Search

G protein-coupled receptors (GPCRs), critical for cellular communication and signaling, represent the largest cell surface protein family and play important roles in numerous pathophysiological processes. Consequently, GPCRs have become a primary focus in drug discovery efforts. Beyond their traditional G protein-dependent signaling pathways, GPCRs are also capable of activating alternative signaling mechanisms, including G protein-independent signaling, biased signaling, and signaling crosstalk. A particularly novel signaling mode employed by these receptors is GPCR transactivation, which enables cross-communication between GPCRs and other receptor types. Intriguingly, GPCR transactivation by distinct GPCRs has also been identified. In this review, I provide an overview of the known GPCR transactivation mechanisms and explore recently uncovered GPCR transactivation mediated by adhesion-class GPCRs (aGPCRs). These aGPCR-GPCR transactivation processes regulate unique cell type-specific functions, offering an exciting opportunity to develop therapies that precisely modulate specific GPCR-mediated biological effects.

Foamy viruses are members of the Retroviridae family's Spumaretrovirinae subfamily. They induce cell vacuolation and exhibit a foamy pathogenic impact after infecting cells. DACH1 (dachshund family transcription factor 1) is a crucial cytokine linked to tumor development, and is associated with the growth of many different malignant tumor cells. Additionally, DACH1 suppresses pancreatic cell proliferation and is involved in diabetes insulin signaling. Prototype foamy viruses (PFVs) were used for the investigation of the regulatory mechanism of FVs on cellular DACH1 expression. The results show that DACH1 expression in PFV-infected cells was inconsistent at both the transcriptional and protein levels. At the transcriptional level, DACH1 was significantly activated by PFV transactivator Tas, and dual-luciferase reporter gene tests, EMSA, and ChIP assays found a Tas response element of 21 nucleotides in the DACH1 promoter. PFV and Tas did not boost the levels of DACH1 protein in a manner consistent with the high levels of DACH1 transcription expression. It was noted that Tas increased the expression of the Ser/Thr protein phosphatase PPM1E, causing PPM1E-mediated post-translational SUMOylation alterations of DACH1 to prompt DACH1 to degrade. The reason for DACH1 protein degradation is that DACH1 inhibits PFV replication. To sum up, these findings show that PFV upregulated the transcription of DACH1, while urging its protein into PPM1E-mediated SUMOylation, to eliminate the adverse effect of DACH1 overexpression of host cells on viral replication and promote virus survival.

The BcWCL1 protein is a blue-light photoreceptor from the fungus Botrytis cinerea . This protein has a central role in B. cinerea circadian regulation and is an ortholog to WC-1 from Neurospora crassa . The BcWCL1 and WC-1 proteins have similar protein domains, including a LOV (Light Oxygen Voltage) domain for light sensing, two PAS (Per Arnt Sim) domains for protein-protein interaction, and a DNA binding domain from the GATA family. Recently, the blue-light response of BcWCL1 was demonstrated in a version without PAS domains (BcWCL1 PAS∆ ). Here, we demonstrated that BcWCL1 PAS∆ is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1 PAS∆ enables transcriptional activation as a single component in yeast. By using chimeric transcription factors and the luciferase reporter gene, we assessed the transcriptional activity of different fragments of the N-terminal and C-terminal regions of BcWCL1 PAS∆ , identifying a functional transcriptional activation domain (AD) in the N-terminal region that belongs to the 9aaTAD family. Finally, we determined that the transcriptional activation levels of BcWCL1 PAS∆ AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1 PAS∆ protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics.

Background: Ulcerative colitis is a chronic inflammatory disease affecting the colon. During chronic inflammation of epithelial cells, lipid metabolism via pro-inflammatory eicosanoids is known to modify the immune response., Methods: Starting from the Mammalian Metabolic Database, the expression of metabolic enzymes was investigated in two independent cohorts from transcriptome datasets GSE38713 and GSE11223, which analyzed ulcerative colitis tissue samples from the digestive tract., Results: In the first cohort, 145 differentially expressed enzymes were identified as significantly regulated between ulcerative colitis tissues and normal controls. Overexpressed enzymes were selected to tune an Elastic Net model in the second cohort. Using the best parameters, the model achieved a prediction accuracy for ulcerative colitis with an area under the curve (AUC) of 0.79. Twenty-two metabolic enzymes were found to be commonly overexpressed in both independent cohorts, with decreasing Elastic Net predictive coefficients as follows: LIPG (3.98), PSAT1 (3.69), PGM3 (2.74), CD38 (2.28), BLVRA (1.99), CBR3 (1.94), NT5DC2 (1.76), PHGDH (1.71), GPX7 (1.58), CASP1 (1.56), ASRGL1 (1.4), SOD3 (1.25), CHST2 (0.965), CHST11 (0.95), KYNU (0.94), PLAG2G7 (0.92), SRM (0.87), PTGS2 (0.80), LPIN1 (0.47), ME1 (0.31), PTGDS (0.14), and ADA (0.13). Functional enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database highlighted the main implications of these enzymes in cysteine and methionine metabolism (adjusted p -value = 0.01), arachidonic acid and prostaglandin metabolism (adjusted p -value = 0.01), and carbon metabolism (adjusted p -value = 0.04). A metabolic score based on the transcriptional activation of the validated twenty-two enzymes was found to be significantly greater in Ulcerative colitis samples compared to healthy donor samples ( p -value = 1.52 × 10 -8 )., Conclusions: A metabolic expression score was established and reflects the implications of heterogeneous metabolic pathway deregulations in the digestive tract of patients with ulcerative colitis.

The transcription factor E2F1 is the principal target of the tumor suppressor pRB. E2F1 promotes cell proliferation by activating growth-promoting genes upon growth stimulation. In contrast, E2F1 contributes to tumor suppression by activating tumor suppressor genes, such as ARF , upon loss of pRB function, a major oncogenic change. The transactivation domain of E2F1 has previously been mapped to the C-terminal region. We show here that the N-terminal region of E2F1 is critical for the activation of tumor suppressor genes. Deletion of the N-terminal region dramatically compromised E2F1 activation of tumor suppressor genes. The N-terminal region showed transactivation ability when fused to the DNA-binding domain of GAL4. A search for novel interacting factors with the N-terminal region, using a yeast two-hybrid system, identified the general transcription factor GTF2H2. Overexpression of GTF2H2 enhanced E2F1 activation of tumor suppressor genes and induction of cell death. Conversely, the knockdown of GTF2H2 compromised both. E2F1 binding enhanced the binding of GTF2H2 to target promoters depending on the integrity of the N-terminal region. Taken together, these results suggest that the N-terminal region of E2F1 contains a novel transactivation domain that mediates the activation of tumor suppressor genes, at least in part, by recruiting GTF2H2.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections that are part the human genome and are normally silenced through epigenetic mechanisms. However, HERVs can be induced by various host and environmental factors, including viral infection, and transcriptionally active HERVs have been implicated in various physiological processes. In this review, we summarize mounting evidence of transactivation of HERVs by a wide range of DNA and RNA viruses. Though a mechanistic understanding of this phenomenon and the biological implications are still largely missing, the link between exogenous and endogenous viruses is intriguing. Considering the increasing recognition of the role of viral infections in disease, understanding these interactions provides novel insights into human health.

