scholarly journals The Sox-2 Regulatory Regions Display Their Activities in Two Distinct Types of Multipotent Stem Cells

2004 ◽  
Vol 24 (10) ◽  
pp. 4207-4220 ◽  
Author(s):  
Satoru Miyagi ◽  
Tetsuichiro Saito ◽  
Ken-ichi Mizutani ◽  
Norihisa Masuyama ◽  
Yukiko Gotoh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Regulatory Region ◽  
Embryonic Stem ◽  
Specific Expression ◽  
Regulatory Regions ◽  
Multipotent Stem Cells ◽  
Differentiated Cells

ABSTRACT The Sox-2 gene is expressed in embryonic stem (ES) cells and neural stem cells. Two transcription enhancer regions, Sox-2 regulatory region 1 (SRR1) and SRR2, were described previously based on their activities in ES cells. Here, we demonstrate that these regulatory regions also exert their activities in neural stem cells. Moreover, our data reveal that, as in ES cells, both SRR1 and SRR2 show their activities rather specifically in multipotent neural stem or progenitor cells but cease to function in differentiated cells, such as postmitotic neurons. Systematic deletion and mutation analyses showed that the same or at least overlapping DNA elements of SRR2 are involved in its activity in both ES and neural stem or progenitor cells. Thus, SRR2 is the first example of an enhancer in which a single regulatory core sequence is involved in multipotent-state-specific expression in two different stem cells, i.e., ES and neural stem cells.

Human embryonic stem cells: challenges and opportunities

2006 ◽  
Vol 18 (8) ◽  
pp. 839 ◽  
Author(s):  
Steven L. Stice ◽  
Nolan L. Boyd ◽  
Sujoy K. Dhara ◽  
Brian A. Gerwe ◽  
David W. Machacek ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Cell Line ◽  
Cell Fate ◽  
Neural Development ◽  
Es Cells ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Es Cell ◽  
Cell Therapies

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.

scholarly journals Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

2019 ◽  
Author(s):  
Aseda Tena ◽  
Yuxiang Zhang ◽  
Nia Kyritsis ◽  
Anne Devorak ◽  
Jeffrey Zurita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Progenitors ◽  
Neural Genes ◽  
Genome Wide ◽  
Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

scholarly journals The Profile of Post-translational Modifications of Histone H1 in Chromatin of Mouse Embryonic Stem Cells

Acta Naturae ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 82-91 ◽  
Author(s):  
T. Yu. Starkova ◽  
T. O. Artamonova ◽  
V. V. Ermakova ◽  
E. V. Chikhirzhina ◽  
M. A. Khodorkovskii ◽  
...  
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histone H1 ◽  
Ion Cyclotron Resonance ◽  
Compact Structure ◽  
Post Translational Modifications ◽  
Differentiated Cells ◽  
Eukaryotic Dna ◽  
Nih 3T3

Linker histone H1 is one of the main chromatin proteins which plays an important role in organizing eukaryotic DNA into a compact structure. There is data indicating that cell type-specific post-translational modifications of H1 modulate chromatin activity. Here, we compared histone H1 variants from NIH/3T3, mouse embryonic fibroblasts (MEFs), and mouse embryonic stem (ES) cells using matrix-assisted laser desorption/ ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS). We found significant differences in the nature and positions of the post-translational modifications (PTMs) of H1.3-H1.5 variants in ES cells compared to differentiated cells. For instance, methylation of K75 in the H1.2-1.4 variants; methylation of K108, K148, K151, K152 K154, K155, K160, K161, K179, and K185 in H1.1, as well as of K168 in H1.2; phosphorylation of S129, T146, T149, S159, S163, and S180 in H1.1, T180 in H1.2, and T155 in H1.3 were identified exclusively in ES cells. The H1.0 and H1.2 variants in ES cells were characterized by an enhanced acetylation and overall reduced expression levels. Most of the acetylation sites of the H1.0 and H1.2 variants from ES cells were located within their C-terminal tails known to be involved in the stabilization of the condensed chromatin. These data may be used for further studies aimed at analyzing the functional role played by the revealed histone H1 PTMs in the self-renewal and differentiation of pluripotent stem cells.

