scholarly journals Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering

2021 ◽  
Author(s):  
Daisylyn Senna Tan ◽  
Yanpu Chen ◽  
Ya Gao ◽  
Anastasia Bednarz ◽  
Yuanjie Wei ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Phenotypic Selection ◽  
Biochemical Assays ◽  
Genome Wide ◽  
Dna Elements ◽  
Pioneer Factor ◽  
Selection Of

Abstract Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.

scholarly journals A Genome-Wide RNA Interference Screen Identifies a Differential Role of the Mediator CDK8 Module Subunits for GATA/ RUNX-Activated Transcription in Drosophila

2010 ◽  
Vol 30 (11) ◽  
pp. 2837-2848 ◽  
Author(s):  
Vanessa Gobert ◽  
Dani Osman ◽  
Stéphanie Bras ◽  
Benoit Augé ◽  
Muriel Boube ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Rna Interference ◽  
Cell Fate ◽  
Gata Factor ◽  
Hematopoietic System ◽  
Crystal Cell ◽  
Genome Wide ◽  
A Genome

ABSTRACT Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. We used Serpent/Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genome-wide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory “CDK8 module,” composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, our data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, our in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. We thus propose that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, we suggest that the set of conserved factors identified herein may regulate GATA/RUNX activity in mammals.

scholarly journals Functional correlation of H3K9me2 and nuclear compartment formation

2020 ◽  
Author(s):  
Kei Fukuda ◽  
Chikako Shimura ◽  
Hisashi Miura ◽  
Akie Tanigawa ◽  
Takehiro Suzuki ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Epigenetic Mark ◽  
3 Dimensional ◽  
3D Genome ◽  
Functional Correlation ◽  
Genome Wide

AbstractBackgroundHistone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and correlates well with lamina-associated domains and the B compartment. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear.ResultsWe investigated the genome-wide H3K9me2 distribution, the transcriptome and 3D genome organization in mouse embryonic stem cells (mESCs) upon the inhibition or depletion of H3K9 methyltransferases (MTases) G9a/GLP, SETDB1, and SUV39H1/2. We found that H3K9me2 is regulated by these five MTases; however, H3K9me2 and transcription in the A and B compartments were largely regulated by different sets of the MTases: H3K9me2 in the A compartments were mainly regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments were regulated by all five H3K9 MTases. Furthermore, decreased H3K9me2 correlated with the changes to the more active compartmental state that accompanied transcriptional activation.ConclusionOur data showed that H3K9me2 domain formation is functionally linked to 3D genome organization.

scholarly journals Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate

Nature ◽  
2010 ◽  
Vol 463 (7282) ◽  
pp. 808-812 ◽  
Author(s):  
Ye Zheng ◽  
Steven Josefowicz ◽  
Ashutosh Chaudhry ◽  
Xiao P. Peng ◽  
Katherine Forbush ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Fate ◽  
Regulatory T Cell ◽  
Foxp3 Gene ◽  
Dna Elements

scholarly journals Elaborating the Role of Natural Products-Induced Autophagy in Cancer Treatment: Achievements and Artifacts in the State of the Art

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Ning Wang ◽  
Yibin Feng
Keyword(s):  
Cell Death ◽  
Natural Products ◽  
Cancer Treatment ◽  
Tumor Cells ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Apoptotic Cell ◽  
Rapid Expansion ◽  
Drug Study

Autophagy is a homeostatic process that is highly conserved across different types of mammalian cells. Autophagy is able to relieve tumor cell from nutrient and oxidative stress during the rapid expansion of cancer. Excessive and sustained autophagy may lead to cell death and tumor shrinkage. It was shown in literature that many anticancer natural compounds and extracts could initiate autophagy in tumor cells. As summarized in this review, the tumor suppressive action of natural products-induced autophagy may lead to cell senescence, provoke apoptosis-independent cell death, and complement apoptotic cell death by robust or target-specific mechanisms. In some cases, natural products-induced autophagy could protect tumor cells from apoptotic death. Technical variations in detecting autophagy affect data quality, and study focus should be made on elaborating the role of autophagy in deciding cell fate. In vivo study monitoring of autophagy in cancer treatment is expected to be the future direction. The clinical-relevant action of autophagy-inducing natural products should be highlighted in future study. As natural products are an important resource in discovery of lead compound of anticancer drug, study on the role of autophagy in tumor suppressive effect of natural products continues to be necessary and emerging.

scholarly journals Regulation of mammalian 3D genome organization and histone H3K9 dimethylation by H3K9 methyltransferases

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Kei Fukuda ◽  
Chikako Shimura ◽  
Hisashi Miura ◽  
Akie Tanigawa ◽  
Takehiro Suzuki ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Epigenetic Mark ◽  
3 Dimensional ◽  
3D Genome ◽  
Genome Wide ◽  
H3k9 Dimethylation

AbstractHistone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and overlaps well with lamina-associated domains and the B compartment defined by Hi-C. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. Here, we investigated genome-wide H3K9me2 distribution, transcriptome, and 3D genome organization in mouse embryonic stem cells following the inhibition or depletion of H3K9 methyltransferases (MTases): G9a, GLP, SETDB1, SUV39H1, and SUV39H2. We show that H3K9me2 is regulated by all five MTases; however, H3K9me2 and transcription in the A and B compartments are regulated by different MTases. H3K9me2 in the A compartments is primarily regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments is regulated by all five MTases. Furthermore, decreased H3K9me2 correlates with changes to more active compartmental state that accompanied transcriptional activation. Thus, H3K9me2 contributes to inactive compartment setting.

