Elevated Transcriptional Activity Research Articles

Andean Enriched NFKB1 Haplotype Reduces Inflammation and Improves Response to Ropeginterferon Alfa-2b in Polycythemia Vera (PV) and Essential Thrombocythemia (ET)

Chronic inflammation is a feature of polycythemia vera (PV), essential thrombocythemia (ET), and other myeloproliferative neoplasms (MPNs). NF-κB plays a key role in regulating pro-inflammatory cytokines. In PV and ET, activation of the NF-κB pathway is a primary driver of inflammation and augments hypoxia-inducible factors (HIFs) activity, increasing transcription of prothrombotic genes. Suppression of the NF-κB pathway has been shown to reduce disease burden in MPNs (Laranjeira, Blood, 2024).We investigated the genetic basis underlying elevated hemoglobin in Andean natives, Aymara, by using granulocyte transcriptome data from both Aymara and Europeans from La Paz, Bolivia. We found their differentially expressed and spliced genes are associated with inflammatory and HIFs pathways. We identified three novel, alternatively spliced NFKB1 transcripts (AS-NFKB1) in Aymara, with skipped exon 4, exon 5, or both. The AS-NFKB1 transcript with exon 5 skipped was translated into protein but showed a defect in nuclear translocation, while the other two AS-NFKB1 transcripts were not translated, indicating a partial or complete loss of NFKB1's function as an NF-κB suppressor. AS-NFKB1 also augmented activity of HIFs, explaining high Aymara hemoglobin. Using integrative analysis of whole-genome sequencing and granulocyte transcriptome, we identified splicing quantitative trait locus associated with AS-NFKB1; NFKB1 rs230511 is linked to high AS-NFKB1 expression. Its T allele (C/T or T/T genotype) is present in approximately 90% of Aymara but is also present in ~30% of Europeans, Hispanics, and Asians. Under TNF-induced inflammatory stress, unlike wild-type NFKB1, AS-NFKB1 suppressed inflammatory gene expression, suggesting a protective mechanism against inflammation (under review, Nature Communication).Thus, we examined NFKB1 rs230511 association with inflammatory markers and response to ropeginterferon alfa-2b (Ropeg) therapy in PV and ET. We genotyped 30 PV and 15 ET patients, assessing inflammatory gene expression (IL1B, CXCL8, IL6, IL15, and TNF) in neutrophils and platelets. In platelets, individuals with the C/T genotype showed lower levels of these inflammatory transcripts compared to those with the C/C genotype and IL1B, CXCL8, and TNF levels were also reduced in the T/T genotype. In neutrophils, IL1B and CXCL8 were decreased in the T/T genotype, and IL15 and TNF levels were lower in the C/T genotype.Given that AS-NFKB1 and NFKB1 rs230511 are linked to elevated transcriptional activity of HIFs, we analyzed the expression of HIF-target genes. LDHA, SLC2A1, and VEGFA transcripts were significantly lower in the C/T genotype in both PV and ET neutrophils and platelets. Since increased inflammation and HIF transcriptional activity are associated with thrombosis in PV and ET (Gangaraju, Blood Advances, 2020), we also measured expression levels of prothrombotic genes (F3, THBD, SELP, SERPINE1). In both platelets and granulocytes, F3 and THBD transcript levels were significantly lower in individuals with the C/T genotype compared to those with the C/C genotype.Ropeg modulates inflammation via interferon signaling and attacks PV and ET clones (Verger, Blood Cancer J., 2018). We found that the C/T genotype (58.6%) was more prevalent among patients who achieved complete hematological response (CHR) compared to the non-CHR cohort, which included patients who did not reach CHR or had cytopenia (33.3%) (p<0.0001). This finding also correlated with lower transcript levels of inflammatory, HIF-targeted, and thrombotic genes in the CHR group compared to non-CHR group.Our study indicates that presence of Aymara enriched NFKB1 rs230511 haplotype decreases PV and ET inflammatory, prothrombotic, and HIF's activity compared to NFKB1 wild allele. The NFKB1 rs230511 haplotype also modulates response to Ropeg in PV and ET. The presence of NFKB1 rs230511 T allele is associated with reduced expression of inflammatory and HIF-targeted genes, potentially decreasing inflammation and thrombosis risk in PV and ET. Patients with the C/T genotype were more likely to achieve CHR, suggesting this NFKB1 haplotype may impact Ropeg therapy efficacy and serve as a potential biomarker for response assessment in PV and ET patients. These novel insights offer valuable implications for both of our understanding of inflammatory and prothrombotic processes and may also lead to novel PV and ET therapies.

