Transcription Of Endogenous Genes Research Articles

Involvement of GATA transcription factors in the regulation of endogenous bovine interferon‐Tau gene transcription

Expression of interferon-tau (IFNT), necessary for pregnancy establishment in ruminant ungulates, is regulated in a temporal and spatial manner. However, molecular mechanisms by which IFNT gene transcription is regulated in this manner have not been firmly established. In this study, DNA microarray/RT-PCR analysis between bovine trophoblast CT-1 and Mardin-Darby bovine kidney (MDBK) cells was initially performed, finding that transcription factors GATA2, GATA3, and GATA6 mRNAs were specific to CT-1 cells. These mRNAs were also found in Days 17, 20, and 22 (Day 0 = day of estrus) bovine conceptuses. In examining other bovine cell lines, ovary cumulus granulosa (oCG) and ear fibroblast (EF) cells, GATA2 and GATA3, but not GATA6, were found specific to the bovine trophoblast cells. In transient transfection analyses using the upstream region (-631 to +59 bp) of bovine IFNT gene (bIFNT, IFN-tau-c1), over-expression of GATA2/GATA3 did not affect the transcription of bIFNT-reporter construct in human choriocarcinoma JEG3 cells. Transfection of GATA2, GATA3, ETS2, and/or CDX2, however, was effective in the up-regulation of the bIFNT construct transfected into bovine oCG and EF cells. One Point mutation studies revealed that among six potential GATA binding sites located on the upstream region of the bIFNT gene, the one next to ETS2 site exhibited reduced luciferase activity. In CT-1 cells, endogenous bIFNT gene transcription was up-regulated by over-expression of GATA2 or GATA3, but down-regulated by siRNA specific to GATA2 mRNA. These data suggest that GATA2/3 is involved in trophoblast-specific regulation of bIFNT gene transcription.

Nuclear Factor I Regulates Brain Fatty Acid-Binding Protein and Glial Fibrillary Acidic Protein Gene Expression in Malignant Glioma Cell Lines

Glial fibrillary acidic protein (GFAP), an intermediate filament protein normally found in astrocytes, and the radial glial marker brain fatty acid-binding protein (B-FABP; also known as FABP7) are co-expressed in malignant glioma cell lines and tumors. Nuclear factor I (NFI) recognition sites have been identified in the B-FABP and GFAP promoters, and transcription of both genes is believed to be regulated by NFI. Here, we study the role of the different members of the NFI family in regulating endogenous and ectopic B-FABP and GFAP gene transcription in human malignant glioma cells. We show by gel shifts that all four members of the NFI family (NFIA, NFIB, NFIC, and NFIX) bind to B-FABP and GFAP NFI consensus sites. Over-expression of NFIs, in conjunction with mutation analysis of NFI consensus sites using a reporter gene assay, supports a role for all four NFIs in the regulation of the GFAP and B-FABP genes. Knock-down of single or combined NFIs reveals promoter-dependent and promoter-context-dependent interaction patterns and suggests cross talk between the different members of the NFI family. Our data indicate that the NFI family of transcription factors plays a key role in the regulation of both the B-FABP and GFAP genes in malignant glioma cells.

The Rtt106 Histone Chaperone Is Functionally Linked to Transcription Elongation and Is Involved in the Regulation of Spurious Transcription from Cryptic Promoters in Yeast*♦

Rtt106 is a histone chaperone that has been suggested to play a role in heterochromatin-mediated silencing in Saccharomyces cerevisiae. It interacts physically and functionally with the chromatin assembly factor-1 (CAF-1), which is associated with replication-coupled nucleosomal deposition. In this work, we have taken several approaches to study Rtt106 in greater detail and have identified a previously unknown function of Rtt106. We found genetic interactions between rtt106Delta and mutations in genes encoding transcription elongation factors, including Spt6, TFIIS, and members of the PAF and yeast DSIF complexes. In addition, chromatin immunoprecipitation analysis indicates that Rtt106 is associated with transcribed regions of active genes. Furthermore, our results show that Rtt106 is required for the repression of transcription from a cryptic promoter within a coding region. This observation strongly suggests that Rtt106 is involved in the regulation of chromatin structure of transcribed regions. Finally, we provide evidence that Rtt106 plays a role in regulating the levels of histone H3 transcription-coupled deposition over transcribed regions. Taken together, our results indicate a direct link for Rtt106 with transcription elongation and the chromatin dynamics associated with RNA polymerase II passage.

