scholarly journals Synthetic and genomic regulatory elements reveal aspects of cis regulatory grammar in Mouse Embryonic Stem Cells

2018 ◽  
Author(s):  
Dana M. King ◽  
Brett B. Maricque ◽  
Barak A. Cohen
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Binding Sites ◽  
Genomic Sequence ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Site Quality ◽  
Genomic Sequences ◽  
Position Effects

In embryonic stem cells (ESCs), a core network of transcription factors establish and maintain the gene expression program necessary to grow indefinitely in cell culture and generate all three primary germ layers. To understand how interactions between four key pluripotency transcription factors (TFs), SOX2, POU5F1 (OCT4), KLF4, and ESRRB, contribute to cis-regulation in mouse ESCs, we assayed two massively parallel reporter assay (MPRA) libraries composed of different combinations of binding sites for these TFs. One library was an exhaustive set of synthetic cis-regulatory elements and the second was a set of genomic sequences with comparable configurations of binding sites. Comparisons between the libraries allowed us to determine the regulatory grammar requirements for these binding sites in constrained synthetic contexts versus genomic sequence contexts. We found that binding site quality is a common attribute for active elements in both the synthetic and genomic contexts. For synthetic regulatory elements, the level of expression is mostly determined by the number of binding sites but is tuned by a grammar that includes position effects. Surprisingly, this grammar appears to only play a small role in setting the output levels of genomic sequences. The relative activity of genomic sequences is best explained by the predicted affinity of binding sites, regardless of identity, and optimized spacing between sites. Our findings highlight the need for detailed examinations of complex sequence space when trying to understand cis-regulatory grammar in the genome.

scholarly journals Synthetic and genomic regulatory elements reveal aspects of cis-regulatory grammar in mouse embryonic stem cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Dana M King ◽  
Clarice Kit Yee Hong ◽  
James L Shepherdson ◽  
David M Granas ◽  
Brett B Maricque ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Binding Site ◽  
Binding Sites ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Genomic Sequences ◽  
Primary Determinant ◽  
Massively Parallel Reporter Assay

In embryonic stem cells (ESCs), a core transcription factor (TF) network establishes the gene expression program necessary for pluripotency. To address how interactions between four key TFs contribute to cis-regulation in mouse ESCs, we assayed two massively parallel reporter assay (MPRA) libraries composed of binding sites for SOX2, POU5F1 (OCT4), KLF4, and ESRRB. Comparisons between synthetic cis-regulatory elements and genomic sequences with comparable binding site configurations revealed some aspects of a regulatory grammar. The expression of synthetic elements is influenced by both the number and arrangement of binding sites. This grammar plays only a small role for genomic sequences, as the relative activities of genomic sequences are best explained by the predicted occupancy of binding sites, regardless of binding site identity and positioning. Our results suggest that the effects of transcription factor binding sites (TFBS) are influenced by the order and orientation of sites, but that in the genome the overall occupancy of TFs is the primary determinant of activity.

The Deposition of H3.3 Mediated by Transcription Factors Determines Hematopoietic Cell Fate

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1193-1193
Author(s):  
Jun Odawara ◽  
Kohta Miyawaki ◽  
Kentaro Kohno ◽  
Takahiro Shima ◽  
Kazumitsu Maehara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Binding Sites ◽  
Embryonic Stem ◽  
Marker Genes ◽  
Open Chromatin ◽  
Differentiation Marker ◽  
Almost All

