Transcription Initiation Factor Research Articles

Design of a tunable bacterial gene expression system using engineered σ factors.

The synthesis of organic compounds involves the action of multiple enzymes in a biosynthetic pathway. Incorporating such biosynthetic pathways into microbes often leads to substantial cellular and metabolic stress resulting in low titers of the target compound. This limitation can be offset, in part, by optimizing enzyme efficiency and cellular enzyme concentration. The former involves significant efforts to achieve improvements in catalytic efficiency with the caveat that the metabolic load on a microbial cell imposed by the overexpression of the exogenous enzyme could result in reduced cell fitness. Here, we demonstrate the feasibility of engineered σ factors to modulate gene expression levels without significant genetic engineering. We note that changing the sequence of two flexible polypeptide loops without any changes to the structural scaffold of the transcription initiation factor σE could modulate the expression levels of the target genes. This ability provides a route to improve the efficiency of a biosynthetic pathway without altering the overall genomic makeup. The σE chimera library thus provides an avenue for pre-determined conditional gene expression of specific genes in Escherichia coli.

Identification of Salt-Sensitive and Salt-Tolerant Genes through Weighted Gene Co-Expression Networks across Multiple Datasets: A Centralization and Differential Correlation Analysis.

Salt stress is a significant challenge that severely hampers rice growth, resulting in decreased yield and productivity. Over the years, researchers have identified biomarkers associated with salt stress to enhance rice tolerance. However, the understanding of the mechanism underlying salt tolerance in rice remains incomplete due to the involvement of multiple genes. Given the vast amount of genomics and transcriptomics data available today, it is crucial to integrate diverse datasets to identify key genes that play essential roles during salt stress in rice. In this study, we propose an integration of multiple datasets to identify potential key transcription factors. This involves utilizing network analysis based on weighted co-expression networks, focusing on gene-centric measurement and differential co-expression relationships among genes. Consequently, our analysis reveals 86 genes located in markers from previous meta-QTL analysis. Moreover, six transcription factors, namely LOC_Os03g45410 (OsTBP2), LOC_Os07g42400 (OsGATA23), LOC_Os01g13030 (OsIAA3), LOC_Os05g34050 (OsbZIP39), LOC_Os09g29930 (OsBIM1), and LOC_Os10g10990 (transcription initiation factor IIF), exhibited significantly altered co-expression relationships between salt-sensitive and salt-tolerant rice networks. These identified genes hold potential as crucial references for further investigation into the functions of salt stress response in rice plants and could be utilized in the development of salt-resistant rice cultivars. Overall, our findings shed light on the complex genetic regulation underlying salt tolerance in rice and contribute to the broader understanding of rice's response to salt stress.

Promoting Nucleic Acid Synthesis in Saccharomyces cerevisiae through Enhanced Expression of Rrn7p, Rrn11p, IMPDH, and Pho84p.

Saccharomyces cerevisiae has emerged as a preferred source for industrial production of ribonucleic acids (RNAs) and their derivatives, which find wide applications in the food and pharmaceutical sectors. In this study, we employed a modified RNA polymerase I-mediated green fluorescent protein expression system, previously developed by our team, to screen and identify an industrial S. cerevisiae strain with an impressive 18.2% increase in the RNA content. Transcriptome analysis revealed heightened activity of genes and pathways associated with rRNA transcription, purine metabolism, and phosphate transport in the high nucleic acid content mutant strains. Our findings highlighted the crucial role of the transcription factor Sfp1p in enhancing the expression of two key components of the transcription initiation factor complex, Rrn7p and Rrn11p, thereby promoting rRNA synthesis. Moreover, elevated expression of 5'-inosine monophosphate dehydrogenases, regardless of the specific isoform (IMD2, 3, or 4), resulted in increased rRNA synthesis through heightened GTP levels. Additionally, exogenous phosphate application, coupled with overexpression of the phosphate transporter PHO84, led to a 61.4% boost in the RNA yield, reaching 2050.4 mg/L. This comprehensive study provides valuable insights into the mechanism of RNA synthesis and serves as a reference for augmenting RNA production in the food industry.