Rac1 GTPase and the Rac1 exchange factor Tiam1 associate with Wnt-responsive promoters to enhance beta-catenin/TCF-dependent transcription in colorectal cancer cells

Backgroundβ-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with β-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the β-catenin/TCF transcription factor complex.ResultsWe employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the β-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and β-catenin. Furthermore, endogenous Tiam1 associated with endogenous β-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner.ConclusionTaken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active β-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.

The Positive Transcription Elongation Factor b Is an Essential Cofactor for the Activation of Transcription by Myocyte Enhancer Factor 2

The positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1, stimulates the elongation of transcription by hyperphosphorylating the C-terminal region of RNA polymerase II. Aberrant activation of P-TEFb results in manifestations of cardiac hypertrophy in mice, suggesting that P-TEFb is an essential factor for cardiac myocyte function and development. Here, we present evidence that P-TEFb selectively activates transcription mediated by the myocyte enhancer factor 2 (MEF2) family of transcription factors, key regulatory factors for myocyte development. Knockdown of endogenous cyclin T1 in murine C2C12 cells abolishes MEF2-dependent reporter gene expression as well as transcription of endogenous MEF2 target genes, whereas overexpression of P-TEFb enhances MEF2-dependent transcription. P-TEFb interacts with MEF2 both in vitro and in vivo. Activation of MEF2-dependent transcription induced by serum starvation is mediated by a rapid dissociation of P-TEFb from its inhibitory subunit, HEXIM1, and a subsequent recruitment of P-TEFb to MEF2 binding sites in the promoter region of MEF2 target genes. These results indicate that recruitment of P-TEFb is a critical step for stimulation of MEF2-dependent transcription, therefore providing a fundamentally important regulatory mechanism underlying the transcriptional program in muscle cells.

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

New strategy for increasing the specific and efficient expression of transgene in plastids

Plastid gene engineering has become a new way for plant genetic improvements, particularly showing a unique application value in the use of plants as reactors to produce biopharmaceuticals and other important organic compounds. However, plastids only have a semi-autonomous transcription and translation machinery. The transcription of endogenous plastid genes is largely dependent on nuclear-encoded transcription factors. Regulation of foreign gene expression in plastids is influenced by various factors. Several technique strategies for regulation of transgene expression in plastids were reported recently, such as hybrid transcription factor-mediated system, phage T7-based transcription system and bacterial lac suppressor-based system. The application and improvement of these systems will greatly enhance the specific and effective expression of the transgenes, and achieve high-level accumulations of foreign proteins in plastids.

The Cell Cycle Factor E2F-1 Activates Bnip3 and the Intrinsic Death Pathway in Ventricular Myocytes

The cell cycle factor E2F-1 is known to regulate a variety of cellular processes including apoptosis. Previously we showed that disruption of Rb-E2F-1 complexes provoked apoptosis of postmitotic adult and neonatal ventricular myocytes; however, the underlying mechanism was undetermined. In this report, we show that E2F-1 provokes cell death of ventricular myocytes through a mechanism that directly impinges on the intrinsic death pathway. Furthermore, we show mechanistically that the hypoxia-inducible death factor Bnip3 is a direct transcriptional target of E2F-1 that is necessary and sufficient for E2F-1-induced cell death. Expression of E2F-1 resulted in a 4.9-fold increase (P<0.001) in nucleosomal DNA fragmentation and cell death by Hoechst 33258 dye and vital staining. E2F-1 provoked mitochondrial perturbations that were consistent with permeability transition pore opening. As determined by quantitative real-time PCR analysis, a 6.2-fold increase (P<0.001) in endogenous Bnip3 gene transcription was observed in cells expressing wild-type E2F-1 but not in cells expressing a mutation of E2F-1 defective for DNA binding. Rb, the principle regulator of cellular E2F-1 activity, was proteolytically cleaved and inactivated in ventricular myocytes during hypoxia. Consistent with the proteolytic cleavage of Rb, chromatin immunoprecipitation analysis revealed increased binding of E2F-1 to the Bnip3 promoter during hypoxia, a finding concordant with the induction of Bnip3 gene transcription. The Bnip3 homolog Nix/Bnip3L was unaffected in ventricular myocytes by either E2F-1 or hypoxia. Genetic knockdown of E2F-1 or expression of a caspase-resistant form of Rb suppressed basal and hypoxia-inducible Bnip3 gene transcription. Loss-of-function mutations of Bnip3 defective for mitochondrial membrane insertion or small interference RNA directed against Bnip3 suppressed cell death signals elicited by E2F-1. To our knowledge, the data provide the first direct evidence that activation of the intrinsic mitochondrial death pathway by E2F-1 is mutually dependent on and obligatorily linked to the transcriptional activation of Bnip3.

