DNA methylation and gene expression changes during human embryonic stem cell differentiation
ABSTRACT: Investigate the genome-wide DNA methylation and gene expression changes during human embryonic stem cell differentiation. hESCs (H1 and H13) DNA samples for 3 different differentian stage (D0, undifferentiated;D21, 21 days after undirected differentiation; D90, 90 days after undirected differentiation); two-color experiment, paired comparison (differentiated vs undifferentiated; A & B: two biological replicates). Differentian-stage variation in methylation profiling of human DNA samples were detected by MSAM. 2µg of DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification. Analysis of differentian-stage variation in gene expression of human mRNA samples were implementd following standard Agilent protocol.
Project description:Investigate the genome-wide DNA methylation changes in the mouse hypothalamus during the suckling period. Hypothalami were collected from new born (P0) mice, or mice of 21 day-old (P21). Two-color experiment was performed as paired comparison of P21 vs. P0 with two biological replicates. Genome-wide DNA methylation changes from P0 to P21 were detected by MSAM. As a brief description of the MSAM: 500ng of genomic DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification and then cohybridization.
Project description:Interindividual variation in methylation profiling of human DNA samples were detected using two-tissue screening by MSAM. 0.5ug of DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification HF (hair follicle) and PBL (Peripheral Blood Leukocyte) DNA samples for 8 different individuals, two-color experiment, interindividual paired comparison (same sex and age)
Project description:The goal is to compare the DNA methylation patterns between ARH and PVH to examine to what extent DNA methylation is region specific in genome scale. ARH and PVH were micro-dissected from mice brains. Each 7 pieces of ARH or PVH were pooled and used for DNA methylation comparison by methylation specific amplification microarray (MSAM). As a brief description of the MSAM: 500ng of genomic DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification and then cohybridization. Two-color cohybridizations were performed as paired comparison of PVH vs. ARH with two biological replicates including dye swap.
Project description:Investigate the persistent effects of early postnatal overnutrition on the developmental establishment of the DNA methylation in the mouse hypothalamus. Early postnatal overnutrition was induced in mice by reducing the litter size from normally 9 (C) to 4 (SL) pups per litter. Hypothalami were collected from both C and SL mice at the age of postnatal day 180 (P180). Genome-wide DNA methylation difference between SL and C were detected by MSAM. Equal amount of genomic DNA from 5 hypothalami of the same group were pooled as one MSAM sample. Two pooled DNA samples for each group were used for comparison that meant total 10 hypothalami for each group. 500ng pooled DNA was serially digested with SmaI and XmaI followed by adaptor ligation and PCR amplification. Two cohybridizations were performed to compare DNA methylation between SL and C hypothalami, with day swap.
Project description:DNA methylation profiling of colonic mucosal DNA between P90 and P30 mice. 0.5ug of DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification Two independent P90 to P30 comparisons were performed as follows. Samples were labelled with Cy3 (P30) and Cy5 (P90) and two independent P90 to P30 comparisons were done on a 2x105k methylation specific amplification microarray (MSAM) containing 90,535 probes, covering 77% of the 31,019 SmaI intervals between 200 bp and 2 kb in the mouse genome (average 3.8 probes per interval)
Project description:Studies in yeast have demonstrated that signalling kinases with well known cytoplasmic functions have a surprisingly active role in the nucleus, where they are tethered to chromatin and modulate gene expression programs. Here we provide evidence for a novel function of the signal transduction kinase, protein kinase C-theta (PKC-0) that physically associates with the proximal regulatory regions of key inducible immune response genes in human T cells. Chromatin-anchored PKC-q forms hitherto undescribed nuclear complexes by interacting with active RNA polymerase II, the histone kinase MSK-1 and the adaptor molecule 14-3-3z. ChIP-on-Chip analysis reveals that PKC-0 binds directly to both the promoter and transcribed regions of genes, as well as to the promoters of microRNA genes implicated in cell migration and invasion. Moreover, enforced expression of these microRNAs is associated with heightened production of mRNAs encoding a distinct subset of inducible immune response genes. Collectively, these data suggest that in addition to its well known role as a cytoplasmic signalling kinase, PKC-0 controls immune gene expression within the nucleus of T cells by participating in chromatin-associated signalling complexes PKC-0 and Pol II ChIP DNA were pooled from five independent ChIP assay experiments that were first individually validated by real-time PCR. Pooled ChIP samples were subsequently amplified based on one round of the whole genome amplification method using the WGA2 kit (Sigma-Aldrich), as described previously (O'Geen, Hollenhorst, Dindot). An alternate random primed and linker-mediated PCR amplification protocol was compared with the WGA2 kit using quantitative real-time PCR, which generated similar yields of amplified material, but failed to preserve the degree of enrichment after the linker-mediated PCR amplification (data not shown). Labelling, hybridization and scanning were performed as described in the Mammalian ChIP-on-chip protocol (version 9.1; Agilent Technologies). Briefly, the control and PKC-0 or Pol II ChIP DNA were labelled with 5 ug cyanine-3 and 5 ug cyanine-5 (Invitrogen BioPrime CGH labeling kit), respectively. Samples were hybridised on each microarray slide for 40 h at 65°C. Agilent human promoter microarrays were utilised comprising of two slides per set defined to cover ~17,000 promoters of human transcripts from -5.5 to +2.5 Kb relative to transcriptional start site. The microarrays were scanned on an Agilent scanner (G2565BA) at 100% PMT gain. Two replicates of each ChIP-on-chip experiment were performed.
