Molecular mechanisms controlling the tissue-specific activity of the nutritionally regulated promoter I of the acetyl-CoA carboxylase-alpha encoding gene in cattle

We analyzed molecular mechanisms controlling the activity of the nutritionally regulated promoter I (PI) of the bovine acetyl-CoA-carboxylase-alpha gene. PI-activity is driven by NF-Y and C/EBP factors having bound to high affinity sites on the proximal PI. However, both factors may also bind to low affinity sites on the distal PI. From there, they act as repressors of PI. We found that NF-Y may diminish and block C/EBP-beta mediated PI activation, even in trans, through direct factor interaction. Alterations in the concentrations of both factors apparently regulate the tissue specific PI-activity.Es wurden die molekularen Mechanismen untersucht, welche die Aktivität des ernährungsabhängig regulierten Promotors I (PI) des bovinen Acetyl-CoA-carboxylase-alpha Gens bestimmen. NF-Y und C/EBP Faktoren treiben die PI-Aktivität, wenn sie an proximale Bindungsstellen hoher Affinität gebunden haben. Sie drosseln jedoch PI-Aktivität nach Bindung an distale Ansatzstellen geringer Bindungsaffinität. NF-Y blockiert die Aktivierungsfähigkeit von C/EBP-beta, auch im Tran, durch direkte Faktoren-Interaktion. Veränderungen der Konzentrationen beider Faktoren bestimmen die gewebespezifische PI-Aktivität

Dual effect of a single nucleotide polymorphism in the first intron of the porcine Secreted phosphoprotein 1 gene: allele-specific binding of C/EBP beta and activation of aberrant splicing

<p>Abstract</p> <p>Background</p> <p>Secreted phosphoprotein 1 (SPP1 or Osteopontin, OPN) is a multifunctional matricellular glycoprotein involved in development and regeneration of skeletal muscle. Previously, we have demonstrated that porcine <it>SPP1 </it>shows breed-related differential mRNA expression during myogenesis. With the aim to identify putative contributing <it>cis</it>-regulatory DNA variation we resequenced the 5' upstream region of the gene in the respective breeds Pietrain and Duroc. We found two single nucleotide polymorphisms (SNP; [GenBank:<ext-link ext-link-id="M84121" ext-link-type="gen">M84121</ext-link>]: g.1804C>T and g.3836A>G). We focused our investigation on the SNP g.3836A>G, because <it>in silico </it>analysis and knowledge about the regulation of <it>SPP1 </it>suggested an effect of this SNP on a CCAAT/enhancer binding protein beta (C/EBPβ) responsive transcriptional enhancer.</p> <p>Results</p> <p>Using electrophoretic mobility shift assay we demonstrated that, similar to human <it>SPP1</it>, the 3' terminal end of the first intron of porcine <it>SPP1 </it>harbors a C/EBPβ binding site and showed that this binding site is negatively affected by the mutant G allele. Genotyping of 48 fetuses per breed revealed that the G allele segregated exclusively in Duroc fetuses with a frequency of 57 percent. Using real-time quantitative PCR we showed that, consistent with its negative effect on a transcriptional enhancer element, the G allele tends to decrease mRNA abundance of <it>SPP1 </it>in the fetal <it>musculus longissimus dorsi </it>(~1.3 fold; P ≥ 0.1).</p> <p>Moreover, we showed that the SNP g.3836A>G leads to ubiquitous aberrant splicing of the first intron by generating a <it>de novo </it>and activating a cryptic splice acceptor site. Aberrantly spliced transcripts comprise about half of the <it>SPP1 </it>messages expressed by the G allele. Both aberrant splice variants differ from the native transcript by insertions in the leader sequences which do not change the reading frame of <it>SPP1</it>.</p> <p>Conclusion</p> <p>At the 3' terminal end of the first intron of the porcine <it>SPP1 </it>we identified a unique, dually functional SNP g.3836A>G. This SNP affects the function of the <it>SPP1 </it>gene at the DNA level by affecting a C/EBPβ binding site and at the RNA level by activating aberrant splicing of the first intron, and thus represents an interesting DNA-marker to study phenotypic effects of <it>SPP1 </it>DNA-variation.</p

An epigenetic switch induced by Shh signalling regulates gene activation during development and medulloblastoma growth

The Sonic hedgehog (Shh) signalling pathway plays important roles during development and in cancer. Here we report a Shh-induced epigenetic switch that cooperates with Gli to control transcription outcomes. Before induction, poised Shh target genes are marked by a bivalent chromatin domain containing a repressive histone H3K27me3 mark and an active H3K4me3 mark. Shh activation induces a local switch of epigenetic cofactors from the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) to an H3K27me3 demethylase Jmjd3/Kdm6b-centred coactivator complex. We also find that non-enzymatic activities of Jmjd3 are important and that Jmjd3 recruits the Set1/MLL H3K4 methyltransferase complexes in a Shh-dependent manner to resolve the bivalent domain. In vivo, changes of the bivalent domain accompanied Shh-activated cerebellar progenitor proliferation. Overall, our results reveal a regulatory mechanism that underlies the activation of Shh target genes and provides insight into the causes of various diseases and cancers exhibiting altered Shh signalling

Interaction of C/EBP-beta and NF-Y factors constrains activity levels of the nutritionally controlled promoter IA expressing the acetyl-CoA carboxylase-alpha gene in cattle

BACKGROUND: The enzyme acetyl-CoA carboxylase-alpha (ACC-α) is rate limiting for de novo fatty acid synthesis. Among the four promoters expressing the bovine gene, promoter IA (PIA) is dominantly active in lipogenic tissues. This promoter is in principal repressed but activated under favorable nutritional conditions. Previous analyses already coarsely delineated the repressive elements on the distal promoter but did not resolve the molecular nature of the repressor. Knowledge about the molecular functioning of this repressor is fundamental to understanding the nutrition mediated regulation of PIA activity. We analyzed here the molecular mechanism calibrating PIA activity. RESULTS: We finely mapped the repressor binding sites in reporter gene assays and demonstrate together with Electrophoretic Mobility Shift Assays that nuclear factor-Y (NF-Y) and CCAAT/enhancer binding protein-β(C/EBPβ) each separately repress PIA activity by binding to their cognate low affinity sites, located on distal elements of the promoter. Simultaneous binding of both factors results in strongest repression. Paradoxically, over expression of NFY factors, but also - and even more so - of C/EBPβ significantly activated the promoter when bound to high affinity sites on the proximal promoter. However, co-transfection experiments revealed that NF-Y may eventually diminish the strong stimulatory effect of C/EBPβ at the proximal PIA in a dose dependent fashion. We validated by chromatin immunoprecipitation, that NF-Y and C/EBP factors may physically interact. CONCLUSION: The proximal promoter segment of PIA appears to be principally in an active state, since even minute concentrations of both, NF-Y and C/EBPβ factors can saturate the high affinity activator sites. Higher factor concentrations will saturate the low affinity repressive sites on the distal promoter resulting in reduced and calibrated promoter activity. Based on measurements of the mRNA concentrations of those factors in different tissues we propose that the interplay of both factors may set tissue-specific limits for PIA activity

Recombinant Expression of a Novel Fungal Immunomodulatory Protein with Human Tumor Cell Antiproliferative Activity from Nectria haematococca

To our best knowledge, all of the fungal immunomodulatory proteins (FIPs) have been successfully extracted and identified in Basidomycetes, with only the exception of FIP from ascomycete Nectria haematococca (FIP-nha) discovered through homology alignment most recently. In this work, a gene encoding FIP-nha was synthesized and recombinantly expressed in an Escherichia coli expression system. SDS-PAGE and MALDI-MS analyses of recombinant FIP-nha (rFIP-nha) indicated that the gene was successfully expressed. The yield of the bioactive FIP-nha protein was 42.7 mg/L. In vitro assays of biological activity indicated that the rFIP-nha caused hemagglutination of human and rabbit red blood cells, significantly stimulated mouse spleen lymphocyte proliferation, and enhanced expression of interleukin-2 (IL-2) released from mouse splenocytes, revealing a strong antitumor effect against HL60, HepG2 and MGC823. Through this work, we constructed a rapid and efficient method of FIP production, and suggested that FIP-nha is a valuable candidate for use in future medical care and pharmaceutical products

Effect of Polymer Addition on the Structure and Hydrogen Evolution Reaction Property of Nanoflower-Like Molybdenum Disulfide

Nano-structured molybdenum disulfide (MoS2) catalysts have been extensively developed for the hydrogen evolution reaction (HER). Herein, a novel hydrothermal intercalation approach is employed to fabricate nanoflower-like 2H–MoS2 with the incorporation of three polymers, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and polyethylenimine (PEI). The as-prepared MoS2 specimens were characterized by techniques of scanning electron microscope (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), together with Raman and Fourier transform infrared spectroscopy (FTIR). The HER properties of these lamellar nanoflower-like composites were evaluated using electrochemical tests of linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS). The existent polymer enlarges the interlayer spacing of the lamellar MoS2, and reduces its stacked thickness. The lamellar MoS2 samples exhibit a promoting activity in HER at low additions of these three polymers (0.04 g/g MoS2 for PVA and PEI, and 0.08 g/g MoS2 for PVP). This can be attributed to the fact that the expanded interlayer of MoS2 can offer abundant exposed active sites for HER. Conversely, high additions of the polymers exert an obvious interference in the HER activity of the lamellar MoS2. Compared with the samples of MoS2/PVP–0.08 and MoS2/PEI–0.04, the MoS2/PVA–0.04 composite exhibits excellent activity in HER, in terms of higher current density and lower onset potential

Regulation of Gli ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-β2 nuclear import system

<div><p>Hedgehog (Hh) signaling in vertebrates depends on primary cilia. Upon stimulation, Hh pathway components, including Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting remains largely unknown. Here, we show that the PY-type nuclear localization signal (PY-NLS)/karyopherinβ2 (Kapβ2) nuclear import system regulates Gli ciliary localization and Hh pathway activation. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization. Kapβ2 is required for the formation of Gli activator (Gli^A) in wild-type but not in <i>Sufu</i> mutant cells. Knockdown of Kapβ2 affected Hh signaling in zebrafish embryos, as well as in vitro cultured cerebellum granule neuron progenitors (CGNPs) and SmoM2-driven medulloblastoma cells. Furthermore, Kapβ2 depletion impaired the growth of cultured medulloblastoma cells, which was rescued by Gli overexpression. Interestingly, Kapβ2 is a transcriptional target of the Hh pathway, thus forming a positive feedback loop for Gli activation. Our study unravels the molecular mechanism and cellular machinery regulating Gli ciliary localization and identifies Kapβ2 as a critical regulator of the Hh pathway and a potential drug target for Hh-driven cancers.</p></div