Transcriptional Transactivation Research Articles

Protein Kinase C Beta II Inhibitor Conjugated with Myristic Acid and Trans-Activator of Transcription improves intracellular delivery and cardiovascular protection in porcine myocardial ischemia/reperfusion injury

Introduction: While timely reperfusion of oxygenated blood is critical during resuscitation of ischemic myocardium, oxidative stress during reperfusion still leads to ischemia/reperfusion (I/R) injury and ultimately, cardiomyocyte death. The major sources of oxidative stress targeted in this study are NADPH oxidase (NOX-2) and mitochondria, which are both activated by protein kinase C beta II (PKCβII). In previous studies with an ex-vivo rat heart I/R model, Myristic Acid (Myr) and Trans-Activator Transcription (Tat; YGRKKRRQRRR) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-; N-Myr-Tat-CC-SLNPEWNET) showed cardioprotective effects and decreased infarct size. In this study, we hypothesized that Myr-Tat-PKCβII- will offer cardioprotective effects in comparison with a scrambled peptide control (Myr-Tat-PKCβII-scram; N-Myr-Tat-CC-WNPESLNTE) in an in-vivo porcine myocardial I/R model. Methods: Regional I(1 hour)/R(3 hours) was induced in Male Yorkshire pigs (38-50kg) by inflating a balloon to occlude the Left Anterior Descending Artery (LAD) at the level of the second diagonal branch (supplies 40% of anterior left ventricle) and then deflating it to allow for reperfusion. Then, a bolus of either Myr-Tat-PKCβII- or Myr-Tat-PKCβII-scram (20ng/kg; ~100pmol in blood) was immediately administered into the LAD. Cardiac function and injury were measured by monitoring changes in ejection fraction (EF) and through routine measurements of serum creatine phosphokinase (CPK), troponin I, and myoglobin, respectively. After I/R, the hearts were stained with 1% Evans Blue dye to identify the area at risk (AR) and 1% triphenyltetrazolium chloride to determine the area of necrosis (AN). Infarct size was then quantified (AN/AR) and all data was analyzed via Student’s t-test. Results: Analysis showed that Myr-Tat-PKCβII- significantly restored EF to within 1.40.7% of baseline compared to controls which only restored EF to within 6.42.1% (p<0.05) of baseline EF. Myr-Tat-PKCβII- showed a significant increase in serum myoglobin levels at 1 hr of reperfusion (1362.1 83.4 μg/mL, n=4) compared to scrambled control (202.1197.3 μg/mL, n=3 p<0.05). Myr-Tat-PKCβII- reduced infarct size to 10.0±2.8%; n=4; compared to scrambled control hearts (28.5±8.3%; n=6; p<0.05). CPK and Troponin I levels were comparable in both groups. These results suggest that Myr-Tat-PKCβII- can help prevent cardiac injury when given immediately after an ischemic event. Conclusions: Data from ex-vivo rat heart I/R model, coupled with data from this in-vivo porcine myocardial I/R model indicate the effcacy of Myr-Tat-PKCβII- in preventing oxidative stress-induced myocardial I/R injury when given at the beginning of reperfusion. These findings suggest that Myr-Tat-PKCβII- could be useful in the clinical setting when administered immediately after cardiac resuscitation. Future studies include the treatment of rat cardiomyocyte cells with Myr-Tat-PKCβII- prior to 24 hr hypoxia/24 hr reoxygenation, followed by cell viability assays compared to untreated control. This additional data can help determine the optimal dose to use in a 12 week survival study using the same porcine myocardial I/R protocol. Funding: This study was funded by the Philadelphia College of Osteopathic Medicine, Department of Biomedical Sciences, Division of Research, and the Center for Chronic Diseases of Aging, the National Heart, Lung & Blood Institute at NIH (grant # 1R43HL160338-01) and Young Therapeutics, LLC. 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.

Co-regulatory Proteins Integrate Metabolic Signals to Regulate ESRRG Transcriptional Transactivation in Stomach Parietal Cell Differentiation