This study describes, to some extent, the VCC contribution as an early stimulation of the macrophage lineage. Regarding the onset of the innate immune response caused by infection, the β form of IL-1 is the most important interleukin involved in the onset of the inflammatory innate response. Activated macrophages treated in vitro with VCC induced the activation of the MAPK signaling pathway in a one-hour period, with the activation of transcriptional regulators for a surviving and pro-inflammatory response, suggesting an explanation inspired and supported by the inflammasome physiology. The mechanism of IL-1β production induced by VCC has been gracefully outlined in murine models, using bacterial knockdown mutants and purified molecules; nevertheless, the knowledge of this mechanism in the human immune system is still under study. This work shows the soluble form of 65 kDa of the Vibrio cholerae cytotoxin (also known as hemolysin), as it is secreted by the bacteria, inducing the production of IL-1β in the human macrophage cell line THP-1. The mechanism involves triggering the early activation of the signaling pathway MAPKs pERK and p38, with the subsequent activation of (p50) NF-κB and AP-1 (cJun and cFos), determined by real-time quantitation. The evidence shown here supports that the monomeric soluble form of the VCC in the macrophage acts as a modulator of the innate immune response, which is consistent with the assembly of the NLRP3 inflammasome actively releasing IL-1β.

Metabolic syndrome (MetS) is a non-communicable disease characterized by a cluster of metabolic irregularities. Alarmingly, the prevalence of MetS in people living with Human Immunodeficiency Virus (HIV) and antiretroviral (ARV) usage is increasing rapidly. Insulin resistance is a common characteristic of MetS that leads to the development of Type 2 diabetes mellitus (T2DM). The progression of insulin resistance is strongly linked to inflammasome activation. This study aimed to draw links between the combinational use of Tenofovir disoproxil fumarate (TDF), Lamivudine (3TC), and Dolutegravir (DTG), and inflammasome activation and subsequent promotion of insulin resistance following a 120 h treatment period in HepG2 liver in vitro cell model. Furthermore, we assess microRNA (miR-128a) expression as a negative regulator of the IRS1/AKT signaling pathway. The relative expression of phosphorylated IRS1 was determined by Western blot. Transcript levels of NLRP3, IL-1β, JNK, IRS1, AKT, PI3K, and miR-128a were assessed using quantitative PCR (qPCR). Caspase-1 activity was measured using luminometry. Following exposure to ARVs for 120 h, NLRP3 mRNA expression ( p = 0.0500) and caspase-1 activity ( p < 0.0001) significantly increased. This was followed by a significant elevation in IL-1β in mRNA expression ( p = 0.0015). Additionally, JNK expression ( p = 0.0093) was upregulated with coinciding increases in p-IRS1 protein expression ( p < 0.0001) and decreased IRS1 mRNA expression ( p = 0.0004). Consequently, decreased AKT ( p = 0.0005) and PI3K expressions ( p = 0.0007) were observed. Interestingly miR-128a expression was significantly upregulated. The results indicate that combinational use of ARVs upregulates inflammasome activation and promotes insulin resistance through dysregulation of the IRS1/PI3K/AKT insulin signaling pathway.

Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1 (DYRK1) encodes a conserved protein kinase that is indispensable to neuron development. However, whether DYRK1 possesses additional functions apart from kinase function remains poorly understood. In this study, we firstly demonstrated that the C-terminal of ascidian Ciona robusta DYRK1 (CrDYRK1) showed transcriptional activation activity independent of its kinase function. The transcriptional activation activity of CrDYRK1 could be autoinhibited by a repression domain in the N-terminal. More excitingly, both activation and repression domains were retained in HsDYRK1A in humans. The genes, activated by the activation domain of HsDYRK1A, are mainly involved in ion transport and neuroactive ligand-receptor interaction. We further found that numerous mutation sites relevant to the DYRK1A -related intellectual disability syndrome locate in the C-terminal of HsDYRK1A. Then, we identified several specific DNA motifs in the transcriptional regulation region of those activated genes. Taken together, we identified a conserved transcription activation domain in DYRK1 in urochordates and vertebrates. The activation is independent of the kinase activity of DYRK1 and can be repressed by its own N-terminal. Transcriptome and mutation data indicate that the transcriptional activation ability of HsDYRK1A is potentially involved in synaptic transmission and neuronal function related to the intellectual disability syndrome.

Dengue virus (DENV), an arbovirus, strongly activates mast cells (MCs), which are key immune cells for pathogen immune surveillance. In animal models, MCs promote clearance of local peripheral DENV infections but, conversely, also promote pathological vascular leakage when widely activated during systemic DENV infection. Since DENV is a human pathogen, we sought to ascertain whether a similar phenomenon could occur in humans by characterizing the products released by human MCs (huMCs) upon direct (antibody-independent) DENV exposure, using the phenotypically mature huMC line, ROSA. DENV did not productively infect huMCs but prompted huMC release of proteases and eicosanoids and induced a Th1-polarized transcriptional profile. In co-culture and trans-well systems, huMC products activated human microvascular endothelial cells, involving transcription of vasoactive mediators and increased monolayer permeability. This permeability was blocked by MC-stabilizing drugs, or limited by drugs targeting certain MC products. Thus, MC stabilizers are a viable strategy to limit MC-promoted vascular leakage during DENV infection in humans.

To test whether homologous recombination repair (HRR) depends on FOXO3a, a cellular aging model of human dermal fibroblast (HDF) and tet-on flag-h-FOXO3a transgenic mice were studied. HDF cells transfected with over-expression of wt-h-FOXO3a increased the protein levels of MRE11, BRCA1, BRIP1, and RAD50, while knock-down with siFOXO3a decreased them. The protein levels of MRE11, BRCA1, BRIP1, RAD50, and RAD51 decreased during cellular aging. Chromatin immunoprecipitation (ChIP) assay was performed on FOXO3a binding accessibility to FOXO consensus sites in human MRE11, BRCA1, BRIP1, and RAD50 promoters; the results showed FOXO3a binding decreased during cellular aging. When the tet-on flag-h-FOXO3a mice were administered doxycycline orally, the protein and mRNA levels of flag-h-FOXO3a, MRE11, BRCA1, BRIP1, and RAD50 increased in a doxycycline-dose-dependent manner. In vitro HRR assays were performed by transfection with an HR vector and I-SceI vector. The mRNA levels of the recombined GFP increased after doxycycline treatment in MEF but not in wt-MEF, and increased in young HDF comparing to old HDF, indicating that FOXO3a activates HRR. Overall, these results demonstrate that MRE11, BRCA1, BRIP1, and RAD50 are transcriptional target genes for FOXO3a, and HRR activity is increased via transcriptional activation of MRE11, BRCA1, BRIP1, and RAD50 by FOXO3a.

The steroid hormone ecdysone regulates insect development via its nuclear receptor (the EcR protein), which functions as a ligand-dependent transcription factor. The EcR regulates target gene expression by binding to ecdysone response elements (EcREs) in their promoter or enhancer regions. Its role in epigenetic regulation and, particularly, in histone acetylation remains to be clarified. Here, we analyzed the dynamics of histone acetylation and demonstrated that the acetylation of histone H3 on lysine 27 (H3K27) at enhancers was required for the transcriptional activation of ecdysone-responsive genes. Western blotting and ChIP-qPCR revealed that ecdysone altered the acetylation of H3K27. For E75B and Hr4 , ecdysone-responsive genes, enhancer activity, and transcription required the histone acetyltransferase activity of the CBP. EcR binding was critical in inducing enhancer activity and H3K27 acetylation. The CREB-binding protein (CBP) HAT domain catalyzed H3K27 acetylation and CBP coactivation with EcR, independent of the presence of ecdysone. Increased H3K27 acetylation promoted chromatin accessibility, with the EcR and CBP mediating a local chromatin opening in response to ecdysone. Hence, epigenetic mechanisms, including the modification of acetylation and chromatin accessibility, controlled ecdysone-dependent gene transcription.

