Contrasting effects of whole-body and hepatocyte-specific deletion of the RNA polymerase III repressor Maf1 in the mouse.

MAF1 is a nutrient-sensitive, TORC1-regulated repressor of RNA polymerase III (Pol III). MAF1 downregulation leads to increased lipogenesis in Drosophila melanogaster, Caenorhabditis elegans, and mice. However, Maf1 -/- mice are lean as increased lipogenesis is counterbalanced by futile pre-tRNA synthesis and degradation, resulting in increased energy expenditure. We compared Chow-fed Maf1 -/- mice with Chow- or High Fat (HF)-fed Maf1 hep-/- mice that lack MAF1 specifically in hepatocytes. Unlike Maf1 -/- mice, Maf1 hep-/- mice become heavier and fattier than control mice with old age and much earlier under a HF diet. Liver ChIPseq, RNAseq and proteomics analyses indicate increased Pol III occupancy at Pol III genes, very few differences in mRNA accumulation, and protein accumulation changes consistent with increased lipogenesis. Futile pre-tRNA synthesis and degradation in the liver, as likely occurs in Maf1 hep-/- mice, thus seems insufficient to counteract increased lipogenesis. Indeed, RNAseq and metabolite profiling indicate that liver phenotypes of Maf1 -/- mice are strongly influenced by systemic inter-organ communication. Among common changes in the three phenotypically distinct cohorts, Angiogenin downregulation is likely linked to increased Pol III occupancy of tRNA genes in the Angiogenin promoter.

Peptides designed from a bacteriophage capsid protein function as synthetic transcription repressors.

The bacteriophage capsid protein, Psu (polarity suppression), inhibits the bacterial transcription terminator, Rho. In an effort to find nontraditional antibacterial agents, we previously designed peptides from the Psu C terminus that function as inhibitors of Rho. Here, we demonstrated that these peptides have positive surface-charge densities, and they downregulate many genes in Escherichia coli. We hypothesized that these peptides could bind to nucleic acids and repress gene expression. One of these peptides, peptide 33, represses in vitro transcription from the T7A1 and Plac promoters efficiently by blocking the access of RNA polymerase to the promoter, a mode of transcription repression akin to many bacterial repressors. In vivo, expressions of the peptides reduce the total RNA level as well as transcription from Plac and Posm promoters significantly. However, they are less efficient in repressing transcription from the rRNA promoters with a very high turnover of RNA polymerase. The peptide 33 binds to both single and dsDNA as well as to RNA with dissociation constants ranging from 1 to 5 µM exhibiting preferences for the single-stranded DNA and RNAs. These interactions are salt-resistant and not sequence-specific. Interactions with dsDNA are entropy-driven, while it is enthalpy-driven for the ssDNA. This mode of interaction with nucleic acids is similar to many nonspecific ssDNA-binding proteins. Expression of peptide 33 induces cell elongation and impaired cell division, possibly due to the dislodging of the DNA-binding proteins. Overall, we surmised that these synthetic transcription repressors would function like bacterial nucleoid-associated proteins.

Transcription factor mce3R modulates antibiotics and disease persistence in Mycobacteriumtuberculosis.

Transcription factors (TFs) of Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis, regulate a network of pathways that help prolong the survival of Mtb inside the host. In this study, we have characterized a transcription repressor gene (mce3R) from the TetR family, that encodes for Mce3R protein in Mtb. We demonstrated that the mce3R gene is dispensable for the growth of Mtb on cholesterol. Gene expression analysis suggests that the transcription of genes belonging to the mce3R regulon is independent of the carbon source. We found that, in comparison to the wild type, the mce3R deleted strain (Δmce3R) generated more intracellular ROS and demonstrated reduced susceptibility to oxidative stress. Total lipid analysis suggests that mce3R regulon encoded proteins modulate the biosynthesis of cell wall lipids in Mtb. Interestingly, the absence of Mce3R increased the frequency of generation of antibiotic persisters in Mtb and imparted in-vivo growth advantage phenotype in guinea pigs. In conclusion, genes belonging to the mce3R regulon modulate the frequency of generation of persisters in Mtb. Hence, targeting mce3R regulon encoded proteins could potentiate the current regimen by eliminating persisters during Mtb infection.