Expression of tomato susceptibility genes triggered by Xanthomonas euvesicatoria pv. perforans and disease control by short antisense oligodeoxynucleotide gene silencing

Sources of durable resistance against bacterial spot of tomato have been prioritized for disease management. However, traditional resistance through resistance genes is scarce and does not always confer a broad and lasting protection to this disease. An alternative is a recessive resistance, mediated by mutation or silencing of host genes that confer susceptibility, known as S-genes. Some bacterial pathogens induce the expression of such genes to promote host colonization, and their identification is necessary to implement alternative management strategies, such as short antisense oligodeoxynucleotides (ASOs) capable of silencing a specific target gene. The approach taken in this study helps to understand the behavior of S-genes during the interaction of tomato with Xanthomonas euvesicatoria pv. perforans (Xep). We compared the expression pattern of 17 S-genes selected from the literature in response to others pathogen infections, including Transcription initiation factor IIA subunit 2 (gamma) (SlTFIIAγ), Defense No Death 1 (DND1) and several Sugars Will Eventually Be Exported Transporters (SWEET), that have not been shown yet as tomato S-genes during Xep interaction. Our data reveal that the expression of several putative host S-genes was positively modulated during bacterial infection. In addition, we showed that an ASO targeting the SlTFIIAγ gene remarkably improved plant resistance against bacterial attack.

Arsenic trioxide-induced apoptosis contributes to suppression of viral reservoir in SIV-infected rhesus macaques.

Latent viral reservoir is recognized as the major obstacle to achieving a functional cure for HIV infection. We previously reported that arsenic trioxide (As2O3) combined with antiretroviral therapy (ART) can reactivate the viral reservoir and delay viral rebound after ART interruption in chronically simian immunodeficiency virus (SIV)-infected macaques. In this study, we further investigated the effect of As2O3 independent of ART in chronically SIV-infected macaques. We found that As2O3-only treatment significantly increased the CD4/CD8 ratio, improved SIV-specific T cell responses, and reactivated viral latency in chronically SIVmac239-infected macaques. RNA-sequencing analysis revealed that As2O3 treatment downregulated the expression levels of genes related to HIV entry and infection, while the expression levels of genes related to transcription initiation, cell apoptosis, and host restriction factors were significantly upregulated. Importantly, we found that As2O3 treatment specifically induced apoptosis of SIV-infected CD4+ T cells. These findings revealed that As2O3 might not only impact viral latency, but also induce the apoptosis of HIV-infected cells and thus block the secondary infection of bystanders. Moreover, we investigated the therapeutic potential of this regimen in acutely SIVmac239-infected macaques and found that As2O3 + ART treatment effectively restored the CD4+ T cell count, delayed disease progression, and improved survival in acutely SIV-infected macaques. In sum, this work provides new insights to develop As2O3 as a component of the "shock-and-kill" strategy toward HIV functional cure. IMPORTANCE Although antiretroviral therapy (ART) can effectively suppress the viral load of AIDS patients, it cannot functionally cure HIV infection due to the existence of HIV reservoir. Strategies toward HIV functional cure are still highly anticipated to ultimately end the pandemic of AIDS. Herein, we investigated the direct role of As2O3 independent of ART in chronically SIV-infected macaques and explored the underlying mechanisms of the potential of As2O3 in the treatment of HIV/SIV infection. Meanwhile, we investigated the therapeutic effects of ART+As2O3 in acutely SIVmac239-infected macaques. This study showed that As2O3 has the potential to be launched into the "shock-and-kill" strategy to suppress HIV/SIV reservoir due to its latency-reversing and apoptosis-inducing properties.

Molecular characterization, transcriptional regulation of sea perch Moloney leukemia virus 10 and its antiviral function against VHSV.

