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Cell-cycle control of developmentally regulated transcription factors accounts for heterogeneity in human pluripotent cells.

ABSTRACT: Heterogeneity within pluripotent stem cell (PSC) populations is indicative of dynamic changes that occur when cells drift between different states. Although the role of metastability in PSCs is unclear, it appears to reflect heterogeneity in cell signaling. Using the Fucci cell-cycle indicator system, we show that elevated expression of developmental regulators in G1 is a major determinant of heterogeneity in human embryonic stem cells. Although signaling pathways remain active throughout the cell cycle, their contribution to heterogeneous gene expression is restricted to G1. Surprisingly, we identify dramatic changes in the levels of global 5-hydroxymethylcytosine, an unanticipated source of epigenetic heterogeneity that is tightly linked to cell-cycle progression and the expression of developmental regulators. When we evaluated gene expression in differentiating cells, we found that cell-cycle regulation of developmental regulators was maintained during lineage specification. Cell-cycle regulation of developmentally regulated transcription factors is therefore an inherent feature of the mechanisms underpinning differentiation.

SUBMITTER: Singh AM

PROVIDER: S-EPMC3871385 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Cell-cycle control of developmentally regulated transcription factors accounts for heterogeneity in human pluripotent cells.

Singh Amar M AM Chappell James J Trost Robert R Lin Li L Wang Tao T Tang Jie J Matlock Brittany K BK Weller Kevin P KP Wu Hao H Zhao Shaying S Jin Peng P Dalton Stephen S

Stem cell reports 20131205 6

Heterogeneity within pluripotent stem cell (PSC) populations is indicative of dynamic changes that occur when cells drift between different states. Although the role of metastability in PSCs is unclear, it appears to reflect heterogeneity in cell signaling. Using the Fucci cell-cycle indicator system, we show that elevated expression of developmental regulators in G1 is a major determinant of heterogeneity in human embryonic stem cells. Although signaling pathways remain active throughout the ce ...[more]

PMID: 24371808

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Project description:Genetically identical cells exhibit extensive phenotypic variation even under constant and benign conditions. This so-called nongenetic heterogeneity has important clinical implications: within tumors and microbial infections, cells show nongenetic heterogeneity in growth rate and in susceptibility to drugs or stress. The budding yeast, Saccharomyces cerevisiae, shows a similar form of nongenetic heterogeneity in which growth rate correlates positively with susceptibility to acute heat stress at the single-cell level. Using genetic and chemical perturbations, combined with high-throughput single-cell assays of yeast growth and gene expression, we show here that heterogeneity in intracellular cyclic AMP (cAMP) levels acting through the conserved Ras/cAMP/protein kinase A (PKA) pathway and its target transcription factors, Msn2 and Msn4, underlies this nongenetic heterogeneity. Lower levels of cAMP correspond to slower growth, as shown by direct comparison of cAMP concentration in subpopulations enriched for slower vs. faster growing cells. Concordantly, an endogenous reporter of this pathway's activity correlates with growth in individual cells. The paralogs Msn2 and Msn4 differ in their roles in nongenetic heterogeneity in a way that demonstrates slow growth and stress tolerance are not inevitably linked. Heterogeneity in growth rate requires each, whereas only Msn2 is required for heterogeneity in expression of Tsl1, a subunit of trehalose synthase that contributes to acute-stress tolerance. Perturbing nongenetic heterogeneity by mutating genes in this pathway, or by culturing wild-type cells with the cell-permeable cAMP analog 8-bromo-cAMP or the PKA inhibitor H89, significantly impacts survival of acute heat stress. Perturbations that increase intracellular cAMP levels reduce the slower-growing subpopulation and increase susceptibility to acute heat stress, whereas PKA inhibition slows growth and decreases susceptibility to acute heat stress. Loss of Msn2 reduces, but does not completely eliminate, the correlation in individual cells between growth rate and acute-stress survival, suggesting a major role for the Msn2 pathway in nongenetic heterogeneity but also a residual benefit of slow growth. Our results shed light on the genetic control of nongenetic heterogeneity and suggest a possible means of defeating bet-hedging pathogens or tumor cells by making them more uniformly susceptible to treatment.

Project description:BackgroundThe cell cycle has long been an important model to study the genome-wide transcriptional regulation. Although several methods have been introduced to identify cell cycle regulated genes from microarray data, they can not be directly used to investigate cell cycle regulated transcription factors (CCRTFs), because for many transcription factors (TFs) it is their activities instead of expressions that are periodically regulated across the cell cycle. To overcome this problem, it is useful to infer TF activities across the cell cycle by integrating microarray expression data with ChIP-chip data, and then examine the periodicity of the inferred activities. For most species, however, large-scale ChIP-chip data are still not available.ResultsWe propose a two-step method to identify the CCRTFs by integrating microarray cell cycle data with ChIP-chip data or motif discovery data. In S. cerevisiae, we identify 42 CCRTFs, among which 23 have been verified experimentally. The cell cycle related behaviors (e.g. at which cell cycle phase a TF achieves the highest activity) predicted by our method are consistent with the well established knowledge about them. We also find that the periodical activity fluctuation of some TFs can be perturbed by the cell synchronization treatment. Moreover, by integrating expression data with in-silico motif discovery data, we identify 8 cell cycle associated regulatory motifs, among which 7 are binding sites for well-known cell cycle related TFs.ConclusionOur method is effective to identify CCRTFs by integrating microarray cell cycle data with TF-gene binding information. In S. cerevisiae, the TF-gene binding information is provided by the systematic ChIP-chip experiments. In other species where systematic ChIP-chip data is not available, in-silico motif discovery and analysis provide us with an alternative method. Therefore, our method is ready to be implemented to the microarray cell cycle data sets from different species. The C++ program for AC score calculation is available for download from URL http://leili-lab.cmb.usc.edu/yeastaging/projects/project-base/.

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Cell-cycle control of developmentally regulated transcription factors accounts for heterogeneity in human pluripotent cells.

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Cell-cycle control of developmentally regulated transcription factors accounts for heterogeneity in human pluripotent cells.

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