Brassica napus BnaC9.DEWAX1 Negatively Regulates Wax Biosynthesis via Transcriptional Suppression of BnCER1-2.

Very-long-chain alkane plays an important role as an aliphatic barrier. We previously reported that BnCER1-2 was responsible for alkane biosynthesis in Brassica napus and improved plant tolerance to drought. However, how the expression of BnCER1-2 is regulated is still unknown. Through yeast one-hybrid screening, we identified a transcriptional regulator of BnCER1-2, BnaC9.DEWAX1, which encodes AP2\ERF transcription factor. BnaC9.DEWAX1 targets the nucleus and displays transcriptional repression activity. Electrophoretic mobility shift and transient transcriptional assays suggested that BnaC9.DEWAX1 repressed the transcription of BnCER1-2 by directly interacting with its promoter. BnaC9.DEWAX1 was expressed predominantly in leaves and siliques, which was similar to the expression pattern of BnCER1-2. Hormone and major abiotic stresses such as drought and high salinity affected the expression of BnaC9.DEWAX1. Ectopic expression of BnaC9.DEWAX1 in Arabidopsis plants down-regulated CER1 transcription levels and resulted in a reduction in alkanes and total wax loads in leaves and stems when compared with the wild type, whereas the wax depositions in the dewax mutant returned to the wild type level after complementation of BnaC9.DEWAX1 in the mutant. Moreover, both altered cuticular wax composition and structure contribute to increased epidermal permeability in BnaC9.DEWAX1 overexpression lines. Collectively, these results support the notion that BnaC9.DEWAX1 negatively regulates wax biosynthesis by binding directly to the BnCER1-2 promoter, which provides insights into the regulatory mechanism of wax biosynthesis in B. napus.

ASR1 and ASR2, Two Closely Related ABA-Induced Serine-Rich Transcription Repressors, Function Redundantly to Regulate ABA Responses in Arabidopsis.

The plant hormone abscisic acid (ABA) is able to regulate the expression of ABA-responsive genes via signaling transduction, and thus plays an important role in regulating plant responses to abiotic stresses. Hence, characterization of unknown ABA response genes may enable us to identify novel regulators of ABA and abiotic stress responses. By using RT-PCR analysis, we found that the expression levels of ABA-induced Serine-rich Repressor 1 (ASR1)and ASR2, two closely related unknown function genes, were increased in response to ABA treatment. Amino acid sequence analyses show that ASR1 contains an L×L×L motif and both ASR1 and ASR2 are enriched in serine. Transfection assays in Arabidopsis leaf protoplasts show that ASR1 and ASR2 were predominantly localized in the nucleus and were able to repress the expression of the reporter gene. The roles of ASRs in regulating ABA responses were examined by generating transgenic Arabidopsis plants over-expressing ASR1 and ASR2, respectively, and CRISPR/Cas9 gene-edited single and double mutants for ASR1 and ASR2. In both the seed germination and cotyledon greening assays, ABA sensitivity remained largely unchanged in the over-expression transgenic plants and the single mutants of ASR1 and ASR2, but greatly increased ABA sensitivity was observed in the asr1 asr2 double mutants. In root elongation assays, however, decreased ABA sensitivity was observed in the 35S:ASR1 and 35S:ASR2 transgenic plants, whereas increased ABA sensitivity was observed in the asr1 and asr2 single mutants, and ABA sensitivity was further increased in the asr1 asr2 double mutants. Transcriptome analysis show that the differentially expressed genes (DEGs) down-regulated in the 35S:ASR1 transgenic plant seedlings, but up-regulated in the asr1 asr2 double mutant seedlings were highly enriched in processes including responses to plant hormones and stress stimuli. Taken together, our results show that ASR1 and ASR2 are closely related ABA response genes, ASR1 and ASR2 are serine-rich novel transcription repressors, and they negatively regulate ABA responses in Arabidopsis in a redundant manner.

A p-Coumaroyl-CoA Biosensor for Dynamic Regulation of Naringenin Biosynthesis in Saccharomyces cerevisiae.

