting, and ELISA. The binding of MEX3b with TGFBR3was test by improved RNA immunoprecipitation–coupled high-throughput sequencing (iRIP-Seq). Results: Compared to control subjects, mNRA and protein expression of Mex3b in nasal epithelial cells from CRSwNP patients are upregulated. And Mex3b mRNA and protein levels in cultured human nasal epithelial cells(HNECs) obtained from control subjects were upregulated by LPS, CpG and IL-1β. Incresed Mex3b downregulates TGFBR3expression in airway epithelial cells by binding to TGFBR3mRNA, which results in downregulated level of TGFBR3in CRS compared to control subjects. Loss of TGFBR3 inhibits TGF-β2 induced gene transcription in nasal epithelial cells. And Mex3b suppresses TGF-β2 signaling activation by downregulating TGFBR3 expression in nasal epithelial cells. Mex3b and TGFBR3 expression levels are correlated with tissue edema in CRSwNP. Conclusions: The upregulation of Mex3b in CRSwNP suppresses TGF-β2 signaling activation by downregulating TGFBR3 expression in nasal epithelial cells, and thefore regulates tissue remodeling of CRS.

Abstract LB134: Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth

Abstract Background: In-frame chimeric proteins that are encoded by ESR1 exons 1 to 6 (ESR1-e6) fused to C-terminal gene partners can induce hormone-independent growth and endocrine therapy (ET) resistance of estrogen receptor-α (ERα) positive breast cancer by driving a unique transcriptional signature (PMID: 34711608). However, the mechanism how active ESR1 fusions reprogram transcription is not understood. Moreover, the lack of targeted treatment due to the loss of ligand binding domain (LBD) in ESR1 gene fusions necessitates the need to develop new therapies targeting active ESR1 fusions that promote tumorigenesis. Methods: An estrogen response element (ERE) DNA pull down assay was performed to identify coactivators recruited by active ESR1 fusions. An alamar blue assay was utilized to assess cell growth after treatment with pharmacological inhibitors to the coactivators. Single cells isolated from ERα+ patient-derived xenografts (PDX) were used to form organoids (PDxOs). A CellTiter-Glo 3D assay was conducted to measure the growth of PDxOs after treatment with coactivator inhibitors. Results: Using ERE DNA pull down assays, we identified bromodomain-containing proteins (BRDs) and steroid receptor coactivators (SRCs/p160 family members) as key regulators recruited by active ESR1 fusions from nuclear extracts of breast cancer cells. A pan-BET family inhibitor (BETi), birabresib, and pan-SRC inhibitors (SRCi), SI-2 and SI-12, inhibited the growth of ERα positive breast cancer cells and PDxOs expressing an active ESR1 fusion or LBD point mutation. Importantly, the combination of birabresib and a CDK4/6 kinase inhibitor (palbociclib) conferred the largest growth reduction of PDxOs expressing an ESR1-YAP1 fusion. RNA sequencing showed BETi and SRCi led to downregulation of ERα target gene expression and cell cycle pathways, with upregulation of apoptosis pathways. Here, we propose a model whereby BRD and SRC protein families are major regulators of active ESR1 fusion-driven gene transcription and growth and positioned the potential therapeutic significance of BETi and SRCi to be tailored to treat active ESR1-translocated breast tumors. Conclusions: Active ESR1 fusions cannot be treated with standard-of-care ET due to the lack of the LBD of ERα. Therefore, it is critical to investigate how active, in-frame ESR1 fusion proteins induce gene transcription and to develop molecular mechanism-based targeted therapies. Here, we revealed two groups of coactivators (BRDs and SRCs) as critical transcriptional regulators of active ESR1 fusion-driven tumor biology. Our study demonstrated the efficacy of a BETi (birabresib) and SRCi (SI-2/SI-12) in inhibiting the viability of active ESR1 fusion/LBD mutation expressing breast cancer cells and 3D grown PDxOs. Importantly, birabresib combined with a standard-of-care CDK4/6 inhibitor conferred the best PDxO growth reduction. Citation Format: Xuxu Gou, Craig T. Vollert, Yue Chen, Diana Fandino, Matthew V. Holt, Beom-Jun Kim, Qin Feng, Jin Wang, David M. Lonard, Bert W. O'Malley, Matthew J. Ellis, Charles E. Foulds. Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB134.

