Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors

Salomonis, Nathan; Nelson, Brandon; Vranizan, Karen; Pico, Alexander R.; Hanspers, Kristina; Kuchinsky, Allan; Ta, Linda; Mercola, Mark; Conklin, Bruce R.

doi:10.1371/journal.pcbi.1000553

Cited by 89 publications

(90 citation statements)

References 62 publications

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“…Recently, it has been shown that the large repertoire of splice isoforms expressed in ES cells is gradually reduced in absolute number during neural differentiation (44). In addition, during both neural and cardiac differentiation, the specific repertoire of splice isoforms present in the pluripotent versus committed lineages changes (36). These changes in splice isoform repertoire imply that ES cells likely utilize their large and unique set of splice isoforms to sustain a pluripotent state.…”

Section: Discussionmentioning

confidence: 99%

Differential Roles of Sall4 Isoforms in Embryonic Stem Cell Pluripotency

Rao

Shao

Rumyantsev

et al. 2010

Molecular and Cellular Biology

156

203

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Murine embryonic stem (ES) cells are defined by continuous self-renewal and pluripotency. A diverse repertoire of protein isoforms arising from alternative splicing is expressed in ES cells without defined biological roles. Sall4, a transcription factor essential for pluripotency, exists as two isoforms (Sall4a and Sall4b). Both isoforms can form homodimers and a heterodimer with each other, and each can interact with Nanog. By genomewide location analysis, we determined that Sall4a and Sall4b have overlapping, but not identical binding sites within the ES cell genome. In addition, Sall4b, but not Sall4a, binds preferentially to highly expressed loci in ES cells. Sall4a and Sall4b binding sites are distinguished by both epigenetic marks at target loci and their clustering with binding sites of other pluripotency factors. When ES cells expressing a single isoform of Sall4 are generated, Sall4b alone could maintain the pluripotent state, although it could not completely suppress all differentiation markers. Sall4a and Sall4b collaborate in maintenance of the pluripotent state but play distinct roles. Our work is novel in establishing such isoform-specific differences in ES cells.

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Section: Discussionmentioning

confidence: 99%

Differential Roles of Sall4 Isoforms in Embryonic Stem Cell Pluripotency

Rao

Shao

Rumyantsev

et al. 2010

Molecular and Cellular Biology

156

203

View full text Add to dashboard Cite

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“…Positive splicing indexes were associated mainly with genes with a high level of expression (P = 1.39 × 10 −22 ; hypergeometric test), whereas negative splicing indexes were present mostly in genes with medium or low levels of transcription (P = 2.04 × 10 −24 ) ( A and B), suggesting that the effect of canonical histone depletion on splicing depends on the transcription intensity. As a second tool for studying alternative splicing, we used AltAnalyze software (31). In this case, using the default parameters of the MiDAS algorithm, 875 probe sets with splicing defects were identified in 702 genes (Dataset S1).…”

Section: Slbp Depletion Disrupts Chromatin Structure and Alters Histonementioning

confidence: 99%

Defective histone supply causes changes in RNA polymerase II elongation rate and cotranscriptional pre-mRNA splicing

Jimeno-González

Payán-Bravo

Muñoz-Cabello

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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RNA polymerase II (RNAPII) transcription elongation is a highly regulated process that greatly influences mRNA levels as well as pre-mRNA splicing. Despite many studies in vitro, how chromatin modulates RNAPII elongation in vivo is still unclear. Here, we show that a decrease in the level of available canonical histones leads to more accessible chromatin with decreased levels of canonical histones and variants H2A.X and H2A.Z and increased levels of H3.3. With this altered chromatin structure, the RNAPII elongation rate increases, and the kinetics of pre-mRNA splicing is delayed with respect to RNAPII elongation. Consistent with the kinetic model of cotranscriptional splicing, the rapid RNAPII elongation induced by histone depletion promotes the skipping of variable exons in the CD44 gene. Indeed, a slowly elongating mutant of RNAPII was able to rescue this defect, indicating that the defective splicing induced by histone depletion is a direct consequence of the increased elongation rate. In addition, genome-wide analysis evidenced that histone reduction promotes widespread alterations in pre-mRNA processing, including intron retention and changes in alternative splicing. Our data demonstrate that premRNA splicing may be regulated by chromatin structure through the modulation of the RNAPII elongation rate. T he transcription process comprises several steps, including preinitiation complex formation, promoter escape, elongation, and termination (1). Recent reports indicate that elongation rates of RNA polymerase II (RNAPII) in mammals range from 0.5 to 4 kb/min, but which factors are responsible for these differences is still unclear (2-4). One obvious candidate for affecting transcription elongation is chromatin structure. The building block of chromatin is the nucleosome comprising 147 bp of DNA around a histone octamer formed by two H2A-H2B dimers and one H3-H4 tetramer. In vitro experiments have demonstrated that nucleosomes are a barrier for RNAPII transcription elongation (5, 6). We have reported that a nucleosome positioned in the body of a transcription unit impairs RNAPII progression in vivo (7). Furthermore, Weber et al. (8) have shown recently that RNAPII stalls in vivo at the entry site of essentially every transcribed nucleosome in Drosophila. Despite this evidence, it is still unclear whether changes in chromatin structure in different regions of a gene or between different genes can regulate the rate of transcription elongation.Transcription and splicing are coupled processes (9, 10). Splicing occurs cotranscriptionally, and multiple lines of evidence indicate that transcription elongation and splicing influence each other. On one hand, it has been suggested that splicing factors are recruited to the template by the transcription machinery (11, 12). On the other hand, the rate of RNAPII elongation influences splicing. The kinetic model proposes that a slow elongation rate facilitates weak splice-site recognition, promoting the inclusion of alternative exons (13, 14). However, recent studies have ex...