In vivo biosensors that can convert metabolite concentrations into measurable output signals are valuable tools for high-throughput screening and dynamic pathway control in the field of metabolic engineering. Here, we present a novel biosensor in Saccharomyces cerevisiae that is responsive to p-coumaroyl-CoA, a central precursor of many flavonoids. The sensor is based on the transcriptional repressor CouR from Rhodopseudomonas palustris and was applied in combination with a previously developed malonyl-CoA biosensor for dual regulation of p-coumaroyl-CoA synthesis within the naringenin production pathway. Using this approach, we obtained a naringenin titer of 47.3 mg/L upon external precursor feeding, representing a 15-fold increase over the nonregulated system.

Two Arabidopsis MYB-SHAQKYF transcription repressors regulate leaf wax biosynthesis via transcriptional suppression on DEWAX.

Plant cuticular wax accumulation limits nonstomatal transpiration and is regulated by external environmental stresses. DEWAX (DECREASE WAX BIOSYNTHESIS) plays a vital role in diurnal wax biosynthesis. However, how DEWAX expression is controlled and the molecular mechanism of wax biosynthesis regulated by the diurnal cycle remains largely unknown. Here, we identified two Arabidopsis MYB-SHAQKYF transcription factors, MYS1 and MYS2, as new regulators in wax biosynthesis and drought tolerance. Mutations of both MYS1 and MYS2 caused significantly reduced leaf wax, whereas overexpression of MYS1 or MYS2 increased leaf wax biosynthesis and enhanced drought tolerance. Our results demonstrated that MYS1 and MYS2 act as transcription repressors and directly suppress DEWAX expression via ethylene response factor-associated amphiphilic repression motifs. Genetic interaction analysis with DEWAX, SPL9 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9), and CER1 (ECERIFERUM 1) in wax biosynthesis and under drought stresses demonstrated that MYS1 and MYS2 act upstream of the DEWAX-SPL9 module, thus regulating CER1 expression. Expression analysis suggested that the diurnal expression pattern of DEWAX is partly regulated by MYS1 and MYS2. Our findings demonstrate the roles of two unidentified transcription repressors, MYS1 and MYS2, in wax biosynthesis and provide insights into the mechanism of diurnal cycle-regulated wax biosynthesis.

JA-induced FtBPM3 accumulation promotes FtERF-EAR3 degradation and rutin biosynthesis in Tartary buckwheat.

Buckwheat accumulates abundant flavonoids, which exhibit excellent health-promoting value. Flavonoids biosynthesis is mediated by a variety of phytohormones, among which jasmonates (JAs) induce numerous transcription factors, taking part in regulation of flavonoids biosynthesis genes. However, some transcriptional repressors appeared also induced by JAs. How these transcriptional repressors coordinately participate in JA signaling remains unclear. Here, we found that the disruption of the GCC-box in FtF3H promoter was associated with flavonoids accumulation in Tartary buckwheat. Further, our study illustrated that the nucleus-localized FtERF-EAR3 could inhibit FtF3H expression and flavonoids biosynthesis through binding the GCC-box in the promoter of FtF3H. The JA induced FtERF-EAR3 gene expression while facilitating FtERF-EAR3 protein degradation via the FtBPM3-dependent 26S proteasome pathway. Overall, these results illustrate a precise modulation mechanism of JA-responsive transcription suppressor participating in flavonoid biosynthesis, and will further help to improve the efficiency of flavonoids biosynthesis in Tartary buckwheat.

A novel Cbx1, PurB, and Sp3 complex mediates long-term silencing of tissue- and lineage-specific genes.

miRNA-based cellular fate reprogramming offers an opportunity to investigate the mechanisms of long-term gene silencing. To further understand how genes are silenced in a tissue-specific manner, we leveraged our miRNA-based method of reprogramming fibroblasts into cardiomyocytes. Through screening approaches, we identified three proteins that were downregulated during reprogramming of fibroblasts into cardiomyocytes: heterochromatin protein Cbx1, transcriptional activator protein PurB, and transcription factor Sp3. We show that knockdown of Cbx1, PurB, and Sp3 was sufficient to induce cardiomyocyte gene expression in fibroblasts. Similarly, gene editing to ablate Cbx1, PurB, and Sp3 expression induced fibroblasts to convert into cardiomyocytes in vivo. Furthermore, high-throughput DNA sequencing and coimmunoprecipitation experiments indicated that Cbx1, PurB, and Sp3 also bound together as a complex and were necessary to localize nucleosomes to cardiomyocyte genes on the chromosome. Finally, we found that the expression of these genes led to nucleosome modification via H3K27me3 (trimethylated histone-H3 lysine-27) deposition through an interaction with the polycomb repressive PRC2 complex. In summary, we conclude that Cbx1, PurB, and Sp3 control cell fate by actively repressing lineage-specific genes.