Type 1 diabetes (T1D) results from the progressive loss of pancreatic beta cells as a result of autoimmune destruction. We recently reported that during the natural history of T1D in humans and the female nonobese diabetic (NOD) mouse model, beta cells acquire a senescence-associated secretory phenotype (SASP) that is a major driver of disease onset and progression, but the mechanisms that activate SASP in beta cells were not explored. Here, we show that the SASP in islet cells is transcriptionally controlled by Bromodomain ExtraTerminal (BET) proteins, including Bromodomain containing protein 4 (BRD4). A chromatin analysis of key beta cell SASP genes in NOD islets revealed binding of BRD4 at active regulatory regions. BET protein inhibition in NOD islets diminished not only the transcriptional activation and secretion of SASP factors, but also the non-cell autonomous activity. BET protein inhibition also decreased the extent of SASP induction in human islets exposed to DNA damage. The BET protein inhibitor iBET-762 prevented diabetes in NOD mice and also attenuated SASP in islet cells in vivo. Taken together, our findings support a crucial role for BET proteins in the activation of the SASP transcriptional program in islet cells. These studies suggest avenues for preventing T1D by transcriptional inhibition of SASP.

Insulin resistance in skeletal muscle in type 2 diabetes (T2D) is characterized by more pronounced metabolic and molecular defects than in obesity per se . There is increasing evidence that adipose tissue dysfunction contributes to obesity-induced insulin resistance in skeletal muscle. Here, we used an unbiased approach to examine if adipose tissue dysfunction is exaggerated in T2D and linked to diabetes-related mechanisms of insulin resistance in skeletal muscle. Transcriptional profiling and biological pathways analysis were performed in subcutaneous adipose tissue (SAT) and skeletal muscle biopsies from 17 patients with T2D and 19 glucose-tolerant, age and weight-matched obese controls. Findings were validated by qRT-PCR and western blotting of selected genes and proteins. Patients with T2D were more insulin resistant and had lower plasma adiponectin than obese controls. Transcriptional profiling showed downregulation of genes involved in mitochondrial oxidative phosphorylation and the tricarboxylic-acid cycle and increased expression of extracellular matrix (ECM) genes in SAT in T2D, whereas genes involved in proteasomal degradation were upregulated in the skeletal muscle in T2D. qRT-PCR confirmed most of these findings and showed lower expression of adiponectin in SAT and higher expression of myostatin in muscle in T2D. Interestingly, muscle expression of proteasomal genes correlated positively with SAT expression of ECM genes but inversely with the expression of ADIPOQ in SAT and plasma adiponectin. Protein content of proteasomal subunits and major ubiquitin ligases were unaltered in the skeletal muscle of patients with T2D. A transcriptional signature of exaggerated adipose tissue dysfunction in T2D, compared with obesity alone, is linked to low plasma adiponectin and increased transcriptional activation of proteasomal degradation in skeletal muscle.

In breast cancer, epithelial-mesenchymal transition (EMT) is positively associated with programmed death ligand 1 (PD-L1) expression and immune escape, and TWIST1 silences ERα expression and induces EMT and cancer metastasis. However, how TWIST1 regulates PD-L1 and immune evasion is unknown. This study analyzed TWIST1 and PD-L1 expression in breast cancers, investigated the mechanism for TWIST1 to regulate PD-L1 transcription, and assessed the effects of TWIST1 and PD-L1 in cancer cells on cytotoxic CD8+ T cells. Interestingly, TWIST1 expression is correlated with high-level PD-L1 expression in ERα-negative breast cancer cells. The overexpression and knockdown of TWIST1 robustly upregulate and downregulate PD-L1 expression, respectively. TWIST1 binds to the PD-L1 promoter and recruits the TIP60 acetyltransferase complex in a BRD8-dependent manner to transcriptionally activate PD-L1 expression, which significantly accelerates the exhaustion and death of the cytotoxic CD8+ T cells. Accordingly, knockdown of TWIST1 or BRD8 or inhibition of PD-L1 significantly enhances the tumor antigen-specific CD8+ T cells to suppress the growth of breast cancer cells. These results demonstrate that TWIST1 directly induces PD-L1 expression in ERα-negative breast cancer cells to promote immune evasion. Targeting TWIST1, BRD8, and/or PD-L1 in ERα-negative breast cancer cells with TWIST1 expression may sensitize CD8+ T-cell-mediated immunotherapy.

BRIL (bone restricted ifitm-like; also known as IFITM5) is a transmembrane protein expressed in osteoblasts. Although its role in skeletal development and homeostasis is unknown, mutations in BRIL result in rare dominant forms of osteogenesis imperfecta. The pathogenic mechanism has been proposed to be a gain-of or neomorphic function. To understand the function of BRIL and its OI type V mutant (MALEP BRIL) and whether they could activate signaling pathways in osteoblasts, we performed a luciferase reporter assay screen based on the activity of 26 transcription factors. When overexpressed in MC3T3-E1 and MLO-A5 cells, the MALEP BRIL activated the reporters dependent on MEF2, NFATc, and NR4A significantly more. Additional co-transfection experiments with MEF2C and NFATc1 and a number of their modulators (HDAC4, calcineurin, RCAN, FK506) confirmed the additive or synergistic activation of the pathways by MALEP, and suggested a coordinated regulation involving calcineurin. Endogenous levels of Nr4a members, as well as Ptgs2 , were upregulated by MALEP BRIL. Y2H and co-immunoprecipitation indicated that BRIL interacted with CAML, but its contribution as the most upstream stimulator of the Ca 2+ -calcineurin-MEF2/NFATc cascade was not confirmed convincingly. Altogether the data presented provide the first ever readout to monitor for BRIL activity and suggest a potential gain-of-function causative effect for MALEP BRIL in OI type V, leading to perturbed signaling events and gene expression.

Atherosclerosis is the main cause of cardiovascular diseases with high prevalence worldwide. A promising therapeutic strategy to reverse atherosclerotic process is to improve the athero-protective potential of high-density lipoproteins (HDL). Since the small intestine is a source of HDL, we aimed to activate transcription of the endogenous HDL major proteins, apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1), in enterocytes, and to evaluate their potential to correct the pro-inflammatory status of endothelial cells (EC). Caco-2 enterocytes were transfected with CRISPR activation plasmids targeting ApoAI or PON1, and their gene and protein expression were measured in cells and conditioned medium (CM). ATP binding cassette A1 and G8 transporters (ABCA1, ABCG8), scavenger receptor BI (SR-BI), and transcription regulators peroxisome proliferator-activated receptor γ (PPARγ), liver X receptors (LXRs), and sirtuin-1 (SIRT1) were assessed. Anti-inflammatory effects of CM from transfected enterocytes were estimated through its ability to inhibit tumor necrosis factor α (TNFα) activation of EC. Transcriptional activation of ApoAI or PON1 in enterocytes induces: (i) increase of their gene and protein expression, and secretion in CM; (ii) stimulation of ABCA1/G8 and SR-BI; (iii) upregulation of PPARγ, LXRs, and SIRT1. CM from transfected enterocytes attenuated the TNFα-induced inflammatory and oxidative stress in EC, by decreasing TNF receptor 1, monocyte chemoattractant protein-1, and p22phox. In conclusion, transcriptional activation of endogenous ApoAI or PON1 in enterocytes by CRISPR/dCas9 system is a realistic approach to stimulate biogenesis and function of major HDL proteins which can regulate cholesterol efflux transporters and reduce the inflammatory stress in activated EC.

