Knocking down gene function with an RNA aptamer expressed as part of an intron

We developed a powerful expression system to produce aptamers and other types of functional RNA in yeast to examine their effects. Utilizing the intron homing process, the aptamer-coding sequences were integrated into hundreds of rRNA genes, and the aptamers were transcribed at high levels by RNA polymerase I without any additional promoter being introduced into the cell. We used this system to express an aptamer against the heat shock factor 1 (HSF1), a conserved transcription factor responsible for mobilizing specific genomic expression programs in response to stressful conditions such as elevated temperature. We observed a temperature sensitive growth retardation phenotype and specific decrease of heat shock gene expression. As HSF1 enables and promotes malignant growth and metastasis in mammals, and this aptamer binds yeast HSF1 and its mammalian ortholog with equal affinity, the results presented here attest to the potential of this aptamer as a specific and effective inhibitor of HSF1 activity

Protein Kinase A Binds and Activates Heat Shock Factor 1

BACKGROUND. Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1), a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. METHODOLOGY/PRINCIPAL FINDINGS. In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA). HSF1 binds avidly to the catalytic subunit of PKA, (PKAca) and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320), both in vitro and in vivo. Intracellular PKAca levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1) and activate hsp70.1 after stress. Reduction in PKAca levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. CONCLUSIONS. These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.National Institutes of Health (2RO1CA047407, RO1CA077465

Expression of estrogen receptors in the hypothalamo-pituitary-ovarian axis in middle-aged rats after re-instatement of estrus cyclicity

During reproductive aging female rats enter an anovulatory state of persistent estrus (PE). In an animal model of re-instatement of estrus cyclicity in middle-aged PE rats we injected the animals with progesterone (0.5 mg progesterone/kg body weight) at 12:00 for 4 days whereas control animals received corn oil injections. After the last injection animals were analyzed at 13:00 and 17:00. Young regular cycling rats served as positive controls and were assessed at 13:00 and 17:00 on proestrus. Progesterone treatment of middle-aged PE rats led to occurrence of luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin surges in a subset of animals that were denoted as responders. Responding middle-aged rats displayed a reduction of ER-β mRNA in the preoptic area which was similar to the effect in young rats. Within the mediobasal hypothalamus, only young rats showed a decline of ER-α mRNA expression. A decrease of ER-α mRNA levels in the pituitary was observed in progesterone-responsive rats and in young animals. ER-β mRNA expression was reduced in young regular cycling rats. ER-β mRNA levels in the ovary were reduced following progesterone treatment in PE rats and in young rats. Taken together our data show that cyclic administration of progesterone reinstates ovulatory cycles in intact aging females which have already lost their ability to display spontaneous cyclicity. This treatment leads to the occurrence of preovulatory LH, FSH and prolactin surges which are accompanied by differential modulation of ERs in the hypothalamus, the pituitary and the ovary

Regulatory elements and transcriptional control of chicken vasa homologue (CVH) promoter in chicken primordial germ cells

BACKGROUND: Primordial germ cells (PGCs), the precursors of functional gametes, have distinct characteristics and exhibit several unique molecular mechanisms to maintain pluripotency and germness in comparison to somatic cells. They express germ cell-specific RNA binding proteins (RBPs) by modulating tissue-specific cis- and trans-regulatory elements. Studies on gene structures of chicken vasa homologue (CVH), a chicken RNA binding protein, involved in temporal and spatial regulation are thus important not only for understanding the molecular mechanisms that regulate germ cell fate, but also for practical applications of primordial germ cells. However, very limited studies are available on regulatory elements that control germ cell-specific expression in chicken. Therefore, we investigated the intricate regulatory mechanism(s) that governs transcriptional control of CVH. RESULTS: We constructed green fluorescence protein (GFP) or luciferase reporter vectors containing the various 5′ flanking regions of CVH gene. From the 5′ deletion and fragmented assays in chicken PGCs, we have identified a CVH promoter that locates at −316 to +275 base pair fragment with the highest luciferase activity. Additionally, we confirmed for the first time that the 5′ untranslated region (UTR) containing intron 1 is required for promoter activity of the CVH gene in chicken PGCs. Furthermore, using a transcription factor binding prediction, transcriptome analysis and siRNA-mediated knockdown, we have identified that a set of transcription factors play a role in the PGC-specific CVH gene expression. CONCLUSIONS: These results demonstrate that cis-elements and transcription factors localizing in the 5′ flanking region including the 5′ UTR and an intron are important for transcriptional regulation of the CVH gene in chicken PGCs. Finally, this information will contribute to research studies in areas of reproductive biology, constructing of germ cell-specific synthetic promoter for tracing primordial germ cells as well as understanding the transcriptional regulation for maintaining germness in PGCs