The OsSRO1c-OsDREB2B complex undergoes protein phase transition to enhance cold tolerance in rice

Cold stress is one of the major abiotic stress factors affecting rice growth and development, leading to significant yield loss in the context of global climate change. Exploring natural variants that confer cold resistance and the underlying molecular mechanism responsible for this is the major strategy to breed cold-tolerant rice varieties. Here, we show that natural variations of a SIMILAR to RCD ONE (SRO) gene, OsSRO1c, confer cold tolerance in rice at both seedling and booting stages. Our in vivo and in vitro experiments demonstrated that OsSRO1c possesses intrinsic liquid–liquid phase-separation ability and recruits OsDREB2B, an AP2/ERF transcription factor that functions as a positive regulator of cold stress, into its biomolecular condensates in the nucleus, resulting in elevated transcriptional activity of OsDREB2B. We found that the OsSRO1c-OsDREB2B complex directly responds to low temperature through dynamic phase transitions and regulates key cold-response genes, including COLD1. Furthermore, we showed that introgression of an elite haplotype of OsSRO1c into a cold-susceptible indica rice could significantly increase its cold resistance. Collectively, our work reveals a novel cold-tolerance regulatory module in rice and provides promising genetic targets for molecular breeding of cold-tolerant rice varieties.

Elucidating the Role of Transcriptomic Networks and DNA Methylation in Collagen Deposition of Dezhou Donkey Skin.

DNA methylation represents a predominant epigenetic modification with broad implications in various biological functions. Its role is particularly significant in the process of collagen deposition, a fundamental aspect of dermal development in donkeys. Despite its critical involvement, the mechanistic insights into how DNA methylation influences collagen deposition in donkey skin remain limited. In this study, we employed whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) to investigate the epigenetic landscape and gene expression profiles in the dorsal skin tissues of Dezhou donkeys across three developmental stages: embryonic (YD), juvenile (2-year-old, MD), and mature (8-year-old, OD). Our analysis identified numerous differentially methylated genes that play pivotal roles in skin collagen deposition and overall skin maturation, including but not limited to COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, GLUL, SFRP2, FOSL1, SERPINE1, MMP1, MMP2, MMP9, and MMP13. Notably, we observed an inverse relationship between gene expression and DNA methylation proximal to transcription start sites (TSSs), whereas a direct correlation was detected in regions close to transcription termination sites (TTSs). Detailed bisulfite sequencing analyses of the COL1A1 promoter region revealed a low methylation status during the embryonic stage, correlating with elevated transcriptional activity and gene expression levels. Collectively, our findings elucidate key genetic markers associated with collagen deposition in the skin of Dezhou donkeys, underscoring the significant regulatory role of DNA methylation. This research work contributes to the foundational knowledge necessary for the genetic improvement and selective breeding of Dezhou donkeys, aiming to enhance skin quality attributes.

Abstract C062: Defining the interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. Non-Hispanic Black men are 1.8 times more likely to develop PCa and 2.3 times more likely to die from PCa. Tumor suppressor retinoblastoma (RB1) is frequently deleted in PCa. RB represses the transcriptional activity of E2Fs and the phosphorylation of RB by CDK4/6 results in dissociation of the complex and a subsequent increase in E2F transcriptional activity, leading to cell cycle progression. RB loss is associated with tumor aggressiveness occurring in 10-15% of castration-resistant prostate cancer (CRPC) tumors and corresponds to a decrease in overall survival. RB depletion leads to increased expression of DNA repair proteins due to elevated transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in DNA repair. Elevated PARP-1 enzymatic activity is associated with PCa progression and correlates with poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, however the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry. Defining the interplay between PARP and RB/E2F is likely to contribute to the identification of novel treatment strategies and/or biomarker response. Canonically, E2F1-3 are transcription factors that positively regulate gene expression programs associated with pro-proliferative signals. Prior research by other groups has shown that E2F target genes expression is elevated in tumors excised from Black men compared to Asian and White men. There was an increase in germline RAD51 mutations observed. RAD51 is an E2F target gene involved in DDR, therefore attenuating PARP enzymatic activity may be a potential mode of targeting this axis. These data suggest that there may be differential DNA damage response (DDR) in African American men driven by aberrations in the RB/E2F signaling axis, however the mechanism is yet to be fully explored. Analysis of publicly available patient datasets were performed and demonstrate that RB1 deletions is more frequent than RB1/BRCA2 co-deletion in metastatic PCa. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 mRNA and PARP-1 protein are elevated upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. In hormone-depleted conditions, results suggest a differential response between RB depleted models compared to the control when challenged with PARP inhibition. Furthermore, an IC50 dose of Olaparib elicits differential PARP enzymatic response in the RB1 depleted model. RB1 loss is associated with an enrichment in E2F1 binding at the PARP-1 locus. Future studies have been designed to determine the impact of the RB/E2F pathway alterations on downstream consequences PARP enzymatic function and establish the contribution of PARP and the RB/E2F axis on racial disparity in PCa. Citation Format: Latese Evans. Defining the interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C062.