Chrysanthemum embryo development is negatively affected by a novel ERF transcription factor, CmERF12.

Embryo abortion often occurs during distant hybridization events. Apetala 2/ethylene-responsive factor (AP2/ERF) proteins are key transcription factor (TF) regulators of plant development and stress resistance, but their roles in hybrid embryo development are poorly understood. In this study, we isolated a novel AP2/ERF TF, CmERF12, from chrysanthemum and show that it adversely affects embryo development during distant hybridization. Transcriptome and real-time quantitative PCR demonstrate that CmERF12 is expressed at significantly higher levels in aborted ovaries compared with normal ones. CmERF12 localizes to the cell nucleus and contains a conserved EAR motif that mediates its transcription repressor function in yeast and plant cells. We generated artificial microRNA (amiR) CmERF12 transgenic lines of Chrysanthemum morifolium var. 'Yuhualuoying' and conducted distant hybridization with the wild-type tetraploid, Chrysanthemum nankingense, and found that CmERF12-knock down significantly promoted embryo development and increased the seed-setting rates during hybridization. The expression of various genes related to embryo development was up-regulated in developing ovaries from the cross between female amiR-CmERF12 C. morifolium var. 'Yuhualuoying'× male C. nankingense. Furthermore, CmERF12 directly interacted with CmSUF4, which is known to affect flower development and embryogenesis, and significantly reduced its ability to activate its target gene CmEC1 (EGG CELL1). Our study provides a novel method to overcome barriers to distant hybridization in plants and reveals the mechanism by which CmERF12 negatively affects chrysanthemum embryo development.

Structural Insight into the Binding of TGIF1 to SIN3A PAH2 Domain through a C-Terminal Amphipathic Helix.

TGIF1 is a transcriptional repressor playing crucial roles in human development and function and is associated with holoprosencephaly and various cancers. TGIF1-directed transcriptional repression of specific genes depends on the recruitment of corepressor SIN3A. However, to date, the exact region of TGIF1 binding to SIN3A was not clear, and the structural basis for the binding was unknown. Here, we demonstrate that TGIF1 utilizes a C-terminal domain (termed as SIN3A-interacting domain, SID) to bind with SIN3A PAH2. The TGIF1 SID adopts a disordered structure at the apo state but forms an amphipathic helix binding into the hydrophobic cleft of SIN3A PAH2 through the nonpolar side at the holo state. Residues F379, L382 and V383 of TGIF1 buried in the hydrophobic core of the complex are critical for the binding. Moreover, homodimerization of TGIF1 through the SID and key residues of F379, L382 and V383 was evidenced, which suggests a dual role of TGIF1 SID and a correlation between dimerization and SIN3A-PAH2 binding. This study provides a structural insight into the binding of TGIF1 with SIN3A, improves the knowledge of the structure-function relationship of TGIF1 and its homologs and will help in recognizing an undiscovered SIN3A-PAH2 binder and developing a peptide inhibitor for cancer treatment.

Down-Regulation of CXXC5 De-Represses MYCL1 to Promote Hepatic Stellate Cell Activation.

Liver fibrosis is mediated by myofibroblasts, a specialized cell type involved in wound healing and extracellular matrix production. Hepatic stellate cells (HSC) are the major source of myofibroblasts in the fibrotic livers. In the present study we investigated the involvement of CXXC-type zinc-finger protein 5 (CXXC5) in HSC activation and the underlying mechanism. Down-regulation of CXXC5 was observed in activated HSCs compared to quiescent HSCs both in vivo and in vitro. In accordance, over-expression of CXXC5 suppressed HSC activation. RNA-seq analysis revealed that CXXC5 influenced multiple signaling pathways to regulate HSC activation. The proto-oncogene MYCL1 was identified as a novel target for CXXC5. CXXC5 bound to the proximal MYCL1 promoter to repress MYCL1 transcription in quiescent HSCs. Loss of CXXC5 expression during HSC activation led to the removal of CpG methylation and acquisition of acetylated histone H3K9/H3K27 on the MYCL1 promoter resulting in MYCL1 trans-activation. Finally, MYCL1 knockdown attenuated HSC activation whereas MYCL1 over-expression partially relieved the blockade of HSC activation by CXXC5. In conclusion, our data unveil a novel transcriptional mechanism contributing to HSC activation and liver fibrosis.