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“…In addition, the study may provide an insight for regenerative medicine since cardiomyocytes cannot be reproduced in patients with ischemic damage. Our results are consistent with those obtained by Salomonis et al (Salomonis, Nelson et al 2009) that…”

Section: Mesendodermal or Cardiomyocyte Differentiationsupporting

confidence: 94%

Regulation of mitochondrial biogenesis in human embryonic stem cells

Kao¹

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Human embryonic stem cells (hESCs) are established from the inner cell mass of the pre-implantation blastocyst and are both pluripotent (being able to be differentiated into all cell types of the body) and immortal (being able to proliferate indefinitely). When grown in culture, they provide a unique in vitro model system that allows us to study the earliest steps of human embryogenesis, a developmental process that is otherwise inaccessible to experimentation.Mitochondria are not only the essential organelles for generating energy in cells but also generate reactive oxygen species (ROS) as side-products of aerobic energy production. The presence of ROS leads to the progressive accumulation of mutations within mitochondrial DNA (mtDNA), resulting in dysfunctional mitochondria. To counteract this, it is thought that a selective amplification of healthy mitochondria occurs during pre-implantation development, aiming at maintaining the mitochondrial fitness in the germline (the ‗bottleneck' theory). Embryonic stem cells harvested from the pre-implantation blastocyst can be cultured indefinitely in vitro and contain metabolically-active mitochondria. Therefore, in this study, we wished to investigate how the mtDNA copy number is regulated in cultured pluripotent stem cells.Initially, the mitochondrial-encoded as well as the mitochondrial biogenesis-related gene expression were analysed in spontaneously differentiating hESCs and during different lineage-specific differentiation of hESCs in a time-dependent manner. The spontaneously-differentiated hESCs possess more copies of mitochondrial genome in mitochondria and early-differentiated hESCs display more mature mitochondrial morphology and a higher mitochondrial membrane potential as compared to 3 undifferentiated hESCs. After 7 days of spontaneous differentiation of hESCs, the transcript levels of D-loop and PGC1α, the master regulatory gene of the mitochondrial biogenesis, were upregulated. We also demonstrated that the early ectoderm and cardiomyocyte differentiation is accompanied by an upregulation of PGC1α. While this shows that mitochondrial gene expression and biogenesis changes upon differentiation it remained to be determined what role mitochondria play in pluripotent stem cells and their differentiated derivatives.To address this question, we investigated the effect of interference with mitochondrial DNA replication (using EtBr) or mitochondrial protein synthesis (using chloramphenicol) in fibroblasts. Compared with untreated primary human foreskin fibroblasts, the mitochondrial impaired rho-minus fibroblasts showed a lower rate of proliferation and a decreased level of mitochondrial gene expression, as well as lower mitochondrial membrane potential and greater rate of lactate production. Moreover, the mitochondrial-encoded and mitochondrial biogenesis-related genes were upregulated after the treatments had been removed. While fibroblasts were able to tolerate mitochondrial depletion or inhibition of mitochondrial protein synthesis repeated attempt...

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Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors

Cited by 89 publications

References 62 publications

Differential Roles of Sall4 Isoforms in Embryonic Stem Cell Pluripotency

Differential Roles of Sall4 Isoforms in Embryonic Stem Cell Pluripotency

Defective histone supply causes changes in RNA polymerase II elongation rate and cotranscriptional pre-mRNA splicing

Regulation of mitochondrial biogenesis in human embryonic stem cells

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