Acid-producing stomach parietal cells (PC) are metabolically active and energy demanding cells. Aberrant PC function and census leads to complications ranging from peptic ulcers to stomach adenocarcinoma. Effcient energy expenditure and catabolic metabolism are required for normal PC differentiation from progenitor cells. Here, our data show that the master metabolic transcriptional factor, estrogen-related receptor gamma ( ESRRG), governs murine and human PC differentiation. We are further investigating how known metabolically controlled co-regulatory proteins — Sirtuin 1 (SIRT1), Nuclear Receptor Subfamily 0 Group B Member 2 (NR0B2), and CREB/ATF BZIP Transcription Factor (CREBZF, or SMILE) — integrate stimuli to enable ESRRG transcriptional activation during PC emergence from precursors. Specifically, our lab has developed an acute and reversible PC ablation model to monitor PC recovery in which Simian diphtheria toxin receptor (DTR) expression under the Atp4b promoter targets PC-specific ablation by diphtheria toxin (DT). Additionally, we employ high-dose tamoxifen (HD TAM), which causes a more general gastric injury (independent of tamoxifen effects on estrogen) from PC death along with reactive spasmolytic polypeptide expressing metaplasia (SPEM) of remaining cells. Immunofluorescence, western blotting, and qRT-PCR revealed SIRT1 is upregulated before emergence of early PC clones post-DT and decreases later, suggesting positive regulation of early PC lineage-commitment from stem cells. During the same early time frame, factors NR0B2 and CREBZF decreased, suggesting contrasting roles. Administration of Resv to mice accelerated PC recovery after injury and expanded PCs even at homeostasis. ChIP and ongoing ChIP-seq and Cut&Run assays show ESRRG binds cis-regulatory elements of the metabolic genes and the co-regulatory proteins to promote PC lineage commitment in both mice and in a human gastric cell line in which stable ESRRG-GFP is expressed under a doxycycline-inducible promoter. In other ongoing work, we are generating mice with PC-specific Sirt1/ Nr0b2 ablation to further dissect PC regeneration dynamics and conducting gastric organoid experiments with NR0B2/SIRT1 pharmacological modulators to monitor PC differentiation effects. These organoid samples will be further subjected to epigenetic analysis (Cut&Run, ATACseq) to study chromatin landscape changes. We conclude that cellular metabolic integration by co-regulators enables ESRRG-mediated transactivation during PC differentiation, and SIRT1 promotes early PC lineage-commitment that might offer a promising pro-PC differentiation therapeutic strategy. This work is supported by funding from the NIH through the National Institute of Diabetes and Digestive and Kidney Diseases and the National Cancer Institute. 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.

TAT38 and TAT38 mimics potently inhibit adipogenesis by repressing C/EBPα, PPARγ, Pi-PPARγ, and SREBP1 expression

Antiretroviral therapy-naive people living with HIV possess less fat than people without HIV. Previously, we found that HIV-1 transactivator of transcription (TAT) decreases fat in ob/ob mice. The TAT38 (a.a. 20–57) is important in the inhibition of adipogenesis and contains three functional domains: Cys-ZF domain (a.a. 20–35 TACTNCYCAKCCFQVC), core-domain (a.a. 36–46, FITKALGISYG), and protein transduction domain (PTD)(a.a. 47–57, RAKRRQRRR). Interestingly, the TAT38 region interacts with the Cyclin T1 of the P-TEFb complex, of which expression increases during adipogenesis. The X-ray crystallographic structure of the complex showed that the Cys-ZF and the core domain bind to the Cyclin T1 via hydrophobic interactions. To prepare TAT38 mimics with structural and functional similarities to TAT38, we replaced the core domain with a hydrophobic aliphatic amino acid (from carbon numbers 5 to 8). The TAT38 mimics with 6-hexanoic amino acid (TAT38 Ahx (C6)) and 7-heptanoic amino acid (TAT38 Ahp (C7)) inhibited adipogenesis of 3T3-L1 potently, reduced cellular triglyceride content, and decreased body weight of diet-induced obese (DIO) mice by 10.4–11 % in two weeks. The TAT38 and the TAT38 mimics potently repressed the adipogenic transcription factors genes, C/EBPα, PPARγ, and SREBP1. Also, they inhibit the phosphorylation of PPARγ. The TAT peptides may be promising candidates for development into a drug against obesity or diabetes.

Transactivator of Transcription (Tat)-Induced Neuroinflammation as a Key Pathway in Neuronal Dysfunction: A Scoping Review.

The activity of HIV-1 and its viral proteins within the central nervous system (CNS) is responsible for a wide array of neuropathological effects, resulting in a spectrum of neurocognitive deficits defined as HIV-associated neurocognitive disorders (HAND). Amongst the various viral proteins, the transactivator of transcription (Tat) remains detectable even with effective antiretroviral therapy (ART) and suppressed viremia, highlighting the significance of this protein in the modern ART era. Tat has been extensively researched in both fundamental and clinical settings due to its role in neuroinflammation, neuronal damage, and neurocognitive impairment amongst people living with HIV (PLHIV). To date, numerous fundamental studies have explored Tat-induced neuroinflammation. However, there is no clear consensus on the most frequently studied inflammatory markers or the consistency in the levels of these Tat-induced inflammatory marker levels across different studies. Therefore, we conducted a scoping review of studies investigating Tat-induced neuroinflammation. We conducted searches in PubMed, Scopus, and Web of Science databases using a search protocol tailored specifically to adhere to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for scoping reviews (PRISMA-ScR) guidelines. From the 22 included studies, findings suggest that the HIV-1 Tat protein amplifies levels of neuroinflammatory markers. Amongst the vast array of inflammatory markers explored in the included studies, consistent results point to higher levels of CCL2, IL-6, IL-8, and TNF-α in primary cells and cell lines exposed to or transfected with HIV-1 Tat. These markers are regulated by key inflammatory pathways, such as the extracellularsignal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, the p38 MAPK pathway, and nuclear factor-kB (NF-kB). Furthermore, Tat has been shown to induce neuronal apoptosis, both directly and indirectly. With regards to study designs, utilizing full-length Tat101 at concentrations ranging from 100 to 1000 ng/ml and durations of 24 and 48 h appears optimal for investigating Tat-induced neuroinflammation. In this context, we highlight specific inflammatory markers and pathways that are potentially pivotal in Tat-induced neuroinflammation and subsequent neuronal damage. A deeper investigation into these markers and pathways is crucial to better understand their roles in the development of HAND.

