TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novel Nanog regulators in mouse embryonic stem cells

AbstractCellular identity is ultimately controlled by transcription factors (TFs), which bind to specific regulatory elements (REs) within the genome to regulate gene expression and cell fate changes. While recent advances in genome-wide epigenetic profiling techniques have significantly increased our understanding of which REs are utilized in which cell type, it remains largely unknown which TFs and cofactors interact with these REs to modulate gene expression. A major hurdle in dissecting the whole composition of a multi-protein complex formed at a specific RE is the shortage of appropriate techniques. We have developed a novel method termed TALE-mediated Isolation of Nuclear Chromatin (TINC). TINC utilizes epitope-tagged TALEs to isolate a specific genomic region from the mammalian genome and includes a nuclei isolation and chromatin enrichment step for increased specificity. Upon cross-linking of the cells and isolation of the chromatin, the target region is purified based on affinity purification of the TALE and associated nucleic acid and protein molecules can be subjected to further analyses. A key TF in the pluripotency network and therefore in embryonic stem cells (ESCs) is NANOG. It is currently not fully understood how Nanog expression is regulated and consequently it remains unclear how the ESC state is maintained. Using TINC we dissected the protein complex formed at the Nanog promoter in mouse ESCs and identified many known and numerous novel factors.

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Genetic control of pluripotency epigenome determines differentiation bias in mouse embryonic stem cells

10.1101/2021.01.15.426861 ◽

2021 ◽

Author(s):

Candice Byers ◽

Catrina Spruce ◽

Haley J. Fortin ◽

Anne Czechanski ◽

Steven C. Munger ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Genetic Regulation ◽

Embryonic Stem ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Embryoid Bodies ◽

Cell Fate Decisions

AbstractGenetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naïve networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naïve pluripotency with little bias in differentiation. Genetic mapping identifies 6 major trans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. One trans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

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The combined use of viral transcriptional and post-transcriptional regulatory elements to improve baculovirus-mediated transient gene expression in human embryonic stem cells

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2009.06.017 ◽

2010 ◽

Vol 109 (1) ◽

pp. 1-8 ◽

Cited By ~ 15

Author(s):

Juan Du ◽

Jieming Zeng ◽

Ying Zhao ◽

Jerome Boulaire ◽

Shu Wang

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Transient Gene Expression ◽

Embryonic Stem ◽

Regulatory Elements ◽

Transcriptional Regulatory Elements ◽

Transcriptional Regulatory ◽

Combined Use

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Abstract 14565: Comparative Transcriptomic Analysis of Multiple Cardiovascular Fates From Embryonic Stem Cells Predicts Regulatory Genes, Alternative Splicing and Long Non-coding RNAs in Human Cardiogenesis

Circulation ◽

10.1161/circ.132.suppl_3.14565 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Yang Li ◽

Bo Lin ◽

Lei Yang

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Embryonic Stem ◽

Cardiovascular Development ◽

Rna Seq ◽

Comparison Analysis ◽

Lineage Specificity ◽

Early Human

Introduction: Dissecting the gene expression programs which control the early stage cardiovascular development is essential for understanding the molecular mechanisms of human heart development and heart disease. Many lineage-specific genes are involved in the differentiation into specific cell fate. Hypothesis: The lineage-specificity of the genes may predict regulatory potential during human cardiovascular differentiation. Methods: Here, we performed transcriptome sequencing (RNA-seq) of highly purified human Embryonic Stem Cells (hESCs), hESC-derived Multipotential Cardiovascular Progenitors (MCPs) and MCP-specified three cardiovascular lineages. A novel algorithm, named as Gene Expression Pattern Analyzer (GEPA), was developed to obtain a refined lineage-specificity map of all sequenced genes, which reveals dynamic changes of transcriptional factor networks underlying early human cardiovascular development. Results: The GEPA predictions captured ∼90% of top-ranked regulatory cardiac genes that were previously predicted based on chromatin signature changes in hESCs, and further defined their cardiovascular lineage-specificities, indicating that our multi-fate comparison analysis could predict novel regulatory genes. Furthermore, GEPA analysis revealed the MCP-specific expressions of genes in ephrin signaling pathway, positive role of which in cardiomyocyte differentiation was further validated experimentally. By using RNA-seq plus GEPA workflow, we also identified stage-specific RNA splicing switch and lineage-enriched long noncoding RNAs during human cardiovascular differentiation. Conclusions: Overall, our study utilized multi-cell-fate transcriptomic comparison analysis to establish a lineage-specific gene expression map for predicting and validating novel regulatory mechanisms underlying early human cardiovascular development.

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Polycomb proteins as organizers of 3D genome architecture in embryonic stem cells

Briefings in Functional Genomics ◽

10.1093/bfgp/elz022 ◽

2019 ◽

Cited By ~ 2

Author(s):

Tomas Pachano ◽

Giuliano Crispatzu ◽

Alvaro Rada-Iglesias

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Regulatory Elements ◽

Polycomb Group ◽

Differentiation Potential ◽

3D Genome ◽

Additional Layer ◽

Mammalian Embryogenesis

Abstract Polycomb group proteins (PcGs) control the epigenetic and transcriptional state of developmental genes and regulatory elements during mammalian embryogenesis. Moreover, PcGs can also contribute to 3D genome organization, adding an additional layer of complexity to their regulatory functions. Understanding the mechanistic basis and the dynamics of PcG-dependent chromatin structures will help us untangle the full complexity of PcG function during development. Since most studies concerning the 3D organization of PcG-bound chromatin in mammals have been performed in embryonic stem cells (ESCs), here we will focus on this cell type characterized by its unique self-renewal and pluripotency properties. More specifically, we will highlight recent findings and discuss open questions regarding how PcG-dependent changes in 3D chromatin architecture control gene expression, cellular identity and differentiation potential in ESCs. We believe that this can serve to illustrate the diverse regulatory mechanisms by which PcG proteins control the proper execution of gene expression programs during mammalian embryogenesis.