Moloney leukemia virus 10 (MOV10) is a conserved RNA helicase and has multiple biological functions in mammals, but its role remains poorly understood in bony fish. Here, we cloned a MOV10 homolog from sea perch (Lateolabrax japonicus), which contained 23 exons and 22 introns, with an open reading frame of 3000 bp encoding 1000 amino acids. Tissue distribution analysis showed that MOV10 was high expressed in blood of sea perch. Promoter analysis revealed several putative multiple transcription factors binding sites, including upstream transcription factor 1, GATA-box, transcription initiation factor IIB, activator protein 1 and two interferon (IFN) stimulated response elements. Further analysis found that IFNc, IFNh, and IFNγ could not only activate IFN regulatory factor (IRF) 1 expression which in turn led to the induction of MOV10, but also prompted the expression of IRF10 to hinder excessive MOV10 expression. Moreover, IRF2 also suppressed MOV10 expression that was initiated by IRF1. Viral hemorrhagic septicemia virus (VHSV) infection upregulated MOV10 expression in vivo and in vitro, which in turn, enhanced IFNh expression and exhibited strong antiviral activity against VHSV proliferation. This study provides a basis to investigate the immune escape of VHSV by affecting the biological function of transcription factors in the signaling pathways associated with antiviral molecules.

RNA polymerase sliding on DNA can couple the transcription of nearby bacterial operons

DNA transcription initiates after an RNA polymerase (RNAP) molecule binds to the promoter of a gene. In bacteria, the canonical picture is that RNAP comes from the cytoplasmic pool of freely diffusing RNAP molecules. Recent experiments suggest the possible existence of a separate pool of polymerases, competent for initiation, which freely slide on the DNA after having terminated one round of transcription. Promoter-dependent transcription reinitiation from this pool of posttermination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance and timescales needed for it to function widely on a bacterial genome in vivo. Here, we mathematically model the hypothesized reinitiation mechanism as a diffusion-to-capture process and compute the distances over which significant interoperon coupling can occur and the time required. These quantities depend on molecular association and dissociation rate constants between DNA, RNAP, and the transcription initiation factor σ70; we measure these rate constants using single-molecule experiments in vitro. Our combined theory/experimental results demonstrate that efficient coupling can occur at physiologically relevant σ70 concentrations and on timescales appropriate for transcript synthesis. Coupling is efficient over terminator-promoter distances up to ∼1,000 bp, which includes the majority of terminator-promoter nearest neighbor pairs in the Escherichia coli genome. The results suggest a generalized mechanism that couples the transcription of nearby operons and breaks the paradigm that each binding of RNAP to DNA can produce at most one messenger RNA.

Mutational and biophysical analyses reveal a TFIIIC binding region in the TFIIF-related Rpc53 subunit of RNA polymerase III

The TFIIF-like Rpc53/Rpc37 heterodimer of RNA polymerase (pol) III is involved in various stages of transcription. The C-terminal region of Rpc53 dimerizes with Rpc37 to anchor on the lobe domain of the pol III cleft. However, structural and functional features of the Rpc53 N-terminal region had not been characterized previously. Here, we conducted site-directed alanine replacement mutagenesis on the Rpc53 N-terminus, generating yeast strains that exhibited a cold-sensitive growth defect and severely compromised pol III transcriptional activity. Circular dichroism and NMR spectroscopy revealed a highly disordered 57-amino acid polypeptide in the Rpc53 N-terminus. This polypeptide is a versatile protein-binding module displaying nanomolar-level binding affinities for Rpc37 and the Tfc4 subunit of the transcription initiation factor TFIIIC. Accordingly, we denote this Rpc53 N-terminus polypeptide as the TFIIIC-binding region or CBR. Alanine replacements in the CBR significantly reduced its binding affinity for Tfc4, highlighting its functional importance to cell growth and transcription invitro. Our study reveals the functional basis for Rpc53's CBR in assembly of the pol III transcription initiation complex.

High RRN3 expression is associated with malignant characteristics and poor prognosis in pancreatic cancer.