Nanoscale detection and real-time monitoring of free radicals in a single living cell under the stimulation of targeting moieties using a nanodiamond quantum sensor

Intracellular radicals play important roles in cell signaling and regulation of growth factors, cytokines, transcription, apoptosis, and immunomodulation, among others. To gain a more comprehensive understanding of their biological functions from a spatio-temporal pers­pective, there is a great need for nanoscale sensitive tools that allow real-time detection of these reactive species. Currently, intracellular radical probes are based on chemical reactions that could significantly alter radical levels during detection. Due to the excellent bio­compatibility and favorable photophysical properties of nitrogen-vacancy (NV–) centers in fluorescent nanodiamonds (fNDs), the fNDs can serve as a powerful and chemically inert nanotool for intracellular radical detection. In this study, a positively charged nanogel (NG) coating was prepared to prevent the precipitation of fNDs and promote cellular internalization. After internalization of nanodiamond-nanogels (fND-NGs), different stimulators, namely somatostatin (SST), triphenylphosphonium (TPP), and trans-activator of transcription (TAT) peptide, which are widely used cell- or organelle-targeting ligands in medicine, drug delivery, and diagnostics, were applied to stimulate the cells. In parallel, the intracellular radical changes under stimulation of SST, TPP, and TAT ligands were monitored by fND-NGs in a home-built optically detected magnetic resonance (ODMR) microscope. Our method allows for detecting intracellular radicals in-situ and monitoring their real-time changes during incubation with the targeting ligands in a single living cell. We believe that our method will provide insights into the generation of radical stress in cells, which could improve our fundamental understanding of the pharmacology and signaling pathways of widely used cell- and organelle-targeting ligands associated with free radicals.

SP1 transcriptionally activates HTR2B to aggravate traumatic spinal cord injury by shifting microglial M1/M2 polarization.

Spinal cord injury (SCI) can result in structural and functional damage to the spinal cord, which may lead to loss of limb movement and sensation, loss of bowel and bladder control, and other complications. Previous studies have revealed the critical influence of trans-acting transcription factor 1 (SP1) in neurological pathologies, however, its role and mechanism in SCI have not been fully studied. The study was performed using mouse microglia BV2 stimulated using lipopolysaccharide (LPS) and male adult mice subjected to spinal hitting. Western blotting was performed to detect protein expression of SP1, 5-hydroxytryptamine (serotonin) receptor 2B (HTR2B), BCL2-associated x protein (Bax), B-cell lymphoma-2 (Bcl-2), inducible nitric oxide synthase (iNOS), clusters of differentiation 86 (CD86), Arginase 1 (Arg-1) and clusters of differentiation 206 (CD206). Cell viability and apoptosis were analyzed by MTT assay and TUNEL assay. mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-4 (IL-4) and tumor necrosis factor-β (TNF-β) were quantified by quantitative real-time polymerase chain reaction. The association of SP1 and HTR2B was identified by chromatin immunoprecipitation assay and dual-luciferase reporter assay. HE staining assay was performed to analyze the pathological conditions of spinal cord tissues. LPS treatment induced cell apoptosis and inhibited microglia polarization from M1 to M2 phenotype, accompanied by an increase of Bax protein expression and a decrease of Bcl-2 protein expression, however, these effects were relieved after SP1 silencing. Mechanism assays revealed that SP1 transcriptionally activated HTR2B in BV2 cells, and HTR2B knockdown rescued LPS-induced effects on BV2 cell apoptosis and microglial M1/M2 polarization. Moreover, SP1 absence inhibited BV2 cell apoptosis and promoted microglia polarization from M1 to M2 phenotype by decreasing HTR2B expression. SCI mouse model assay further showed that SP1 downregulation could attenuate spinal hitting-induced promoting effects on cell apoptosis of spinal cord tissues and microglial M1 polarization. SP1 transcriptionally activated HTR2B to aggravate traumatic SCI by shifting microglial M1/M2 polarization.

Acute Administration of HIV-1 Tat Protein Drives Glutamatergic Alterations in a Rodent Model of HIV-Associated Neurocognitive Disorders

HIV-1-associated neurocognitive disorders (HAND) are a major comorbidity of HIV-1 infection, marked by impairment of executive function varying in severity. HAND affects nearly half of people living with HIV (PLWH), with mild forms predominating since the use of anti-retroviral therapies (ART). The HIV-1 transactivator of transcription (Tat) protein is found in the cerebrospinal fluid of patients adherent to ART, and its administration or expression in animals causes cognitive symptoms. Studies of Tat interaction with the N-methyl-D-aspartate receptor (NMDAR) suggest that glutamate toxicity contributes to Tat-induced impairments. To identify changes in regional glutamatergic circuitry underlying cognitive impairment, we injected recombinant Tat86 or saline to medial prefrontal cortex (mPFC) of male Sprague–Dawley rats. Rats were assessed with behavioral tasks that involve intact functioning of mPFC including the novel object recognition (NOR), spatial object recognition (SOR), and temporal order (TO) tasks at 1 and 2 postoperative weeks. Following testing, mPFC tissue was collected and analyzed by RT-PCR. Results showed Tat86 in mPFC-induced impairment in SOR, and upregulation of Grin1 and Grin2a transcripts. To further understand the mechanism of Tat toxicity, we assessed the effects of full-length Tat101 on gene expression in mPFC by RNA sequencing. The results of RNAseq suggest that glutamatergic effects of Tat86 are maintained with Tat101, as Grin2a was upregulated in Tat101-injected tissue, among other differentially expressed genes. Spatial learning and memory impairment and Grin2a upregulation suggest that exposure to Tat protein drives adaptation in mPFC, altering the function of circuitry supporting spatial learning and memory.