BMP4 and TGFbeta Differentially Regulate CD34+ Progenitor Development in Human Embryonic Stem Cells through SMAD-Dependent Pathway

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 889-889
Author(s):  
ZacK Z. Wang ◽  
Hao Bai ◽  
Melanie Arzigian ◽  
Yong-Xing Gao ◽  
Wen-Shu Wu
Keyword(s):  
Stem Cells ◽  
Smooth Muscle ◽  
Growth Factors ◽  
Progenitor Cells ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Bmp Signaling ◽  
Cd34 Cells

Abstract Pluripotent stem cells derived from patients, including embryonic stem (ES) cells and “induced pluripotent stem” (iPS) cells, are a promising area of regenerative medical research. A major roadblock toward human clinical therapies using ES cells or iPS cells is to define the factors that direct ES cell differentiation into lineage specific cells. We previously established a simple and efficient human embryonic stem cell (hESC) differentiation system to generate CD34+/CD31+ progenitor cells that gave rise to hematopoietic and endothelial cells (Nat Biotech.25:317, 2007). To advance potential clinical application and to define the effects of growth factors on hematopoietic and vascular differentiation, we assessed hESC differentiation on human feeder cells in serum-free condition without intermediate embryoid body (EB) formation. We investigated the roles of BMPs, TGFbeta, VEGF, and FGF2 in directing hESC differentiation. Growth factors were added into culture at different time points to test their stage specific roles. Our study demonstrated that BMP proteins, including BMP2, BMP4, and BMP7, but not BMP9, had synergic effects to VEGF and FGF-2 on hESC differentiation to CD34+/CD31+ progenitor cells. BMP4 was essential to initial CD34+/CD31+ cell development, whereas VEGF and FGF2 promoted the differentiation in later stage, suggesting the sequential roles of BMP4, VEGF and FGF2 in directing hESC differentiation to CD34+/CD31+ progenitor cells. TGFbeta or activin promoted hESC differentiation into CD34+/CD31− cells that were unable to give rise to hematopoietic, endothelial, and smooth muscle cells. Furthermore, TGFbeta or activin activated Smad2/3 signaling, and suppressed BMP4-induced CD34+/CD31+ cells. Microarray analysis revealed that BMP4-induced CD34+ cells expressed hematopoietic, endothelial and smooth muscle genes, including GATA2, gamma globins, VE-Cad, KDR, CD31, Tie2, and aortic smooth muscle actin, whereas TGFbeta-induced CD34+ cells expressed pluripotent markers and endoderm markers, including Oct3/4, Sox2, and Nanog, HHEX, GATA6, and FoxA2. Both canonical BMP signaling (Smad1/5/8-dependent) and non-canonical BMP signaling (p38 MAPK and p42 ERK pathway) were activated by BMP4 in hESCs. Dorsomorphin specifically inhibited BMP4-mediated phosphorylation of Smad1/5/8, and blocked hESC differentiation into CD34+/CD31+ cells. In summary, BMPs and TGFbeta regulate distinct populations of CD34+ cells in hESCs. BMP-Smad1/5/8 pathway is critical for hematopoietic and vascular progenitor development.

scholarly journals 170 ASSESSING THE POTENTIAL OF STEM CELLS TO GENERATE CHIMERIC RATS

2005 ◽  
Vol 17 (2) ◽  
pp. 236
Author(s):  
J. Guo ◽  
S. Fida ◽  
K. Gou ◽  
C. Zhang ◽  
J. Morrison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Development ◽  
Neural Stem Cells ◽  
Embryo Transfer ◽  
Coat Color ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dark Agouti ◽  
Inbred Rat ◽  
Lac Z

