Embryonic Stem Cells Escs Proteins

 Embryonic Stem Cells Escs Proteins Background

A lot of recent work has focused on embryonic stem cells (ESCs): cells from the inner cell mass of the mammalian blastocyst that go on to form the embryo proper. ESCs have two unique properties: (1) they can self-renew indefinitely and (2), they are pluripotent, meaning they have the potential to differentiate into every cell type of the developing and adult organism, save the extraembryonic tissues. Together, these features make ESCs a unique resource for the study of the control, stability, and flexibility of cell identity, as well as the development of regenerative medicine and patient-specific disease modeling.
ESC identity, like cell identity in general, is largely understood in terms of a gene expression profile: what subset of the genome is expressed, what is silenced, and to what degree. This gene expression program determines not only a cell’s physiological properties, but also establishes a gene regulatory network to maintain the program, thereby defining a stable, coherent, and distinct cell identity. This network can be roughly subdivided into three major components: (1) sequence-specific DNA-binding transcription factors (TFs), which control the activation and repression of gene transcription, often hierarchically coordinated with a small group of “master” TFs; (2) chromatin regulatory factors that realize and sustain the TFs’ regulatory influence; and (3) signal transduction pathways that incorporate signals from environmental stimuli into the regulatory network. These will each be discussed in the following sections. There are also important contributions from microRNAs and RNA-binding proteins that regulate mRNA turnover and translation post-transcriptionally, as well as mechanisms of post-translational regulation, but these will not be considered here.

Regulation of ESC Identity: Transcription Factors
In ESCs, the ability of TFs to control cell identity is especially well established, and most forcefully demonstrated by the TF-driven reprogramming of somatic cells into a pluripotent state. The three factors OCT4, SOX2, and NANOG (together, OSN) form the master regulatory group that governs the core GE program. They are each necessary for the maintenance and establishment of pluripotency, and act cooperatively to activate the expression of several hundred pluripotency genes, including themselves, and repress several hundred others, largely involved in differentiation. However, OSN are not individually sufficient: each factor, if overexpressed relative to the others, ceases to promote pluripotency and instead triggers development of a differentiated lineage. Thus, their cooperative activity and positive autoregulation is a crucial component to their pluripotency-promoting function, as it allows the factors to keep each other balanced, and maintain mutual repression of their lineage-specifying activities, thereby stabilizing the ESC state. This cooperatively enforced ESC transcriptional program also depends on the presence of a number of other TFs, including SALL4, SALL1, cMYC, KLF4, KLF5, ESSRB, STAT3, and SMAD1, as well as a large number of transcription cofactors, including the Mediator complex, which connect sequence-specific TFs to the transcription machinery.

Regulation of ESC Identity: Chromatin
The scaffolding of genomic DNA onto histones to form nucleosomes, and packaging of nucleosomes into chromatin, allows eukaryotic cells to control gene expression by regulating the accessibility of genes to the transcription machinery. In general, chromatin regulators do not have sequence-specific DNA-binding activities themselves, but rather depend on TFs or histone marks, discussed below, for targeting—indeed, many regulators described below have been shown to interact directly and co-occupy regulatory regions with OSN themselves. Nonetheless, because chromatin regulation involves covalent modifications and ATP-dependent remodeling, often over kilobase-scale domains, and with direct, local mechanisms for self-reinforcement, it provides a power and stability to GE programs that TFs alone do not.
Loose or “open” chromatin, known as euchromatin, allows higher accessibility and is associated with transcriptional activity, while condensed areas, known as heterochromatin, are involved in transcriptional silencing. In ESCs, however, many silent genes, including those involved in development and lineage specification, are maintained in an unusual form of open chromatin, known as bivalent, that carries hallmarks of both transcriptional silencing and activity. Meanwhile, many active regions, including core pluripotency genes, are subject to chromatin based repression despite their transcriptional activity. These conflicting features are thought to be required to balance ESCs’ opposing requirements of maintaining a stable identity and being disposed to promptly differentiate into any of several mutually exclusive lineages. Accordingly, the regulation of chromatin is equally important once differentiation does proceed, to ensure that genes of the chosen lineage are stably activated, while pluripotency genes and “poised” genes of other lineages are resolved to a repressive state. In fact, stable, unambiguous silencing may be more essential to differentiation than pluripotency per se, since loss of function of polycomb group (PcG) or nucleosome remodeling and deacetylase (NuRD) complexes, described below, does not disturb ESC self-renewal, but does result in overexpression of OSN and impairment of differentiation.
The regulation of chromatin depends on the action of many different regulatory factors, with two most prevalent types: histone modifying enzymes and ATP-dependent chromatin remodeling complexes.
Histone modifying enzymes catalyze the addition of modifying groups to residues in the extended tails of histone subunits, which may affect DNA accessibility directly, by altering the strength of DNA-histone association, or indirectly, by recruiting other regulatory factors. The two most common such modifications are lysine methylation and acetylation. For example, the Trithorax group (TrxG) complex trimethylates H3K4 at the promoters of active genes. It is antagonized by the repressive PcG repressive complexes (PRC1 or PRC2), which trimethylates H3K27, and is required for repression of differentiation genes in ESCs and of pluripotency genes in differentiation. Similarly, lysine acetylation and deacetylation are important mechanisms of activation and repression, respectively, and are regulated by multiple histone acetylase (HAT), such as the p300 complex, and histone deacetylase (HDAC) complexes, such as the NuRD. Both p300 and NuRD are also each required in differentiation.
Nucleosome-remodeling complexes move histones along DNA to alter the nucleosome density—inversely related to accessibility—of the underlying genes. The ESC-specific es-Brg1-Associated Factors (esBAF) complex, for example, is required for both activation of genes that maintain self-renewal and repression of developmental genes. The NuRD complex mentioned above also includes the nucleosome remodeling CHD4/Mi-2 subunit alongside HDAC1/2, and has been thoroughly implicated in the repression of both differentiation and pluripotency genes in pluripotent stem cells. These functions are thought to facilitate stable pluripotency and competency to differentiate, respectively.
Interestingly, chromatin regulatory complexes of both types often display flexibility in their subunit composition, giving rise to biochemically and functionally distinct versions. In particular, esBAF is a variant of the BAF complex which, with the addition of a different specificity subunit or transcriptional cofactor, can become important to the biology of cardiac progenitors, neuronal progenitors or postmitotic neurons. Similarly, the PcG complex chromobox domain (Cbx) subunits can direct the Polycomb Repressive Complex 1 (PRC1) to either maintain pluripotency, via CBX7, or orchestrate differentiation, via CBX2 and CBX4. And the NuRD complex is perhaps most flexible of all, as it is able to substitute different paralogs of any of its subunits. For example, MBD2 and MBD3 are thought to participate mutually exclusively in the NuRD complex, with the two complexes regulating distinct subsets of NuRD targets. With Mbd3, the NuRD complex behaves as described above, while MBD2, which has retained a functional methyl-binding domain unlike MBD3, directs the complex to specifically interact with methylated CpG dinucleotides and generate tight heterochromatin. Different MTA subunits can also change NuRD’s gene regulatory behavior, with MTA1 facilitating a contribution to metastatic growth that both MTA2 and MTA3 lack. 