Retinoic Acid Signalling in the Pineal Gland Is Conserved across Mammalian Species and Its Transcriptional Activity Is Inhibited by Melatonin.

The pineal gland is integral to the circadian timing system due to its role in nightly melatonin production. Retinoic acid (RA) is a potent regulator of gene transcription and has previously been found to exhibit diurnal changes in synthesis and signalling in the rat pineal gland. This study investigated the potential for the interaction of these two systems. PCR was used to study gene expression in mouse and human pineal glands, ex-vivo organotypic cultured rat pineal gland and cell lines. The mouse and human pineal glands were both found to express the necessary components required for RA signalling. RA influences the circadian clock in the brain, therefore the short-term effect of RA on clock gene expression was determined in ex vivo rat pineal glands but was not found to rapidly regulate Per1, Per2, Bmal1, or Cry1. The interaction between RA and melatonin was also investigated and, unexpectedly, melatonin was found to suppress the induction of gene transcription by RA. This study demonstrates that pineal expression of the RA signalling system is conserved across mammalian species. There is no short-term regulation of the circadian clock but an inhibitory effect of melatonin on RA transcriptional activity was demonstrated, suggesting that there may be functional cross-talk between these systems.

PSUN375 What Blindness and Paraganglioma Have in Common? A Rare Case of Paraganglioma Syndrome

Abstract Background Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors with an annual incidence in the U.S. of 500-1600 cases per year. Approximately 30-35% of PPGLs harbor germline mutations, while 35-40% are due to somatic mutations. Recent studies have identified the association between somatic mutations of the hypoxia-inducible factor 2 alpha (HIF2A) gene and a new syndrome of PPGLs associated with congenital polycythemia. Patients present with recurrent PPGLs, congenital malformation of intracranial veins, malformation of macula and retina, and type I Chiari malformations. Case A 44-year-old man with history of in-utero stroke, congenital blindness, polycythemia and spinal stenosis had an incidental para-aortic lesion found during workup of back pain. He experienced episodic palpitations associated with diaphoresis, headache and uncontrolled hypertension for the past eight year. There was no family history of neuroendocrine tumors. Dedicated abdominal computer tomography with contrast showed a right adrenal enhancing lesion measuring 1.4×1.2 cm and a conglomerate of heterogenous enhancing periaortic lesions measuring up to 5 cm in the mid-abdomen. Biochemical work up revealed plasma free normetanephrine of 27.5 nmol/L (0.00-0.89) and plasma free metanephrines of 0.49 nmol/L (0.00-0.49). Gallium 68 DOTATATE scan showed multiple avid retroperitoneal lesions measuring up to 4.4 cm and an avid right adrenal nodule. He underwent right adrenalectomy and para-aortic lesion resection with perio-operative alpha blockade. Pathology confirmed synchronous pheochromocytoma (1.5 cm, Ki-67&amp;lt; 1%, with capsular and periadrenal fat invasion) and paraganglioma (9.3 cm, Ki-67 &amp;lt; 1%, with lymphovascular invasion and one out of nine lymph nodes positive for metastatic disease). Post-operatively, his metanephrines normalized and a six-months positron emission tomography scan showed no evidence of disease. Genetic testing has been delayed. Discussion This case illustrates a rare syndrome of PPGLs, congenital blindness, and polycythemia, likely due to somatic mutations of HIF2a. HIF is an alpha/beta heterodimer. HIF2a becomes a transcription factor after dimerization with HIF2b. Any mutations that stabilize HIF protein leads to transcription of hypoxia inducible genes resulting in angiogenesis, polycythemia, and tumorigenesis. In our patient, we suspect the stroke in-utero may have been related to abnormal intracranial angiogenesis and/or polycythemia. Recognition of this syndrome and lifelong surveillance is imperative due to risk of recurrence of PPGL. Targeted therapies such as a HIF2a inhibitor have been proposed and trials are ongoing. Our patient remains disease free on radiographic and biochemical surveillance. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

The chromatin factor ROW cooperates with BEAF‐32 in regulating long‐range inducible genes