Embryonic stem (ES) cells are pluripotent cells derived from inner cell masses (ICMs) of blastocysts. The capacity of pluripotency in differentiation is assumed to contribute to embryonic development to form a chimeric individual when these cells are reintroduced into embryos. Chimeric mice can be routinely generated by aggregation of ES cells with morulae or injection into blastocysts, which are then implanted in pseudopregnant foster mothers. Furthermore, recent studies have demonstrated that bone marrow-derived stem cells and neural stem cells can integrate into the embryonic development in mouse (Geiger et al. 1998 Cell 93, 1055–1065; Clarke et al. 2000 Science 288, 1660–1663). We therefore tried to assess the ability of rat ICMs and neural stem cells to form chimeras by injecting these cells into rat blastocysts. Forty-two rat ICMs from Day 6 blastocysts of Dark Agouti (DA) inbred rat were injected into Day 5 blastocysts of Sprague-Dawleyd (SD) outbred rats; 14 pups were born following embryo transfer of these blastocysts injected into Hooded Wistar (HW) recipients. One male of the 14 pups was coat color-patched and displayed germline transmission. Following embryo transfer of 22 SD blastocysts injected by Day 5 DA ICMs, 7 pups were born and 2 of them were coat color-patched. Nine pups were obtained from 23 DA blastocysts injected by Day 5 SD ICMs; 4 of them were coat color-patched. The ICM cells were isolated and cultured for 6 days. No chimeras were generated by injection of the cultured ICM cells, as assessed by coat color patching. These results suggest that rat embryonic ICMs have potential to develop into chimeras, but the chimeric potential of ICMs was rapidly lost in our culture system. Investigation of potential chimeric development of rat fetal neural stem (rFNS) cells transfected with Lac Z was carried out. Staining was observed in tissues from 2 of 41 E14 fetuses. These results demonstrated that rFNS cells can integrate into the early embryonic environment although the ability of these cells to contribute to chimeric formation was marginal. No coat color chimerism was observed in any of the 88 pups generated from the LacZ-rFNS cell experiments.

scholarly journals Host cis-Mediated Extinction of a Retrovirus Permissive for Expression in Embryonal Stem Cells during Differentiation

1998 ◽  
Vol 72 (1) ◽  
pp. 339-348 ◽  
Author(s):  
Christine Laker ◽  
Johann Meyer ◽  
Arndt Schopen ◽  
Jutta Friel ◽  
Christoph Heberlein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Time Window ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Retroviral Vectors ◽  
Early Embryo ◽  
Differentiated Cells ◽  
Secondary Mechanism ◽  
Embryonal Stem

ABSTRACT The use of retroviral vectors for gene transfer into animals has been severely hampered by the lack of provirus transcription in the early embryo and embryonic stem (ES) cells. This primary block in provirus expression is maintained in differentiated cells by acis-acting mechanism that is not well characterized. Retroviral vectors based on the murine embryonal stem cell virus (MESV), which overcome the transcriptional block in ES cells, were constructed to investigate this secondary mechanism. These vectors transferred G418 resistance to ES cells with the same efficiency as to fibroblasts, but overall transcript levels were greatly reduced. A mosaic but stable expression pattern was observed when single cells from G418-resistant clones were replated in G418 or assayed for expression of LacZ or interleukin-3. The expression levels in independent clones were variable and correlated inversely with methylation. However, a second, more pronounced, block to transcription was found upon differentiation induction. Differentiation of the infected ES cells to cells permissive for retroviral expression resulted in repression and complete extinction of provirus expression. Extinction was not accompanied by increased levels of methylation. Provirus expression is thus regulated by two independentcis-acting mechanisms: (i) partial repression in the undifferentiated state, accompanied by increased methylation but compatible with long-term, low expression of retroviral genes, and (ii) total repression and extinction during early stages of differentiation, apparently independent of changes in methylation. These results indicate a time window early during the transition from an undifferentiated to a differentiated stage in which provirus expression is silenced. The mechanisms are presently unknown, but elucidation of these events will have an important impact on vector development for targeting stem cells and for gene therapy.

HoxA11 Is Expressed in the Developing Hemangioblast as Well as Early Hematopoietic Precursor Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4206-4206
Author(s):  
Regina D. Horvat-Switzer ◽  
Alexis A. Thompson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Hox Genes ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Rt Pcr ◽  
Hematopoietic Precursor