Genic male sterility increases rice drought tolerance.

Plant fertility and resistance to stress environments are antagonistic to each other. At booting stage, fertility is often sacrificed for survive in rice under abiotic stress. However, the relationship between fertility and resistance at molecular level remains elusive. Here, we identified a transcription factor, OsAlfin like 5, which regulates the OsTMS5 and links both the drought stress response and thermosensitive genic male sterility. The OsAL5 overexpression plants (OE-OsAL5) became sensitive to temperature owning to the OsTMS5 that the OE-OsAL5 plants were fertile under low temperature (23 °C) and sterile under high temperature (28 °C). Significantly, the survival rate of OE-OsAL5 lines was higher than that of the wide-type (WT) under drought stress. Further experiments confirmed that the OsAL5 regulated both of the OsTMS5 and the down-stream drought-related genes by binding to the 'GTGGAG' element in vivo, revealing that the OsAL5 participated both in the drought stress response and thermosensitive genic male sterility in rice. These findings open up the possibility of breeding elite TGMS lines with strong drought tolerance by manipulating the expression of OsAL5.

Binding of Tetracyclines to Acinetobacter baumannii TetR Involves Two Arginines as Specificity Determinants.

Acinetobacter baumannii is an important nosocomial pathogen that requires thoughtful consideration in the antibiotic prescription strategy due to its multidrug resistant phenotype. Tetracycline antibiotics have recently been re-administered as part of the combination antimicrobial regimens to treat infections caused by A. baumannii. We show that the TetA(G) efflux pump of A. baumannii AYE confers resistance to a variety of tetracyclines including the clinically important antibiotics doxycycline and minocycline, but not to tigecycline. Expression of tetA(G) gene is regulated by the TetR repressor of A. baumannii AYE (AbTetR). Thermal shift binding experiments revealed that AbTetR preferentially binds tetracyclines which carry a O-5H moiety in ring B, whereas tetracyclines with a 7-dimethylamino moiety in ring D are less well-recognized by AbTetR. Confoundingly, tigecycline binds to AbTetR even though it is not transported by TetA(G) efflux pump. Structural analysis of the minocycline-bound AbTetR-Gln116Ala variant suggested that the non-conserved Arg135 interacts with the ring D of minocycline by cation-π interaction, while the invariant Arg104 engages in H-bonding with the O-11H of minocycline. Interestingly, the Arg135Ala variant exhibited a binding preference for tetracyclines with an unmodified ring D. In contrast, the Arg104Ala variant preferred to bind tetracyclines which carry a O-6H moiety in ring C except for tigecycline. We propose that Arg104 and Arg135, which are embedded at the entrance of the AbTetR binding pocket, play important roles in the recognition of tetracyclines, and act as a barrier to prevent the release of tetracycline from its binding pocket upon AbTetR activation. The binding data and crystal structures obtained in this study might provide further insight for the development of new tetracycline antibiotics to evade the specific efflux resistance mechanism deployed by A. baumannii.

XRE-Type Regulator BioX Acts as a Negative Transcriptional Factor of Biotin Metabolism in Riemerella anatipestifer.