Negative lipid membranes enhance the adsorption of TAT-decorated elastin-like polypeptide micelles

A cell-penetrating peptide (CPP) is a short amino-acid sequence capable of efficiently translocating across the cellular membrane of mammalian cells. However, the potential of CPPs as a delivery vector is hampered by the strong reduction of its translocation efficiency when it bears an attached molecular cargo. To overcome this problem, we used previously developed diblock copolymers of elastin-like polypeptides (ELPBCs), which we end functionalized with TAT (transactivator of transcription), an archetypal CPP built from a positively charged amino acid sequence of the HIV-1 virus. These ELPBCs self-assemble into micelles at a specific temperature and present the TAT peptide on their corona. These micelles can recover the lost membrane affinity of TAT and can trigger interactions with the membrane despite the presence of a molecular cargo. Herein, we study the influence of membrane surface charge on the adsorption of TAT-functionalized ELP micelles onto giant unilamellar vesicles (GUVs). We show that the TAT-ELPBC micelles show an increased binding constant toward negatively charged membranes compared to neutral membranes, but no translocation is observed. The affinity of the TAT-ELPBC micelles for the GUVs displays a stepwise dependence on the lipid charge of the GUV, which, to our knowledge, has not been reported previously for interactions between peptides and lipid membranes. By unveiling the key steps controlling the interaction of an archetypal CPP with lipid membranes, through regulation of the charge of the lipid bilayer, our results pave the way for a better design of delivery vectors based on CPPs.

Acute Effects of Monoacylglycerol Lipase Inhibitor ABX1431 on Neuronal Hyperexcitability, Nociception, Locomotion, and the Endocannabinoid System in HIV-1 Tat Male Mice.

Background: Evidence suggests that monoacylglycerol lipase (MAGL) inhibitors can potentially treat HIV symptoms by increasing the concentration of 2-arachidonoylglycerol (2-AG). We examined a selective MAGL inhibitor ABX1431 in the context of neuroHIV. Methods: To assess the effects of ABX1431, we conducted in vitro and in vivo studies. In vitro calcium imaging on frontal cortex neuronal cultures was performed to evaluate the role of ABX1431 (10, 30, 100 nM) on transactivator of transcription (Tat)-induced neuronal hyperexcitability. Following in vitro experiments, in vivo experiments were performed using Tat transgenic male mice. Mice were treated with 4 mg/kg ABX1431 and assessed for antinociception using tail-flick and hot plate assays followed by locomotor activity. After the behavioral experiments, their brains were harvested to quantify endocannabinoids (eCB) and related lipids through mass spectrometry, and cannabinoid type-1 and -2 receptors (CB1R and CB2R) were quantified through western blot. Results: In vitro studies revealed that adding Tat directly to the neuronal cultures significantly increased intracellular calcium concentration, which ABX1431 completely reversed at all concentrations. Preincubating the cultures with CB1R and CB2R antagonists showed that ABX1431 exhibited its effects partially through CB1R. In vivo studies demonstrated that acute ABX1431 increased overall total distance traveled and speed of mice regardless of their genotype. Mass spectrometry and western blot analyses revealed differential effects on the eCB system based on Tat expression. The 2-AG levels were significantly upregulated following ABX1431 treatment in the striatum and spinal cord. Arachidonic acid (AA) was also upregulated in the striatum of vehicle-treated Tat(+) mice. No changes were noted in CB1R expression levels; however, CB2R levels were increased in ABX1431-treated Tat(-) mice only. Conclusion: Findings indicate that ABX1431 has potential neuroprotective effects in vitro partially mediated through CB1R. Acute treatment of ABX1431 in vivo shows antinociceptive effects, and seems to alter locomotor activity, with upregulating 2-AG levels in the striatum and spinal cord.

Hmo1 Promotes Efficient Transcription Elongation by RNA Polymerase I in Saccharomyces cerevisiae.

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA.

Determination of molecular epidemiologic pattern of human T-lymphotropic virus type 1 (HTLV-1) in Alborz province, Iran.

Human T-cell lymphotropic virus type 1 (HTLV-1) is linked to two debilitating diseases, adult T-cell leukemia/lymphoma (ATLL) and HTLV-1 associated myelopathy tropical spastic paraparesis (HAM/TSP), which are prevalent in various parts of the world, including the Alborz province in Iran. Understanding the prevalence and evolutionary relationships of HTLV-1 infections in these endemic areas is of utmost importance. In the realm of phylogenetic studies, long terminal repeat (LTR) region of HTLV-1 stands out as highly conserved, yet more variable compared to other gene segments. Consequently, it is the primary focus for phylogenetic analyses. Additionally, trans-activator of transcription (Tax), an oncoprotein, holds a pivotal role in the regulation of gene expression. This cross-sectional study delved into the phylogenetic analysis of HTLV-1 among individuals in Alborz province of Iran. To confirm infection, we amplified partial sequence LTR (PLTR) and HTLV-1 bZIP factor (PHBZ). For phylogenetic analysis, we sequenced the full sequence LTR (FLTR) andfull Tax sequence (FTax). The FLTR and FTax sequences underwent analysis using BioEdit, and phylogenetic trees were constructed using MEGA-X software. Out of the roughly 15,000 annual blood donors in Alborz, 19 samples tested positive for HTLV-1, indicating a 0.13% HTLV-1 positivity rate among blood donors. Furthermore, the HTLV-1 virus prevalent in the Alborz province belongs to subtype A (cosmopolitan) subgroup A. The findings revealed that while mutations were observed in both the LTR and Tax genes, they were not significant enough to bring about fundamental alterations. Despite positive selection detected in three Alborz isolates, it has not led to mutations affecting Tax function and virulence.