Project description:The transcription factor Zic3 is required for maintenance of embryonic stem (ES) cell pluripotency (Lim LS et al, Mol Biol Cell. 2007;18:1348-1358). By genome-wide chromatin immunoprecipitation (ChIP-chip) in ES cells, we have identified 379 direct Zic3 targets, many of which are functionally associated with pluripotency, cell cycle, proliferation, oncogenesis and early embryogenesis. E14 cells were cultured to a density of 1 x 108 cells for each IP. Two biological replicates were performed per experiment. Cells were cross-linked for 10 minutes at room temperature with 1% (w/v) formaldehyde and the reaction subsequently quenched with 125mM glycine. Nuclear fractions were isolated and the DNA sheared to average lengths of 200-500 bp. For analysis on mouse promoter arrays, purified ChIP material was processed according to the Agilent ChIP-on-chip protocol, and labeled DNA was hybridized to Agilent mouse promoter ChIP-on-chip arrays for 40 hours at 65oC (G4490A; Agilent Technologies, Santa Clara, CA). Chips were washed and scanned as per manufacturer’s protocol and the results were processed with Agilent's ChIP Analytics software v1.3. A p-value cutoff <0.001 was specified in our analysis. To further minimize false positives, we applied a “neighborhood voting” algorithm  to filter for high confidence Zic3-enriched sites, wherein binding was considered genuine only in the presence of a second, significantly enriched, neighboring probe (p < 0.005).
Project description:The TEAD (1-4) transcription factors comprise the conserved TEA/ATTS DNA binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy amongst the family members. Expression of the TEA/ATTS DNA binding domain that acts a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, while selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation (ChIP-chip) shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors cooperate with MYOD1 to directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation and fusion. RNA-seq identifies a novel set of TEAD4 target genes encoding muscle structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF and Cyclin D1 to promote differentiation. Together these results show that TEAD factor activity is essential for C2C12 cell differentiation and define a novel and nonredundant role for TEAD4 in regulating the unfolded protein response. C2C12 cells were infected with retrotiviral vector expressing Flag-HA-Tagged TEAD4 or with empty control vector and selected in the continouos presence of puromycin. Infected cell populations were then differentiated for 5 days in DMEM medium with 2% horse serum and fixed in 0.4% formaldehyde.
Project description:Background: Previously we reported extensive gene expression reprogramming during epithelial to mesenchymal transition (EMT) of primary prostate cells. Here we investigated the hypothesis that specific histone and DNA methylations are involved in coordination of gene expression during EMT and early stages of transformation. Results: Genome-wide profiling of histone methylations (H3K4me3 and H3K27me3) and DNA methylation (DNAMe) was applied on a prostate cell model during EMT and malignant transformation. Integrated analyses of promoter epigenetic modifications and gene expression changes revealed strong correlations between the dynamic changes of histone methylations and gene expression. DNA methylation was weakly associated with global gene repression, but strongly correlated to gene silencing when genes co-modified by H3K4me3 were excluded. In genes labeled with multiple epigenetic marks in their promoters, the level of transcription was associated with the net signal intensity of the activating mark H3K4me3 minus the repressive mark H3K27me3 or DNAMe, indicating that the effect on gene expression of bivalent marks (H3K4/K27me3 or H3K4me3/DNAMe) depends on relative modification intensities. Sets of genes, including epithelial cell junction and EMT associated fibroblast growth factor receptor genes, showed corresponding changes concerning epigenetic modifications and gene expression during EMT. Conclusions: This work presents the first blueprint of epigenetic modifications during EMT in prostate cells and shows that specific histone methylations are extensively involved in gene expression reprogramming during EMT and carcinogenesis. The observation that transcription activity of bivalently marked genes depends on the relative labeling intensity of individual marks provides a new view of quantitative regulation of epigenetic modification.