Transcriptional And Epigenetic Dynamics During Specification Of Human Embryonic Stem Cells Pdf
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- Transcriptional and Epigenetic Dynamics during Specification of Human Embryonic Stem Cells
- The Epigenomics of Embryonic Stem Cell Differentiation
- Epigenetic dynamics during preimplantation development
Mammalian preimplantation development is a time of dynamic change in which the fertilized egg undergoes cleavage divisions developing into a morula and then a blastocyst with the first two distinct cell lineages inner cell mass ICM and trophectoderm TE. This developmental period is characterized by three major transitions, each of which entails pronounced changes in the pattern of gene expression. The first transition is the maternal-to-zygotic transition MZT , which serves three functions: i to destroy oocyte-specific transcripts e.
Metrics details. Epigenome is highly dynamic during the early stages of embryonic development. Epigenetic modifications provide the necessary regulation for lineage specification and enable the maintenance of cellular identity. Given the rapid accumulation of genome-wide epigenomic modification maps across cellular differentiation process, there is an urgent need to characterize epigenetic dynamics and reveal their impacts on differential gene regulation. We proposed DiffEM, a computational method for differential analysis of epigenetic modifications and identified highly dynamic modification sites along cellular differentiation process.
Transcriptional and Epigenetic Dynamics during Specification of Human Embryonic Stem Cells
International Journal of Medical Sciences. Journal of Cancer. Journal of Genomics. Global reach, higher impact. Journal of Genomics - Submit manuscript now Int J Biol Sci ; 9 10 Daniel C. Kraushaar , Keji Zhao. Embryonic stem cells ESCs possess an open and highly dynamic chromatin landscape, which underlies their plasticity and ultimately maintains ESC pluripotency.
The ESC epigenome must not only maintain the transcription of pluripotency-associated genes but must also, through gene priming, facilitate rapid and cell type-specific activation of developmental genes upon lineage commitment. Trans-generational inheritance ensures that the ESC chromatin state is stably transmitted from one generation to the next; yet at the same time, epigenetic marks are highly dynamic, reversible and responsive to extracellular cues.
Once committed to differentiation, the ESC epigenome is remodeled and resolves into a more compact chromatin state. A thorough understanding of the role of chromatin modifiers in ESC fate and differentiation will be important if they are to be used for therapeutic purposes.
Recent technical advances, particularly in next-generation sequencing technologies, have provided a genome-scale view of epigenetic marks and chromatin modifiers. More affordable and faster sequencing platforms have led to a comprehensive characterization of the ESC epigenome and epigenomes of differentiated cell types.
In this review, we summarize and discuss the recent progress that has highlighted the central role of histone modifications, histone variants, DNA methylation and chromatin modifiers in ESC pluripotency and ESC fate. We provide a detailed and comprehensive discussion of genome-wide studies that are pertinent to our understanding of mammalian development.
Embryonic stem cells ESCs are derived from the inner cell mass of the d4. They are defined by their unique properties of self-renewal and pluripotency, which make them an invaluable tool for studies of development and therapeutic medicine. Continuous self-renewal can be readily achieved in culture and maintains the pluripotent plasticity of ESCs. When transplanted into blastocyst-stage embryos, ESCs can contribute to somatic tissues from all three germ layers as well as the germ line of the developing mouse demonstrating the retention of their pluripotent potential during culture [ 1 ].
As ESCs exit self-renewal and commit to differentiation into cell types representative of the three germ layers ectoderm, mesoderm and endoderm they recapitulate embryonic differentiation programs that take place during in vivo development.
For this reason, ESCs provide a valid model for the study of early development. Furthermore, defined culture protocols have been developed that allow for the differentiation of ESCs into cell types that may be used for cell therapeutic purposes. The transition of a pluripotent stem cell to a committed and more developmentally restricted cell type is accompanied by rapid and global changes in transcription that result in stable silencing of pluripotency genes and activation of lineage-specific genes.