Strawberry is a soft fruit with short postharvest life, due to a rapid loss of firmness. Pectin methylesterase (PME)-mediated cell wall remodeling is important to determine fruit firmness and softening. Previously, we have verified the essential role of FvPME38 in regulation of PME-mediated strawberry fruit softening. However, the regulatory network involved in PME-mediated fruit softening is still largely unknown. Here, we identified an R2R3-type MYB transcription factor FvMYB79, which activates the expression level of FvPME38 , thereby accelerating fruit softening. During fruit development, FvMYB79 co-expressed with FvPME38 , and this co-expression pattern was opposite to the change of fruit firmness in the fruit of 'Ruegen' which significantly decreased during fruit developmental stages and suddenly became very low after the color turning stage. Via transient transformation, FvMYB79 could significantly increase the transcriptional level of FvPME38 , leading to a decrease of firmness and acceleration of fruit ripening. In addition, silencing of FvMYB79 showed an insensitivity to ABA-induced fruit ripening, suggesting a possible involvement of FvMYB79 in the ABA-dependent fruit softening process. Our findings suggest FvMYB79 acts as a novel regulator during strawberry ripening via transcriptional activation of FvPME38 , which provides a novel mechanism for improvement of strawberry fruit firmness.

HIV-1 latency maintenance and reactivation are regulated by several viral and host factors. One such factor is Krüppel-associated box (KRAB)-associated protein 1 (KAP1: also named TRIM28 or TIF1β). While initial studies have revealed KAP1 to be a positive regulator of latency reversal in transformed and primary CD4 + T cells, subsequent studies have proposed KAP1 to be a repressor required for latency maintenance. Given this discrepancy, in this study, we re-examine KAP1 transcription regulatory functions using a chemical genetics strategy to acutely deplete KAP1 expression to avoid the accumulation of indirect effects. Notably, KAP1 acute loss partially decreased HIV-1 promoter activity in response to activating signals, a function that can be restored upon complementation with exogenous KAP1, thus revealing that KAP1-mediated activation is on target. By combining comprehensive KAP1 domain deletion and mutagenesis in a cell-based reporter assay, we genetically defined the RING finger domain and an Intrinsically Disordered Region as key activating features. Together, our study solidifies the notion that KAP1 activates HIV-1 transcription by exploiting its multi-domain protein arrangement via previously unknown domains and functions.

Despite advances in the preparation of metal oxide (MO) nanoparticles (NPs) as catalysts for various applications, concerns about the biosafety of these particles remain. In this study, we prepared transition metal-doped cerium oxide (TM@CeO 2 ; TM = Cr, Mn, Fe, Co, or Ni) nanoparticles and investigated the mechanism underlying dopant-dependent toxicity in HaCaT human keratinocytes. We show that doping with Cr or Co but not Fe, Mn, or Ni increased the toxicity of CeO 2 NPs in dose- and time-dependent manners and led to apoptotic cell death. Interestingly, while both undoped and transition metal-doped NPs increased intracellular reactive oxygen species (ROS), toxic Cr@CeO 2 and Co@CeO 2 NPs failed to induce the expression of NRF2 (nuclear factor erythroid 2-related factor 2) as well as its downstream target genes involved in the antioxidant defense system. Moreover, activation of NRF2 transcription was correlated with dynamic changes in H3K4me3 and H3K27me3 at the promoter of NRF2, which was not observed in cells exposed to Cr@CeO 2 NPs. Furthermore, exposure to relatively non-toxic Fe@CeO 2 NPs, but not the toxic Cr@CeO 2 NPs, resulted in increased binding of MLL1 complex, a major histone lysine methylase mediating trimethylation of histone H3 lysine 4, at the NRF2 promoter. Taken together, our findings strongly suggest that failure of cells to respond to oxidative stress is critical for dopant-dependent toxicity of CeO 2 NPs and emphasize that careful evaluation of newly developed NPs should be preceded before industrial or biomedical applications.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal gastrointestinal malignancies due to its characteristics of local invasion and distant metastasis. Purine element binding protein α (PURα) is a DNA and RNA binding protein, and recent studies have showed that abnormal expression of PURα is associated with the progression of some tumors, but its oncogenic function, especially in ESCC progression, has not been determined. Based on the bioinformatic analysis of RNA-seq and ChIP-seq data, we found that PURα affected metabolic pathways, including oxidative phosphorylation and fatty acid metabolism, and we observed that it has binding peaks in the promoter of mitochondrial phosphoenolpyruvate carboxykinase (PCK2). Meanwhile, PURα significantly increased the activity of the PCK2 gene promoter by binding to the GGGAGGCGGA motif, as determined though luciferase assay and ChIP-PCR/qPCR. The results of Western blotting and qRT-PCR analysis showed that PURα overexpression enhances the protein and mRNA levels of PCK2 in KYSE510 cells, whereas PURα knockdown inhibits the protein and mRNA levels of PCK2 in KYSE170 cells. In addition, measurements of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) indicated that PURα promoted the metabolism of ESCC cells. Taken together, our results help to elucidate the molecular mechanism by which PURα activates the transcription and expression of PCK2, which contributes to the development of a new therapeutic target for ESCC.

Single-minded homologue 1 (SIM1) is a transcription factor with numerous different physiological and developmental functions. SIM1 is a member of the class I basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor family, that includes several other conserved proteins, including the hypoxia-inducible factors, aryl hydrocarbon receptor, neuronal PAS proteins, and the CLOCK circadian regulator. Recent studies of HIF-a-ARNT and CLOCK-BMAL1 protein complexes have revealed the organization of their bHLH, PASA, and PASB domains and provided insight into how these heterodimeric protein complexes form; however, experimental structures for SIM1 have been lacking. Here, we describe the first full-length atomic structural model for human SIM1 with its binding partner ARNT in a heterodimeric complex and analyze several pathogenic variants utilizing state-of-the-art simulations and algorithms. Using local and global positional deviation metrics, deductions to the structural basis for the individual mutants are addressed in terms of the deleterious structural reorganizations that could alter protein function. We propose new experiments to probe these hypotheses and examine an interesting SIM1 dynamic behavior. The conformational dynamics demonstrates conformational changes on local and global regions that represent a mechanism for dysfunction in variants presented. In addition, we used our ab initio hybrid model for further prediction of variant hotspots that can be engineered to test for counter variant (restoration of wild-type function) or basic research probe.