Insulator proteins located at the boundaries of topological associated domains (TAD) are involved in higher‐order chromatin organization and transcription regulation. However, it is still not clear how long‐range contacts contribute to transcriptional regulation. Here, we show that relative‐of‐WOC (ROW) is essential for the long‐range transcription regulation mediated by the boundary element‐associated factor of 32kD (BEAF‐32). We find that ROW physically interacts with heterochromatin proteins (HP1b and HP1c) and the insulator protein (BEAF‐32). These proteins interact at TAD boundaries where ROW, through its AT‐hook motifs, binds AT‐rich sequences flanked by BEAF‐32‐binding sites and motifs. Knockdown of row downregulates genes that are long‐range targets of BEAF‐32 and bound indirectly by ROW (without binding motif). Analyses of high‐throughput chromosome conformation capture (Hi‐C) data reveal long‐range interactions between promoters of housekeeping genes bound directly by ROW and promoters of developmental genes bound indirectly by ROW. Thus, our results show cooperation between BEAF‐32 and the ROW complex, including HP1 proteins, to regulate the transcription of developmental and inducible genes through long‐range interactions.

Epigenetics: An opportunity to shape innate and adaptive immune responses.

Epigenetics connects genetic and environmental factors: it includes DNA methylation, histone post-translational modifications and the regulation of chromatin accessibility by non-coding RNAs, all of which control constitutive or inducible gene transcription. This plays a key role in harnessing the transcriptional programs of both innate and adaptive immune cells due to its plasticity and environmental-driven nature, piloting myeloid and lymphoid cell fate decisions with no change in their genomic sequence. In particular, epigenetic marks at the site of lineage-specific transcription factors and maintenance of cell type-specific epigenetic modifications, referred to as 'epigenetic memory', dictate cell differentiation, cytokine production and functional capacity following repeated antigenic exposure in memory T cells. Moreover, metabolic and epigenetic reprogramming occurring during a primary innate immune response leads to enhanced responses to secondary challenges, a phenomenon known as 'trained immunity'. Here, we discuss how stable and dynamic epigenetic states control immune cell identity and plasticity in physiological and pathological conditions. Dissecting the regulatory circuits of cell fate determination and maintenance is of paramount importance for understanding the delicate balance between immune cell activation and tolerance, in healthy conditions and in autoimmune diseases.

Super-enhancer-controlled positive feedback loop BRD4/ERα-RET-ERα promotes ERα-positive breast cancer.

Estrogen and estrogen receptor alpha (ERα)-induced gene transcription is tightly associated with ERα-positive breast carcinogenesis. ERα-occupied enhancers, particularly super-enhancers, have been suggested to play a vital role in regulating such transcriptional events. However, the landscape of ERα-occupied super-enhancers (ERSEs) as well as key ERα-induced target genes associated with ERSEs remain to be fully characterized. Here, we defined the landscape of ERSEs in ERα-positive breast cancer cell lines, and demonstrated that bromodomain protein BRD4 is a master regulator of the transcriptional activation of ERSEs and cognate ERα target genes. RET, a member of the tyrosine protein kinase family of proteins, was identified to be a key ERα target gene of BRD4-regulated ERSEs, which, in turn, is vital for ERα-induced gene transcriptional activation and malignant phenotypes through activating the RAS/RAF/MEK2/ERK/p90RSK/ERα phosphorylation cascade. Combination therapy with BRD4 and RET inhibitors exhibited additive effects on suppressing ERα-positive breast cancer both in vitro and in vivo, comparable with that of standard endocrine therapy tamoxifen. Furthermore, combination therapy re-sensitized a tamoxifen-resistant ERα-positive breast cancer cell line to tamoxifen treatment. Taken together, our data uncovered the critical role of a super-enhancer-associated positive feedback loop constituting BRD4/ERα–RET–ERα in ERα-positive breast cancer, and suggested that targeting components in this loop would provide a new therapeutic avenue for treating ERα-positive breast cancer in the clinic.

Local Ca2+ Signals within Caveolae Cause Nuclear Translocation of CaMK1α in Mouse Vascular Smooth Muscle Cells.