Pancreatic cancer has an extremely poor prognosis and is one of the most chemoresistant cancers. Targeting cancer cell transcriptional complexes may enhance chemotherapy effectiveness. RNA-polymerase I (Pol-I)-mediated transcription is an essential initial step for ribosome biogenesis and is related to cancer cell proliferation. RRN3 is a Pol-I-specific transcription initiation factor. In this study, we aimed to elucidate the function and clinical significance of RRN3 in pancreatic cancer. We performed immunohistochemical staining to detect RRN3 protein expression in 96 pancreatic cancer tissues and analyzed the relationship between RRN3 protein expression, clinicopathological factors, and cancer patient prognosis. Moreover, we evaluated RRN3 function in vitro and in vivo using proliferation, invasion, and chemosensitivity assays in PANC-1 and SW1990 cell lines, with/without depleting RRN3 expression. RRN3 was mainly expressed in cancer cell nuclei. High levels of RRN3 expression were associated with Ki-67 expression and shorter overall survival. Additionally, proliferation and invasion ability were decreased when RRN3 was silenced with siRNA, compared to non-targeting siRNA-transfected cells. Chemosensitivity analysis showed that inhibition of RRN3 enhanced the sensitivity of pancreatic cancer cell lines to gemcitabine and paclitaxel. RRN3 siRNA-transfected PANC-1 tumors showed significantly reduced tumor volumes and high gemcitabine sensitivity compared to the control in a mouse xenograft model. High levels of RRN3 expression are associated with poor prognosis and cancer malignancy, such as proliferation, invasion ability, and chemosensitivity in pancreatic cancer. RRN3 targeting with anticancer drugs may be a promising therapeutic strategy to overcome refractory pancreatic cancer.

Acetylation and phosphorylation of mitochondrial transcription factor a promote mitochondrial transcription processivity.

Mitochondrial transcription factor A (TFAM) is the main component in mitochondrial nucleoids. TFAM serves as the mitochondrial DNA (mtDNA) compaction protein and as an essential transcription initiation factor. The compaction level of mtDNA has been proposed to regulate processes involving mtDNA like transcription and replication processivity. One mechanism to regulate TFAM binding to mtDNA within the mitochondria is via post-translational modifications (PTMs), such as phosphorylation by protein kinase A (PKA) and nonenzymatic acetylation by acetyl-CoA. Here, we demonstrate that DNA-bound TFAM is less susceptible to these modifications. We confirm using electrophoretic mobility shift assays that phosphorylated or acetylated TFAM compacted circular double-stranded DNA just as well as unmodified TFAM. Additionally, we provide an in-depth analysis of TFAM's lysine reactivity towards acetyl-CoA using isotopic labelling and liquid chromatography-tandem mass spectrometry. We show that both modifications to TFAM increase the processivity of transcription through TFAM-imposed barriers on DNA, but that TFAM bearing either modification maintains full activity in transcription initiation. We conclude that phosphorylation by PKA and nonenzymatic acetylation by acetyl-CoA are unlikely to occur on TFAM that are bound to the mitochondrial DNA. Instead, we propose that PTMs increase transcription output by modifying TFAM before it binds mtDNA. These findings call into question the PTM model of mitochondrial nucleoid regulation.

MoSnf5 Regulates Fungal Virulence, Growth, and Conidiation in Magnaporthe oryzae.

Snf5 (sucrose nonfermenting) is a core component of the SWI/SNF complexes and regulates diverse cellular processes in model eukaryotes. In plant pathogenic fungi, its biological function and underlying mechanisms remain unexplored. In this study, we investigated the biological roles of MoSnf5 in plant infection and fungal development in the rice blast pathogen Magnaporthe oryzae. The gene deletion mutants of MoSNF5 exhibited slower vegetative hyphal growth, severe defects in conidiogenesis, and impaired virulence and galactose utilization capacities. Domain dissection assays showed that the Snf5 domain and the N- and C-termini of MoSnf5 were all required for its full functions. Co-immunoprecipitation and yeast two-hybrid assays showed that MoSnf5 physically interacts with four proteins, including a transcription initiation factor MoTaf14. Interestingly, the ∆MoTaf14 mutants showed similar phenotypes as the ∆Mosnf5 mutants on fungal virulence and development. Moreover, assays on GFP-MoAtg8 expression and localization showed that both the ∆Mosnf5 and ∆MoTaf14 mutants were defective in autophagy. Taken together, MoSnf5 regulates fungal virulence, growth, and conidiation, possibly through regulating galactose utilization and autophagy in M. oryzae.