Downy Mildew Resistance 6-like (DMR6-like) genes are identified as salicylic acid (SA) hydroxylases and negative regulators of plant immunity. Previously, we identified two rice DMR6-like genes, OsF3H03g, and OsF3H04g , that act as susceptible targets of transcription activator-like effectors (TALEs) from Xanthomonas oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak (BLS) in rice. Furthermore, all four homologs of rice DMR6-like proteins were identified to predominantly carry the enzyme activity of SA 5-hydroxylase (S5H), negatively regulate rice broad-spectrum resistance, and cause the loss of function of these OsDMR6s , leading to increased resistance to rice blast and bacterial blight (BB). Here, we curiously found that an OsF3H04g knock-out mutant created by T-DNA insertion, osf3h04g , was remarkedly susceptible to BLS and BB and showed an extreme reduction in SA content. OsF3H04g knock-out rice lines produced by gene-editing were mildly susceptible to BLS and reduced content of SA. To explore the susceptibility mechanism in OsF3H04g loss-of-function rice lines, transcriptome sequencing revealed that another homolog, OsS3H , had induced expression in the loss-of-function OsF3H04g rice lines. Furthermore, we confirmed that a great induction of OsS3H downstream and genomically adjacent to OsF3H04g in osf3h04g was primarily related to the inserted T-DNA carrying quadruple enhancer elements of 35S , while a slight induction was caused by an unknown mechanism in gene-editing lines. Then, we found that the overexpression of OsS3H increased rice susceptibility to BLS, while gene-editing mediated the loss-of-function OsS3H enhanced rice resistance to BLS. However, the knock-out of both OsF3H04g and OsS3H by gene-editing only neutralized rice resistance to BLS. Thus, we concluded that the knock-out of OsF3H04g activated the expression of the OsS3H , partially participating in the susceptibility to BLS in rice.

The SMYD family is a unique class of lysine methyltransferases (KMTases) whose catalytic SET domain is split by a MYND domain. Among these, Smyd1 was identified as a heart- and skeletal muscle-specific KMTase and is essential for cardiogenesis and skeletal muscle development. SMYD1 has been characterized as a histone methyltransferase (HMTase). Here we demonstrated that SMYD1 methylates is the Skeletal muscle-specific splice variant of the Nascent polypeptide-Associated Complex (skNAC) transcription factor. SMYD1-mediated methylation of skNAC targets K1975 within the carboxy-terminus region of skNAC. Catalysis requires physical interaction of SMYD1 and skNAC via the conserved MYND domain of SMYD1 and the PXLXP motif of skNAC. Our data indicated that skNAC methylation is required for the direct transcriptional activation of myoglobin ( Mb ), a heart- and skeletal muscle-specific hemoprotein that facilitates oxygen transport. Our study revealed that the skNAC, as a methylation target of SMYD1, illuminates the molecular mechanism by which SMYD1 cooperates with skNAC to regulate transcriptional activation of genes crucial for muscle functions and implicates the MYND domain of the SMYD-family KMTases as an adaptor to target substrates for methylation.

Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is highly correlated with many human diseases, but the underlying molecular mechanisms remain to be fully uncovered. GATA4 is a conserved GATA family transcription factor that is essential for cardiac and dorsal epidermal development. Here, we describe a novel interaction between Drosophila Nrf2 and GATA4 proteins, i.e., cap'n'collar C (CncC) and Pannier (Pnr), respectively. Using the bimolecular fluorescence complementation (BiFC) assay-a unique imaging tool for probing protein complexes in living cells-we detected CncC-Pnr complexes in the nuclei of Drosophila embryonic and salivary gland cells. Visualization of CncC-Pnr BiFC signals on the polytene chromosome revealed that CncC and Pnr tend to form complexes in euchromatic regions, with a preference for loci that are not highly occupied by CncC or Pnr alone. Most genes within these loci are activated by the CncC-Pnr BiFC, but not by individually expressed CncC or Pnr fusion proteins, indicating a novel mechanism whereby CncC and Pnr interact at specific genomic loci and coactivate genes at these loci. Finally, CncC-induced early lethality can be rescued by Pnr depletion, suggesting that CncC and Pnr function in the same genetic pathway during the early development of Drosophila . Taken together, these results elucidate a novel crosstalk between the Nrf2 xenobiotic/oxidative response factor and GATA factors in the transcriptional regulation of development. This study also demonstrates that the polytene chromosome BiFC assay is a valuable tool for mapping genes that are targeted by specific transcription factor complexes.

Histone acetylation or deacetylation is closely associated with the progression of multiple myeloma (MM). Currently, many histone deacetylase (HDAC) inhibitors have been approved for being used in clinical trials, but theirtherapeutic effectsarestill not ideal. As a novel HDAC inhibitor, hydroxamicacid-based small-molecule N -hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB)’s possible roles in MM have not been studied. In this present study, the effect of HPOB as a potential anti-tumor agent in preventingproliferation and inducing apoptosis of MM cells had been investigated in detail. Our results showed that HPOB decreased the survival of MM cells in dose- and time-dependent manner. In addition, HPOB caused the accumulation of MM cells in G1 phase compared with the dimethylsulfoxide (DMSO) control group. Interestingly, we found that HPOB could overcome bortezomib (BTZ) resistance inMM cells and combining HPOB with BTZ could further sensitize MM cells. Certainly, our data illuminated that HPOB-mediated cell death occurs via transcriptional activation of p21, which was associated with an elevated level of global histone 3 acetylation (H3Ac) modification. Therefore, HPOB could be a potential candidate for MM treatment and the combination of HPOB and bortezomibcould bea possible therapeutic strategy for relapsed and refractory MM.

Kojic acid (KA) is a fungal metabolite and has a variety of applications in the cosmetics and food industries. Aspergillus oryzae is a well-known producer of KA, and its KA biosynthesis gene cluster has been identified. In this study, we showed that nearly all section Flavi aspergilli except for A. avenaceus had complete KA gene clusters, and only one Penicillium species, P. nordicum, contained a partial KA gene cluster. Phylogenetic inference based on KA gene cluster sequences consistently grouped section Flavi aspergilli into clades as prior studies. The Zn(II)2Cys6 zinc cluster regulator KojR transcriptionally activated clustered genes of kojA and kojT in Aspergillus flavus. This was evidenced by the time-course expression of both genes in kojR-overexpressing strains whose kojR expression was driven by a heterologous Aspergillus nidulans gpdA promoter or a homologous A. flavus gpiA promoter. Using sequences from the kojA and kojT promoter regions of section Flavi aspergilli for motif analyses, we identified a consensus KojR-binding motif to be an 11-bp palindromic sequence of 5′-CGRCTWAGYCG-3′ (R = A/G, W = A/T, Y = C/T). A CRISPR/Cas9-mediated gene-targeting technique showed that the motif sequence, 5′-CGACTTTGCCG-3′, in the kojA promoter was critical for KA biosynthesis in A. flavus. Our findings may facilitate strain improvement and benefit future kojic acid production. [ABSTRACT FROM AUTHOR]

Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53–Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it. [ABSTRACT FROM AUTHOR]

Non-viral gene delivery has become a popular approach in tissue engineering, as it permits the transient delivery of a therapeutic gene, in order to stimulate tissue repair. However, the efficacy of non-viral delivery vectors remains an issue. Our lab has created gene-activated scaffolds by incorporating various non-viral delivery vectors, including the glycosaminoglycan-binding enhanced transduction (GET) peptide into collagen-based scaffolds with proven osteogenic potential. A modification to the GET peptide (FLR) by substitution of arginine residues with histidine (FLH) has been designed to enhance plasmid DNA (pDNA) delivery. In this study, we complexed pDNA with combinations of FLR and FLH peptides, termed GET* nanoparticles. We sought to enhance our gene-activated scaffold platform by incorporating GET* nanoparticles into collagen-nanohydroxyapatite scaffolds with proven osteogenic capacity. GET* N/P 8 was shown to be the most effective formulation for delivery to MSCs in 2D. Furthermore, GET* N/P 8 nanoparticles incorporated into collagen-nanohydroxyapatite (coll-nHA) scaffolds at a 1:1 ratio of collagen:nanohydroxyapatite was shown to be the optimal gene-activated scaffold. pDNA encoding stromal-derived factor 1α (pSDF-1α), an angiogenic chemokine which plays a role in BMP mediated differentiation of MSCs, was then delivered to MSCs using our optimised gene-activated scaffold platform, with the aim of significantly increasing angiogenesis as an important precursor to bone repair. The GET* N/P 8 coll-nHA scaffolds successfully delivered pSDF-1α to MSCs, resulting in a significant, sustained increase in SDF-1α protein production and an enhanced angiogenic effect, a key precursor in the early stages of bone repair.