Peptide-Mediated Disruption of NFκB/NRF Interaction Inhibits IL-8 Gene Activation by IL-1 or Helicobacter pylori

Selective inhibition of proinflammatory chemokines such as IL-8 is an important approach to combat inflammatory and infection diseases. Previous studies suggested that interaction of transcription factors NFkappaB repressing factor (NRF) and NFkappaB play a crucial role in activation of IL-8 gene expression. In a search for a specific inhibitor of IL-8 expression, we applied tandem affinity purification to investigate interaction of NRF and NFkappaB p65 in cells. We identified a synthetic peptide corresponding to aa 223-238 of NRF interfering with binding of endogenous p65 to NRF. Furthermore, nucleofection experiments were established to introduce this inhibitory peptide into the nucleus of IL-1 stimulated human cervical and Helicobacter pylori infected gastric epithelial cells. Our data clearly show that the specific peptide disturbing NRF/NFkappaB interaction is able to significantly decrease endogenous IL-8 gene transcription in response to IL-1 or Helicobacter pylori infection. Thus, our study provides novel insights into NRF and NFkappaB interaction in vivo and may facilitate the design of new anti-IL-8 drugs based on novel strategies.

Transcription MRI: A New View of the Living Brain

Altered gene activities are underlying causes of many neurological disorders. The ability to detect, image, and report endogenous gene transcription using magnetic resonance (MR) holds great potential for providing significant clinical benefits. In this review, we present the development of conjugates consisting of gene-targeting short nucleic acids (oligodeoxynucleotides, or sODN) and superparamagnetic iron oxide nanoparticles (SPION, an MR susceptibility T2 agent) for reporting gene activity using transcription MRI (tMRI). We will discuss 1) the target specificity of sODN, 2) selection of contrast agents for tMRI, 3) the distribution and uptake, 4) sequence specificity, 5) histology of SPION and sODN, 6) data acquisition and quantitative analysis for tMRI, and 7) application of gene transcript—targeting nanoparticles in biology and medicine. We will also discuss methods of validating the correlation between results from conventional assays (in situ hybridization, PCR, histology Prussian blue stain and immunohistochemistry) in postmortem samples and retention of SPION-sODN using tMRI. The application of our novel contrast probe to report and target gene transcripts in the mesolimbic pathways of living mouse brains after amphetamine exposure will be discussed. Because of the targeting ability in the nucleic acid sequence, the concept of tMRI probes with complementary nucleic acid (antisense DNA or short interfering RNA) allows not only tracking, targeting, binding to intracellular mRNA, and manipulating gene action but also tracing cells with specific gene action in living brains. Transcription MRI will lend itself to myriad applications in living organs. NEUROSCIENTIST 14(5):503—520, 2008. DOI: 10.1177/1073858407309746

Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors

Small-molecule-regulated gene expression offers the promise of titrating the dose and duration of action of DNA-based therapies. To this end, we show that engineered zinc-finger protein transcription factors (ZFP TFs) can be coupled with a drug-inducible regulatory domain to permit small-molecule control of endogenous gene transcription. We constructed a drug-responsive ZFP TF via the fusion of a ZFP DNA-binding domain (DBD) targeting the human VEGF-A gene and an effector domain containing a truncated progesterone receptor ligand-binding domain linked to the NFkappaB p65 activation domain. Introduction of this engineered ZFP TF into human or murine cells allowed expression of the chromosomal VEGF-A gene to be induced upon addition of mifepristone, a synthetic steroid analog. Mifepristone-dependent VEGF-A induction was rapid, dose-dependent and reversible. Moreover, stable lines expressing the drug-responsive ZFP TF could be maintained in a state of continuous induction for at least 30 days without loss of viability. Potent VEGF-A induction was demonstrated using different engineered ZFP DBDs, thus this approach may represent a general solution to small-molecule regulation of targeted endogenous genes.

A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5

AbstractWe report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3′ rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre–B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.

A plant lignan, 3′‐O‐Methyl‐Nordihydroguaiaretic acid, suppresses papillomavirus E6 protein function, stabilizes p53 protein, and induces apoptosis in cervical tumor cells

Persistent infection with oncogenic human papillomaviruses (HPVs) is the most important factor in the induction of uterine cervical cancer, a leading cause of cancer mortality in women worldwide. Upon cell transformation, continual expression of the viral oncogenes is required to maintain the transformed phenotype. The viral E6 protein forms a ternary complex with the cellular E6-AP protein and p53 protein which promotes the rapid degradation of p53. Recent studies have revealed that lignans from the creosote bush (3'-O-methyl-nordihydroguaiaretic acid) can repress the viral promoter responsible for E6 gene expression. Work reported here shows that the lignan can subvert viral oncogene function resulting in stabilized p53 protein within treated HPV-containing tumor cells. The stabilized p53 is transcriptionally active as demonstrated by a luciferase reporter vector and induction of genes for Bax and PUMA proteins. Apoptosis is detected by annexin V binding to treated cells as analyzed by flow cytometry. Programmed cell death is confirmed by the induction of active caspases and TUNEL assay. Initiator caspase-9 is activated first, followed later by the effector caspase-3 enzyme. The stabilization and induced apoptosis are not observed within treated HPV-negative cervical tumor cells. Quantitative real time RT-PCR analysis of endogenous E6 gene transcription from the integrated HPV 16 promoter shows at least a fivefold repression of expression as compared to untreated cells. These results indicate that the loss of E6 protein in treated cells could be, in part, responsible for the stabilization of p53 within the lignan treated cells.

Targeting HSP70 to motoneurons protects locomotor activity from hyperthermia in Drosophila

Heat shock preconditioning can enhance locomotor and synaptic performance during subsequent hyperthermia. The molecular basis underlying this neural phenotypic modification is largely unknown. Here we report that directing the expression of the 70 kDa heat shock protein (HSP70) to motoneurons protected larval locomotor activity of Drosophila. Tissue-specific expression showed that motoneurons were critical for developing HSP70-mediated thermoprotection of locomotor activity, whereas peripheral sensory neurons, dopaminergic neurons, serotonergic neurons, and muscle cells alone were insufficient. Targeting HSP70 to motoneurons caused structural plasticity of axonal terminals associated with increased transmitter release at neuromuscular junctions at high temperature. The thermoprotection induced by motoneuronal expression of HSP70 mimicked the protective effect of a prior heat shock (36 degrees C, 1 h; 25 degrees C, 1 h) but the effects of heat shock and motoneuronal expression of HSP70 were not additive. In the absence of heat shock pretreatment, ubiquitously expressed transgenic HSP70 activated the transcription of endogenous hsp70 genes. These results demonstrate that motoneurons were critical for HSP70-mediated thermoprotection, and that transgenic HSP70 activated the transcription of endogenous hsp70 in motoneurons with the result that a mix of transgenic and endogenous HSP70 conferred thermoprotection in Drosophila larva.