Abstract Congenital amegakaryocytic thrombocytopenia with radio-ulnar synostosis is associated with mutations in the HOXA11 gene, suggesting that HoxA11 may play a role in megakaryocytic lineage commitment or differentiation. The HOX genes encode transcription factors that are involved in cellular differentiation in embryonic as well as adult tissues. Numerous studies have identified HOX genes as important regulators of various aspects of hematopoiesis including self-renewal, proliferation, differentiation and leukemogenesis. Our initial studies failed to identify the expression of HoxA11 in platelets, TPO-induced CD34+ umbilical cord stem cells or normal bone marrow. More recently our lab has detected a small amount of HoxA11 mRNA in cells isolated from unfractionated human cord blood, suggesting the expression of HoxA11 may occur in a small subset of early hematopoietic or stromal cells. To test this hypothesis we have employed a murine embryonic stem (ES) cell culture system. Co-culture of ES cells and the bone marrow stromal cell line, OP9, can give rise to primitive as well as definitive hematopoietic progenitors in the absence of leukemia inhibitory factor (LIF). By day 6, ES cells on OP9 can differentiate into mesodermal colonies, which contain a bi-potential progenitor known as the hemangioblast. The hemangioblast can further differentiate into either a hematopoietic or endothelial lineage. To determine when HoxA11 is expressed we have employed this model using green fluorescent protein (GFP) expressing ES cells grown on OP9 and differentiated into hematopoietic precursors in the absence of LIF. Nested RT-PCR revealed that HoxA11 mRNA is highly expressed in ES cells following 6 days (D6) on OP9. HoxA11 expression was restricted to D6 ES cells, as HoxA11 mRNA was not found in OP9 cells alone or ES cells differentiated on OP9 for 0, 3, or 9 days. RT-PCR revealed HoxA11 mRNA expression coincided with the expression of flk-1, a marker for the hemangioblast. Since HoxA11 expression is concurrent with hemangioblast differentiation, we sought to determine if the hemangioblast is the cell that expressed HoxA11. Using flow cytometery and fluorescence activated cell sorting (FACS) analysis we separated D6 ES cells into flk-1 positive (flk-1+) and negative (flk-1−) populations and investigated which population expressed HoxA11. Nested RT-PCR revealed that HoxA11 mRNA expression is found in both the flk-1+ and flk-1- fractions. We further analyzed these fractions by RT-PCR for SCL/Tal-1. SCL/Tal-1 is a transcription factor that plays a critical role in the commitment of mesoderm into hematopoietic progenitor cells. We find SCL/Tal-1 mRNA also expressed in both flk-1+ and flk-1- fractions, which parallels HoxA11 mRNA expression. These data suggest HoxA11 expression occurs in the flk-1+ hemangioblast but also possibly in a flk-1-/SCL+ hematopoietic precursor cell population. Current studies are underway to determine the cell fate and role of the HoxA11 expressing progenitor cell. Taken together, these data are the first findings of HoxA11 expression in early progenitor cells as well as the first evidence of controlled HoxA11 regulation during early hematopoietic development.

Maintenance of pluripotential embryonic stem cells by stem cell selection

1998 ◽  
Vol 10 (8) ◽  
pp. 527 ◽  
Author(s):  
Peter Mountford ◽  
Jennifer Nichols ◽  
Branko Zevnik ◽  
Carmel O'Brien ◽  
Austin Smith
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Stem Cell Population ◽  
Cell Selection ◽  
Es Cell ◽  
Mammalian Embryo ◽  
Differentiated Cells

As gastrulation proceeds, pluripotential stem cells with the capacity to contribute to all primary germ layers disappear from the mammalian embryo. The extinction of pluripotency also occurs during the formation of embryoid bodies from embryonic stem (ES) cells. In this report we show that if the initial differentiated progeny are removed from ES cell aggregates, further differentiation does not proceed and the stem cell population persists and expands. Significantly, the presence of even minor populations of differentiated cells lead to the complete loss of stem cells from the cultures. This finding implies that the normal elimination of pluripotent cells is dictated by inductive signals provided by differentiated progeny. We have exploited this observation to develop a strategy for the isolation of pluripotential cells. This approach, termed stem cell selection, may have widespread applicability to the derivation and propagation of stem cells.

scholarly journals Cellular Reprogramming Using Defined Factors and MicroRNAs

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Takanori Eguchi ◽  
Takuo Kuboki
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dna Methyltransferases ◽  
Culture Conditions ◽  
Cellular Reprogramming ◽  
Multipotent Stem Cells ◽  
Cell Tissue ◽  
Induced Pluripotent ◽  
Review Reports

Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming.

scholarly journals Ανάλυση γονιδιακών ρυθμιστικών δικτύων που ενέχονται στην εμβρυική ανάπτυξη του παγκρέατος των θηλαστικών

2011 ◽  
Author(s):  
Μαρία Καπασά
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Embryonic Development ◽  
Progenitor Cells ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Regulatory Regions

