Project description:PGCs undergo two distinct stages of demethylation before reaching a hypomethylated ground state at E13.5. Stage 1 occurs between E7.25- E9.5 in which PGCs experience a global loss of cytosine methylation. However, discreet loci escape this global loss of methylation and between E10.5-E13.5, stage 2 of demethylation takes place. In this stage these loci are targeted by Tet1 and Tet2 leading to the loss of the remaining methylation and resulting in the epigenetic ground state. Our data shows that Dnmt1 is responsible for maintaining the methylation of loci that escape stage 1 demethylation, and that it functions in a UHRF1 independent manner. Our data further demonstrates that when these loci lose methylation prior to stage 2 it results in early activation of the meiotic program, which leads to precocious differentiation of the germ line resulting in a decreased pool of PGCs in the embryo and subsequent infertility in adult mice.
Project description:We created mice, which are deficient for Myc specifically in cardiac myocytes by crossing crossed Myc-floxed mice (Mycfl/fl) and MLC-2VCre/+ mice. Serial analysis of earlier stages of gestation revealed that Myc-deficient mice died prematurely at E13.5-14.5. Morphological analyses of E13.5 Myc-null embryos showed normal ventricular size and structure; however, decreased cardiac myocyte proliferation and increased apoptosis was observed. BrdU incorporation rates were also decreased significantly in Myc-null myocardium. Myc-null mice displayed a 3.67-fold increase in apoptotic cardiomyocytes by TUNEL assay. We examined global gene expression using oligonucleotide microarrays. Numerous genes involved in mitochondrial death pathways were dysregulated including Bnip3L and Birc2. Keywords: wildtype vs Myc-null
Project description:We created mice, which are deficient for Myc specifically in cardiac myocytes by crossing crossed Myc-floxed mice (Mycfl/fl) and MLC-2VCre/+ mice. Serial analysis of earlier stages of gestation revealed that Myc-deficient mice died prematurely at E13.5-14.5. Morphological analyses of E13.5 Myc-null embryos showed normal ventricular size and structure; however, decreased cardiac myocyte proliferation and increased apoptosis was observed. BrdU incorporation rates were also decreased significantly in Myc-null myocardium. Myc-null mice displayed a 3.67-fold increase in apoptotic cardiomyocytes by TUNEL assay. We examined global gene expression using oligonucleotide microarrays. Numerous genes involved in mitochondrial death pathways were dysregulated including Bnip3L and Birc2. Hearts were taken from wide type and Myc-null Mouse embryos at E13.5 under the dissecting scope. Cardiac myocyte RNA was isolated using TRIZOL®Reagent Total RNA (100 ng) was hybridized to the Sentrix® MouseRef-8 Expression BeadChip that contains probes for ~24,000 transcripts. GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A. The data were analyzed with Illumina Inc. BeadStudio version 1.5.0.34 and normalized by rank invariant method.
Project description:We analyzed gene expression in human fibroblasts stimulated by platelet-derived growth factor-BB (PDGF-BB) or basic fibroblast growth factor (bFGF) for 1h and 24h. The results of two independent experiments were merged. SAM analysis identified 116 relevant probe sets. Hierarchical clustering of these probe sets showed divergent early gene regulation by PDGF and FGF but overlapping late response. We first analyzed genes commonly regulated by PDGF-BB and b-FGF more than 2 fold after 24h of stimulation and we found that these two growth factors activated SREBP and E2F and repressed FOXO. We then focused on the early gene expression response induced by both growth factors. We performed a fold change analysis and found 114 probe sets regulated by PDGF-BB and 42 probe sets regulated by b-FGF, 37 of which were shared between the two gene lists . We found by data mining that both PDGF-BB and b-FGF activated AP-1 and NF-kB. Next we analyzed genes specifically regulated by PDGF-BB and found that STATs are specifically activated by PDGF and not by FGF. Experiment Overall Design: Human foreskin fibroblasts (AG01518) were cultured (1.5E6 cells/10 cm dish) in MEM medium with 10% fetal calf serum and glutamine for 24h, then washed and incubated for 47h in serum-free medium. Cells were stimulated for 1h by PDGF-BB (25ng/ml) or b-FGF (10ng/ml, + heparine 50 microg/ml). Alternatively, cells were incubated for 24h in starvation medium and treated for 24h with growth factors, or left untreated for 48h in serum-free medium (control treatment). Experiment Overall Design: Two biological replicates were performed