Transcriptional Regulatory Network in Embryonic Stem Cell

Stem Cells are undifferentiated cells that can give rise to several lineages of differentiated cell types. They are the founder cells for every organ, tissue and cell in the body. Stem cells are characterized by the ability to self-renew and maintain Pluripotency. These features allow Stem Cells to fulfill their multiple functions, namely to provide enough cells during organogenesis, to control tissue homeostasis and, in addition, to ensure regeneration and repair. ESCs (Embryonic Stem Cells) are derived from the ICM (Inner Cell Mass) of the developing Blastocysts, multicellular structures originating from four (Human) to five (Mouse) cleavages of fertilized oocytes. In Human, the fertilization of an Egg by a Sperm generates a Zygote that thirty hours later begins to divide. By the third to fourth day the Embryo develops to a compact ball of twelve or more cells called a Morula. After several more divisions, the Morula cells begin to specialize and form a hollow sphere of cells called a Blastocyst. The outer layer of the Blastocyst is named the Trophectoderm and the cells inside ICM. The cells of the ICM are Pluripotent stem cells that can give rise to all cell types of the three embryonic germ layers, i.e., Ectoderm, Mesoderm, and Endoderm, and the Germ Cell lineage. The recent derivation of human ESCs provides a unique opportunity to study early development and is thought to hold great promise for regenerative medicine. An understanding of the transcriptional regulatory circuitry that is responsible for Pluripotency and self-renewal in Human ES cells is fundamental to understanding Human development and realizing the therapeutic potential of these cells (Ref.1 & 2).

ESCs maintain their pluripotent state by expressing a battery of transcription factors including Oct4 (Octamer Binding Transcription Factor-4), SOX2 (SRY (Sex Determining Region Y) Box-2) and Nanog (Nanog Homeobox). Other factors like FoxD3 (Forkhead Box-D3), FGF4 (Fibroblast Growth Factor-4) and H2AZ (H2A histone family, member-Z) are also believed to play important role in Pluripotency and Self-renewal. Oct3/4, SOX2 and Nanog form a regulatory feedback circuit that maintains Pluripotency in Human and Mouse ESCs; in this circuit, all three transcription factors regulate themselves, as well as each other. The regulatory circuit formed by Oct4, Nanog, and SOX2 have been identified to be essential to the ESC identity. The frequency with which these three factors co-occupy within the same gene region is very high. Among Oct4 bound genes, half of them are also bound by SOX2. More than 90% of the promoter region bound by Oct4 and SOX2 are also bound by Nanog. Oct4, SOX2 and Nanog are also bound to their own promoters, thus forming an interconnected autoregulation loop to maintain the ESC identity. Recently, a negative feedback loop formed by Nanog, Oct4 and another Pluripotent factor, FoxD3 have been reported. Oct4 maintains Nanog expression by directly binding to a Nanog promoter when present at a sub-steady level, but represses it when Oct4 is above the normal level. On the other hand, FoxD3 positively regulates Nanog to counter the repression effect of excess Oct4. Conversely, Nanog and FoxD3 function as activators for Oct4 expression. When the expression level of Oct4 rises above a steady level, it represses its own promoter also, thus exerting a negative feedback regulation loop to limit its own expression. This negative feedback regulation loop keeps the expression of Oct4 at a steady level, thus maintaining the ESC properties (Ref.3 & 4).

Besides forming the regulatory circuit, the three core factors Oct4, Nanog and SOX2 contribute to the hallmark characteristics of ESCs by activation of target genes that encode Pluripotency and Self-renewal mechanisms and repression of signaling pathways that promote differentiation. In total, 352 genes are bound by Oct4, Nanog and SOX2 simultaneously in undifferentiated Human ESCs, which may be expressed or repressed. The active targets include genes encoding components of Chromatin Remodeling and Histone-modifying complexes (e.g., SMARCAD1 (SWI/SNF-Related, Matrix-Associated Actin-Dependent Regulator of Chromatin, Subfamily-A, Containing DEAD/H Box-1), MYST3 (MYST Histone Acetyltransferase (Monocytic Leukemia)-3), and SET (SET Translocation (Myeloid Leukemia Associated))), which have general roles in transcriptional regulation, and genes encoding transcription factors (e.g., REST (RE1 Silencing Transcription Factor), SKIL (SKI-Like Oncogene), HESX1 (HESX Homeobox-1), and STAT3 (Signal Transducer and Activator of Transcription-3)), which themselves are known to regulate specific genes. REST has recently been shown to be highly abundant in ESCs and functions in part to repress neuronal specific genes like CALB1 (Calbindin-1, 28kDa), L1CAM (L1 Cell Adhesion Molecule), GRIN1 (Glutamate Receptor, Ionotropic, N-methyl D-aspartate-1) and Cx36 (Connexin-36). Besides, RIF1 (RAP1 Interacting Factor Homolog (Yeast)) gene, which has been implicated in regulating telomere length and considered important for self-renewal as well as JARID2 (Jumonji, AT Rich Interactive Domain-2) gene, which is believed to have important roles in development, is also positively regulated (Ref.5 & 6). Another active gene induced by Oct4, Nanog and SOX2 simultaneously is Zic3 (Zic Family Member-3 Heterotaxy-1 (Odd-Paired Homolog, Drosophila)). ZIC3 belongs to the GLI superfamily of transcription factors and is a vertebrate homologue of the Drosophila pair rule gene OPa (Odd-Paired). The expression of ZIC3 in the Embryonic Ectoderm and Mesoderm during Gastrulation, and throughout the tail bud, retina and limb bud during Neurulation and Organogenesis suggests an important role for this transcription factor in Embryonic Ectoderm and Mesoderm development. ZIC3 is preferentially expressed in Pluripotent state. ZIC3 shares significant overlap with the Oct4, Nanog, and SOX2 transcriptional networks and is important in maintaining ESC Pluripotency by preventing differentiation of cells into Endodermal lineages. Few other genes known to be activated by Oct4, Nanog and SOX2 include CDYL (Chromodomain Protein, Y-Like), GBX2 (Gastrulation Brain Homeobox-2), TIF1 (Transcription Intermediary Factor-1-α), GSH2 (GS Homeobox-2), TCF7L1 (Transcription Factor-7 Like-1), FoxC1 (Forkhead Box-C1), RFX4 (Regulatory FactorX-4) and SalL1 (Sal Like-1 (Drosophila)). Functions of these genes are still not known (Ref.1, 7 & 8).

