Cell type-specific regulation of choline acetyltransferase gene expression - Role of the neuron-restrictive silencer element and cholinergic-specific enhancer

This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pair-long region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. A 21-base pair-long neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region. This element was recognized by the neuron-restrictive silencer factor (NRSF), previously shown to regulate expression of other neuron-specific genes. The ChAT NRSE was inactive in both cholinergic and non-cholinergic neuronal cells, but repressed expression from a heterologous promoter in non-neuronal cells. Specific deletion of this element allowed ChAT gene promoter activity in non-neuronal cells, and overexpression of NRSF repressed ChAT gene promoter activity in cholinergic cells. The distal part of the ChAT gene promoter showed cholinergic-specific enhancing activity, which stimulated promoter activity in cholinergic cells, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. Thus, at least two kinds of regulatory elements cooperate to direct ChAT gene expression to cholinergic neurons, namely a neuron-restrictive silencer element and a cholinergic-specific enhancer

IGF-II transcription in skeletal myogenesis is controlled by mTOR and nutrients

Insulin-like growth factors (IGFs) are essential for skeletal muscle development, regeneration, and hypertrophy. Although autocrine actions of IGF-II are known to initiate myoblast differentiation, the regulatory elements and upstream signaling pathways for myogenic expression of IGF-II remain elusive. Here, we report the regulation of IGF-II transcription by mTOR, as well as by amino acid sufficiency, through the IGF-II promoter 3 and a downstream enhancer during C2C12 myoblast differentiation. Furthermore, we present evidence that IGF production, and not IGF signaling, is the primary target for mTOR's function in the initiation of differentiation. Moreover, myogenic signaling by mTOR is independent of its kinase activity and mediated by the PI3K–Akt pathway. Our findings represent the first identification of a signaling pathway that regulates IGF-II expression in myogenesis and implicate the mTOR–IGF axis as a molecular link between nutritional levels and skeletal muscle development

The Igf2/H19 imprinting control region exhibits sequence-specific and cell-type-dependent DNA methylation-mediated repression

Methylation of CpGs is generally thought to repress transcription without significant influence from the sequence surrounding the methylated dinucleotides. Using the mouse Igf2/H19 imprinting control region (ICR), Igf2r differentially methylated region 2 (DMR2) and bacterial sequences, we addressed how methylation-dependent repression (MDR) from a distance varies with CpG number, density and surrounding sequence. In stably transfected F9 cells, the methylated ICR repressed expression from a CpG-free reporter plasmid more than 1000-fold compared with its unmethylated control. A segment of pBluescript, with a CpG number equal to the ICR's but with a higher density, repressed expression only 70-fold when methylated. A bacteriophage lambda fragment and the Igf2r DMR2 showed minimal MDR activity, despite having CpG numbers and densities similar to or greater than the ICR. By rearranging or deleting CpGs, we identified CpGs associated with three CTCF sites in the ICR that are necessary and sufficient for sequence-specific MDR. In contrast to F9 cells, the methylated ICR and pBS fragments exhibited only 3-fold reporter repression in Hela cells and none in Cos7. Our results show that the strength of MDR from a distance can vary a 1000-fold between different cell types and depends on the sequence surrounding the methylated CpGs, but does not necessarily increase with CpG number or density

Loss of Maternal CTCF Is Associated with Peri-Implantation Lethality of Ctcf Null Embryos

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5–E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16–32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays

BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care

The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes

The neuron-restrictive silencer factor (NRSF) binds a DNA sequence element, called the neuron-restrictive silencer element (NRSE), that represses neuronal gene transcription in nonneuronal cells. Consensus NRSEs have been identified in 18 neuron-specific genes. Complementary DNA clones encoding a functional fragment of NRSF were isolated and found to encode a novel protein containing eight noncanonical zinc fingers. Expression of NRSF mRNA was detected in most nonneuronal tissues at several developmental stages. In the nervous system, NRSF mRNA was detected in undifferentiated neuronal progenitors, but not in differentiated neurons. NRSF represents the first example of a vertebrate silencer protein that potentially regulates a large battery of cell type-specific genes, and therefore may function as a master negative regulator of neurogenesis

A common silencer element in the SCG10 and type II Na^+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells

We have localized a cell type-specific silencer element in the SCG10 gene by deletion analysis. This neural-restrictive silencer element (NRSE) selectively represses SCG10 expression in nonneuronal cells and tissues. The NRSE contains a 21 by region with striking homology to a sequence present in a silencer domain of the rat type II sodium channel (Nall), another neuron-specific gene. We have identified a sequence-specific protein(s) that binds the SCG10 NRSE, as well as the homologous element in the Nall gene. A point mutation in the NRSE that abolishes binding of this neural-restrictive silencerbinding factor (NRSBF) in vitro also eliminates silencing activity in vivo. NRSBF is present in nuclear extracts from nonneuronal cells but not in extracts from neuronal cells, suggesting that the neuron-specific expression of SCG10 reflects, at least in part, the absence or inactivity of this protein. These data identify the NRSE as a potentially general DNA element for the control of neuron-specific gene expression in vertebrates

mTOR regulates skeletal muscle regeneration in vivo through kinase-dependent and kinase-independent mechanisms

Rapamycin-sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (mTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, the kinase-independent function of mTOR remains controversial, and no in vivo studies have examined those mTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, we have generated and characterized transgenic mice expressing two mutants of mTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of mTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, mTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively