Nuclear Reprogramming and Mitosis--how does mitosis enhance changes in gene expression?
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ABSTRACT:
SUBMITTER: Halley-Stott RP
PROVIDER: S-EPMC4422820 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, it is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming and show that mitosis may be a driving force of reprogramming.
| S-EPMC4786677 | biostudies-literature
Project description:It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, It is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming, and show that mitosis may be a driving force of reprogramming. Human BJ cells transduced with lentiviral particles of the conventional reprogramming factors (OCT3/4, SOX2 and KLF4) were used as controls. Two types of controls were used: 1) BJ transduced with OSK (OCT4, SOX2 and KFL4) viruses; 2) BJ cells transduced with OSK plus GFP viruses. Experimental treatment was BJ cells transduced with OSK plus BRD3R viruses. RNA was extracted from cells at day 3 of reprogramming because the reprogramming cells are still homogeneous and transgenes are well expressed at this time point.
2016-03-01 | E-GEOD-66798 | biostudies-arrayexpress
Project description:The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in disease mechanisms and regenerative medicine. Epigenetic modifications enable differentiated cells to perpetuate molecular memory to retain their identity. Therefore, the aim of this study was to investigate the reprogramming modification of yak fibroblast cells that were permeabilized and incubated in the extracts of mesenchymal stem cells derived from mice adipose tissue [adipose-derived stem cells (ADSCs)]. According to the results, the treatment of ADSC extracts promoted colony formation. Moreover, pluripotent gene expression was associated with the loss of repressive histone modifications and increased global demethylation. The genes Col1a1 and Col1a2, which are typically found in differentiated cells only, demonstrated decreased expression and increased methylation in the 5'-flanking regulatory regions. Moreover, yak fibroblast cells that were exposed to ADSC extracts resulted in significantly different eight-cell and blastocyst formation rates of cloned embryos compared with their untreated counterparts. This investigation provides the first evidence that nuclear reprogramming of yak fibroblast cells is modified after the ADSC extract treatment. This research also presents a methodology for studying the dedifferentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells toward a pluripotent state without genetic alteration.
| S-EPMC4030660 | biostudies-literature
Project description:BackgroundSomatic cells can be reprogrammed to induced-pluripotent stem cells (iPSCs) by introducing few reprogramming factors, which challenges the long held view that cell differentiation is irreversible. However, the mechanism of induced pluripotency is still unknown.MethodsInspired by the phenomenological reprogramming model of Artyomov et al (2010), we proposed a novel Markov model, stepwise reprogramming Markov (SRM) model, with simpler gene regulation rules and explored various properties of the model with Monte Carlo simulation. We calculated the reprogramming rate and showed that it would increase in the condition of knockdown of somatic transcription factors or inhibition of DNA methylation globally, consistent with the real reprogramming experiments. Furthermore, we demonstrated the utility of our model by testing it with the real dynamic gene expression data spanning across different intermediate stages in the iPS reprogramming process.ResultsThe gene expression data at several stages in reprogramming and the reprogramming rate under several typically experiment conditions coincided with our simulation results. The function of reprogramming factors and gene expression change during reprogramming could be partly explained by our model reasonably well.ConclusionsThis lands further support on our general rules of gene regulation network in iPSC reprogramming. This model may help uncover the basic mechanism of reprogramming and improve the efficiency of converting somatic cells to iPSCs.
| S-EPMC3287488 | biostudies-literature
Project description:Nuclear transfer (NT) has potential applications in agriculture and biomedicine, but the technology is hindered by low efficiency. Global gene expression analysis of clones is important for the comprehensive study of nuclear reprogramming. Here, we compared global gene expression profiles of individual bovine NT blastocysts with their somatic donor cells and fertilized control embryos using cDNA microarray technology. The NT embryos' gene expression profiles were drastically different from those of their donor cells and closely resembled those of the naturally fertilized embryos. Our findings demonstrate that the NT embryos have undergone significant nuclear reprogramming by the blastocyst stage; however, problems may occur during redifferentiation for tissue genesis and organogenesis, and small reprogramming errors may be magnified downstream in development.
| S-EPMC1308920 | biostudies-literature
Project description:It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, It is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming, and show that mitosis may be a driving force of reprogramming.
2016-03-01 | GSE66798 | GEO
Project description:Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.
| S-EPMC2715272 | biostudies-literature
Project description:Various cellular stresses including oxidative stress induce a collapse of the Ran gradient, which causes accumulation of importin ? in the nucleus and a subsequent block of nuclear protein import. However, it is unknown whether accumulated importin ? performs roles in the nucleus after its migration in response to stress. In this study, we found that nuclear-retained importin ?2 binds with DNase I-sensitive nuclear component(s) and exhibits selective upregulation of mRNA encoding Serine/threonine kinase 35 (STK35) by microarray analysis. Chromatin immunoprecipitation and promoter analysis demonstrated that importin ?2 can access to the promoter region of STK35 and accelerate its transcription in response to hydrogen peroxide exposure. Furthermore, constitutive overexpression of STK35 proteins enhances caspase-independent cell death under oxidative stress conditions. These results collectively reveal that nuclear-localized importin ?2 influences gene expression and contributes directly to cell fate outcomes including non-apoptotic cell death.
| S-EPMC3252573 | biostudies-literature
Project description:While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism.
| S-EPMC3367913 | biostudies-literature
Project description:It is thought that most cell types of the human body share the same genetic information as that contained in the zygote from which they originate. Consistent with this view, animal cloning studies demonstrated that the intact genome of a differentiated cell can be reprogrammed to support the development of an entire organism and allow the production of pluripotent stem cells. Recent progress in reprogramming research now points to an important role for transcription factors in the establishment and the maintenance of cellular phenotypes, and to cell division as a mediator of transitions between different states of gene expression.
| S-EPMC7250051 | biostudies-literature