Smad7 has been identified as a negative regulator of the transforming growth factor TGF-β pathway by direct interaction with the TGF-β type I receptor (TβR-I). Although Smad7 has also been shown to play TGF-β unrelated functions in the cytoplasm and in the nucleus, a comprehensive analysis of its nuclear function has not yet been performed. Here, we show that in ESCs Smad7 is mainly nuclear and acts as a general transcription factor regulating several genes unrelated to the TGF-β pathway. Loss of Smad7 results in the downregulation of several key stemness master regulators, including Pou5f1 and Zfp42 , and in the upregulation of developmental genes, with consequent loss of the stem phenotype. Integrative analysis of genome-wide mapping data for Smad7 and ESC self-renewal and pluripotency transcriptional regulators revealed that Smad7 co-occupies promoters of highly expressed key stemness regulators genes, by binding to a specific consensus response element NCGGAAMM. Altogether, our data establishes Smad7 as a new, integral component of the regulatory circuitry that controls ESC identity.

Endogenous DNA damage occurs throughout the cell cycle, with cells responding differently at various stages. The base excision repair (BER) pathway predominantly repairs damaged bases in the genome. While extensively studied in interphase cells, it is unknown if BER operates in mitosis and how apurinic/apyrimidinic (AP) sites, intermediates in the BER pathway that inhibit transcriptional elongation, are processed for post-mitotic gene reactivation. In this study, using an alkaline comet assay, we demonstrate that BER is inefficient in mitosis and that AP endonuclease 1 (APE1), a key BER enzyme, is required for the repair of damage post-mitosis. We previously demonstrated that APE1 is acetylated (AcAPE1) in the chromatin. Using high-resolution microscopy, we show that AcAPE1 remains associated with specific regions in the condensed chromatin in each of the phases of mitosis. This association presumably occurs via the binding of APE1 to the G-quadruplex structure, a non-canonical DNA structure predominantly present in the transcribed gene regions. Additionally, using a nascent RNA detection strategy, we demonstrate that the knockdown of APE1 delayed the rapid post-mitotic transcriptional reactivation of genes. Our findings highlight the functional importance of APE1 in the mitotic chromosomes to facilitate faster repair of endogenous damage and rapid post-mitotic gene reactivation in daughter cells.

Plants may encounter abiotic stresses, such as drought, flooding, salinity, and extreme temperatures, thereby negatively affecting their growth, development, and reproduction. In order to enhance their tolerance to such stresses, plants have developed intricate signaling networks that regulate stress-responsive gene expression. For example, Arabidopsis Enhanced Drought Tolerance1/HOMEODOMAIN GLABROUS 11 ( AtEDT1/HDG11 ), one of the transcription factor genes from the group IV of homeodomain-leucine zipper (HD-ZIP) gene family, has been shown to increase drought tolerance in various transgenic plants. However, the underlying molecular mechanisms of enhanced stress tolerance remain unclear. In this study, we identified a homologous gene related to AtEDT1/HDG11 , named FaTEDT1L , from the transcriptome sequencing database of cultivated strawberry. Phylogenetic analysis revealed the close relationship of FaTEDT1L with AtEDT1/HDG11, which is one of the group IV members of the HD-ZIP gene family. Yeast one-hybrid analysis showed that FaTEDT1L functions as a transcriptional activator. Transgenic Arabidopsis plants overexpressing FaTEDT1L under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited significantly enhanced tolerance to osmotic stress (both drought and salinity) when compared to the wild-type (WT) plants. Under osmotic stress, the average root length was 3.63 ± 0.83 cm, 4.20 ± 1.03 cm, and 4.60 ± 1.14 cm for WT, 35S::FaTEDT1L T 2 #3 , and 35S:: FaTEDT1L T 2 #5 , respectively. Substantially increased root length in 35S::FaTEDT1L T 2 #3 and 35S::FaTEDT1L T 2 #5 was noted when compared to the WT. In addition, the average water loss rates were 64%, 57.1%, and 55.6% for WT, 35S::FaTEDT1L T2 #3 , and 35S::FaTEDT1L T 2 #5 , respectively, after drought treatment, indicating a significant decrease in water loss rate of 35S:: FaTEDT1L T 2 #3 and 35S::FaTEDT1L T 2 #5 is a critical factor in enhancing plant drought resistance. These findings thus highlight the crucial role of FaTEDT1L in mitigating drought and salt stresses and regulating plant osmotic stress tolerance. Altogether, FaTEDT1L shows its potential usage as a candidate gene for strawberry breeding in improving crop resilience and increasing agricultural productivity under adverse environmental conditions.

RNA activation (RNAa) is a mechanism whereby RNA oligos complementary to genomic sequences around the promoter region of genes increase the transcription output of their target gene. Small activating RNA (saRNA) mediate RNAa through interaction with protein co-factors to facilitate RNA polymerase II activity and nucleosome remodeling. As saRNA are small, versatile and safe, they represent a new class of therapeutics that can rescue the downregulation of critical genes in disease settings. This review highlights our current understanding of saRNA biology and describes various examples of how saRNA are successfully used to treat various oncological, neurological and monogenic diseases. MTL-CEBPA, a first-in-class compound that reverses CEBPA downregulation in oncogenic processes using CEBPA-51 saRNA has entered clinical trial for the treatment of hepatocellular carcinoma (HCC). Preclinical models demonstrate that MTL-CEBPA reverses the immunosuppressive effects of myeloid cells and allows for the synergistic enhancement of other anticancer drugs. Encouraging results led to the initiation of a clinical trial combining MTL-CEBPA with a PD-1 inhibitor for treatment of solid tumors.

Activating transcription factor 3 (ATF3), a transcription factor and acute stress sensor, is rapidly induced by a variety of pathophysiological signals and is essential in the complex processes in cellular stress response. FOXP3, a well-known breast and prostate tumor suppressor from the X chromosome, is a novel transcriptional repressor for several oncogenes. However, it remains unknown whether ATF3 is the target protein of FOXP3. Herein, we demonstrate that ATF3 expression is regulated by FOXP3. Firstly, we observed that overexpression of FOXP3 reduced ATF3 protein level. Moreover, knockdown FOXP3 by siRNA increased ATF3 expression. Secondly, FOXP3 dose-dependently reduced ATF3 promoter activity in the luciferase reporter assay. Since FOXP3 is regulated by post-translational modifications (PTMs), we next investigated whether PTMs affect FOXP3-mediated ATF3 expression. Interestingly, we observed that phosphorylation mutation on FOXP3 (Y342F) significantly abolished FOXP3-mediated ATF3 expression. However, other PTM mutations on FOXP3, including S418 phosphorylation, K263 acetylation and ubiquitination, and K268 acetylation and ubiquitination, did not alter FOXP3-mediated ATF3 expression. Finally, the FOXP3 binding site was found on ATF3 promoter region by deletion and mutagenesis analysis. Taken together, our results suggest that FOXP3 functions as a novel regulator of ATF3 and that this novel event may be involved in tumor development and progression.