AMPK Downregulates Histone Gene Expression Via Dissociation of a Chromatin-Associated Transcription Complex in Response to Metabolic Stress in Acute Lymphoblastic Leukemia

We and others have reported that activation of the AMP-activated protein kinase (AMPK) mediates cell death in ALL. AMPK has been reported to interact with chromatin-associated proteins to epigenetically regulate gene expression in response to environmental/energy/metabolic stress, however, little is known about AMPK's epigenetic targets. To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we performed ChIP-seq with AMPKα2 and RNA pol II antibodies in Bp-ALL (KASUMI-2) and T-ALL (KE-37) cells treated with or without glucose or AICAR, a well known AMPK activator. ChIP-seq identified 132 and 167 significant AMPKα2 peaks located at gene promoters in AICAR-treated KASUMI-2 and KE-37 cells, respectively. GO terms analysis in KASUMI-2 control cells showed that AMPKα2 target set was enriched for genes involved in regulation of hematopoietic cell differentiation, DNA metabolism, DNA topology, and histone H3-K4 methylation, whereas in AICAR-treated cells it included the histone H3-K4 methylation, protein localization, Notch signaling, and mRNA destabilization. In AICAR-treated cells, we found that AMPKα2 was recruited to regions that were enriched for TAF1 and MYC targets, and to the promoter of the epigenetic regulators KMT2A and SETD1A. Among AMPKα2 targets, we identified a cluster of histone genes in which recruitment of AMPKα2 was significantly decreased at gene promoters. ChIP-seq data were validated by ChIP-qPCR on histone genes H1-2, H1-3, and H4C4, and the occupancy of AMPKα2 on histone gene loci in other Bp- and T-ALL cells confirmed. To correlate the level of gene mRNA expression and recruitment of AMPKα2 to chromatin we performed RNA-seq in KASUMI-2 and KE-37 cells treated ± AICAR for 24h. RNA-seq identified 1342 differential expressed genes in at least one of the cell lines, of which 57 were histone genes and 1285 non-histone genes. RT-qPCR confirmed histone gene mRNA downregulation in AICAR-treated cells. In addition, consistent with an epigenetic role for AMPK in ALL cells in response to energy/metabolic stress, we found that expression of the H3-K4 histone methyltransferase KMT2A and SETD1A genes were upregulated in KASUMI-2 cells treated with AICAR. To further investigate the effect of AMPK on histone gene expression, we generated HEK293 cells expressing single knockout (KO) or double knockout (DKO) of AMPKα1 and AMPKα2 genes using CRISPR/Cas9, and determined the level of selected histone gene mRNA expression using RT-qPCR. Our data show that both AMPKα1 and AMPKα2 participate in regulating histone gene expression, and these findings were confirmed using MEF WT, AMPKα1/KO, AMPKα2/KO, and AMPKα1/α2/DKO cells. These data support our previous findings indicating that AMPK kinase activity was required for histone genes’ regulation in response to metabolic stress. Next, we examined the cluster of non-histone genes altered by AICAR, and found that downregulated genes were enriched in MYC, Myc-associated factor X, and transcription initiation factor TFIID subunit 1 (TAF1) targets, and involved in pathways such as gene expression and protein metabolism. In contrast, the upregulated genes were enriched in RUNX1, SPI1, and GATA1 targets, and involved in pathways related to immune system signaling, regulation by TP53, and apoptosis. Using HEK293 CRISPR AMPKα1/KO, AMPKα2/KO and AMPKα1/α2/DKO cells, we demonstrated that AMPK is also required for MYC expression. In conclusion, our data show that (i) in response to metabolic stress, AMPKα2 is recruited to or released from a chromatin-associated complex to regulate gene transcription in ALL, (ii) AMPKα2 targets include the histone, MYC-target, and the epigenetic regulator KMT2A and SETD1A genes (H3-K4 histone methyltransferases), and (iii) AICAR treatment represses histone gene transcription via an AMPK-dependent mechanism. Further elucidation of AMPK's interactions with members of a putative AMPK/chromatin-associated transcription complex may lead to unique opportunities for epigenetic-based therapeutic interventions to exploit synthetic lethality in relapse/refractory ALL and other hematological malignancies.