Biotin is essential for the growth and pathogenicity of microorganisms. Damage to biotin biosynthesis results in impaired bacterial growth and decreased virulence in vivo. However, the mechanisms of biotin biosynthesis in Riemerella anatipestifer remain unclear. In this study, two R. anatipestifer genes associated with biotin biosynthesis were identified. AS87_RS05840 encoded a BirA protein lacking the N-terminal winged helix-turn-helix DNA binding domain, identifying it as a group I biotin protein ligase, and AS87_RS09325 encoded a BioX protein, which was in the helix-turn-helix xenobiotic response element family of transcription factors. Electrophoretic mobility shift assays demonstrated that BioX bound to the promoter region of bioF. In addition, the R. anatipestifer genes bioF (encoding 7-keto-8-aminopelargonic acid synthase), bioD (encoding dethiobiotin synthase), and bioA (encoding 7,8-diaminopelargonic acid synthase) were in an operon and were regulated by BioX. Quantitative reverse transcription-PCR showed that transcription of the bioFDA operon increased in the mutant Yb2ΔbioX in the presence of excessive biotin, compared with that in the wild-type strain Yb2, suggesting that BioX acted as a repressor of biotin biosynthesis. Streptavidin blot analysis showed that BirA caused biotinylation of BioX, indicating that biotinylated BioX was involved in metabolic pathways. Moreover, as determined by the median lethal dose, the virulence of Yb2ΔbioX was attenuated 500-fold compared with that of Yb2. To summarize, the genes birA and bioX were identified in R. anatipestifer, and BioX was found to act as a repressor of the bioFDA operon involved in the biotin biosynthesis pathway and identified as a bacterial virulence factor. IMPORTANCE Riemerella anatipestifer is a causative agent of diseases in ducks, geese, turkeys, and various other domestic and wild birds. Our study reveals that biotin synthesis of R. anatipestifer is regulated by the BioX through binding to the promoter region of the bioF gene to inhibit transcription of the bioFDA operon. Moreover, bioX is required for R. anatipestifer pathogenicity, suggesting that BioX is a potential target for treatment of the pathogen. R. anatipestifer BioX has thus been identified as a novel negative regulator involved in biotin metabolism and associated with bacterial virulence in this study.

Analysis of mouse male germ cell-specific or -predominant Tex13 family genes encoding proteins with transcriptional repressor activity.

Mammalian spermatogenesis is a highly organized process with successive mitotic, meiotic, and postmeiotic phases. This unique developmental process is characterized by the involvement of spermatogenic cell-specific genes. In this study, we identified and investigated testis expressed gene 13 (Tex13) family genes, consisting of Tex13a, Tex13b, Tex13c1, and Tex13d, in mice. All of these genes were transcribed specifically or predominantly in male germ cells, and their transcription was developmentally regulated. Proteins encoded by the Tex13 genes were predicted to have a conserved domain of ~ 145 amino acids. Tex13a, Tex13c1, and Tex13d encode additional C-terminal regions containing a short conserved sequence termed a zinc finger-RAN binding protein 2 (zf-RanBP2) or zf-RanBP2-like domain. As TEX13B reportedly has transcriptional repressor activity, we examined the effect of the TEX13 proteins on transcriptional regulation using a reporter assay. All of the TEX13 proteins exhibited transcriptional repressor activity. This activity was revealed to reside in the TEX13B-corresponding regions of TEX13A, TEX13C1, and TEX13D. Further, we found that the C-terminal regions of TEX13A, TEX13C1, and TEX13D also have inhibitory activities. These results suggest that male germ cell-specific or -predominant TEX13 proteins commonly function in transcriptional repression as transcription cofactors and/or RNA binding proteins.

Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes.

Male and female gametocytes are sexual precursor cells essential for mosquito transmission of malaria parasite. Differentiation of gametocytes into fertile gametes (known as gametogenesis) relies on the gender-specific transcription program. How the parasites establish distinct repertoires of transcription in the male and female gametocytes remains largely unknown. Here, we report that an Apetala2 family transcription factor AP2-O3 operates as a transcription repressor in the female gametocytes. AP2-O3 is specifically expressed in the female gametocytes. AP2-O3-deficient parasites produce apparently normal female gametocytes. Nevertheless, these gametocytes fail to differentiate into fully fertile female gametes, leading to developmental arrest in fertilization and early development post-fertilization. AP2-O3 disruption causes massive upregulation of transcriptionally dormant male genes and simultaneously downregulation of highly transcribed female genes in the female gametocytes. AP2-O3 targets a substantial proportion of the male genes by recognizing an 8-base DNA motif. In addition, the maternal AP2-O3 is removed after fertilization, which is required for the zygote to ookinete development. Therefore, the global transcriptional repression of the male genes in the female gametocytes is required for safeguarding female-specific transcriptome and essential for the mosquito transmission of Plasmodium.