Histone acetylation-independent transcription stimulation by a histone chaperone

Histone chaperones are thought to be important for maintaining the physiological activity of histones; however, their exact roles are not fully understood. The physiological function of template activating factor (TAF)-I, one of the histone chaperones, also remains unclear; however, its biochemical properties have been well studied. By performing microarray analyses, we found that TAF-I stimulates the transcription of a sub-set of genes. The transcription of endogenous genes that was up-regulated by TAF-I was found to be additively stimulated by histone acetylation. On performing an experiment with a cell line containing a model gene integrated into the chromosome, TAF-I was found to stimulate the model gene transcription in a histone chaperone activity-dependent manner additively with histone acetylation. TAF-I bound to the core histones and remodeled the chromatin structure independent of the N-terminal histone tail and its acetylation level in vitro. These results suggest that TAF-I remodel the chromatin structure through its interaction with the core domain of the histones, including the histone fold, and this mechanism is independent of the histone acetylation status.

Differential regulation of the tyrosine hydroxylase and enkephalin neuropeptide transmitter genes in rat PC12 cells by short chain fatty acids: Concentration-dependent effects on transcription and RNA stability

At physiologic concentrations, butyrate regulates the expression of individual genes involving at least three mechanisms: (i) through induction of cis- and trans-acting butyrate-dependent transcription factors for selected genes, (ii) by inhibition of histone deacetylation and attendant chromatin remodeling and (iii) by affecting turnover of mRNAs. Our previous work illustrated gradual accumulation of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis and the neuropeptide transmitter proenkephalin (ppEnk) in butyrate-differentiated PC12 cells (Nankova, B.B., Chua, J., Mishra, R., Kobasiuk, C.D., La Gamma, E.F. 2003. Nicotinic induction of preproenkephalin and tyrosine hydroxylase gene expression in butyrate-differentiated rat PC12 cells: a model for adaptation to gut-derived environmental signals. Pediatr. Res. 53, 113–118.). However, at higher physiological concentrations (6 mM), TH mRNA levels are significantly reduced while ppEnk mRNA transcripts remained elevated. These differential effects suggest suppression of endogenous TH gene transcription, targeted degradation of TH mRNA or both. By using nuclear run-on assays, we found that transcription increased for both endogenous TH and ppEnk genes, even at time points and concentrations when reduced steady-state levels of TH mRNA were observed. The reduction in TH mRNA was blocked by cycloheximide consistent with a protein-dependent mechanism. We also observed a dose-dependent accumulation of luciferase reporter molecules driven by TH promoter in transient transfection experiments, data that provide additional support for separate regulatory pathways. Significantly, butyrate-dependent decreases in TH mRNA were also reflected in a reduction in TH protein. Our results suggest a novel mode of regulation for TH by butyrate operating via both transcriptional and post-transcriptional mechanisms. We speculate that, depending on plasma concentrations of butyrate, this naturally occurring signaling molecule can function as an in vivo molecular switch to alter levels of TH mRNA, its protein and thus the biosynthesis of endogenous catecholamines.

Selective Activation of Estrogen Receptor-β Transcriptional Pathways by an Herbal Extract

Novel estrogenic therapies are needed that ameliorate menopausal symptoms and have the bone-sparing effects of endogenous estrogens but do not promote breast or uterine cancer. Recent evidence suggests that selective activation of the estrogen receptor (ER)-beta subtype inhibits breast cancer cell proliferation. To establish whether ERbeta-selective ligands represent a viable approach to improve hormone therapy, we investigated whether the estrogenic activities present in an herbal extract, MF101, used to treat hot flashes, are ERbeta selective. MF101 promoted ERbeta, but not ERalpha, activation of an estrogen response element upstream of the luciferase reporter gene. MF101 also selectively regulates transcription of endogenous genes through ERbeta. The ERbeta selectivity was not due to differential binding because MF101 binds equally to ERalpha and ERbeta. Fluorescence resonance energy transfer and protease digestion studies showed that MF101 produces a different conformation in ERalpha from ERbeta when compared with the conformations produced by estradiol. The specific conformational change induced by MF101 allows ERbeta to bind to an estrogen response element and recruit coregulatory proteins that are required for gene activation. MF101 did not activate the ERalpha-regulated proliferative genes, c-myc and cyclin D1, or stimulate MCF-7 breast cancer cell proliferation or tumor formation in a mouse xenograft model. Our results demonstrate that herbal ERbeta-selective estrogens may be a safer alternative for hormone therapy than estrogens that nonselectively activate both ER subtypes.