Agronomic and Proteomic Assessment of Salt Stress Responses in <i>Pennisetum glaucum</i> (Pearl Millet) Genotypes

Salinity is one of the environmental stresses that alter protein expression. To evaluate that, two ICRISAT Pennisetum glaucum (pearl millet) genotypes (IP 19586 and IP 22269) were subjected to 0.3 dSm-1 and 10 dSm-1 salinities. The agronomic parameters and ion content were assessed to find the tolerance level of the genotype to salinity. Differential expression of proteins on salt stress in both genotypes were analyzed by 2-dimensional gel electrophoresis. MALDI–MS/MS-TOF-TOF analysis helped in ascertain the amino acid sequences and the MASCOT search suggested the biological process of the predicted proteins. IP 22269 exclude the accumulation of toxic level of sodium in leaf. Based on physiological responses, IP 22269 was proposed to be salt-sensitive, and IP 19586 salt-tolerant genotypes. 67 proteins were responsive either less or more abundant to salinity. As per findings, the proteins responsive to salt stress are granule-bound starch synthase 1, alpha-amylase, Meiotic recombination protein SPO11, flavonoid 3'- monooxygenase, ethylene receptor 4, protein transcription initiation factor Y 11g, and auxin-responsive protein IAA16. Further, based on the STRING analysis these proteins form a protein-protein interaction network with 171 proteins. This research elucidates few salt stress tolerance traits and network between the salt stress-responsive mechanisms in pearl millet.

Structural and mechanistic basis of σ-dependent transcriptional pausing

In σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific protein–DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway “backtracked” states that are substrates for the transcript-cleavage factors of the Gre family and on-pathway “scrunched” states that mediate pause escape. Here, using site-specific protein–DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR′ promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2–4 bp, and σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2–3 bp. Analogous experiments with a library of 47 (∼16,000) transcribed-region sequences show that the state scrunched by 2–3 bp—and only that state—is associated with the consensus sequence, T−3N−2Y−1G+1, (where −1 corresponds to the position of the RNA 3′ end), which is identical to the consensus for pausing in initial transcription and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T−3 resides in the DNA nontemplate strand. A cryoelectron microscopy structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T−3 of the consensus sequence exerts its effects by facilitating scrunching.

Structural Features of the Nucleosomal DNA Modulate the Functional Binding of a Transcription Factor and Productive Transcription.

A small non-histone protein of budding yeast, Nhp6 has been reported to specifically influence the transcription of a yeast gene, SNR6. The gene is essential, transcribed by the enzyme RNA polymerase III, and codes for the U6snRNA required for mRNA splicing. A translationally positioned nucleosome on the gene body enables the assembly factor TFIIIC binding by juxtaposing its otherwise widely separated binding sites, boxes A and B. We found histone depletion results in the loss of U6 snRNA production. Changing the rotational phase of the boxes and the linear distance between them with deletions in 5 bp steps displayed a helical periodicity in transcription, which gradually reduced with incremental deletions up to 40 bp but increased on further deletions enclosing the pseudoA boxes. Nhp6 influences the transcription in a dose-dependent manner, which is modulated by its previously reported co-operator, an upstream stretch of seven T residues centered between the TATA box and transcription start site. Nhp6 occupancy on the gene in vivo goes up at least 2-fold under the repression conditions. Nhp6 absence, T7 disruption, or shorter A–B box distance all cause the downstream initiation of transcription. The right +1 site is selected with the correct placement of TFIIIC before the transcription initiation factor TFIIIB. Thus, the T7 sequence and Nhp6 help the assembly and placement of the transcription complex at the right position. Apart from the chromatin remodelers, the relative rotational orientation of the promoter elements in nucleosomal DNA, and Nhp6 regulate the transcription of the SNR6 gene with precision.

Glutamate-aspartate transporter 1 attenuates oxygen-glucose deprivation-induced injury by promoting glutamate metabolism in primary cortical neurons.

