Dissecting the action of an evolutionary conserved non-coding region on renin promoter activity.

Elucidating the mechanisms of the human transcriptional regulatory network is a major challenge of the post-genomic era. One important aspect is the identification and functional analysis of regulatory elements in non-coding DNA. Genomic sequence comparisons between related species can guide the discovery of cis-regulatory sequences. Using this technique, we identify a conserved region CNSmd of approximately 775 bp in size, approximately 14 kb upstream of the renin gene. Renin plays a pivotal role for mammalian blood pressure regulation and electrolyte balance. To analyse the cis-regulatory role of this region in detail, we perform 132 combinatorial reporter gene assays in an in vitro Calu-6 cell line model. To dissect the role of individual subregions, we fit several mathematical models to the experimental data. We show that a multiplicative switch model fits best the experimental data and that one subregion has a dominant effect on promoter activity. Mapping of the sub-sequences on phylogenetic conservation data reveals that the dominant regulatory region is the one with the highest multi-species conservation score.

Wilms' tumor protein (-KTS) modulates renin gene transcription.

Renin plays a crucial role in the control of various physiological processes such as blood pressure and body fluid homeostasis. Here, we show that a splice variant of the Wilms' tumor protein lacking three amino acids WT1(-KTS) suppresses renin gene transcription. Using bioinformatics tools, we initially predicted that a WT1-binding site exists in a regulatory region about 12 kb upstream of the renin promoter; this was confirmed by reporter gene assays and gel shift experiments in heterologous cells. Co-expression of Wt1 and renin proteins was found in rat kidney sections, mouse kidney blood vessels, and a cell line derived from the juxtaglomerular apparatus that produces renin. Knockdown of WT1 protein by siRNA significantly increased the cellular renin mRNA content, while overexpression of WT1(-KTS) reduced renin gene expression in stable and transiently transfected cells. A mutant WT1(-KTS) protein found in Wilms' tumors failed to suppress renin gene reporter activity and endogenous renin expression. Our findings show that renin gene transcription is regulated by the WT1(-KTS) protein and this may explain findings in patients with WT1 gene mutations of increased plasma renin and hypertension.

Role of nucleolin in posttranscriptional control of MMP-9 expression.

Matrix-metalloproteinases (MMPs), which are able to degrade extra cellular matrix (ECM) components, are crucial in ECM-remodeling, under physiological (e.g., embryogenesis, wound healing, angiogenesis) or pathophysiological conditions (e.g., arthritis, cancer progression and metastasis, fibrosis). Treating HT1080 cells, a human fibrosarcoma cell line, with the iron chelator 2,2-Dipyridyl, which mimics certain aspects of hypoxia, leads to a 3-fold elevated Matrix-metalloproteinase-9 (MMP-9) protein level. This elevation occurs within 3 h, without any change of mRNA-concentration. The rapid increase in MMP-9 expression is caused by an enhancement of translational efficiency characterized by a recruitment of translationally inactive MMP-9 mRNP-complexes into the rough endoplasmatic reticulum (rER). Reporter gene assays, which depend on the untranslated regions (UTR) of MMP-9 mRNA, reveal that the posttranscriptional regulation is mainly attributed to the 3'UTR. RNA/protein interaction studies indicate that the elevated binding of nucleolin ( approximately 64 kDa form) to the 3'UTR may be of major importance for the increased efficiency of MMP-9 translation. The results show that MMP-9 expression can be regulated posttranscriptionally, affecting the efficiency of translation and localization of the mRNA.

Translational regulation of the human achaete-scute homologue-1 by fragile X mental retardation protein.

Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5'-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)(10) sequence located in the 5'-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation.

Translational regulation of glutathione peroxidase 4 expression through guanine-rich sequence-binding factor 1 is essential for embryonic brain development.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a moonlighting selenoprotein, which has been implicated in basic cell functions such as anti-oxidative defense, apoptosis, and gene expression regulation. GPx4-null mice die in utero at midgestation, and developmental retardation of the brain appears to play a major role. We investigated post-transcriptional mechanisms of GPx4 expression regulation and found that the guanine-rich sequence-binding factor 1 (Grsf1) up-regulates GPx4 expression. Grsf1 binds to a defined target sequence in the 5'-untranslated region (UTR) of the mitochondrial GPx4 (m-GPx4) mRNA, up-regulates UTR-dependent reporter gene expression, recruits m-GPx4 mRNA to translationally active polysome fractions, and coimmunoprecipitates with GPx4 mRNA. During embryonic brain development, Grsf1 and m-GPx4 are coexpressed, and functional knockdown (siRNA) of Grsf1 prevents embryonic GPx4 expression. When compared with mock controls, Grsf1 knockdown embryos showed significant signs of developmental retardations that are paralleled by apoptotic alterations (TUNEL staining) and massive lipid peroxidation (isoprostane formation). Overexpression of m-GPx4 prevented the apoptotic alterations in Grsf1-deficient embryos and rescued them from developmental retardation. These data indicate that Grsf1 up-regulates translation of GPx4 mRNA and implicate the two proteins in embryonic brain development.