Decoding the multifaceted interventions between human sirtuin 2 and dynamic hepatitis B viral proteins to confirm their roles in HBV replication.

The human sirtuin 2 gene (SIRT2) encodes a full-length Sirt2 protein (i.e., the Sirt2 isoform 1), which primarily functions as a cytoplasmic α-tubulin deacetylase, and which promotes the growth of hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) replication itself, or HBV X (HBx) protein-mediated transcriptional transactivation, enhances Sirt2.1 expression; therefore, Sirt2.1 itself is capable of positively increasing HBV transcription and replication. Sirt2.1 is linked to liver fibrosis and epithelial-to-mesenchymal transition and, consequently, augments the risk of HCC. The Sirt2.1 protein enhances the HBV replication cycle by activating the AKT/glycogen synthase kinase 3 beta (GSK3β)/β-catenin pathway. It also activates the transcription of the viral enhancer I/HBx promoter (EnI/Xp) and enhancer II/HBc promoter (EnII/Cp) by targeting the transcription factor p53. The Sirt2 isoform 2 (Sirt2.2) is mainly localized in the cytoplasm, and the N-terminus is shorter by 37 amino acids than that of Sirt2.1. Despite the truncation of the N-terminal region, Sirt2.2 is still capable of enhancing HBV replication and activating the AKT/GSK3β/β-catenin signaling pathway. The Sirt2 isoform 5 (Sirt2.5) is primarily localized to the nucleus, it lacks a nuclear export signal (NES), and the catalytic domain (CD) is truncated. Upon HBV replication, expression of the Sirt2 isoforms is also enhanced, which further upregulates the HBV replication, and, therefore, supports the vicious cycle of viral replication and progression of the disease. Sirt2 diversely affects HBV replication such that its isoform 1 intensely augments HBV replication and isoform 2 (despite of the truncated N-terminal region) moderately enhances HBV replication. Isoform 5, on the other hand, tends to protect the cell (for smooth long-term continued viral replication) from HBV-induced extreme damage or death via a discrete set of regulatory mechanisms impeding viral mRNAs, the hepatitis B core/capsid protein (HBc), core particles, replicative intermediate (RI) DNAs, and covalently closed circular DNA (cccDNA) levels, and, consequently, limiting HBV replication. In contrast to Sirt2.1 and Sirt 2.2, the Sirt2.5-mediated HBV replication is independent of the AKT/GSK3β/β-catenin signaling cascade. Sirt2.5 is recruited more at cccDNA than the recruitment of Sirt2.1 onto the cccDNA. This recruitment causes the deposition of more histone lysine methyltransferases (HKMTs), including SETDB1, SUV39H1, EZH2, and PR-Set7, along with the respective corresponding transcriptional repressive markers such as H3K9me3, H3K27me3, and H4K20me1 onto the HBV cccDNA. In HBV-replicating cells, Sirt2.5 can also make complexes with PR-Set7 and SETDB1. In addition, Sirt2.5 has the ability to turn off transcription from cccDNA through epigenetic modification via either direct or indirect interaction with HKMTs.

Forced intercalation-induced light-up peptides as fluorogenic indicators for the HIV-1 TAR RNA-ligand assay.

Fluorescence indicators capable of binding to human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA are powerful tools for the exploratory studies of the identification of anti-HIV drug candidates. This work presents a new design strategy for fluorogenic indicators with a transactivator of transcription (Tat)-derived peptide based on the forced intercalation of thiazole orange (TO) dyes (FIT). The developed 9-mer FIT peptide (RKKRR-TO-RRR: named FiLuP) features the TO unit integrated onto a Dap (2,3-diaminopropionic acid) residue in the middle of the Tat peptide sequence; the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. This facilitates a significant light-up response (450-fold at λem = 541 nm, Φfree = 0.0057, and Φbound = 0.61) upon binding to TAR RNA. The response of FiLuP is highly selective to TAR RNA over other non-cognate RNAs, and FiLuP maintains strong binding affinity (Kd = 1.0 ± 0.6 nM). Significantly, in contrast to previously developed Tat peptide-based FRET probes, FiLuP is able to discriminate between "competitive" and "noncompetitive" inhibitors when used in the fluorescence indicator displacement (FID) assay. The FID assay under stringent screening conditions is also possible, enabling super-strong competitive binders toward TAR RNA to be sieved out.

Reprogramming of endothelial gene expression by tamoxifen inhibits angiogenesis and ERα-negative tumor growth.