Regulation of ESC Identity: Signaling
ESC identity also depends on signaling from the extracellular environment that can direct cells to either maintain pluripotency or differentiate. There are four major, well-studied signaling pathways that affect maintenance of pluripotent mESCs in vitro. LIF activates a Jak/STAT pathway leading to phosphorylation and nuclear translocation of the transcription factor STAT3, which supports the ESC state by promoting the expression of secondary pluripotency factors, including Klf4 and Klf5, and repressing endoderm differentiation. Similarly, BMP4 signaling promotes pluripotency by activating SMAD1 and SMAD4, which in turn activate expression of ID, a repressor of genes involved in neurectoderm differentiation. WNT signaling can also support pluripotency, by directly activating expression of Oct4 and Nanog and inhibiting neural differentiation, although the cooperation of the Wnt transcriptional effector, β-catenin, with Tcf3 may also repress expression of these genes. And, lastly, signaling from FGF4 to the MAPK/ERK pathway promotes differentiation and loss of pluripotency. These signaling pathways contain many points of interdependence and cross-talk. For example, BMP4 is no longer able to support pluripotency if the LIF pathway is inactivated. On the other hand, while LIF and BMP4 were originally thought to both be required for maintenance of pluripotency, it has since been shown that, in the absence of both extrinsic LIF and BMP4, a Wnt agonist and ERK inhibitor can cooperate to prevent differentiation. Moreover, mESCs have been shown to engage in autocrine signaling with FGF4 and BMP4, making it difficult to truly isolate the effect of any one pathway. In fact, it has so far been impossible to culture ESCs in the total absence of ESC-promoting extracellular signaling, suggesting that the self-stabilizing pluripotency TF network governed by OCT4, SOX2, and NANOG is not strictly sufficient to maintain the ESC state without support from these pathways. This is consistent with the observation that the TFs associated with these pathways can interact and often co-occupy the same regulatory sites as OSN, suggesting a direct connection to the core TF circuitry.
It should also be noted that signaling requirements of pluripotent cells change in vivo, where the inner cell mass from which ESCs are derived is only a transient state. Accordingly, different pluripotent cell types derived from subtly different developmental stages can have different signaling requirements in vitro. For example, mouse epiblast stem cells (EpiSCs) require Activin or Nodal signaling and activation of SMAD2 and SMAD3 to maintain pluripotency and are not maintained by LIF, whereas ESCs do not require these signaling factors but do require LIF for maintaining the pluripotent state. Incidentally, human “ESCs” share both of these features of mouse EpiSCs, suggesting that they may be more closely related to epiblast-derived stem cells. Nonetheless, these two cell types do differ in other important characteristics—e.g. EpiSCs do not express alkaline phosphatase (AP) or Klf4, whereas h“ESCs” and mESCs both do—suggesting that there may also be species-specific differences.

ESC Identity: Stability vs Flexibility
The indispensible importance of signaling to the maintenance of ESCs has led to contention about whether the ESC state represents a self-sufficient, stable “ground state” of cell identity, or only a precarious stalemate between competing, mutually exclusive differentiated lineages, requiring continual external support. Although the property of indefinite self-renewal and the auto-regulatory TF circuitry argue for the former, the latter view is more consistent with several other features, including highly heterogenous expression of key pluripotency genes in ESCs, the differentiation-inducing capacities of individual pluripotency factors, and the moderating repression of OSN by esBAF and NuRD. Thus, the ESC regulatory circuitry seems to contain two mutually exclusive tendencies: one for stable self-renewal, and the other for stable and multipotent differentiation. A major question in the future of the field will be to understand how these two objectives are divided up among regulatory components, and how they manage to balance each other.

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