scholarly journals LncRNA Mrhl orchestrates differentiation programs in mouse embryonic stem cells through chromatin mediated regulation

2019 ◽  
Author(s):  
Debosree Pal ◽  
C V Neha ◽  
Utsa Bhaduri ◽  
Zenia ◽  
Subbulakshmi Chidambaram ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Loss Of Function ◽  
Genome Wide ◽  
Development And Differentiation ◽  
Lineage Specific Genes

AbstractLong non-coding RNAs (lncRNAs) have been well-established to act as regulators and mediators of development and cell fate specification programs. LncRNA Mrhl (meiotic recombination hotspot locus) has been shown to act in a negative feedback loop with WNT signaling to regulate male germ cell meiotic commitment. In our current study, we have addressed the role of Mrhl in development and differentiation using mouse embryonic stem cells (mESCs) as our model system of study. We found Mrhl to be a nuclear-localized, chromatin-bound lncRNA with moderately stable expression in mESCs. Transcriptome analyses and loss-of-function phenotype studies revealed dysregulation of developmental processes and lineage-specific genes along with aberrance in specification of early lineages during differentiation of mESCs. Genome-wide chromatin occupancy studies suggest regulation of chromatin architecture at key target loci through triplex formation. Our studies thus reveal a role for lncRNA Mrhl in regulating differentiation programs in mESCs in the context of appropriate cues through chromatin-mediated responses.

scholarly journals Monoubiquitylation of H2A.Z Distinguishes Its Association with Euchromatin or Facultative Heterochromatin

2007 ◽  
Vol 27 (18) ◽  
pp. 6457-6468 ◽  
Author(s):  
Elizabeth Sarcinella ◽  
Philip C. Zuzarte ◽  
Priscilla N. I. Lau ◽  
Ryan Draker ◽  
Peter Cheung
Keyword(s):  
Mammalian Cells ◽  
Regulation Of Gene Expression ◽  
Histone H2a ◽  
Facultative Heterochromatin ◽  
X Chromosomes ◽  
Genome Wide ◽  
New Findings ◽  
Mouse Cells ◽  
Heterochromatin Silencing

ABSTRACT H2A.Z is a histone H2A variant that is essential for viability in organisms such as Tetrahymena thermophila, Drosophila melanogaster, and mice. In Saccharomyces cerevisiae, loss of H2A.Z is tolerated, but proper regulation of gene expression is affected. Genetics and genome-wide localization studies show that yeast H2A.Z physically localizes to the promoters of genes and functions in part to protect active genes in euchromatin from being silenced by heterochromatin spreading. To date, the function of H2A.Z in mammalian cells is less clear, and evidence so far suggests that it has a role in chromatin compaction and heterochromatin silencing. In this study, we found that the bulk of H2A.Z is excluded from constitutive heterochromatin in differentiated human and mouse cells. Consistent with this observation, analyses of H2A.Z- or H2A-containing mononucleosomes show that the H3 associated with H2A.Z has lower levels of K9 methylation but higher levels of K4 methylation than those associated with H2A. We also found that a fraction of mammalian H2A.Z is monoubiquitylated and that, on the inactive X chromosomes of female cells, the majority of this histone variant is modified by ubiquitin. Finally, ubiquitylation of H2A.Z is mediated by the RING1b E3 ligase of the human polycomb complex, further supporting a silencing role of ubiquitylated H2A.Z. These new findings suggest that mammalian H2A.Z is associated with both euchromatin and facultative heterochromatin and that monoubiquitylation is a specific mark that distinguishes the H2A.Z associated with these different chromatin states.

Abstract 199: Dissecting the Shox2-nkx2-5 Antagonistic Mechanism in the Pulmonary Vein Myocardium and Sinoatrial Node

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Wenduo Ye ◽  
Jun Wang ◽  
Yingnan Song ◽  
Diankun Yu ◽  
Cheng Sun ◽  
...  
Keyword(s):  
Cell Fate ◽  
Pulmonary Vein ◽  
Sinoatrial Node ◽  
Venous Return ◽  
Allelic Series ◽  
Expression Of Genes ◽  
Genome Wide ◽  
Systemic Venous Return

Atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. However, the genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. We have identified a Shox2-Nkx2-5 antagonistic mechanism that primes the pacemaking cell fate in the PV myocardium and SAN in the embryonic stage. Specifically, Shox2 deletion in the Nkx2-5+ domain of the SAN caused sick sinus syndrome, associated with the loss of pacemaker program. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. Shox2 interacts with Nkx2-5 directly, and a substantial genome wide co-occupancy of Shox2, Nkx2-5, and Tbx5 suggest a balanced transcription output is essential for the pacemaker cell fate determination. Further characterization of mice carrying allelic series of Shox2 and Nkx2-5 revealed an essential role of both Shox2 and Nkx2-5 in maintain the normal development of pulmonary vein myocardium and the structures in the venous pole, suggesting the importance of transcriptional precision of Shox2 and Nkx2-5 and a role of Shox2-Nkx2-5 antagonistic mechanism in integrating the transcriptional accuracy of Shox2 or Nkx2-5.

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