Cell cycle analysis of histone marks and 4C in human Fucci ESCs
ABSTRACT: Using the Fucci cell cycle indicator system in hESCs, we evaluated the patterns of bivalent histone marks and enhancer histone marks during the cell cycle by ChIP-seq. We further evaluated how the chromatin architecture changed during the cell cycle. We found that bivalent domains are cell cycle regulated, and H3K4me3 specifically peaks during the late G1 stage of the cell cycle. H3K27me3, however, is largely unchanged during the cell cycle. Cell cycle-regulated bivalent domains interact with enhancers and form cell cycle regulated chromatin interactions. FACS-isolated cell cycle fractions (DN, early G1; KO2, late G1; AzL, S-phase; and AzH, G2/M) from Fucci hESCs were subject to ChIP-seq for H3K4me3, H3K27me3, H3K27ac and H3K4me1, and used for sequencing along with input controls for each of the 4 cell cycle fractions (20 samples total), using Illumina platform, or 4C-seq for each cell cycle fraction using viewpoints neighboring the GATA6 or SOX17 promoters.
Project description:By mapping the genomic enrichments of H3K4me3 and H3K27me3 modifications in pure populations of hESCs during the G2, mitotic and G1 phases of the cell cycle, we characterize cell cycle-dependent variations in the epigenetic landscape of bivalent genes, altering the current view of mitotic inheritance in pluripotent cells. We identified novel classes of bivalent domains that are highly enriched with H3K4me3 during mitosis, depleted during G1 only, and ubiquitously bivalent. These bivalent domains are associated with specific genes and expression patterns during differentiation. These cell cycle-dependent epigenetic profiles are unique to hESCs and are not observed following initiation of phenotype commitment. Our results establish a new dimension in cell cycle-dependent chromatin regulation that advances understanding of contributions the pluripotent epigenetic landscape to hESC identity. Study of two histone modifications, H3K4me3 and H3K27me3, during three cell cycle phases in two cell types. Study of gene expression from these two cell types in asynchronous cells.
Project description:hiPS derived cardiomycoytes treated with 4 cell cycle genes to induce cardiomyocyte proliferation and sorted for the different cell cycle stages according to FUCCI dye Overall design: hiPS derived cardiomyocytes infected with adenovirus encoding 4 cell cycle genes in the presence or absence of TGFB inhibitor and Wee1 inhibitor (2i) to induce cardiomyocyte proliferation and sorted for the different cell cycle stages according to FUCCI dye. (-ve:G0 phase, Red:G1 phase, RedGreen: S phase, Green: G2/M phase).
Project description:Heterogeneity in pluripotent cells marks a metastable state where cells may drift between native and lineage-primed populations. While the role for these heterogeneities are unclear, they may reflect the dynamic equilibriums of signaling networks and have a direct effect on differentiation potentialities. Here, we report the role of the cell cycle in establishing heterogeneity of human pluripotent stem cells. By utilizing the FUCCI cell cycle indicator system coupled to fluorescent activated cell sorting (FACS), we have uncovered that the cell cycle drives heterogeneity at the epigenetic, transcriptional and post-transcriptional levels. Our data show widespread dynamics in 5-hydroxymethylcytosine (5hmC) during the cell cycle. Furthermore, transcript profiling by RNA-sequencing identified >500 genes that were cell cycle-regulated, of which the largest cohort of genes were transcriptional regulators. In sum, we demonstrate the role of the cell cycle in coordinating cellular transitions between metastable states in pluripotent stem cells. mRNA sequencing of the cell cycle phases; early & late G1, S and G2/S from human ES cells in triplicate.
Project description:Oscillatory gene expression is fundamental to mammalian development, but technologies to monitor expression oscillations are limited. We have developed a statistical approach called Oscope to identify and characterize the transcriptional dynamics of oscillating genes in single-cell RNA-seq data from an unsynchronized cell population. Applications to a number of data sets, include a single-cell RNA-seq data set of human embroyonic stem cells (hESCs), demonstrate advantages of the approach and also identify a potential artifact in the Fluidigm C1 platform. Total 213 H1 single cells and 247 H1-Fucci single cells were sequenced. The 213 H1 cells were used to evaluate Oscope in identifying oscillatory genes. The H1-Fucci cells were used to confirm the cell cycle gene cluster identified by Oscope in the H1 hESCs.
Project description:Fucci-expressing HCT116 were inoculated into subcutaneous tissue or cecum of NOD/SCID mice. Four weeks after inoculation, Fucci green (mAG; S/G2/M) and red (mKO2;G1) expressing cells were isolated from implanted tumors by FACSAria sorting (BD, Biosciences). Comparative analyses among green and red cells cultured in vivo were performed to elucidate the molecular basis of cell cycle-dependent motility control mechanism. The labeled cRNAs were hybridized on 4X44K Agilent Whole Human Genome dual colour Microarrays (G4112F) in two dye swap experiments, resulting in four individual microarrays.
Project description:Sorting U2OS and HeLa cells genetically modified with the Fucci System allowed us to separate cells according to cell cycle progression followed by RNA Sequencing to characterize the oscillating transcriptome in cells without the need for chemical synchronization. Overall design: HeLa cells were sorted at three timepoints, while U2OS cells were sorted at two timepoints. Each time into three groups, categorized as "G1", "S", and "G2".
Project description:Non mitotic tumor cells are resistant to conventional chemotherapeutic drugs. However, the mechanisms underlying this phenomenon remain unclear. Here, we found a population that is viable but remains in the G1 phase for an extended period of time (up to 48 h) by Long-term time-lapse observations in Fucci-HCT116 cells and then we conducted DNA microarray-based comparative analyses between the RR (the long-term G1-arrested cells) and R (the G1-arrested cells) fractions, to determine the molecular basis of the G1 arrest/maintenance mechanism. This study is related to GSE34940. The labeled cRNAs were hybridized on 4X44K v2 Agilent Whole Human Genome dual color Microarrays (G4845A) in two dye swap experiments, resulting in four individual microarrays.
Project description:To elucidate the Nodal transcriptional network that governs endoderm formation, we used ChIP-Seq to identify genomic targets for SMAD2/3, SMAD3, SMAD4, FOXH1 and the active and repressive chromatin marks, H3K4me3 and H3K27me3, in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that while SMAD2/3, SMAD4 and FOXH1 target binding is highly dynamic, there is an optimal signature for driving endoderm commitment. Initially, this signature is marked by both H3K4me3 and H3K27me3 as a very broad bivalent domain in hESCs. Within the first 24 hours, at a few select promoters, SMAD2/3 accumulation coincides with H3K27me3 depletion so that these loci become selectively monovalent marked only by H3K4me3. The correlation between SMAD2/3 binding, monovalent formation and transcriptional activation suggests a mechanism by which SMAD proteins coordinate with chromatin at critical promoters to drive endoderm specification. Examination of 2 different histone modifications and 4 different transcription factor associations in 2 cell types. For transcription factor analysis, three biological replicate ChIPs were pooled from each antibody, as well as input controls, for both hESCs and derived endoderm. For histone modifications, two biological replicates for H3K4me3 and three for H3K27me3 were used.
Project description:To investigate how histone post-translational modifications (PTMs) change during the cell cycle, we profiled histone H3 and histone H4 in breast cancer and normal breast cell lines arrested in G1/S or G2/M phases.