An RNA aptamer perturbs heat shock transcription factor activity in Drosophila melanogaster

Heat shock transcription factor (HSF1) is a conserved master regulator that orchestrates the protection of normal cells from stress. However, HSF1 also protects abnormal cells and is required for carcinogenesis. Here, we generate an highly specific RNA aptamer (iaRNAHSF1) that binds Drosophila HSF1 and inhibits HSF1 binding to DNA. In Drosophila animals, iaRNAHSF1 reduces normal Hsp83 levels and promotes developmental abnormalities, mimicking the spectrum of phenotypes that occur when Hsp83 activity is reduced. The HSF1 aptamer also effectively suppresses the abnormal growth phenotypes induced by constitutively active forms of the EGF receptor and Raf oncoproteins. Our results indicate that HSF1 contributes toward the morphological development of animal traits by controlling the expression of molecular chaperones under normal growth conditions. Additionally, our study demonstrates the utility of the RNA aptamer technology as a promising chemical genetic approach to investigate biological mechanisms, including cancer and for identifying effective drug targets in vivo

Expanded syringe exchange programs and reduced HIV infection among new injection drug users in Tallinn, Estonia

<p>Abstract</p> <p>Background</p> <p>Estonia has experienced an HIV epidemic among intravenous drug users (IDUs) with the highest per capita HIV prevalence in Eastern Europe. We assessed the effects of expanded syringe exchange programs (SEP) in the capital city, Tallinn, which has an estimated 10,000 IDUs.</p> <p>Methods</p> <p>SEP implementation was monitored with data from the Estonian National Institute for Health Development. Respondent driven sampling (RDS) interview surveys with HIV testing were conducted in Tallinn in 2005, 2007 and 2009 (involving 350, 350 and 327 IDUs respectively). HIV incidence among new injectors (those injecting for < = 3 years) was estimated by assuming (1) new injectors were HIV seronegative when they began injecting, and (2) HIV infection occurred at the midpoint between first injection and time of interview.</p> <p>Results</p> <p>SEP increased from 230,000 syringes exchanged in 2005 to 440,000 in 2007 and 770,000 in 2009. In all three surveys, IDUs were predominantly male (80%), ethnic Russians (>80%), and young adults (mean ages 24 to 27 years). The proportion of new injectors decreased significantly over the years (from 21% in 2005 to 12% in 2009, p = 0.005). HIV prevalence among all respondents stabilized at slightly over 50% (54% in 2005, 55% in 2007, 51% in 2009), and decreased among new injectors (34% in 2005, 16% in 2009, p = 0.046). Estimated HIV incidence among new injectors decreased significantly from 18/100 person-years in 2005 and 21/100 person-years in 2007 to 9/100 person-years in 2009 (p = 0.026).</p> <p>Conclusions</p> <p>In Estonia, a transitional country, a decrease in the HIV prevalence among new injectors and in the numbers of people initiating injection drug use coincided with implementation of large-scale SEPs. Further reductions in HIV transmission among IDUs are still required. Provision of 70 or more syringes per IDU per year may be needed before significant reductions in HIV incidence occur.</p

Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase

The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress

Heat shock proteins in stabilization of spontaneously restored sinus rhythm in permanent atrial fibrillation patients after mitral valve surgery

A spontaneously restored sinus rhythm in permanent atrial fibrillation patients has been often observed after mitral valve (MV) surgery, but persisting duration in sinus rhythm varies from patient to patient. Heat shock proteins (Hsps) may be involved in pathogenesis of atrial fibrillation. We hypothesized that stabilization of restored sinus rhythm is associated with expression of Hsps in the atria. To test this hypothesis, clinical data, biopsies of right atrial appendage, and blood samples were collected from 135 atrial fibrillation patients who spontaneously restored sinus rhythm after conventional isolated MV replacement. Comparison was made between patients who had recurrence of atrial fibrillation within 7 days (AF) vs. patients with persisted sinus rhythm for more than 7 days (SR). Results showed that SR patients had higher activity of heat shock transcription factor 1 (HSF1) as well as upregulated expressions of heat shock cognate 70, Hsp70, and Hsp27 in the tissues. The activation of HSF1–Hsps pathway was associated with less-aggressive pathogenesis as reflected by lower rates of myolysis, apoptosis, interstitial fibrosis, and inflammation in SR patients. However, Hsp60 was lower in both tissue and plasma in SR patients, and was positively correlated with apoptosis, interstitial fibrosis, and inflammation. These findings suggest that the Hsps play important roles in stabilization of restored sinus rhythm after MV surgery by inhibiting AF-related atrial remodeling and arrhythmogenic substrates in atrial fibrillation patients. Low circulating Hsp60 levels preoperatively might predict a stable spontaneously restored sinus rhythm postoperatively