Functional Dissection of Cellular Programs to Uncover Novel Gene Dependencies in AML

Genome-scale CRISPR-Cas9 screens have the power to unveil the Achilles' heel of neoplastic cells. Typically, analyses of such large-scale data sets focus on single gene dependencies. An alternative strategy is to evaluate functional networks enriched in a disease or molecular subset of interest. We applied this strategy to the Broad Institute's Cancer Dependency Map (DepMap) data set consisting of genome-scale CRISPR-Cas9 screens in over 1000 cancer cell line models. We hypothesized that interrogating enriched pathways with context-specific dependencies leads to the discovery of functionally informed gene dependencies. Single sample gene set enrichment analysis (ssGSEA) of over 1000 cancer cell lines in the DepMap identified hematologic malignancies to be highly enriched for signatures associated with elevated transcriptional activity. Notably, within hematologic malignancies, acute myeloid leukemia (AML) with a rearrangement in the KMT2A gene ( KMT2Ar-AML) was the most significantly enriched for dependency on transcriptional activity-associated gene signatures. We next interrogated KMT2Ar-AML gene dependencies (excluding common essential genes) with the human kinase database KinMap (http://www.kinhub.org/kinmap/) to identify readily druggable genes involved in transcriptional activity. Cyclin-dependent kinase 13 ( CDK13) was the only gene meeting these criteria. Further DepMap data analysis confirmed that KMT2Ar-AML cell lines were indeed more strongly dependent on CDK13 than all other cancer cell lines screened. Moreover, when we performed GSEA on the gene dependencies observed in CDK13-dependent versus non-dependent cell lines, we found transcriptional activity and KMT2A-target gene sets as top hits. We next validated AML cell dependency on CDK13 with orthogonal genetic approaches (CRISPR-Cas9 knock-out and shRNA) in vitro in human AML cell lines and cells from patient-derived xenograft (PDX) models of KMT2Ar-AML. Perturbation of CDK13 induced cell death with hallmarks of apoptosis. Doxycycline-inducible shRNA directed against CDK13 also impaired AML progression in the peripheral blood and bone marrow in an MV4-11- KMT2Ar xenograft. Moreover, in an orthotopic PDX model, doxycycline inducible knock-out of CDK13 in mice with established disease significantly reduced leukemia burden in the peripheral blood and bone marrow. CDK13 is reported to regulate transcriptional elongation and the clearance of prematurely terminated RNAs. To decipher why AML cells, particularly KMT2Ar, need CDK13 to survive, we identified the CDK13 binding sites and histone marks involved in transcriptional regulation in AML using CUT and RUN and ChIP-seq: We determined that CDK13 co-localizes with H3K4me3 and H3K27ac at promoter sites. Global gene expression studies following the knock-out of CDK13 revealed that the majority of CDK13 bound genes were also differentially expressed, with significantly more genes decreased than increased in expression after CDK13 knock-out. GSEA of CDK13-bound genes with decreased expression upon CDK13 knock-out identified gene signatures associated with transcriptional activation and KMT2A-targets. We next studied the characteristics of CDK13 target genes and observed a predominance of long genes (total length of gene exons &amp;gt; 10 kb, GRCh38.p12). GSEA of genes ranked by size, confirmed that longer genes were enriched for CDK13 target genes, CDK13 differential AML dependencies, and for KMT2A-target transcriptional signatures. Additionally, following CDK13 perturbation in KMT2Ar-AML we observed a decrease of Pol2/pPol2Ser2 occupancy at the transcription end site of long genes. In summary, using functional pathway analyses, we identified CDK13 as a candidate gene dependency enriched in KMT2Ar-AML, a target not immediately prioritized with single gene analysis. Mechanistically, our data support that CDK13 controls Pol2 processivity, with CDK13 perturbation leading to a decreased occupancy at the 3' end of long genes. KMT2Ar, CDK13 dependent cells are also enriched for dependency on long genes, thereby suggesting a molecular basis for the CDK13 dependency in KMT2Ar-AML.