Aryl hydrocarbon receptors (AHRs), a class of ligand-dependent nuclear receptors that regulate cellular responses by inducing the expression of various target genes in response to external signals, are implicated in maintaining retinal tissue homeostasis. Previous studies have shown that the regulation of AHR-induced gene expression requires transcriptional co-regulators. However, it is not yet clear how chromatin remodelers, histone methyltransferases and coactivators interact during AHR-mediated gene expression in human retinal cells. In this study, we reveal that the histone methyltransferase MLL1 and the coactivator FLII are involved in AHR-mediated gene expression in retinal pigment epithelial cells. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly increased the expression of CYP1A1, CYP1B1 and AHRR in ARPE-19 cells, whereas FLII or MLL1 depletion significantly reduced the expression of these genes induced by TCDD. Mechanistically, FLII binds to AHR in a ligand-dependent manner in ARPE-19 cells. In particular, the binding of FLII to MLL1 occurs through the GelB domain of FLII. In addition, MLL1 binds to AHR in a ligand-independent manner. FLII is involved in the recruitment of the BRG1 chromatin remodeler and MLL1 histone methyltransferase to the AHR-regulated CYP1A1 gene region in ARPE-19 cells and consequently, plays an important role in RNA polymerase II binding and transcriptional activity by modulating chromatin accessibility. Our results identify the functions and mechanisms of action of FLII and MLL1 in AHR-induced gene expression in human retinal pigment epithelial cells.

Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding.

RNA-based regulators are promising tools for building synthetic biological systems that provide a powerful platform for achieving a complex regulation of transcription and translation. Recently, de novo-designed synthetic RNA regulators, such as the small transcriptional activating RNA (STAR), toehold switch (THS), and three-way junction (3WJ) repressor, have been utilized to construct RNA-based synthetic gene circuits in living cells. In this work, we utilized these regulators to construct type 1 incoherent feed-forward loop (IFFL) circuits in vivo and explored their dynamic behaviors. A combination of a STAR and 3WJ repressor was used to construct an RNA-only IFFL circuit. However, due to the fast kinetics of RNA-RNA interactions, there was no significant timescale difference between the direct activation and the indirect inhibition, that no pulse was observed in the experiments. These findings were confirmed with mechanistic modeling and simulation results for a wider range of conditions. To increase delay in the inhibition pathway, we introduced a protein synthesis process to the circuit and designed an RNA-protein hybrid IFFL circuit using THS and TetR protein. Simulation results indicated that pulse generation could be achieved with this RNA-protein hybrid model, and this was further verified with experimental realization in E. coli . Our findings demonstrate that while RNA-based regulators excel in speed as compared to protein-based regulators, the fast reaction kinetics of RNA-based regulators could also undermine the functionality of a circuit (e.g., lack of significant timescale difference). The agreement between experiments and simulations suggests that the mechanistic modeling can help debug issues and validate the hypothesis in designing a new circuit. Moreover, the applicability of the kinetic parameters extracted from the RNA-only circuit to the RNA-protein hybrid circuit also indicates the modularity of RNA-based regulators when used in a different context. We anticipate the findings of this work to guide the future design of gene circuits that rely heavily on the dynamics of RNA-based regulators, in terms of both modeling and experimental realization.

Phospholipids are crucial materials that are not only required for cell membrane construction but also play significant roles as signaling molecules. LPIN1 is an enzyme that displays phosphatidate phosphatase activity in the triglyceride and phospholipid synthesis pathway. Recent studies have shown that overexpression of LPIN1 is involved in breast tumorigenesis, but the underlying mechanism regulating LPIN1 expression has not been elucidated yet. In the present study, we showed that the IL-33-induced COT-JNK1/2 signaling pathway regulates LPIN1 mRNA and protein expression by recruiting c-Jun to the LPIN1 promoter in breast cancer cells. IL-33 dose-dependently and time-dependently increased LPIN1 mRNA and protein expression. Moreover, IL-33 promoted colony formation and mammary tumorigenesis via induction of LPIN1 expression, while inhibition of LPIN1 disturbed IL-33-induced cell proliferation and mammary tumorigenesis. IL-33-driven LPIN1 expression was mediated by the COT-JNK1/2 signaling pathway, and inhibition of COT or JNK1/2 reduced LPIN1 expression. COT-JNK1/2-mediated IL-33 signaling activated c-Jun and promoted its binding to the promoter region of LPIN1 to induce LPIN1 expression. These findings demonstrated the regulatory mechanism of LPIN1 transcription by the IL-33-induced COT/JNK1/2 pathway for the first time, providing a potential mechanism underlying the upregulation of LPIN1 in cancer.

The immune system defends against invading pathogens through the rapid activation of innate immune signaling pathways. Interferon regulatory factor 3 (IRF3) is a key transcription factor activated in response to virus infection and is largely responsible for establishing an antiviral state in the infected host. Studies in Irf3 -/- mice have demonstrated the absence of IRF3 imparts a high degree of susceptibility to a wide range of viral infections. Virus infection causes the activation of IRF3 to transcribe type-I interferon (e.g., IFNβ), which is responsible for inducing the interferon-stimulated genes (ISGs), which act at specific stages to limit virus replication. In addition to its transcriptional function, IRF3 is also activated to trigger apoptosis of virus-infected cells, as a mechanism to restrict virus spread within the host, in a pathway called RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA). These dual functions of IRF3 work in concert to mediate protective immunity against virus infection. These two pathways are activated differentially by the posttranslational modifications (PTMs) of IRF3. Moreover, PTMs regulate not only IRF3 activation and function, but also protein stability. Consequently, many viruses utilize viral proteins or hijack cellular enzymes to inhibit IRF3 functions. This review will describe the PTMs that regulate IRF3's RIPA and transcriptional activities and use coronavirus as a model virus capable of antagonizing IRF3-mediated innate immune responses. A thorough understanding of the cellular control of IRF3 and the mechanisms that viruses use to subvert this system is critical for developing novel therapies for virus-induced pathologies.

MYC is a proto-oncogene regulating a large number of genes involved in a plethora of cellular functions. Its deregulation results in activation of MYC gene expression and/or an increase in MYC protein stability. MYC overexpression is a hallmark of malignant growth, inducing self-renewal of stem cells and blocking senescence and cell differentiation. This review summarizes the latest advances in our understanding of MYC-mediated molecular mechanisms responsible for its oncogenic activity. Several recent findings indicate that MYC is a regulator of cancer genome and epigenome: MYC modulates expression of target genes in a site-specific manner, by recruiting chromatin remodeling co-factors at promoter regions, and at genome-wide level, by regulating the expression of several epigenetic modifiers that alter the entire chromatin structure. We also discuss novel emerging therapeutic strategies based on both direct modulation of MYC and its epigenetic cofactors.

The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A , has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.

Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2 . We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.