Robust Heat Shock Response in Chlamydia Lacking a Typical Heat Shock Sigma Factor.

Cells reprogram their transcriptome in response to stress, such as heat shock. In free-living bacteria, the transcriptomic reprogramming is mediated by increased DNA-binding activity of heat shock sigma factors and activation of genes normally repressed by heat-induced transcription factors. In this study, we performed transcriptomic analyses to investigate heat shock response in the obligate intracellular bacterium Chlamydia trachomatis, whose genome encodes only three sigma factors and a single heat-induced transcription factor. Nearly one-third of C. trachomatis genes showed statistically significant (≥1.5-fold) expression changes 30 min after shifting from 37 to 45°C. Notably, chromosomal genes encoding chaperones, energy metabolism enzymes, type III secretion proteins, as well as most plasmid-encoded genes, were differentially upregulated. In contrast, genes with functions in protein synthesis were disproportionately downregulated. These findings suggest that facilitating protein folding, increasing energy production, manipulating host activities, upregulating plasmid-encoded gene expression, and decreasing general protein synthesis helps facilitate C. trachomatis survival under stress. In addition to relieving negative regulation by the heat-inducible transcriptional repressor HrcA, heat shock upregulated the chlamydial primary sigma factor σ66 and an alternative sigma factor σ28. Interestingly, we show for the first time that heat shock downregulates the other alternative sigma factor σ54 in a bacterium. Downregulation of σ54 was accompanied by increased expression of the σ54 RNA polymerase activator AtoC, thus suggesting a unique regulatory mechanism for reestablishing normal expression of select σ54 target genes. Taken together, our findings reveal that C. trachomatis utilizes multiple novel survival strategies to cope with environmental stress and even to replicate. Future strategies that can specifically target and disrupt Chlamydia's heat shock response will likely be of therapeutic value.

Hepatic miR-192-3p reactivation alleviates steatosis by targeting glucocorticoid receptor.

BACKGROUND & AIMS: The paradox of hepatic insulin resistance describes the inability for liver to respond to bioenergetics hormones in suppressing gluconeogenesis whilst maintaining lipid synthesis. Here, we report the deficiency of miR-192-3p in the livers of mice with diabetes and its role in alleviating hepatic steatosis. METHODS: As conventional pre-microRNA (miRNA) stem-loop overexpression only boosts guiding strand (i.e. miR-192-5p) expression, we adopted an artificial AAV(DJ)-directed, RNA Pol III promoter-driven miRNA hairpin construct for star-strand-specific overexpression in the liver. Liver steatosis and insulin resistance markers were evaluated in primary hepatocytes, mice with diabetes, and mice with excessive carbohydrate consumption. RESULTS: Functional loss of miR-192-3p in liver exacerbated hepatic micro-vesicular steatosis and insulin resistance in either mice with diabetes or wild-type mice with excessive fructose consumption. Liver-specific overexpression of miR-192-3p effectively halted hepatic steatosis and ameliorated insulin resistance in these mice models. Likewise, hepatocytes overexpressing miR-192-3p exhibited improved lipid accumulation, accompanied with decreases in lipogenesis and lipid-accumulation-related transcripts. Mechanistically, glucocorticoid receptor (GCR, also known as nuclear receptor subfamily 3, group C, member 1 [NR3C1]) was demonstrated to be negatively regulated by miR-192-3p. The effect of miR-192-3p on mitigating micro-vesicular steatosis was ablated by the reactivation of NR3C1. CONCLUSIONS: The star strand miR-192-3p was an undermined glycerolipid regulator involved in controlling fat accumulation and insulin sensitivity in liver through blockade of hepatic GCR signalling; this miRNA may serve as a potential therapeutic option for the common co-mobility of diabetic mellitus and fatty liver disease. LAY SUMMARY: The potential regulatory activity of star strand microRNA (miRNA) species has been substantially underestimated. In this study, we investigate the role and mechanism of an overlooked star strand miRNA (miR-192-3p) in regulating hepatic steatosis and insulin signalling in the livers of mice with diabetes and mice under excessive carbohydrate consumption.