Molecular basis for SOX2-dependent regulation of super-enhancer activity.

Pioneer transcription factors (TFs) like SOX2 are vital for stemness and cancer through enhancing gene expression within transcriptional condensates formed with coactivators, RNAsand mediators on super-enhancers (SEs). Despite their importance, how these factors work together for transcriptional condensation and activation remains unclear. SOX2, a pioneer TF found in SEs of pluripotent and cancer stem cells, initiates SE-mediated transcription by binding to nucleosomes, though the mechanism isn't fully understood. To address SOX2's role in SEs, we identified mSE078 as a model SOX2-enriched SE and p300 as a coactivator through bioinformatic analysis. In vitro and cell assays showed SOX2 forms condensates with p300 and SOX2-binding motifs in mSE078. We further proved that SOX2 condensation is highly correlated with mSE078's enhancer activity in cells. Moreover, we successfully demonstrated that p300 not only elevated transcriptional activity but also triggered chromatin acetylation via its direct interaction with SOX2 within these transcriptional condensates. Finally, our validation of SOX2-enriched SEs showcased their contribution to target gene expression in both stem cells and cancer cells. In its entirety, this study imparts valuable mechanistic insights into the collaborative interplay of SOX2 and its coactivator p300, shedding light on the regulation of transcriptional condensation and activation within SOX2-enriched SEs.

Molecular Genetic and Functional Analysis of the Genes Encoding Alkane 1‑Monoozygenase Synthesis in Members of the Genus &lt;i&gt;Rhodococcus&lt;/i&gt;

Abstract—Organization and localization of the alkB genes and of alkane 1-monooxygenases they encode in members of the genus Rhodococcus was investigated. All members of a phylogenetic group were found to possess specific types of the alkB genes (alkB1-type in the operons containing rubredoxin-coding genes, rubredoxin reductase, and a regulatory protein and/or alkB2-type in the operons lacking the rubredoxin-reductase-coding gene, and supplementary, separately located determinants of the alkB3−alkB8 type). The alkB1-type genes were present in the chromosomes of bacteria of groups B1 (R. aetherivorans, R. ruber), С (R. opacus, R. jostii, R. wratislaviensis, R. koreensis), D (R. erythropolis, R. qingshengii), G (R. triatomae), and E (R. fascians). The alkB2-type genes occurred in strictly specified loci of the chromosomes of members of the phylogenetic groups А (R. hoagii/R. equi), В2 (R. coprophilus, R. pyridinivorans, R. rhodochrous), and D (R. erythropolis, R. qingshengii). The separately located alkB3−alkB5 were revealed in the chromosomes of members of the D group D (alkB5 was found only in R. qingshengii), the alkB6 genes occurred in members of the groups B1 and В2, and alkB7−alkB8 were present in members of group Е. The proteins coded by the genes alkB1 and alkB2 belonged to three phylogenetic groups. The first one comprised AlkB1-type proteins of members of groups В1 and С and the AlkB2-type proteins of members of groups D and A. The second one was represented by AlkB2-type proteins of bacteria of group В2. The third group comprised AlkB1-type proteins of members of groups G and D. Alkane 1-monooxygenases encoded by separately localized genes alkB3−alkB8 were represented by three phylogenetic lineages: AlkB3−AlkB5, AlkB6, and AlkB7 and AlkB8. In the genome of R. pyridinivorans 5Ар, the genes alkB2 and alkB6 characteristic of group В1 were revealed. This determinants were shown to be required for biosurfactant synthesis. Emulsifying activity of the mutant with impaired alkB2 gene at 28 and 42°С was 16 and 3 times lower, respectively, while the amount of trehalose-containing surfactants decreased 7 and 3 times, respectively. Independent on the cultivation temperature, the mutant with impaired alkB6 gene exhibited 1.2 times lower emulsifying activity and more than twofold lower decrease of the synthesis of trehalose-containing surfactants. These results indicated the key role of alkB2 in biosurfactant synthesis at different cultivation temperatures. The role of alkB6 increased at 42°С, probably due to its elevated transcriptional activity.

Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering in glioblastoma stem cells.

Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size, and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional hubs, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harbouring distinct ecDNA species (EGFR, CDK4, PDGFRA). We find no evidence that ecDNA routinely cluster with one another or closely interact with transcriptional hubs. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy is similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that it is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with each other or with high concentrations of the transcriptional machinery.

Valproic Acid-Induced Upregulation of Multidrug Efflux Transporter ABCG2/BCRP via PPARα-Dependent Mechanism in Human Brain Endothelial Cells

Despite the progress made in the development of new antiepileptic drugs (AEDs), poor response to them is a rising concern in epilepsy treatment. Of several hypotheses explaining AED treatment failure, the most promising theory is the overexpression of multidrug transporters belonging to ATP-binding cassette (ABC) transporter family at blood-brain barrier. Previous data show that AEDs themselves can induce these transporters, in turn affecting their own brain bioavailability. Presently, this induction and the underlying regulatory mechanism involved at human blood-brain barrier is not well elucidated. Herein, we sought to explore the effect of most prescribed first- and second-line AEDs on multidrug transporters in human cerebral microvascular endothelial cells, hCMEC/D3. Our work demonstrated that exposure of these cells to valproic acid (VPA) induced mRNA, protein, and functional activity of breast cancer resistance protein (BCRP/ABCG2). On examining the substrate interaction status of AEDs with BCRP, VPA, phenytoin, and lamotrigine were found to be potential BCRP substrates. Furthermore, we observed that siRNA-mediated knockdown of peroxisome proliferator-activated receptor alpha (PPARα) or use of PPARα antagonist, resulted in attenuation of VPA-induced BCRP expression and transporter activity. VPA was found to increase PPARα expression and trigger its translocation from cytoplasm to nucleus. Findings from chromatin immunoprecipitation and luciferase assays showed that VPA enhances the binding of PPARα to its response element in the ABCG2 promoter, resulting in elevated ABCG2 transcriptional activity. Taken together, these in vitro findings highlight PPARα as the potential molecular target to prevent VPA-mediated BCRP induction, which may have important implications in VPA pharmacoresistance. SIGNIFICANCE STATEMENT: Induction of multidrug transporters at blood-brain barrier can largely affect the bioavailability of the substrate antiepileptic drugs in the brains of patients with epilepsy, thus affecting their therapeutic efficacy. The present study reports a mechanistic pathway of breast cancer resistance protein (BCRP/ABCG2) upregulation by valproic acid in human brain endothelial cells via peroxisome proliferator-activated receptor alpha involvement, thereby providing a potential strategy to prevent valproic acid pharmacoresistance in epilepsy.

Rb1 negatively regulates bone formation and remodeling through inhibiting transcriptional regulation of YAP in Glut1 and OPG expression and glucose metabolism in male mice

ObjectiveBone is a highly dynamic organ that undergoes constant bone formation and remodeling, and glucose as a major nutrient is necessary for bone formation and remodeling. Retinoblastoma (Rb1) is a critical regulator of mesenchymal stem cells (MSCs) fate, but how Rb1 regulates bone formation and remodeling is poorly understood. MethodsWe generated MSCs- and osteoprogenitors-specific Rb1 knockout mouse models and utilized these models to explore the function and mechanism of Rb1 in regulating bone formation and remodeling in vivo and in vitro primary cell culture. ResultsRb1 deficiency in MSCs significantly increased bone mass and impaired osteoclastogenesis. Consistently, depletion of Rb1 in osteoprogenitors significantly promoted bone formation. Mechanistically, loss of Rb1 in MSCs elevated YAP nuclear translocation and transcriptional activity of YAP/TEAD1 complex, thereby increasing the transcriptional expression of Glut1 and OPG. Moreover Prx1-Cre; Rb1f/f mice displayed hypoglycemia with increased systemic glucose tolerance instead of increased insulin level. In vitro data revealed that Rb1-mutant MSCs enhanced glucose uptake and lactate and ATP production. Increased osteogenesis caused by increased glucose metabolism and decreased osteoclastogenesis caused by increased expression of OPG eventually resulted in increased bone formation and remodeling. ConclusionsCollectively, these findings demonstrated that Rb1 in MSCs inhibits YAP-medicated Glut1 and OPG expression to control glucose metabolism, osteogenesis and osteoclastogenesis during bone formation and remodeling, which provide new insights that controlling Rb1 signaling may be a potential strategy for osteopetrosis.

TDP-43 regulates cholesterol biosynthesis by inhibiting sterol regulatory element-binding protein 2

Dyslipidemia is considered an essential component of the pathological process of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease. Although TAR DNA Binding Protein 43 kDa (TDP-43) links both familial and sporadic forms of ALS and cytoplasmic aggregates are a hallmark of most cases of ALS, the molecular mechanism and the in vivo relation of ALS dyslipidemia with TDP-43 have been unclear. To analyze the dyslipidemia-related gene expression by TDP-43, we performed expression microarray and RNA deep sequencing (RNA-Seq) using cell lines expressing high levels of TDP-43 and identified 434 significantly altered genes including sterol regulatory element-binding protein 2 (SREBP2), a master regulator of cholesterol homeostasis and its downstream genes. Elevated TDP-43 impaired SREBP2 transcriptional activity, leading to inhibition of cholesterol biosynthesis. The amount of cholesterol was significantly decreased in the spinal cords of TDP-43-overexpressed ALS model mice and in the cerebrospinal fluids of ALS patients. These results suggested that TDP-43 could play an essential role in cholesterol biosynthesis in relation to ALS dyslipidemia.

Oxymatrine inhibits the pyroptosis in rat insulinoma cells by affecting nuclear factor kappa B and nuclear factor (erythroid-derived 2)-like 2 protein/heme oxygenase-1 pathways.

As the mechanism underlying glucose metabolism regulation by oxymatrine is unclear, this study investigated the effects of oxymatrine on pyroptosis in INS-1 cells. Flow cytometry was employed to examine cell pyroptosis and reactive oxygen species (ROS) production. Cell pyroptosis was also investigated via transmission electron microscopy and lactate dehydrogenase (LDH) release. Protein levels were detected using western blotting and interleukin (IL)-1β and IL-18 secretion by enzyme-linked immunosorbent assay. The caspase-1 activity and DNA-binding activity of nuclear factor kappa B (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 protein (Nrf2) were also assessed. In the high glucose and high fat-treated INS-1 cells (HG + PA), the caspase-1 activity and LDH content, as well as Nod-like receptor family pyrin domain containing 3, Gsdmd-N, caspase-1, apoptosis-associated speck-like protein containing a CARD, IL-1β, and IL-18 levels were increased. Moreover, P65 protein levels increased in the nucleus but decreased in the cytoplasm. Oxymatrine attenuated these effects and suppressed high glucose and high fat-induced ROS production. The increased levels of nuclear Nrf2 and heme oxygenase-1 (HO-1) in the HG + PA cells were further elevated after oxymatrine treatment, whereas cytoplasmic Nrf2 and Keleh-like ECH-associated protein levels decreased. Additionally, the elevated transcriptional activity of p65 in HG + PA cells was reduced by oxymatrine, whereas that of Nrf2 increased. The results indicate that the inhibition of pyroptosis in INS-1 cells by oxymatrine, a key factor in its glucose metabolism regulation, involves the suppression of the NF-κB pathway and activation of the Nrf2/HO-1 pathway.