Mammalian development occurs by the progressive determination of cells from a pluripotent undifferentiated state through successive states of gradually restricted developmental potential, until the full complement of mature terminally differentiated cells has been specified. Embryonic development is a complex and highly orchestrated process during which multiple cell movements and changes in gene expression must be spatially and temporally coordinated to ensure that embryogenesis proceeds correctly. Complex genetic regulatory networks receive input in the form of extracellular signals and output instructions on the regulated expression of specific genes. The linchpins of the regulatory networks are the cis-regulatory elements that directly control gene expression through interpretation of the tissue-specific transcription factors (trans-elements). Embryonic stem cells are orientated across the dorso-ventral and the anterior-posterior axis of the early embryo. The orientation of progenitor cells along these two axes is thought to influence their fate by defining the identity and concentration of inductive signals to which they are exposed.In an effort to develop cell-based therapies, (i.e. for diabetes) experimental protocols aim to mimic the biological procedures that take place during embryonic development in order to differentiate embryonic stem cells towards specific cell types. One of the foremost challenges towards the development of cell therapies for diabetic people is to achieve the directed differentiation of cells capable of producing insulin. Elucidation of the genetic networks involved in the endocrine pancreas specification are thought to be essential for devising rational protocols to efficiently differentiate embryonic stem cells or pancreas progenitor cells into fully differentiated endocrine subtypes. Computational approaches allow the unravelling of complex regulatory networks including genomic (cis-cis) or proteomic (trans-trans) interactions or a combination (cis-trans) of both. In this study the genomic regulatory regions (cis elements) of several genes known and putative targets of the transcription factor NGN3 were analyzed. The NGN3 transcription factor is the major regulator of “insulin-producing cell” formation. Taking into account data from microarray experiments from pancreas progenitor cells, in which NGN3 has been induced, genes shown to be co-regulated (upregulated or downregulated) by this transcription factor were selected for analysis. Using a combination of sophisticated computational tools for exploiting and analyzing genomic data and developing the suitable algorithms, an extensive in silico analysis of the regulatory regions of these genes was performed.Evolutionarily conserved regions are linked with experimentally identified regulatory elements. Comparative genomics are commonly used in order to identify transcription factor binding sites, which are functionally important regions that are thought to be well-conserved. Analysis of genomic regulatory regions included not only genes corregulated by NGN3, but also their orthologs in several species including the most phylogenetically distant species (fish), which have pancreas. In parallel, housekeeping genes, like B-ACTIN, and those not expressed in embryos and stem cells, like B-GLOBIN, were used as negative controls. Regulatory region analysis revealed the presence of a highly conserved regulatory element, where many transcription factors with established involvement in pancreas development bind, in all the orthologs of several genes co-regulated by NGN3. Furthermore, motif identification in separate clusters of the regulatory elements of either upregulated or downregulated genes revealed the presence of additional binding motifs for the factor AP4 only in downregulated genes. In parallel, the regulatory region analysis of the entire mouse genome and the statistical analysis of the upcoming results showed that both types of regulatory elements (with and without AP4) were non-randomly identified inside the regulatory regions of genes whose transcription is controlled by NGN3. Moreover the selective presence of the AP4 binding sequence into this region renders it a highly specific suppressor found in only a small number of genes downregulated by NGN3. Taking into account that both these regulatory elements were identified at considerable distances from each gene’s transcription start site, it was assumed that they represent enhancers, and those capable of binding AP4 were considered silencers. This conclusion was enforced by the compositional analysis of these regions showing low GC levels, similarly to the majority of the regulatory regions implicated in embryonic development, something that has not been reported for promoter sequences. Moreover, analysis of protein-protein interactions showed that some of the transcription factors, predicted to bind onto these elements, together with other non-specific transcription factors, constitute a core transcription control complex. This protein complex interacts with the remaining members of the predicted cluster of transcription regulators and works either as an inducer or a suppressor of transcription. This is determined by the presence of a HAT and/or an HDAC in this protein complex assumed to locally control chromatin acetylation. Based on these data, we constructed a model of the complex regulatory network that describes how through the transcriptional regulation of the analyzed genes mainly guided by ΝGN3 the gradual differentiation of cells capable of producing insulin takes place.

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