Rationale: 17β-estradiol (E2) can directly promote the growth of ERα-negative cancer cells through activation of endothelial ERα in the tumor microenvironment, thereby increasing a normalized tumor angiogenesis. ERα acts as a transcription factor through its nuclear transcriptional AF-1 and AF-2 transactivation functions, but membrane ERα plays also an important role in endothelium. The present study aims to decipher the respective roles of these two pathways in ERα-negative tumor growth. Moreover, we delineate the actions of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) in ERα-negative tumors growth and angiogenesis, since we recently demonstrated that tamoxifen impacts vasculature functions through complex modulation of ERα activity. Methods: ERα-negative B16K1 cancer cells were grafted into immunocompetent mice mutated for ERα-subfunctions and tumor growths were analyzed in these different models in response to E2 and/or tamoxifen treatment. Furthermore, RNA sequencings were analyzed in endothelial cells in response to these different treatments and validated by RT-qPCR and western blot. Results: We demonstrate that both nuclear and membrane ERα actions are required for the pro-tumoral effects of E2, while tamoxifen totally abrogates the E2-induced in vivo tumor growth, through inhibition of angiogenesis but promotion of vessel normalization. RNA sequencing indicates that tamoxifen inhibits the E2-induced genes, but also initiates a specific transcriptional program that especially regulates angiogenic genes and differentially regulates glycolysis, oxidative phosphorylation and inflammatory responses in endothelial cells. Conclusion: These findings provide evidence that tamoxifen specifically inhibits angiogenesis through a reprogramming of endothelial gene expression via regulation of some transcription factors, that could open new promising strategies to manage cancer therapies affecting the tumor microenvironment of ERα-negative tumors.

Transcriptional regulation of the HIV-1 inhibitory factor human mannose receptor 1 by the myeloid-specific transcription factor PU.1.

HIV-1 infection of human macrophages leads to the downmodulation of human mannose receptor 1 (hMRC1), a cell-surface glycoprotein that is involved in the host innate immune response. We previously reported that downmodulation of hMRC1 involves the transactivator of transcription (Tat)-dependent transcriptional silencing of the hMRC1 promoter. However, the inhibitory effect of Tat on hMRC1 transcription was indirect and involved inhibition of the transcriptional activator PU.1, which normally upregulates hMRC1 expression in macrophages and other myeloid cells. We cloned a 284-bp fragment of the hMRC1 promoter, and within it, we identified four PU.1 box elements. We assessed the relative contribution of each of the four PU.1 boxes to PU.1-dependent transcriptional regulation and, surprisingly, found that only one of the four PU.1 boxes [PU.1(b)] was critically required for PU.1-mediated upregulation of luciferase expression. Transfer of this PU.1 box to a heterologous promoter conferred PU.1 responsiveness to an otherwise PU.1 insensitive promoter. Electrophoretic mobility shift assays identified this PU.1 box as a direct binding site for PU.1 both in the context of the hMRC1 promoter and the heterologous promoter. Furthermore, mutational analysis of the PU.1 protein identified the C-terminal DNA-binding domain in PU.1 as the region responsible for interaction with the PU.1 box. Recombinant HIV-1 Tat protein did not bind to the hMRC1 promoter element but efficiently interfered with the binding of PU.1 protein to the hMRC1 promoter. Thus, Tat is likely to inhibit the formation of active PU.1 transcription complexes, presumably by binding to and depleting common transcriptional cofactors.IMPORTANCEHIV-1 infection of cells results in the modulation of cellular gene expression by virus-encoded proteins in a manner that benefits the virus. We reported that HIV-1 transactivator of transcription (Tat) dysregulates the expression of the human mannose receptor 1 (hMRC1). hMRC1 is involved in the innate immune response of macrophages to foreign pathogens. Tat does not act directly on the hMRC1 promoter but instead inhibits PU.1, a cellular transcription factor regulating hMRC1 gene expression. Here, we characterize the PU.1-dependent regulation of hMRC1 expression. We identified four potential PU.1 binding sites in the hMRC1 promoter region but found that only one, PU.1(b), functioned as a true binding site for PU.1. Transfer of the PU.1(b) box to a heterologous promoter did not activate this promoter per se but rendered it responsive to PU.1. Our results support the view that PU.1 acts as a transcriptional co-factor whose activity can be regulated by HIV-1 Tat.

SRI-30827, a novel allosteric modulator of the dopamine transporter, alleviates HIV-1 Tat-induced potentiation of cocaine conditioned place preference in mice.

HIV-1 Tat (transactivator of transcription) protein disrupts dopaminergic transmission and potentiates the rewarding effects of cocaine. Allosteric modulators of the dopamine transporter (DAT) have been shown to reverse Tat-induced DAT dysfunction. We hypothesized that a novel DAT allosteric modulator, SRI-30827, would counteract Tat-induced potentiation of cocaine reward. Doxycycline (Dox)-inducible Tat transgenic (iTat-tg) mice and their G-tg (Tat-null) counterparts were tested in a cocaine conditioned place preference (CPP) paradigm. Mice were treated 14 days with saline, or Dox (100 mg/kg/day, i.p.) to induce Tat protein. Upon induction, mice were place conditioned two days with cocaine (10 mg/kg/day) after a 1-h daily intracerebroventricular (i.c.v.) pretreatment with SRI-30827 (1 nmol) or a vehicle control, and final place preference assessed as a measure of cocaine reward. Dox-treatment significantly potentiated cocaine-CPP in iTat-tg mice over the response of saline-treated control littermates. SRI-30827 treatment eliminated Tat-induced potentiation without altering normal cocaine-CPP in saline-treated mice. Likewise, SRI-30827 did not alter cocaine-CPP in both saline- and Dox-treated G-tg mice incapable of expressing Tat protein. These findings add to a growing body of evidence that allosteric modulation of DAT could provide a promising therapeutic intervention for patients with comorbid HIV-1 and cocaine use disorder (CUD).