Among transcriptionally inactive genes co-occupied by Oct4, SOX2, and NANOG, genes that specify transcription factors important for differentiation into Extra-embryonic, Endodermal, Mesodermal, and Ectodermal lineages (e.g., ESX1l (Extraembryonic, Spermatogenesis, Homeobox-1 Homolog (Mouse)), HOXB1 (Homeobox-B1), HAND1 (Heart And Neural Crest Derivatives Expressed-1), MEIS1 (Meis Homeobox-1), PAX6 (Paired Box-6), LHX5 (LIM Homeobox-5), LBX1 (Ladybird Homeobox-1), MYF5 (Myogenic Factor-5), ONECUT1 (One Cut Homeobox-1)) are the most common ones. Moreover, nearly half of the transcription factor genes that are bound by the three regulators and transcriptionally inactive encoded developmentally important homeodomain proteins. ONECUT1 takes part in mesoderm differentiation by activating another gene HNF4α (Hepatocyte Nuclear Factor-4, α). Other genes like PAX6, MEIS1, HOXB1, LHX5, OTX1 (Orthodenticle Homeobox-1), HAND1 and MYF5 are believed to be involved in ectoderm differentiation. PAX6 activates SIX3 (SIX Homeobox-3) gene whereas MEIS1 takes part in Ectoderm differentiation via HOXB1 gene. Another transcriptionally inactive gene ISL1 (ISL LIM Homeobox-1) takes part in both Ectoderm as well as Endoderm differentiation, whereas ATBF1 (AT-binding transcription factor 1)/ ZFHX3 (Zinc Finger Homeobox-3) genes take part in Endoderm differentiation only. Other transcriptionally inactive genes include ESXL1 and NeuroG1 (Neurogenin-1). ESXL1 is believed to have Extra Embryonic function whereas NeuroG1 is involved in Neurogenesis by activating NeuroD1 (Neurogenic Differentiation-1) (Ref.9 & 10).

Besides regulating different genes simultaneously, Oct4, SOX2 and Nanog also regulates several genes independently. The balance between the levels of Oct4 and the Caudal-type homeodomain transcription factor Cdx2 has recently been shown to influence the first overt lineage differentiation in the embryo. Oct4 and Cdx2 expression patterns become mutually exclusive during embryogenesis, owing to their ability to reciprocally repress each other’s expression. Oct4 is associated with the establishment of the ICM, whereas Cdx2 is necessary for trophectoderm development. Oct4 and Cdx2 also regulate the T-box transcription factor Eomes (Eomesodermin), which, like Cdx2, is necessary for trophectoderm maintenance. The interaction between these factors is essential for segregation of the ICM and trophectoderm lineages during early development. A similar balance between Nanog levels and the transcription factors GATA4 (GATA Binding Protein-4) and GATA6 (GATA Binding Protein-6) is necessary for differentiation into primitive endoderm, a derivative of the ICM of the developing Blastocyst. Nanog represses GATA6 and, implicitly, GATA4, which is downstream of GATA6. GATA4 and GATA6 expression is also upregulated in the absence of Nanog. Other genetic, epigenetic and environmental factors also play an important role in this process (Ref.11 & 12).

Identifying the target genes for key transcriptional regulators of Human stem cells is a first critical step in the process of understanding these transcriptional regulatory networks and learning how they control cell identity. Mapping Oct4, SOX2, and NANOG to their binding sites within known promoters has revealed that these regulators collaborate to form in ES cells regulatory circuitry consisting of specialized autoregulatory and feed forward loops. Functions of these transcription factors depend on the stage of development of a Pluripotent cell, indicating that these factors function in combination with other processes. The activity of these transcription factors also depends on the accessibility of their target genes, which are made more or less accessible by the modification of their DNA, histones, or chromatin structure. Connecting signaling pathways to transcriptional regulatory circuit map may reveal how these Pluripotent cells can be stimulated to differentiate into different cell types or how to reprogram differentiated cells back to a Pluripotent state (Ref.2, 13 & 14).

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