Medicinal mushrooms of the genus Hericium are known to produce secondary metabolites with homeostatic properties for the central nervous system. We and others have recently demonstrated that among these metabolites cyathane diterpenoids and in particular erinacine C possess potent neurotrophin inducing properties in astrocytic cells. Yet, the signaling events downstream of erinacine C induced neurotrophin acitivity in neural-like adrenal phaeochromocytoma cells (PC12) cells have remained elusive. Similar, signaling events activated by erinacine C in astrocytic cells are unknown. Using a combination of genetic and pharmacological inhibitors we show that erinacine C induced neurotrophic activity mediates PC12 cell differentiation via the TrkA receptor and likely its associated PLCγ-, PI3K-, and MAPK/ERK pathways. Furthermore, a small library of transcriptional activation reporters revealed that erinacine C induces transcriptional activation mediated by DNA consensus binding sites of selected conserved transcription factor families. Among these, transcription is activated from an ETS consensus in a concentration dependent manner. Interestingly, induced ETS-consensus transcription occurs in parallel and independent of neurotrophin induction. This finding helps to explain the many pleiotropic functions of cyathane diterpenoids. Moreover, our studies provide genetic access to cyathane diterpenoid functions in astrocytic cells and help to mechanistically understand the action of cyathanes in glial cells.

Bone morphogenetic protein 6 (BMP6) is a multifunctional growth factor involved in organ development and homeostasis. BMP6 controls expression of the liver hormone, hepcidin, and thereby plays a crucial role in regulating iron homeostasis. BMP6 gene transcriptional regulation in liver is largely unknown, but would be of great help to externally modulate iron load in pathologic conditions. Here, we describe a detailed molecular mechanism of hepatic BMP6 gene expression by an orphan nuclear receptor, estrogen-related receptor γ (ERRγ), in response to the pro-inflammatory cytokine interleukin 6 (IL-6). Recombinant IL-6 treatment increases hepatic ERRγ and BMP6 expression. Overexpression of ERRγ is sufficient to increase BMP6 gene expression in hepatocytes, suggesting that IL-6 is upstream of ERRγ. In line, knock-down of ERRγ in cell lines or a hepatocyte specific knock-out of ERRγ in mice significantly decreases IL-6 mediated BMP6 expression. Promoter studies show that ERRγ directly binds to the ERR response element (ERRE) in the mouse BMP6 gene promoter and positively regulates BMP6 gene transcription in IL-6 treatment conditions, which is further confirmed by ERRE mutated mBMP6-luciferase reporter assays. Finally, an inverse agonist of ERRγ, GSK5182, markedly inhibits IL-6 induced hepatic BMP6 expression in vitro and in vivo. Taken together, these results reveal a novel molecular mechanism on ERRγ mediated transcriptional regulation of hepatic BMP6 gene expression in response to IL-6.

AtERF73/HRE1 is an AP2/ERF transcription factor in Arabidopsis and has two distinct alternative splicing variants, HRE1α and HRE1β. In this study, we examined the differences between the molecular functions of HRE1α and HRE1β. We found that HRE1α and HRE1β are both involved in hypoxia response and root development and have transactivation activity. Two conserved motifs in the C-terminal region of HRE1α and HRE1β, EELL and LWSY-like, contributed to their transactivation activity, specifically the four E residues in the EELL motif and the MGLWS amino acid sequence at the end of the LWSY-like motif. The N-terminal region of HRE1β also showed transactivation activity, mediated by the VDDG motif, whereas that of HRE1α did not. The transactivation activity of HRE1β was stronger than that of HRE1α in Arabidopsis protoplasts. Both transcription factors transactivated downstream genes via the GCC box. RNA-sequencing analysis further supported that both HRE1α and HRE1β might regulate gene expression associated with the hypoxia stress response, although they may transactivate different subsets of genes in downstream pathways. Our results, together with previous studies, suggested that HRE1α and HRE1β differentially transactivate downstream genes in hypoxia response and root development in Arabidopsis .

NF-E2-related factor 2 (NRF2) is a basic leucine zipper transcription factor, a master regulator of redox homeostasis regulating a variety of genes for antioxidant and detoxification enzymes. NRF2 was, therefore, initially thought to protect the liver from oxidative stress. Recent studies, however, have revealed that mutations in NRF2 cause aberrant accumulation of NRF2 in the nucleus and exert the upregulation of NRF2 target genes. Moreover, among all molecular changes in hepatocellular carcinoma (HCC), NRF2 activation has been revealed as a more prominent pathway contributing to the progression of precancerous lesions to malignancy. Nevertheless, how its activation leads to poor prognosis in HCC patients remains unclear. In this review, we provide an overview of how aberrant activation of NRF2 triggers HCC development. We also summarize the emerging roles of other NRF family members in liver cancer development.

The assembly of mitochondrial oxidative phosphorylation (OXPHOS) complexes is an intricate process, which-given their dual-genetic control-requires tight co-regulation of two evolutionarily distinct gene expression machineries. Moreover, fine-tuning protein synthesis to the nascent assembly of OXPHOS complexes requires regulatory mechanisms such as translational plasticity and translational activators that can coordinate mitochondrial translation with the import of nuclear-encoded mitochondrial proteins. The intricacy of OXPHOS complex biogenesis is further evidenced by the requirement of many tightly orchestrated steps and ancillary factors. Early-stage ancillary chaperones have essential roles in coordinating OXPHOS assembly, whilst late-stage assembly factors-also known as the LYRM (leucine-tyrosine-arginine motif) proteins-together with the mitochondrial acyl carrier protein (ACP)-regulate the incorporation and activation of late-incorporating OXPHOS subunits and/or co-factors. In this review, we describe recent discoveries providing insights into the mechanisms required for optimal OXPHOS biogenesis, including the coordination of mitochondrial gene expression with the availability of nuclear-encoded factors entering via mitochondrial protein import systems., Competing Interests: The authors declare no conflicts of interest.

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The trans-activator protein Tax of HTLV-1 plays crucial roles in leukemogenesis by promoting proliferation of virus-infected cells through activation of growth-promoting genes. However, critical target genes are yet to be elucidated. We show here that Tax activates the gene coding for cyclin-dependent kinase 7 (CDK7), the essential component of both CDK-activating kinase (CAK) and general transcription factor TFIIH. CAK and TFIIH play essential roles in cell cycle progression and transcription by activating CDKs and facilitating transcriptional initiation, respectively. Tax induced CDK7 gene expression not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs) along with increased protein expression. Tax stimulated phosphorylation of CDK2 and RNA polymerase II at sites reported to be mediated by CDK7. Tax activated the CDK7 promoter through the NF-κB pathway, which mainly mediates cell growth promotion by Tax. Knockdown of CDK7 expression reduced Tax-mediated induction of target gene expression and cell cycle progression. These results suggest that the CDK7 gene is a crucial target of Tax-mediated trans-activation to promote cell proliferation by activating CDKs and transcription.

Zygotic genome activation (ZGA) is a pivotal event in mammalian embryogenesis, marking the transition from maternal to zygotic control of development. During the ZGA process that is characterized by the intricate cascade of gene expression, who tipped the first domino in a meticulously arranged sequence is a subject of paramount interest. Recently, Dux, Obox and Nr5a2 were identified as pioneer transcription factors that reside at the top of transcriptional hierarchy. Through co-option of retrotransposon elements as hubs for transcriptional activation, these pioneer transcription factors rewire the gene regulatory network, thus initiating ZGA. In this review, we provide a snapshot of the mechanisms underlying the functions of these pioneer transcription factors. We propose that ZGA is the starting point where the embryo's own genome begins to influence development trajectory, therefore in-depth dissecting the functions of pioneer transcription factors during ZGA will form a cornerstone of our understanding for early embryonic development, which will pave the way for advancing our grasp of mammalian developmental biology and optimizing in vitro production (IVP) techniques.