At the same time, stage specific genes of multipotent progenitors have to remain competent for activation during further differentiation whereas tissue-specific genes that are considered outside the progenitor's differentiation potential have to be terminally turned off. A number of cytokines, growth factors, morphogens and their co-factors regulate early lineage fate decisions and ultimately the transcription programs of ESCs and their roles are becoming increasingly defined and often coincide with their functions in vivo [ 2 - 6 ].
Extracellular signals are complemented by the action of intracellular transcription factors that drive gene expression patterns required for cell differentiation and lineage specification. Regulators of chromatin states and epigenetic modifiers have emerged as additional players of cell fate. Numerous genetic studies that have disrupted genes, which encode for chromatin modifiers, have resulted in embryonic lethality [ 7 - 9 ].
These studies have demonstrated the importance of ATP-dependent chromatin remodelers, histone modifying enzymes and DNA methyltransferases in embryonic development. Despite significant advances in the technologies used to map the genome-wide distribution of histone marks and a continuous decrease in required cell numbers the study of dynamic epigenetic chromatin modification during in vivo development remains challenging.
At the same time, ESCs can be cultured in abundance and provide a powerful tool for the study of epigenetic mechanisms and cell fate. Genome-wide maps of epigenetic factors have revealed a unique epigenetic signature in pluripotent ESCs and have contributed models to explain their plasticity.
Also, comparisons of ESCs with cells differentiated from ESCs have proven powerful in understanding the dynamics of epigenetic marks during development.
Thus, we have learned that each specific cell type has not only its own specific expression profile or 'transcriptome' but is also featured by its own epigenetic signature or 'epigenome' that is transmitted as heritable information through cell divisions [ 10 - 12 ].
In , Yamanaka et al. The successful reprogramming into induced pluripotent stem cells iPSCs ignited immense excitement since ethical concerns that arise from the derivation of ESCs may be circumvented using this alternative approach. In addition, a pool of patient-specific stem cells could now be generated with a possibility for autologous regenerative transplants.
Since then, we have come to understand that the epigenome of somatic cells must be reset into an ESC-like state and further highlights the importance of epigenetic regulation in maintaining pluripotency [ 15 - 18 ].
Here, we review our current understanding of the global epigenetic landscape of pluripotent ESCs, how it is set up by chromatin remodeling enzymes, and how it resolves during the course of differentiation.
To this end, we have focused our discussion primarily on studies that have utilized genome-wide sequencing technologies such as ChIP-Chip or ChIP-Seq to map histone modifications and epigenetic modifiers. ESCs are singular in exhibiting higher global transcription levels than somatic cells. High global transcription levels can be attributed to elevated transcription of sequences that are silent in differentiated cells and even higher transcription levels of genes that are constitutively expressed in both ESCs and differentiated cells [ 19 , 20 ].
Given such great differences in transcriptional activity one would anticipate substantial differences in chromatin organization between pluripotent and differentiated cell types. In fact, differences in global chromatin configuration can be readily detected by ultrastructural examination of ESC chromatin as well as chromatin from terminally differentiated cells. Chromatin of undifferentiated ESCs appears decondensed and plastic in structure whereas differentiated cells display distinct foci of heterochromatin [ 21 ].
Overall, ESC differentiation is accompanied by an increase in heterochromatic foci and a decrease in mobility and turnover of chromatin-associated proteins [ 21 - 23 ]. Genome-wide analyses of histone marks show that modifications, generally associated with transcriptional activity such as H3K4me3, H3K9ac, H3K14ac, H3K36me2 and H3K36me3, are present at high levels in ESCs and become reduced upon differentiation. An opposite trend holds true for repressive marks such as H4K20me3, H3K9me2 and H3K9me3that are more abundant in differentiated cells [ 24 , 25 ].
An open chromatin configuration may merely be a reflection of the hyper-transcriptional activity found in ESCs. However, the identification of both active H3K4me3 and repressive H3K27me3 at silent genes lead to the idea, that developmental genes are bivalently marked and thus primed for activation prior to ESC differentiation [ 26 ]. Therefore an open chromatin configuration and abundance of active histone marks is not only a reflection of high transcriptional activity but also of cell plasticity.