Abstract Abstract 1193 Cell differentiation is achieved by sequential gene expression. Late differentiation marker genes are already regulated at the chromatin level prior to differentiation, even in pluripotent stem cells. Therefore, we hypothesized that “stem-ness” in hematopoiesis is already programmed in hematopoietic stem/progenitor cells (HSPCs) and attempted to reveal the molecular mechanisms of epigenetic regulations in hematopoietic gene expression. Histone H3 molecule, which is one of the most basic component of chromatin, has at least three variants:H3.1, H3.2, and H3.3. In previous study, it is known that one of H3 variants H3.3 was consistent with open chromatin structure. Only limited organisms which have complex differentiation mechanisms such as mammals have all three variants, suggesting that these histone variants significantly correlate with differentiation. Therefore, we focused on these H3 variants incorporation patterns in HSPCs, and found that the deposition of histone variant H3.3, that is a marker of open chromatin regions, specifically occurred on hematopoietic genes in HSPCs prior to differentiation. HSPC fractions were purified from C57BL/6J mouse bone marrow, and chromatin immunoprecipitation sequence analyses were performed utilizing monoclonal antibodies that specifically recognize H3.3. Although previous studies demonstrated that H3.3 deposition dominantly occurred in the “gene body”, our informatics analysis revealed that more than half of H3.3 existed in the inter-genic regions around hematopoietic genes. The region of H3.3 incorporation changed during differentiation, i.e., almost all of genes were marked with H3.3 in embryonic stem cells, while almost all of hematopoietic genes were marked with H3.3 in LSK, and more lineage specific genes were marked with H3.3 when the differentiation occurred. To explore the factor dependency of the deposition of H3.3, we examined H3.3 incorporation around binding sites of more than 20 different types of transcription factors. The binding sites of Oct3/4, Klf4, Sox2, and Myc, also known as iPS factors, were strongly correlated with H3.3 incorporation sites in embryonic stem cells, while in HSPCs, the incorporated regions of H3.3 significantly co-localized with the binding sites of several hematopoietic key transcription factors such as Scl and Runx1. When we knocked down Oct3/4 in embryonic stem cells, these H3.3 incorporations and differentiation marker genes expressions were diminished, on the other hand, when we knocked down H3.3 in embryonic stem cells, well-ordered differentiation marker genes expressions were suppressed. These data suggest that H3.3 incorporations by key transcription factors are the essences of the “stem-ness”. Interestingly, in leukemic cells, this selective H3.3 incorporation was not observed, suggesting that H3.3 incorporation could reflect the orderly progression of normal hematopoiesis. In conclusion, we found that the incorporation of H3.3 is the earliest epigenetic event involved in determining hematopoietic cell fate. Disclosures: No relevant conflicts of interest to declare.

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.

scholarly journals Regulation of Cyclin E by transcription factors of the naïve pluripotency network in mouse embryonic stem cells

2019 ◽  
Author(s):  
Fabrice Gonnot ◽  
Diana Langer ◽  
Pierre-Yves Bourillot ◽  
Nathalie Doerflinger ◽  
Pierre Savatier
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Binding Sites ◽  
Cyclin E ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Negative Regulator ◽  
Expression Level ◽  
Luciferase Assay

AbstractContinuous, non-cell cycle-dependent expression of cyclin E is a characteristic feature of mouse embryonic stem cells (ESCs). We studied the 5’ regulatory region of Cyclin E, also known as Ccne1, and identified binding sites for transcription factors of the naïve pluripotency network, including Esrrb, Klf4, and Tfcp2l1 within 1 kilobase upstream of the transcription start site. Luciferase assay and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChiP–qPCR) study highlighted one binding site for Esrrb that is essential to transcriptional activity of the promoter region, and three binding sites for Klf4 and Tfcp2l1. Knockdown of Esrrb, Klf4, and Tfcp2l1 reduced Cyclin E expression whereas overexpression of Esrrb and Klf4 increased it, indicating a strong correlation between the expression level of these factors and that of cyclin E. We observed that cyclin E overexpression delays differentiation induced by Esrrb depletion, suggesting that cyclin E is an important target of Esrrb for differentiation blockade. We observed that mESCs express a low level of miR-15a and that transfection of a miR-15a mimic decreases Cyclin E mRNA level. These results lead to the conclusion that the high expression level of Cyclin E in mESCs can be attributed to transcriptional activation by Esrrb as well as to the absence of its negative regulator, miR-15a.

scholarly journals Endogenous retroviruses co-opted into divergently transcribed regulatory elements shape the regulatory landscape of embryonic stem cells

2021 ◽  
Author(s):  
Stylianos Bakoulis ◽  
Robert Krautz ◽  
Nicolas Alcaraz ◽  
Marco Salvatore ◽  
Robin Andersson
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Transposable Elements ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Endogenous Retroviruses ◽  
Gene Promoters ◽  
Specific Regulation ◽  
Regulatory Landscape

Transcription factor binding to regulatory elements is the key process underlying gene regulation during cellular differentiation. Although the specific regulation of genes by transcription factors is generally conserved, regulatory elements themselves are associated with high evolutionary turnover, a process that has been attributed to transposable elements. However, it is unclear how frequent co-option of transposable elements into regulatory elements is and to which regulatory programs they contribute. Here, we report an in-depth characterization of the transposon-derived regulatory landscape of mouse embryonic stem cells. We demonstrate that a substantial number of endogenous retroviral elements are divergently transcribed into unstable RNAs, and that these elements contribute to a sizable proportion of active enhancers and gene promoters. We further show that transposon subfamilies contribute to specific regulatory programs through their enrichment of binding sites for transcription factors, shedding light on the formation of regulatory programs and the origins of regulatory elements.