An increase in intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+-sensitive enzymes such as Ca2+/calmodulin-dependent kinases (CaMK) and induces gene transcription in various types of cells. This signaling pathway is called excitation-transcription (E-T) coupling. Recently, we have revealed that a L-type Ca2+ channel/CaMK kinase (CaMKK) 2/CaMK1α complex located within caveolae in vascular smooth muscle cells (SMCs) can convert [Ca2+]i changes to gene transcription profiles that are related to chemotaxis. Although CaMK1α is expected to be the key molecular identity that can transport Ca2+ signals originated within caveolae to the nucleus, data sets directly proving this scheme are lacking. In this study, multicolor fluorescence imaging methods were utilized to address this question. Live cell imaging using mouse primary aortic SMCs revealed that CaMK1α can translocate from the cytosol to the nucleus; and that this movement was blocked by nifedipine or a CaMKK inhibitor, STO609. Experiments using two types of Ca2+ chelators, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), combined with caveolin-1 knockout (cav1-KO) mice showed that local Ca2+ events within caveolae are required to trigger this CaMK1α nuclear translocation. Importantly, overexpression of cav1 in isolated cav1-KO myocytes recovered the CaMK1α translocation. In SMCs freshly isolated from mesenteric arteries, CaMK1α was localized mainly within caveolae in the resting state. Membrane depolarization induced both nuclear translocation and phosphorylation of CaMK1α. These responses were inhibited by nifedipine, STO609, cav1-KO, or BAPTA. These new findings strongly suggest that CaMK1α can transduce Ca2+ signaling generated within or very near caveolae to the nucleus and thus, promote E-T coupling.

Insulin prevents fatty acid induced increase of adipocyte size

ABSTRACT Metabolic disorders related to obesity are largely dependent on adipose tissue hypertrophy, which involves adipocyte hypertrophy and increased adipogenesis. Adiposize is regulated by lipid accumulation as a result of increased lipogenesis (mainly lipid uptake in mature adipocytes) and reduced lipolysis. Using realtime 2D cell culture analyses of lipid uptake, we show 1) that high glucose concentration (4.5 g/L) was required to accumulate oleic acid increasing lipid droplet size until unilocularisation similar to mature adipocytes in few days, 2) oleic acid reduced Peroxisome-Proliferator Activated Receptor Gamma (PPARG) gene transcription and 3) insulin counteracted oleic acid-induced increase of lipid droplet size. Although the lipolytic activity observed in high versus low glucose (1g/L) conditions was not altered, insulin was found to inhibit oleic acid induced gene transcription required for lipid storage such as CIDEC (Cell Death Inducing DFFA Like Effectors) and G0S2 (G0 switch gene S2), possibly through PPARA activity. Although this signaling pathway requires more detailed investigation, the results point out the differential mechanisms involved in the pro-adipogenic effect of insulin in absence versus its protective effect on adiposity in presence of oleic acid uptake.

Keap1 moderates the transcription of virus induced genes through G9a-GLP and NFκB p50 recruitment.

Cells must control genes that are induced by virus infection to mitigate deleterious consequences of inflammation. We investigated the mechanisms whereby Keap1 moderates the transcription of genes that are induced by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1-/- deletions increased the transcription of virus induced genes independently of Nrf2. Keap1 moderated early virus induced gene transcription. Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6, and reduced Keap1 binding at Cdkn1a and Ccng1. Virus infection induced G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition. Keap1-/- deletions eliminated G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition, but they did not affect NFκB p65, IRF3 or cJun recruitment. G9a-GLP inhibitors (BIX01294, MS012, BRD4770) enhanced virus induced gene transcription in MEFs with intact Keap1, but not in MEFs with Keap1-/- deletions. G9a-GLP inhibitors augmented Keap1 binding to virus induced genes in infected MEFs, and to cell cycle genes in uninfected MEFs. G9a-GLP inhibitors augmented NFκB subunit recruitment in MEFs with intact Keap1. G9a-GLP inhibitors stabilized Keap1 retention in permeabilized MEFs. G9a-GLP lysine methyltransferase activity was required for Keap1 to moderate transcription, and it moderated Keap1 binding to chromatin. The interdependent effects of Keap1 and G9a-GLP on the recruitment of each other and on the moderation of virus induced gene transcription constitute a feedback circuit. Keap1 and the electrophile tBHQ reduced virus induced gene transcription through different mechanisms, and they regulated the recruitment of different NFκB subunits. Characterization of the mechanisms whereby Keap1, G9a-GLP and NFκB p50 moderate virus induced gene transcription can facilitate the development of immunomodulatory agents.