Cardiac-Targeting Peptide: From Discovery to Applications.

Despite significant strides in prevention, diagnosis, and treatment, cardiovascular diseases remain the number one cause of mortality in the United States, with rates climbing at an alarming rate in the developing world. Targeted delivery of therapeutics to the heart has been a lofty goal to achieve with strategies ranging from direct intra-cardiac or intra-pericardial delivery, intra-coronary infusion, to adenoviral, lentiviral, and adeno-associated viral vectors which have preference, if not complete cardio-selectivity, for cardiac tissue. Cell-penetrating peptides (CPP) are 5-30-amino-acid-long peptides that are able to breach cell membrane barriers while carrying cargoes up to several times their size, in an intact functional form. Identified nearly three decades ago, the first of these CPPs came from the HIV coat protein transactivator of transcription. Although a highly efficient CPP, its clinical utility is limited by its robust ability to cross any cell membrane barrier, including crossing the blood-brain barrier and transducing neuronal tissue non-specifically. Several strategies have been utilized to identify cell- or tissue-specific CPPs, one of which is phage display. Using this latter technique, we identified a cardiomyocyte-targeting peptide (CTP) more than a decade ago, a finding that has been corroborated by several independent labs across the world that have utilized CTP for a myriad of different purposes in pre-clinical animal models. The goal of this publication is to provide a comprehensive review of the identification, validation, and application of CTP, and outline its potential in diagnostic and therapeutic applications especially in the field of targeted RNA interference.

ERG Is a New Predisposition Gene for Bone Marrow Failure and Hematological Malignancy

There remain gaps in our knowledge of hereditary and sporadic causes of hematological malignancy (HM) and bone marrow failure (BMF) that prevent optimal diagnosis, disease surveillance and treatment. Here we report the discovery of ERG as a novel predisposition gene for BMF and HM. ERG is a known oncogene, typically via gene-fusions, leading to dysregulated ERG overexpression in blood and solid cancers. We identified a germline ERG ETS domain variant p.Y373C segregating with thrombocytopenia in a mother, who progressed to AML (27 yr) and then therapy-related MDS (35 yr), and in her 2 sons. All three showed copy neutral loss of heterozygosity of all or part of chromosome 21q, including the ERG locus, with the oldest son showing at least 2 somatic genetic rescue (SGR) events. The possibility of causal RUNX1 variants were ruled out, with the smallest somatic cnLOH event beginning within the RUNX1 gene, but not encompassing the RUNT domain where the majority of pathogenic missense variants are located. ERG, a highly constrained gene (LOEUF <0.33), is critical for definitive hematopoiesis, adult hematopoietic stem cell (HSC) function and platelet maintenance. An identical corresponding heterozygous germline variant (p.Y343C) in ERG’s closest gene by homology, FLI1, causes platelet-type bleeding disorder-21 (BDPLT21, OMIM #617443). Through global collaborations, we have identified 15 heterozygous variants in the ERG gene, 13 of which are missense and 2 truncating variants, in 17 individuals with cytopenia and/or HM (mainly myeloid) or lymphedema (Table). Onset of hematological symptoms ranged from birth to 38 years for truncating and constrained ETS domain variants. Of these 15 variants, 12 have been confirmed germline including 2 de novo. Only 4 meiotic transmissions are observed. None of the missense variants in the highly conserved ETS domain of ERG which mediates DNA binding, protein-protein interactions and nuclear localization, are present in gnomAD. We have functionally characterized 19 ERG variants, 12 potentially pathogenic, 1 known mouse pathogenic variant and 3 population controls demonstrating that most ETS domain missense variants display loss-of-function (LOF) characteristics disrupting transcriptional transactivation (Figure), DNA-binding and/or nuclear localization in vitro. Robust preliminary data from ex vivo models of ERG overexpression in mouse fetal liver cells in tissue culture (cytokine-independence), a mouse transplant assay and previous germline mutant Erg mouse models are concordant with ETS domain missense variants being LOF compared to wildtype ERG and benign controls. Together, these data provide clinical, in vitro and ex vivo functional studies implicating LOF variants in hematological disease predisposition. LOF ERG mutations also occur in sporadic cases of HM. Recently, as part of a Genomics England Research Consortium population study, 4 truncating ERG variants were described in 7 individuals across 4 families with 3 meiotic transmissions and a de novo case with primary lymphedema (1) and we add 2 novel missense variants here. One patient showed SGR across the ERG locus in blood. Blood phenotypes were not described. Our results demonstrate that germline ERG variants predispose to diverse cytopenia, BMF and HM in both children and adults. In our family mentioned above, the mother received an unrelated alloHSCT due to t-MDS while her 2 sons with cytopenias continue to be monitored. The natural history of this new syndrome will require careful identification of germline lesions with additional longitudinal studies in more patients and families needed. This ERG syndrome parallels GATA2 deficiency syndrome (HM and lymphedema) and RUNX1 Familial Platelet disorder-myeloid malignancy (thrombocytopenia and HM). Like the well-known disease genes GATA2 and RUNX1, ERG is also a member of the transcription factor heptad involved in HSC maintenance and differentiation. ERG adds to a growing list of genes whose unregulated expression contributes to HM and other cancers. Identification of causal germline ERG variants like those outlined in this study, has direct clinical implications for patient and family management including diagnosis, counselling, surveillance and treatment strategies such as selection of bone marrow transplant donors and potential for targeted therapies including gene and cell therapy. Reference 1. Greene D et al. Nat.Med. 29:679-688 2023

