An Analysis of the Transcriptional Control Domains of the Human c-myc Proto-Oncogene

Transfection of mammalian cells with recombinant plasmid DNAs containing the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter has been used to analyse genomic sequences regulating the transcription of the human c-myc proto-oncogene. Several regulatory domains 5' to the c-myc coding region have been identified, and their locations defined by deletion analysis. Each of these sites encompass previously identified DNase I in vivo hypersensitive sites. Published data suggested that the c-myc gene may be regulated in vivo by a repressor. The mapping of translocation breakpoints for Burkitt's lymphoma and murine plasmacytoma which involve the c-myc gene, suggests that the cis-acting recognition sequence for this putative repressor is located within the 5' flanking region. I have identified a negative regulatory element (NRE-2) in the 5' flanking region of the gene and localised it to a region between -1052 and -607bp 5' to the PI start site of the c-myc mRNA, by deletion analysis. Subsequent competition experiments showed a 270bp sub-fragment to contain an essential component of the negative regulatory element. This element can function in an orientation independent manner, and has the ability to repress heterologous promoters (both viral and eukaryotic), but to a lesser degree than when acting in cis upon its homologous promoter. My data from both DNA titration and competition transfection analysis indicates that this repression is mediated by at least one trans-acting factor. Since the repression was observed in every cell line used as the transfection recipients, a certain promiscuity in the tissue- and species- specificity of the trans-acting repressor(s) is implied. In vivo footprint analysis tentatively identified two sequence-specific DNA-binding proteins which interact with this domain. Both the CCAAT-binding Transcription Factor (CTF) and Spl bind within the NRE-2 domain. This is the first time either of these DNA-binding factors have been implicated in the transcriptional repression of a gene. In addition, deletion analyses identified an Upstream Promoter Element (UPE), located between the NRE-2 and the c-myc mRNA major cap sites, which is responsible for activation of the high levels of CAT expression observed in cells transfected with the recombinant plasmids. This UPE appears to be a highly complex domain which was shown, by DNase I in vivo footprint analysis, to bind several Spl-like factors. In addition, the UPE is somehow involved in the control of the repression function, although it is not required for the repression of heterologous promoters by the NRE-2. Other data (assayed preliminary) suggested also that two other distal regulatory domains are involved in the control of c-myc expression. The more distal element (PRE) has an activating activity, and was localised to a region which showed sequence homology to enhancer elements. A second element (NRE-1) was tentatively identified which had a negative effect on CAT activity. I conclude that the removal, and/or the rearrangement, of these transcription regulatory domains may play a crucial role in the deregulation of the expression of c-myc that is observed in some neoplastic cells

Case reports: pig 917.

<p>Grouped data for the transgenic pig 917 (3 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 917 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–P) Immunolabeling for M-opsin (E,I), PNA (F,J) and merged picture (G,K), GFAP (H,I) and S-opsin (I,J,O,P) in the central region of the retina of pig 917 (E–J) and a non-transgenic control (K–P). Arrows in E, G, K and M show examples of M-opsin positive outersegment, arrowhead in G shows displaced nuclei, arrowhead in J shows a displaced nucleus in a S-opsin positive cell. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–J represents 50 µm.</p

Range of visual function in GUCY2D^E837D/R838S transgenic pigs.

<p>(A) Traces of photopic electroretinogram recordings (ERG) at 11 weeks are shown for one representative control and several transgenic animals. (A, first column) single flash at 3 cds/m², (A, second column) single flash at 10 cds/m2, (A, third column) flickers at 3 cds/m²,30 Hz. (B) b-wave amplitudes for all examined animals at 11 weeks with single flash at 3 cds/m² and at 10 cds/m². (C) a-wave amplitudes obtained for all examined animals at 11 weeks with single flash at 3 cds/m² and at 10 cds/m². (D) Representation of the time needed to complete the obstacle course at 24 and 52 weeks for transgenic and non-transgenic control animals. (E) Representation of the errors (missing or striking into an obstacle), alternative prospections (the number of times individuals investigated an obstacle by sniffing or licking) and resulting scores from the obstacle course at 24 and 52 weeks for transgenic and non-transgenic control animals. Horizontal bars in B,C represent the mean of the different groups with the SEM; *: p<0.05;***: p<0.001; errors in E: miss or strike into an obstacles; alternative prospection in E: sniff or lick the obstacles; score in E: sum of errors and alternative prospections.</p

Case reports: pig 920.

<p>Grouped data collected for the transgenic pig 920 (4 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 920 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–L) Immunolabeling for M-opsin (E,I), PNA (F,J) and merged picture (G,K), and GFAP (H,I) in the central region of the retina of pig 920 (E–H) and a non-transgenic control (I–L). Arrows in E, G, I and K show examples of M-opsin positive outersegment, arrowhead in G shows a displaced nucleus. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–L represents 50 µm.</p

Expression of GUCY2D^E837D/R838S transcript in transgenic pigs.