Ischemic stroke is a common cerebral disease. However, the treatment for the disease is limited. Daurian ground squirrel (GS;Spermophilus dauricus), a hibernating mammalian species, is highly tolerant to ischemia. In the present study, GS neurons in a non-hibernating state were found to be more resistant to oxygen-glucose deprivation (OGD), an ischemic model in vitro. We leveraged the differences in the endurance capacity of GS and rats to investigate the mechanisms of resistance to ischemia in GSneurons. We first identified glutamate-aspartate transporter 1 (GLAST) as a cytoprotective factor that contributed to tolerance against OGD injury of GS neurons. The expression of GLAST in GS neurons was much higher than that in rat neurons. Overexpression of GLAST rescued viability in rat neurons, and GS neurons exhibited decreased viability following GLAST knockdown under OGD conditions. Mechanistically, more glutamate was transported into neurons after GLAST overexpression and served as substrates for ATP production. Furthermore, eukaryotic transcription initiation factor 4E binding protein 1 was downregulated by GLAST to rescue neuronal viability. Our findings not only revealed an important molecular mechanism underlying the survival of hibernating mammals but also suggested that neuronal GLAST may be a potential target for ischemic stroke therapy.

Mechanisms of mitochondrial promoter recognition in humans and other mammalian species

Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for >50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of the DNA in the initiation complex and permitted investigation of the sequence‐specific protein‐DNA interactions. We determined the molecular basis of promoter recognition by mtRNAP and TFB2M, which cooperatively recognize bases near TSS in a species‐specific manner. Our findings reveal a role of mitochondrial transcription machinery in mitonuclear coevolution and speciation.

Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome.

SummaryFragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.

Synergy of Venetoclax and 8-Chloro-Adenosine in AML: The Interplay of rRNA Inhibition and Fatty Acid Metabolism.

Simple SummaryTreatment failures of acute myeloid leukemia (AML) have been attributed to the persistence of leukemia stem cells (LSCs), which are refractory to conventional treatments. Venetoclax (VEN), currently FDA-approved in combination with low-dose cytarabine or hypomethylating agents, is highly effective in inducing disease remission in patients with de novo AML; however, most of these patients eventually relapse; thus, novel VEN combinations are urgently needed. In this regard, 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets AML cells, including LSCs. We demonstrate that VEN and 8-Cl-Ado cooperate in targeting ribosomal RNA synthesis and mitochondrial metabolism in LSCs, thereby decreasing LSC survival. Given the emerging concept that LSC behavior is strongly associated with protein synthesis regulation and mitochondrial metabolism, our results suggest that the VEN/8-Cl-Ado combination is a promising regimen for the treatment of patients with relapsed AML.It is known that 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets leukemic stem cells (LSCs). In a phase I clinical trial with 8-Cl-Ado in patients with refractory or relapsed (R/R) AML, we observed encouraging but short-lived clinical responses, likely due to intrinsic mechanisms of LSC resistance. LSC homeostasis depends on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. We recently reported that 8-Cl-Ado and the BCL-2-selective inhibitor venetoclax (VEN) synergistically inhibit FAO and OXPHOS in LSCs, thereby suppressing acute myeloid leukemia (AML) growth in vitro and in vivo. Herein, we report that 8-Cl-Ado inhibits ribosomal RNA (rRNA) synthesis through the downregulation of transcription initiation factor TIF-IA that is associated with increasing levels of p53. Paradoxically, 8-Cl-Ado-induced p53 increased FAO and OXPHOS, thereby self-limiting the activity of 8-Cl-Ado on LSCs. Since VEN inhibits amino acid-driven OXPHOS, the addition of VEN significantly enhanced the activity of 8-Cl-Ado by counteracting the self-limiting effect of p53 on FAO and OXPHOS. Overall, our results indicate that VEN and 8-Cl-Ado can cooperate in targeting rRNA synthesis and OXPHOS and in decreasing the survival of the LSC-enriched cell population, suggesting the VEN/8-Cl-Ado regimen as a promising therapeutic approach for patients with R/R AML.

Mechanisms of mitochondrial promoter recognition in humans and other mammalian species.

Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for > 50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of the DNA in the initiation complex and permitted investigation of the sequence-specific protein-DNA interactions. We determined the molecular basis of promoter recognition by mtRNAP and TFB2M, which cooperatively recognize bases near TSS in a species-specific manner. Our findings reveal a role of mitochondrial transcription machinery in mitonuclear coevolution and speciation.