Cre-mediated deletion of SMARCA5 disrupts pluripotency in mouse embryonic stem cells

2020 ◽  
Author(s):  
David P. Cook ◽  
Barbara C. Vanderhyden
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Binding Sites ◽  
Morphological Changes ◽  
Embryonic Stem ◽  
Conditional Knockout ◽  
Mouse Escs ◽  
Conditional Knockout Mouse ◽  
Genome Wide

AbstractIn embryonic stem cells (ESCs), the SWI/SNF, CHD, and INO80 families of ATP-dependent chromatin remodellers have been implicated in maintaining pluripotency-associated gene expression. At the time of this study, the importance of ISWI family remodellers had yet to be defined, and we had sought to assess their involvement. During this time, Barisic et al. (Nature, 2019) elegantly demonstrated that the ISWI homologue SNF2H (Smarca5) is important for nucleosomal periodicity, the binding of select transcription factors, and proper differentiation of mouse ESCs. While we do not dispute their findings to any extent, our experiments have led to slightly different conclusions, and we have chosen to use this platform to share our results.Here, we explore the importance of SNF2H by deriving a conditional knockout mouse ESC line and observing the consequences of SNF2H depletion on the pluripotent state. Cre-mediated deletion of Snf2h disrupts hallmark characteristics of pluripotency, resulting in distinct morphological changes; reduced expression of the master transcription factors Oct4, Sox2, and Nanog; and reduced alkaline phosphatase activity. To understand the mechanisms of SNF2H-mediated regulation, we mapped SNF2H-bound nucleosomes genome-wide. SNF2H is broadly distributed across the genome but is preferentially enriched at active regulatory regions and transcription factor binding sites.

scholarly journals Identification of New Transcription Factors that Can Promote Pluripotent Reprogramming

2021 ◽  
Author(s):  
Ping Huang ◽  
Jieying Zhu ◽  
Yu Liu ◽  
Guihuan Liu ◽  
Ran Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Rna Sequencing ◽  
Human Urine ◽  
Embryonic Stem ◽  
Rna Seq ◽  
Reprogramming Efficiency ◽  
Yamanaka Factors ◽  
Urine Cells

Abstract Background Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). Many studies have provided a number of alternative combinations to the non-Yamanaka factors. However, it is clear that many additional transcription factors that can generate iPSCs remain to be discovered. Methods The chromatin accessibility and transcriptional level of human embryonic stem cells and human urine cells were compared by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to identify potential reprogramming factors. Selected transcription factors were employed to reprogram urine cells, and the reprogramming efficiency was measured. Urine-derived iPSCs were detected for pluripotency by Immunofluorescence, quantitative polymerase chain reaction, RNA sequencing and teratoma formation test. Finally, we assessed the differentiation potential of the new iPSCs to cardiomyocytes in vitro. Results ATAC-seq and RNA-seq datasets predicted TEAD2, TEAD4 and ZIC3 as potential factors involved in urine cell reprogramming. Transfection of TEAD2, TEAD4 and ZIC3 (in the presence of Yamanaka factors) significantly improved the reprogramming efficiency of urine cells. We confirmed that the newly generated iPSCs possessed pluripotency characteristics similar to normal H1 embryonic stem cells. We also confirmed that the new iPSCs could differentiate to functional cardiomyocytes. Conclusions In conclusion, TEAD2, TEAD4 and ZIC3 can increase the efficiency of reprogramming human urine cells into iPSCs, and provides a new stem cell sources for the clinical application and modeling of cardiovascular disease. Graphical abstract

scholarly journals A mini review on production of pluripotency factors (Oct4, Sox2, Klf4 and c-Myc) through recombinant protein technology

2020 ◽  
Vol 5 (1) ◽  
pp. 1-4 ◽  
Author(s):  
David Septian Sumanto Marpaung ◽  
Ayu Oshin Yap Sinaga
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Cell Types ◽  
Induced Pluripotency ◽  
Pluripotency Factors ◽  
Induced Pluripotent

The four transcription factors OCT4, SOX2, KLF4 and c-MYC are highly expressed in embryonic stem cells (ESC) and their overexpression can induce pluripotency, the ability to differentiate into all cell types of an organism. The ectopic expression such transcription factors could reprogram somatic stem cells become induced pluripotency stem cells (iPSC), an embryonic stem cells-like. Production of recombinant pluripotency factors gain interests due to high demand from generation of induced pluripotent stem cells in regenerative medical therapy recently. This review will focus on demonstrate the recent advances in recombinant pluripotency factor production using various host.

Sign in / Sign up

Export Citation Format

Share Document