<p>Assessment of transgene expression by RT-PCR. Transgenic pigs: 904, 907, 908, 914, 915, 917, 918, 920, control non-transgenic pig (929). +: with reverse transcription; −: without reverse transcription; hGUCY2Dmut: GUCY2D^E837D/R838S PCR fragment; pig GUCY2D: pig GUCY2D PCR fragment; GAPDH: pig GAPDH PCR fragment.</p

Abnormal retinal morphology in transgenic pigs.

<p>(A) Morphological examination of a retina from a control animal reveals retinal layers: outer segments (OS), inner segment of photoreceptors (IS), photoreceptor nuclei (ONL). (B) Displaced nuclei were observed in the outer segment layer in transgenic retina (arrows). (C) Quantification of the number of displaced nuclei in transgenic and control animals. (D,E,F) Immunolabeling for specific cone markers PNA and M-opsin in transgenic retina identified most of these displaced cells as cones. (G,H) Immunolabeling for specific cone markers PNA and M-opsin in control retina. (I) Quantitation of relative density of displaced cones as determined by PNA or M-opsin labeling across 100 µm on the section. OS: outer segment; IS: inner segment; ONL: outernuclear layer; PNA: peanut agglutinin; M-opsin: medium wavelength opsin; nb: number. Scale bar in A, B and D to F represents 50 µm.</p

Case report: pig 908.

<p>Grouped data for the transgenic pig 908 (2 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 908 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). For technical reasons, the time to reach the end of the obstacle course has not been measured at 52 weeks. (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–G) Immunolabeling for M-opsin (E), PNA (F) and merged picture (G) in the central region of the retina of pig 908. Arrows in E and G show examples of M-opsin positive outersegment, arrowhead in G shows a displaced nucleus. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–G represents 50 µm.</p

Rapid cohort generation and analysis of disease spectrum of large animal model of cone dystrophy.

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients

<i>C9ORF72 </i>repeat expansion causes vulnerability of motor neurons to Ca²⁺-permeable AMPA receptor-mediated excitotoxicity

Mutations in C9ORF72 are the most common cause of familial amyotrophic lateral sclerosis (ALS). Here, through a combination of RNA-seq and electrophysiological studies on induced pluripotent stem cell (iPSC) derived motor neuron (MNs), we show that increased expression of GluA1 AMPA receptor (AMPAR) subunit occurs in MNs with C9ORF72 mutations that leads to increased Ca2+-permeable AMPAR expression and results in enhanced selective MN vulnerability to excitotoxicity. These deficits are not found in iPSC-derived cortical neurons and are abolished by CRISPR/Cas9-mediated correction of the C9ORF72 repeat expansion in MNs. We also demonstrate that MN-specific dysregulation of AMPAR expression is also present in C9ORF72 patient post mortem material. We therefore present multiple lines of evidence for the specific upregulation of GluA1 subunits in human mutant C9ORF72 MNs that could lead to a potential pathogenic excitotoxic mechanism in ALS

Rationale, design, and baseline characteristics in Evaluation of LIXisenatide in Acute Coronary Syndrome, a long-term cardiovascular end point trial of lixisenatide versus placebo

BACKGROUND: Cardiovascular (CV) disease is the leading cause of morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). Furthermore, patients with T2DM and acute coronary syndrome (ACS) have a particularly high risk of CV events. The glucagon-like peptide 1 receptor agonist, lixisenatide, improves glycemia, but its effects on CV events have not been thoroughly evaluated. METHODS: ELIXA (www.clinicaltrials.gov no. NCT01147250) is a randomized, double-blind, placebo-controlled, parallel-group, multicenter study of lixisenatide in patients with T2DM and a recent ACS event. The primary aim is to evaluate the effects of lixisenatide on CV morbidity and mortality in a population at high CV risk. The primary efficacy end point is a composite of time to CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. Data are systematically collected for safety outcomes, including hypoglycemia, pancreatitis, and malignancy. RESULTS: Enrollment began in July 2010 and ended in August 2013; 6,068 patients from 49 countries were randomized. Of these, 69% are men and 75% are white; at baseline, the mean ± SD age was 60.3 ± 9.7 years, body mass index was 30.2 ± 5.7 kg/m(2), and duration of T2DM was 9.3 ± 8.2 years. The qualifying ACS was a myocardial infarction in 83% and unstable angina in 17%. The study will continue until the positive adjudication of the protocol-specified number of primary CV events. CONCLUSION: ELIXA will be the first trial to report the safety and efficacy of a glucagon-like peptide 1 receptor agonist in people with T2DM and high CV event risk