Inositol triphosphate–triggered calcium release from the endoplasmic reticulum induces lysosome biogenesis via TFEB/TFE3

Lysosomes serve as dynamic regulators of cell and organismal physiology by integrating the degradation of macromolecules with receptor and nutrient signaling. Previous studies have established that activation of the transcription factor EB (TFEB) and transcription factor E3 (TFE3) induces the expression of lysosomal genes and proteins in signaling-inactive starved cells, that is, under conditions when activity of the master regulator of nutrient-sensing signaling mechanistic target of rapamycin complex 1 is repressed. How lysosome biogenesis is triggered in signaling-active cells is incompletely understood. Here, we identify a role for calcium release from the lumen of the endoplasmic reticulum in the control of lysosome biogenesis that is independent of mechanistic target of rapamycin complex 1. We show using functional imaging that calcium efflux from endoplasmic reticulum stores induced by inositol triphosphate accumulation upon depletion of inositol polyphosphate-5-phosphatase A, an inositol 5-phosphatase downregulated in cancer and defective in spinocerebellar ataxia, or receptor-mediated phospholipase C activation leads to the induction of lysosome biogenesis. This mechanism involves calcineurin and the nuclear translocation and elevated transcriptional activity of TFEB/TFE3. Our findings reveal a crucial function for inositol polyphosphate-5-phosphatase A–mediated triphosphate hydrolysis in the control of lysosome biogenesis via TFEB/TFE3, thereby contributing to our understanding how cells are able to maintain their lysosome content under conditions of active receptor and nutrient signaling.

Synovial fluid from end-stage osteoarthritis induces proliferation and fibrosis of articular chondrocytes via MAPK and RhoGTPase signaling

Alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Here, we examined the phenotypic and proliferative behavior of human articular chondrocytes when cultured in vitro for 13 days with synovial fluid derived from end-stage osteoarthritis patients. Chondrocyte proliferation and phenotypical changes induced by osteoarthritic synovial fluid were analyzed using DNA staining, RT-qPCR, immunostainings, and immunoblotting. The molecular mechanisms by which osteoarthritic synovial fluid induced fibrosis and proliferation were studied using a phospho-protein antibody array and luciferase-based transcription factor activity assays. Specific pathway inhibitors were used to probe the involvement of pathways in fibrosis and proliferation. Prolonged stimulation with osteoarthritic synovial fluid sustained chondrocyte proliferation and induced profound phenotypic changes, favoring a fibrotic over a chondrogenic or hypertrophic phenotype. A clear loss of chondrogenic markers at both the transcriptional and protein level was observed, while expression of several fibrosis-associated markers were upregulated over time. Phospho-kinase analysis revealed activation of MAPK and RhoGTPase signaling pathways by osteoarthritic synovial fluid, which was confirmed by elevated transcriptional activity of Elk-1 and SRF. Inhibitor studies revealed that ERK played a central role in the loss of chondrocyte phenotype, while EGFR and downstream mediators p38, JNK and Rac/Cdc42 were essential for fibrosis-associated collagen expression. Finally, we identified EGF signaling as a key activator of chondrocyte proliferation. Osteoarthritic synovial fluid promoted chondrocyte fibrosis and proliferation through EGF receptor activation and downstream MAPK and RhoGTPase signaling.

SNP rs4971059 predisposes to breast carcinogenesis and chemoresistance via TRIM46-mediated HDAC1 degradation.

Identification of the driving force behind malignant transformation holds the promise to combat the relapse and therapeutic resistance of cancer. We report here that the single nucleotide polymorphism (SNP) rs4971059, one of 65 new breast cancer risk loci identified in a recent genome-wide association study (GWAS), functions as an active enhancer of TRIM46 expression. Recreating the G-to-A polymorphic switch caused by the SNP via CRISPR/Cas9-mediated homologous recombination leads to an overt upregulation of TRIM46. We find that TRIM46 is a ubiquitin ligase that targets histone deacetylase HDAC1 for ubiquitination and degradation and that the TRIM46-HDAC1 axis regulates a panel of genes, including ones critically involved in DNA replication and repair. Consequently, TRIM46 promotes breast cancer cell proliferation and chemoresistance invitro and accelerates tumor growth invivo. Moreover, TRIM46 is frequently overexpressed in breast carcinomas, and its expression is correlated with lower HDAC1 expression, higher histological grades, and worse prognosis of the patients. Together, our study links SNP rs4971059 to replication and to breast carcinogenesis and chemoresistance and support thepursuit of TRIM46 as a potential target for breast cancer intervention.

The SRSF4-GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy.

[Figure: see text].