Unlike H3K4me3, which is present at almost three quarters of all gene promoters, H3K27me3 is only present at a small subset of developmental genes but is almost always found together with H3K4me3. The majority of bivalent promoters are transcriptionally initiated but not enriched with the elongating form of Pol II or H3K36me3 [ 20 ].
Besides gene promoters, bivalent histone modifications, in the form of H3K4me1 and H3K27me3, also decorate enhancer regions, which silence proximal gene clusters [ 28 , 29 ].
Lineage-related genes will retain bivalency with the idea that they remain poised for subsequent expression at later developmental stages, and unrelated genes become terminally silenced.
Gene priming and gene bivalency though, are not restricted to ESCs and are found in other cell types including hematopoietic stem cells as well as T-cells where bivalent histone modifications facilitate rapid gene activation upon T-cell differentiation [ 31 - 33 ].
The progressive restriction of differentiation potential alongside loss of gene bivalency at lineage-unrelated loci does not always hold true: hematopoietic stem cells derived from ESCs, retain bivalency on non-related neural genes [ 34 ].
In this case, Tbx21 bivalency translates into additional plasticity as illustrated by the capacity of nTreg cells to express Tbx21 in Th1-inducing conditions [ 35 ]. Some promoters may also gain bivalency during the course of differentiation, illustrating that gene priming is by no means a unidirectional process [ 33 , 36 ].
Based on insights into the mechanisms of gene poising, a model that involves H2A ubiquitination has evolved Fig. Loss of Ring1A or Ring1B that results in reduction of H2AK ubiqitination leads to de-repression of bivalent genes and apparent release of poised Pol II from these sites and replacement with hyperphosphorylated and transcriptionally active Pol II [ 20 , 38 , 39 ]. H3K4me3 mediates gene activation by various mechanisms that include interactions with ATP-dependent remodeling factors as well as recruitment of histone acetyltransferases and Pol II for transcription.
Hence the net result of gene priming equals the recruitment of Pol II, mediated by H3K4me3, and simultaneously prevention of transcriptional elongation by H3K27me3, which in effect results in low-level transcription of bivalent genes. Disruption of PcG functional subunits such as Eed, Ezh2, or Suz12 results in de-repression of bivalent differentiation genes, but typically does not affect ESC self-renewal probably because the expression of pluripotency genes remains largely unaffected [ 26 , 27 , 41 , 42 ].
Upon differentiation, H3K27me3 marks will have to be removed for timely activation of developmental genes, yet the enzyme that removes H3K27me3 is currently unknown. A delay in differentiation is observed upon Dpy knockdown that has been proposed to be a result of ineffective H3K4 methylation needed for upregulation of differentiation markers [ 45 ]. Interestingly, knockdown of MLL2 and reduction of H3K4me3 had no effect on gene induction upon differentiation, challenging the idea of gene bivalency as a catalyst for rapid gene activation [ 47 ].
Resolution of gene bivalency upon cell fate commitment. Primed promoters may remain bivalent during differentiation or bivalency may resolve to monovalency. An important, and easily overlooked factor in gene priming are the relative levels of active and inactive marks that need to be finely balanced in order to maintain gene poising without causing aberrant gene expression. Kdm5b, another H3K4 demethylase is primarily required for removal of H3K4me3 from pluripotency regulators such as Oct4 and Nanog, in order to initiate timely differentiation commitment [ 49 ].
Despite a growing understanding of PcG function, how PcG and Trx proteins become recruited to their target genes and how the rearrangement of bivalent genes is orchestrated during differentiation is still very much central to current investigation. Mechanistically, gene bivalency provides an unstable steady state of gene repression that may allow for rapid transition to gene activation upon the presentation of developmental stimuli.
Thus, a fine-tuned regulation of bivalent domains is necessary for proper ESC differentiation. Histone acetylation neutralizes the positive charge of histones and thereby decreases the affinity between histones and DNA [ 50 ]. At the same time, it generates recognition signals for chromatin proteins containing bromo domains [ 51 ] The steady state of chromatin acetylation levels is modulated by both histone acetyltransferases HATs and histone deacetylases HDACs.