Translational control of collagen prolyl 4-hydroxylase-alpha(I) gene expression under hypoxia.

Hypoxia is a pro-fibrotic stimulus, which is associated with enhanced collagen synthesis, as well as with augmented collagen prolyl 4-hydroxylase (C-P4H) activity. C-P4H activity is controlled mainly by regulated expression of the alpha C-P4H subunit. In this study we demonstrate that the increased synthesis of C-P4H-alpha(I) protein in human HT1080 fibroblasts under long term hypoxia (36 h, 1% oxygen) is controlled at the translational level. This is mediated by an interaction of RNA-binding protein nucleolin (approximately 64 kDa form) at the 5'- and 3'-untranslated regions (UTR) of the mRNA. The 5'/3'-UTR-dependent mechanism elevates the C-P4H-alpha(I) expression rate 2.3-fold, and participates in a 5.3-fold increased protein level under long term hypoxia. The interaction of nucleolin at the 5'-UTR occurs directly and depends on the existence of an AU-rich element. Statistical evaluation of the approximately 64-kDa nucleolin/RNA interaction studies revealed a core binding sequence, corresponding to UAAAUC or AAAUCU. At the 3'-UTR, nucleolin assembles indirectly via protein/protein interaction, with the help of another 3'-UTR-binding protein, presumably annexin A2. The increased protein level of the approximately 64-kDa nucleolin under hypoxia can be attributed to an autocatalytic cleavage of a high molecular weight nucleolin form, without alterations in nucleolin mRNA concentration. Thus, the alteration of translational efficiency by nucleolin, which occurs through a hypoxia inducible factor independent pathway, is an important step in C-P4H-alpha(I) regulation under hypoxia.

Heterogeneous nuclear ribonucleoprotein-A2/B1 modulate collagen prolyl 4-hydroxylase, alpha (I) mRNA stability.

Collagen prolyl 4-hydroxylase (C-P4H) alpha-subunit is of regulatory importance in the assembling of C-P4H tetramers, which are necessary for the hydroxylation of procollagen chains. Change in collagen expression by hypoxia or iron diminishment is a significant issue in extracellular matrix remodeling. It was proposed that C-P4H-alpha (I) is regulated at the posttrancriptional level under these conditions. Here we report that the induction of C-P4H-alpha (I) in human fibrosarcoma cells HT1080 by the iron chelator 2,2-dipyridyl is predominantly caused by an enhancement of mRNA stability. This effect is mediated by an increased synthesis and binding of heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, which interacts with a (U)(16) element located in the 3'-untranslated region of C-P4H-alpha (I) mRNA. Luciferase reporter gene assays depending on C-P4H-alpha (I) 3'-untranslated region and co-transfection with hnRNP-A2/B1 provide evidence that the (U)(16) element is necessary and sufficient for posttranscriptional control of C-P4H-alpha (I) synthesis under the analyzed conditions. Further indication for the significance of hnRNP-A2/B1 in C-P4H-alpha (I) induction was obtained by micro array experiments. In a data set representing 686 independent physiological conditions, we found a significant positive correlation between hnRNP-A2/B1 and C-P4H-alpha (I) mRNAs.

Renal effects of Tamm-Horsfall protein (uromodulin) deficiency in mice.

The Tamm-Horsfall protein (THP; uromodulin), the dominant protein in normal urine, is produced exclusively in the thick ascending limb of Henle's loop. THP mutations are associated with disease; however, the physiological role of THP remains obscure. We generated THP gene-deficient mice (THP -/-) and compared them with wild-type (WT) mice. THP -/- mice displayed anatomically normal kidneys. Steady-state electrolyte handling was not different between strains. Creatinine clearance was 63% lower in THP -/- than in WT mice (P < 0.05). Sucrose loading induced no changes between strains. However, water deprivation for 24 h decreased urine volume from 58 +/- 9 to 28 +/- 4 microl x g body wt(-1) x 24 h(-1) in WT mice (P < 0.05), whereas in THP -/- mice this decrease was less pronounced (57 +/- 4 to 41 +/- 5 microl x g body wt(-1) x 24 h(-1); P < 0.05), revealing significant interstrain difference (P < 0.05). We further used RT-PCR, Northern and Western blotting, and histochemistry to study renal transporters, channels, and regulatory systems under steady-state conditions. We found that major distal transporters were upregulated in THP -/- mice, whereas juxtaglomerular immunoreactive cyclooxygenase-2 (COX-2) and renin mRNA expression were both decreased in THP -/- compared with WT mice. These observations suggest that THP influences transporters in Henle's loop. The decreased COX-2 and renin levels may be related to an altered tubular salt load at the macula densa, whereas the increased expression of distal transporters may reflect compensatory mechanisms. Our data raise the hypothesis that THP plays an important regulatory role in the kidney.