Single-cell Transcriptomes Reveal Characteristics of MicroRNAs in Gene Expression Noise Reduction

Isogenic cells growing in identical environments show cell-to-cell variations because of the stochasticity in gene expression. High levels of variation or noise can disrupt robust gene expression and result in tremendous consequences for cell behaviors. In this work, we showed evidence from single-cell RNA sequencing data analysis that microRNAs (miRNAs) can reduce gene expression noise at the mRNA level in mouse cells. We identified that the miRNA expression level, number of targets, target pool abundance, and miRNA–target interaction strength are the key features contributing to noise repression. miRNAs tend to work together in cooperative subnetworks to repress target noise synergistically in a cell type-specific manner. By building a physical model of post-transcriptional regulation and observing in synthetic gene circuits, we demonstrated that accelerated degradation with elevated transcriptional activation of the miRNA target provides resistance to extrinsic fluctuations. Together, through the integrated analysis of single-cell RNA and miRNA expression profiles, we demonstrated that miRNAs are important post-transcriptional regulators for reducing gene expression noise and conferring robustness to biological processes.

Development of a near-infrared fluorescent ligand that visualizes and stabilizes G-quadruplexes by decorating the triphenylamine scaffold

Abstract G-quadruplex (G4) structures are often enriched in cancer-driving genes with elevated transcriptional activity, which are now treated as potential targets for cancer diagnosis and therapy. Thus, development of dual-function ligands that can simultaneously visualize and stabilize G4s are urgently needed. In this study, two π-extended compounds were designed and synthesized by decorating the triphenylamine scaffold with positively charged indolium moieties, which were then revealed as NIR fluorescent probes possessing AIE characteristics. Next, the more promising compound TAIN-2 was further investigated, demonstrating that TAIN-2 could selectively bind to G4s through an end-stacking mode, which thereby triggered its fluorescence by restricting the intramolecular rotations, and largely stabilized the G4 structures. Finally, we also explored the applications of TAIN-2 in the triple-negative breast cancer (TNBC) cells, and the results suggested that TAIN-2 may have the ability to visualize and stabilize the intracellular G4s, holding great potential to develop into feasible diagnostic/therapeutic dual-function agents for TNBC. This study provides new insights into the development of novel theranostic agents for cancers.

A survivin-driven, tumor-activatable minicircle system for prostate cancer theranostics

Gene vectors regulated by tumor-specific promoters to express transgenes specifically in cancer cells are an emerging approach for cancer diagnosis and treatment. Minicircles are shortened plasmids stripped of prokaryotic sequences that have potency and safety characteristics beneficial for clinical translation. Previously, we developed minicircles driven by the tumor-specific survivin promoter, which exhibits elevated transcriptional activity in aggressive cancers, to express a secreted reporter for blood-based cancer detection. Here we present the first activatable, cancer theranostic minicircle system featuring a pair of diagnostic and therapeutic minicircles expressing Gaussia luciferase for urine-based cancer detection or cytosine deaminase:uracil phosphoribosyltransferase for gene-directed enzyme prodrug therapy. Diagnostic minicircles revealed urinary reporter output related to cellular survivin levels. Notably, mice with aggressive prostate tumors exhibited significantly higher urine reporter activity than mice with non-aggressive tumors and healthy mice after intratumoral minicircle administration. Therapeutic minicircles displayed specific cytotoxicity in survivin-rich cancer cells and significantly attenuated growth of aggressive orthotopic prostate tumors in mice. Use of these minicircles together creates a theranostic system that can first identify individuals carrying aggressive prostate cancer via a urinary test, followed by stringent control of tumor progression in stratified individuals who carry high-risk prostate lesions.

Regulation and Roles of the Nucleolus in Embryonic Stem Cells: From Ribosome Biogenesis to Genome Organization.

The nucleolus is the largest compartment of the eukaryotic cell's nucleus. It acts as a ribosome factory, thereby sustaining the translation machinery. The nucleolus is also the subnuclear compartment with the highest transcriptional activity in the cell, where hundreds of ribosomal RNA (rRNA) genes transcribe the overwhelming majority of RNAs. The structure and composition of the nucleolus change according to the developmental state. For instance, in embryonic stem cells (ESCs), rRNA genes display a hyperactive transcriptional state and open chromatin structure compared with differentiated cells. Increasing evidence indicates that the role of the nucleolus and rRNA genes might go beyond the control of ribosome biogenesis. One such role is linked to the genome architecture, since repressive domains are often located close to the nucleolus. This review highlights recent findings describing how the nucleolus is regulated in ESCs and its role in regulating ribosome biogenesis and genome organization for the maintenance of stem cell identity.