Statistical learning quantifies transposable element-mediated cis-regulation

BackgroundTransposable elements (TEs) have colonized the genomes of most metazoans, and many TE-embedded sequences function as cis-regulatory elements (CREs) for genes involved in a wide range of biological processes from early embryogenesis to innate immune responses. Because of their repetitive nature, TEs have the potential to form CRE platforms enabling the coordinated and genome-wide regulation of protein-coding genes by only a handful of trans-acting transcription factors (TFs).ResultsHere, we directly test this hypothesis through mathematical modeling and demonstrate that differences in expression at protein-coding genes alone are sufficient to estimate the magnitude and significance of TE-contributed cis-regulatory activities, even in contexts where TE-derived transcription fails to do so. We leverage hundreds of overexpression experiments and estimate that, overall, gene expression is influenced by TE-embedded CREs situated within approximately 500 kb of promoters. Focusing on the cis-regulatory potential of TEs within the gene regulatory network of human embryonic stem cells, we find that pluripotency-specific and evolutionarily young TE subfamilies can be reactivated by TFs involved in post-implantation embryogenesis. Finally, we show that TE subfamilies can be split into truly regulatorily active versus inactive fractions based on additional information such as matched epigenomic data, observing that TF binding may better predict TE cis-regulatory activity than differences in histone marks.ConclusionOur results suggest that TE-embedded CREs contribute to gene regulation during and beyond gastrulation. On a methodological level, we provide a statistical tool that infers TE-dependent cis-regulation from RNA-seq data alone, thus facilitating the study of TEs in the next-generation sequencing era.

Abstract 18459: A Cell Permeable Protein Kinase C Beta II Peptide Inhibitor Mitigates the Generation of Reactive Oxygen Species in Rat ex-vivo and Porcine in-vivo Ischemia-Reperfusion Injury

Introduction: Restoration of blood flow following myocardial infarction is necessary to salvage ischemic tissue, but reperfusion is known to cause myocardial ischemia/reperfusion injury (MIR). Cytokine induced translocation of protein kinase C beta II (PKCβII) during reperfusion (R) stimulates the production of reactive oxygen species (ROS). Dual conjugation of PKCβII peptide inhibitor (PKCβII-) with myristic acid (myr) and trans-activator of transcription (Tat) (myr-Tat-CC-SLNPEWNET [myr-Tat-PKCβII-]) enhances intracellular delivery and reduces phorbol ester-induced neutrophil ROS. We hypothesize that 20ng/kg of myr-Tat-PKCβII- would exert cardioprotective effects in porcine MIR in-vivo by reducing stimulation of ROS. Methods: Ex vivo: We tested 100nM - 10fmol myr-Tat-PKCβII- in ex-vivo rat MIR. Isolated hearts from anesthetized male SD rats underwent global I (30-min)/R(50-min). Myr-Tat-PKCβII- was given during first 5min of R. DP/dt max was measured via pressure transducer in the left ventricle. 1% TTC staining was used to determine infarct size. Data was analyzed via Fisher’s PLSD. In vivo: Regional I (1 hr)/R (3 hrs) was induced in male Yorkshire pigs. At R, myr-Tat-PKCβII- or scrambled control peptide (20ng/kg; ~100 pM blood volume [vol.] concentration [conc.]) was given by intra-arterial bolus. Ejection fraction (EF) was calculated. Infarct size was determined with Evans Blue dye and 1% TTC staining. Data was analyzed using Student’s t-test. Results: Ex-vivo: Myr-Tat-PKCβII- (100nM - 100fmol; 10-14±3%; n=3-6) significantly reduced infarct size compared to controls (21±3%; n=21; p<0.05). DP/dt max was significantly improved (100pM-1pM; 961±274; n=5-6) compared to 100nM (163±77, n=3; p<0.05), 100fM (481±86, n=5; p<0.05), and 100pMscrambled myr-Tat-PKCβII- (386±86, n=4; p<0.05). In-vivo: Myr-Tat-PKCβII- 20ng/kg (~100pM blood vol. conc.) significantly restored EF to within 1.4±0.7% of baseline and reduced infarct size to (10.0±2.8%; n=4) compared to control (6.4±2.1%; 28.5±8.3%; n=6; p<0.05). Conclusions: Results suggest 20ng/kg (~100pM blood vol. conc.)) should continue to be used to test in porcine MIR in-vivo. Future studies include a 12-week porcine survival study to evaluate infarct size and cardiac function.