Histone deacetylases HDACs , negatively control histone acetylation by removing acetyl groups from histone tails. HDAC activity at active genes prevents excessive acetylation that would otherwise lead to chromatin instability and cryptic transcription [ 56 ]. In ESCs, HDAC1 is bound predominantly to active genes that include Oct4, Sox2 and Nanog although some lineage-specific genes are also occupied, suggesting both positive and negative regulation of gene expression [ 55 ].
It may be for that reason why studies with chemical HDAC inhibitors have not provided consistent results. HDAC inhibition with TSA treatment has been reported to result in spontaneous differentiation and downregulation of pluripotency genes but also inhibition of differentiation in another case [ 55 , 57 ].
Treatment of fibroblasts with HDAC inhibitors such as trichostatin A, suberoylanilide hydroxamic acid and valproic acid VPA substantially improves their reprogramming efficiency [ 59 - 62 ]. Essentially, HDACs may hold a dual role in ESCs: firstly they repress the expression of lineage-specific genes that need to be inactive and entirely free of acetylation.
Secondly, deacetylation at active genes will prevent excessive acetylation, which could lead to indiscriminate transcription. Z deposition and HAT activities combined. Loss of pluripotency is observed upon knockdown of both p and Tip60 subunits and is caused by upregulation of developmental genes [ 63 ]. Hence, gene repression may be conferred by deposition of H2A.
Z rather than histone acetylation. An examination of H2A. Z's important function in facilitating accessibility of PcG complexes to developmental genes in ESCs see histone variant section. Mof occupies both Oct4 and Nanog and also overlaps substantially with Nanog targets that carry H3K4me3 on their promoters.
Mof facilitates the expression of pluripotency factors and represents an upstream regulator of MLL-associated gene priming. ATP-dependent chromatin modifiers are important regulators of lineage fate and embryonic development. Genetic studies showed that esBAF is required for self-renewal and pluripotency. Brg1, a subunit of the esBAF complex, interacts with Oct4, Sox2 and Nanog, and co-occupies many sites with the same transcription factors [ 68 - 70 ]. Hence, Brg1 function is highly context-dependent and facilitates both gene activation and repression by means of modulating nucleosome stability to maintain 'stemness' and pluripotency-associated chromatin competence.
The PcG complex is not the only chromatin modifier that antagonizes Brg1.
The Epigenomics of Embryonic Stem Cell Differentiation
Pamela Hoodless, PhD. Friday, February 12, - pm. Invited Speaker Seminar. Transcriptional and Epigenetic Dynamics during Hepatic Specification The embryonic liver parenchymal cells emerge from the definitive endoderm in response to signals from adjacent mesenchymal and endothelial cells. These bipotent hepatoblasts will differentiate into hepatocytes and cholangiocytes bile duct cells.
Embryonic stem cells ESCs consist of a population of self-renewing cells displaying extensive phenotypic and functional heterogeneity. Research towards the understanding of the epigenetic mechanisms underlying the heterogeneity among ESCs is still in its initial stage. Key issues, such as how to identify cell-subset specifically methylated loci and how to interpret the biological meanings of methylation variations remain largely unexplored. To fill in the research gap, we implemented a computational pipeline to analyze single-cell methylome and to perform an integrative analysis with single-cell transcriptome data. According to the origins of variation in DNA methylation, we determined the genomic loci associated with allelic-specific methylation or asymmetric DNA methylation, and explored a beta mixture model to infer the genomic loci exhibiting cell-subset specific methylation CSM. More interestingly, the putative CSM loci may be clustered into co-methylated modules enriching the binding motifs of distinct sets of transcription factors. Taken together, our study provided a novel tool to explore single-cell methylome and transcriptome to reveal the underlying transcriptional regulatory networks associated with epigenetic heterogeneity of ESCs.
Epigenetic dynamics during preimplantation development
Gifford, Casey A. Cell, 5. ISSN Differentiation of human embryonic stem cells hESCs provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers.
International Journal of Medical Sciences. Journal of Cancer. Journal of Genomics.
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