Hybrid transcription-mediated transgene regulation in plastids

Plastids are semi-autonomous plant organelles with their own transcription and translation machinery that are suitable for producing high levels of transgenic proteins with a bona fide structure. However, the transcription of endogenous plastid genes is largely dependent on nuclear-encoded transcription factors, and the identification of these factors has implications for transgene regulation. A novel transcription system has recently been reported that works independently of the plastid transcription machinery and is potentially useful for transgene containment and to express genes with the assurance of high-level protein accumulation.

Short chain fatty acids induce TH gene expression via ERK-dependent phosphorylation of CREB protein

Butyrate modulates specific gene expression through various second-messenger signal transduction systems including activation of the PKA/cAMP pathway (Decastro, M., Nankova, B.B., Shah, P., Patel, P., Mally, P.V., Mishra, R., La Gamma, E.F., 2005. Short chain fatty acids regulate tyrosine hydroxylase gene expression through a cAMP-dependent signaling pathway, Brain Res. Mol. Brain Res. 142 28–38; Mally, P., Mishra, R., Gandhi, S., Decastro, M.H., Nankova, B.B., Lagamma, E.F., 2004. Stereospecific regulation of tyrosine hydroxylase and proenkephalin genes by short-chain fatty acids in rat PC12 cells, Pediatr. Res. 55 847–854). In the current report, we provide additional evidence that exposure to butyrate causes a rapid activation of the MAP kinase pathway, associated with increased phosphorylation of CREB. Under these conditions, no changes in relative amounts of CREB protein were observed by Western blot. Pre-treatment with the MAPK specific inhibitor (U0126) or the adenylate cyclase inhibitor dideoxyadenosine (ddA) abolished the butyrate-induced: (i) accumulation of TH mRNA, (ii) the phosphorylation of ERK1/2 as well as (iii) CREB phosphorylation. PC12 cells transfected with a TH promoter-luciferase reporter gene showed a robust induction in response to butyrate that was significantly reduced after co-transfection of either of two dominant-negative CREB expression vectors. Nuclear run-on assays demonstrated that butyrate increases endogenous TH gene transcription. We conclude that the initial steps of butyrate-induced gene activation are mediated through the CREB/CREB family of transcription factors which are coupled to both the MAP kinase and cAMP-dependent second messenger systems. Our data delineate a molecular mechanism through which short chain fatty acid's, their related drug-congeners (e.g., valproate) or even diet-derived butyrate (from fermentation of carbohydrates in the gut) can in principle, modulate brain catecholaminergic systems by modifying TH gene expression, dopaminergic levels and the corresponding animal behavior. These molecular relationships also offer a plausible explanation of how the well-recognized clinical effects of ketogenic diets can alter human behavior via the same central mechanisms.

STABILIZED1, a Stress-Upregulated Nuclear Protein, Is Required for Pre-mRNA Splicing, mRNA Turnover, and Stress Tolerance in Arabidopsis

In plants, many gene transcripts are very unstable, which is important for the tight control of their temporal and spatial expression patterns. To identify cellular factors controlling the stability of unstable mRNAs in plants, we used luciferase imaging in Arabidopsis thaliana to isolate a recessive mutant, stabilized1-1 (sta1-1), with enhanced stability of the normally unstable luciferase transcript. The sta1-1 mutation also causes the stabilization of some endogenous gene transcripts and has a range of developmental and stress response phenotypes. STA1 encodes a nuclear protein similar to the human U5 small ribonucleoprotein-associated 102-kD protein and to the yeast pre-mRNA splicing factors Prp1p and Prp6p. STA1 expression is upregulated by cold stress, and the sta1-1 mutant is defective in the splicing of the cold-induced COR15A gene. Our results show that STA1 is a pre-mRNA splicing factor required not only for splicing but also for the turnover of unstable transcripts and that it has an important role in plant responses to abiotic stresses.