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Mammalian SWI/SNF Chromatin Remodeling Complexes in Embryonic Stem Cells: Regulating the Balance Between Pluripotency and Differentiation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.626383 ◽

2021 ◽

Vol 8 ◽

Author(s):

Ying Ye ◽

Xi Chen ◽

Wensheng Zhang

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Chromatin Remodeling ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Self Renewal ◽

Baf Complex ◽

Chromatin Remodeling Complexes

The unique capability of embryonic stem cells (ESCs) to maintain and adjust the equilibrium between self-renewal and multi-lineage cellular differentiation contributes indispensably to the integrity of all developmental processes, leading to the advent of an organism in its adult form. The ESC fate decision to favor self-renewal or differentiation into specific cellular lineages largely depends on transcriptome modulations through gene expression regulations. Chromatin remodeling complexes play instrumental roles to promote chromatin structural changes resulting in gene expression changes that are key to the ESC fate choices governing the equilibrium between pluripotency and differentiation. BAF (Brg/Brahma-associated factors) or mammalian SWI/SNF complexes employ energy generated by ATP hydrolysis to change chromatin states, thereby governing the accessibility of transcriptional regulators that ultimately affect transcriptome and cell fate. Interestingly, the requirement of BAF complex in self-renewal and differentiation of ESCs has been recently shown by genetic studies through gene expression modulations of various BAF components in ESCs, although the precise molecular mechanisms by which BAF complex influences ESC fate choice remain largely underexplored. This review surveys these recent progresses of BAF complex on ESC functions, with a focus on its role of conditioning the pluripotency and differentiation balance of ESCs. A discussion of the mechanistic bases underlying the genetic requirements for BAF in ESC biology as well as the outcomes of its interplays with key transcription factors or other chromatin remodelers in ESCs will be highlighted.

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Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

eLife ◽

10.7554/elife.40167 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 9

Author(s):

Lucy LeBlanc ◽

Bum-Kyu Lee ◽

Andy C Yu ◽

Mijeong Kim ◽

Aparna V Kambhampati ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Hippo Pathway ◽

Embryonic Stem ◽

Regulatory Elements ◽

Self Renewal ◽

Massive Cell Death ◽

Apoptotic Genes ◽

Massive Cell

Approximately, 30% of embryonic stem cells (ESCs) die after exiting self-renewal, but regulators of this process are not well known. Yap1 is a Hippo pathway transcriptional effector that plays numerous roles in development and cancer. However, its functions in ESC differentiation remain poorly characterized. We first reveal that ESCs lacking Yap1 experience massive cell death upon the exit from self-renewal. We subsequently show that Yap1 contextually protects differentiating, but not self-renewing, ESC from hyperactivation of the apoptotic cascade. Mechanistically, Yap1 strongly activates anti-apoptotic genes via cis-regulatory elements while mildly suppressing pro-apoptotic genes, which moderates the level of mitochondrial priming that occurs during differentiation. Individually modulating the expression of single apoptosis-related genes targeted by Yap1 is sufficient to augment or hinder survival during differentiation. Our demonstration of the context-dependent pro-survival functions of Yap1 during ESC differentiation contributes to our understanding of the balance between survival and death during cell fate changes.

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Transgenic Mice and Pluripotent Stem Cells Express EGFP under the Control of miR-302 Promoter

10.1101/450791 ◽

2018 ◽

Cited By ~ 1

Author(s):

Karim Rahimi ◽

Sara Parsa ◽

Mehrnoush Nikzaban ◽

Seyed Javad Mowla ◽

Fardin Fathi

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Transgenic Mice ◽

Cell Fate ◽

Core Promoter ◽

Expression Patterns ◽

Embryonic Stem ◽

Regulatory Sequences ◽

Somatic Tissues

MicroRNAs are a group of short non-coding RNAs that undertake various roles in different cell signaling pathways and developmental stages. They regulate gene expression levels at the post-transcriptional stage, which results in cleavage of mRNAs or repression of their translation. Some miRNAs, including the miR-302 cluster, are critical regulators for the stemness state of embryonic stem cells and cell fate patterning. The miR-302 cluster is located in the intron of a non-coding gene that has no other reported function, other than hosting miR-302, and grant a complex expression regulation through upstream its regulatory sequences. To date, analysis of the miR-302 expression pattern in a transgenic mouse model has not been reported. In this study, we generated transgenic mice that expressed EGFP driven by miR-302 upstream regulatory sequences that harbored the core promoter of its host gene. We examined the activity of the miR-302 promotor in somatic tissues of transgenic mice, transgenic blastocysts, and embryonic stem cells derived from transgenic blastocysts. Our results showed that miR-302 highly expressed in both blastocysts and the first passages of transgenic embryonic stem cells, and has low expression in the somatic tissues of transgenic mice. It could be concluded that different temporal and spatial gene expression patterns occur during the embryonic and adult stages in mice.

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