60 CRISPR-on, a new tool for activation of endogenous gene expression in bovine embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 155
Author(s):  
V. Savy ◽  
V. Alberio ◽  
N. Canel ◽  
L. Ratner ◽  
M. Gismondi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Regulatory Region ◽  
Bovine Embryos ◽  
Endogenous Gene ◽  
Cell Fate Decisions ◽  
Guide Rnas ◽  
Endogenous Expression ◽  
Mammalian Embryos

The CRISPR-Cas9 system enables precise genome editing in mammalian somatic cells and embryos at a very high efficiency. A modified version of Cas9 (dCas9) was engineered, resulting in a DNA binding protein capable of site-specific target recognition but unable to cut the DNA. By means of dCas9 fusion to heterologous domains, including transcriptional activators or repressors, specific modulation of gene expression has successfully been achieved invitro, making possible the modulation of the cell-differentiation state. However, CRISPR-mediated transcriptional activation (CRISPR-on) has been mainly used invitro, and to our knowledge, there are no reports regarding its use for the activation of endogenous gene expression in mammalian embryos. As a proof of principle, we evaluated the CRISPR-on system in bovine embryos for modulation of endogenous expression of SMARCA4 and TFAP2C, transcription factors implicated in trophoblast lineage commitment. We hypothesised that CRISPR-on may induce SMARCA4 or TFAP2C endogenous expression, enabling the design of strategies to induce trophectoderm proliferation of invitro-derived embryos. To this aim, we designed and synthesised 4 non-overlapping single guide RNAs to target the regulatory region of each of these target genes. Presumptive zygotes were cytoplasmically microinjected with a mix containing dCas9-VP160 mRNA and a pool of 4 single guide RNAs targeting SMARCA4 (dCas9_SM group) or TFAP2C (dCas9_TF group). As control, a non-injected group was also included. Analysis was carried out in pools of 10 early embryos or 5 blastocysts and at least 3 biological replicates were included. Gene expression was assessed by RTqPCR at Days 2, 4, and 7 after microinjection and data were normalized to that obtained for the non-injected group. The CRISPR-on system was efficient to induce expression of SMARCA4 two days after microinjection (dCas9_SM group, Mann-Whitney t-test; P<0.05), but failed to significantly increase TFAP2C expression (dCas9_TF group). Surprisingly, CDX2, which is a downstream effector for trophectoderm maintenance, was induced both in dCas9_SM and dCas9_TF groups, supporting the CRISPR-mediated induction of targeted transcription factors. However, no changes were observed in the endogenous level of NANOG. Additional analysis is currently ongoing to determine whether CRISPR-on mediated induction of SMARCA4 and/or TFAP2C expression affects lineage specification and regulation. To our knowledge, this is the first report on the use of CRISPR-on for modulation of endogenous gene expression in mammalian embryos. Our study lays the foundations for CRISPR-on application in embryos as a useful tool to understand key cell fate decisions and will enable unprecedented studies of significance to embryo development, cell differentiation, and segregation.

scholarly journals CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos

Reproduction ◽  
2020 ◽  
Vol 159 (6) ◽  
pp. 767-778
Author(s):  
Virginia Savy ◽  
Virgilia Alberio ◽  
Natalia G Canel ◽  
Laura D Ratner ◽  
Maria I Gismondi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Embryo Development ◽  
Transcriptional Activation ◽  
Cultured Cells ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Endogenous Gene ◽  
Cell Fate Decisions ◽  
Mammalian Embryos

CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.

The Human Plasma Cell Gene Expression Program.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3242-3242
Author(s):  
John De Vos ◽  
Dirk Hose ◽  
Thierry Reme ◽  
Hartmut Goldschmidt ◽  
Jean-Francois Rossi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Plasma Cell ◽  
Expression Profile ◽  
Plasma Cells ◽  
Plasma Cell Differentiation ◽  
Unfolded Protein ◽  
The Mean ◽  
Protein Response

Abstract Seven purified peripheral blood memory B-cells (BM), seven in-vitro-generated polyclonal plasmablastic cells (PPC) and seven purified bone marrow mature plasma cells (BMPC) were studied by oligonucleotide microarrays. All samples were obtained from healthy volunteers. The gene expression profiling of these samples was determined with Affymetrix pan genomic U133A + B arrays (44 928 oligonucleotide probesets). We determined that 2313 genes were differentially expressed between these three B cell categories (P 〈 0.01 by a Kruskal-Wallis test and a ratio between two categories 〉 3). These 2313 genes were classified into six categories, according to the expression profile: early plasma cell genes (EPC), late plasma cell genes (LPC), genes lost early during plasma cell differentiation (LEPC), genes lost late during plasma cell differentiation (LLPC), genes upregulated only in plasmablasts (PBO) and genes lost only in plasmablasts (LPBO). As expected, Ig transcripts where essentially classified as EPC. As a corollary, genes involved in protein synthesis or degradation, transmembrane transporters and metabolism genes were overrepresented in EPC genes. Interestingly, genes involved in intercellular communication and extracellular matrix were enriched in LPC, highlighting the fact that mature plasma cells develop tight interactions with the bone marrow environment. Of note, genes involved in cell cycle are upregulated mainly in plasmablasts, whereas antiapoptotic genes are lost in plasmablasts only. Mains genes known to be involved in plasma cell differentiation display an expression profile in agreement with published data, as illustrated for transcription factors in Figure 1, validating this DNA microarray dataset. However most of these 2313 genes have either never been described yet or have no yet been linked to plasma cell differentiation. The description of those genes among our genome whose expression vary most during plasma cell differentiation will be an essential step in understanding the biology of a cell type essential to immune defenses and involved in deadly diseases. Figure 1: Transcription factors involved in plasma cell differentiation. Color indicates the expression profile category. For each gene is given the ratio of the mean expression value in plasma cell samples (PPC and BMPC) to the mean expression value in BM. UPR: Unfolded Protein Response. Figure 1:. Transcription factors involved in plasma cell differentiation. Color indicates the expression profile category. For each gene is given the ratio of the mean expression value in plasma cell samples (PPC and BMPC) to the mean expression value in BM. UPR: Unfolded Protein Response.

Mechanisms of MEF2C-Dependent Transcription in Lymphoid Differentiation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4341-4341
Author(s):  
Nikki R. Kong ◽  
Li Chai ◽  
Astar Winoto ◽  
Robert Tjian
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
B Cell ◽  
Progenitor Cells ◽  
Luciferase Reporter ◽  
B Cell Differentiation ◽  
Reporter Assays ◽  
B Lineage ◽  
Transcription Program

Abstract Hematopoiesis is a multi-step developmental process that requires an intricate coordination of signal relays and transcriptional regulation to give rise to all blood lineages in the organism. Hematopoietic stem/progenitor cells (HSPCs) can be driven to differentiate along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for HSPCs is whether to adopt the lymphoid or myeloid fate. Despite the discovery of several transcription factors required for different lineages of hematopoietic differentiation, the understanding of how gene expression allows HSPCs to adopt the lymphoid fate still remains incomplete. A study using an inducible hematopoietic-specific (Mx1-Cre) KO mouse line found that Myocyte Enhancer Factor 2C (MEF2C) is required for multi-potent HSPCs to differentiate into the lymphoid lineage (Stehling-Sun et al, 2009). However, the mechanisms of how MEF2C is activated and in turn, drives lymphoid fate specification are not known. Through a candidates approach with co-expression and co-immunoprecipitation, we have identified Early B Cell Factor 1 (EBF1) to be a specific interacting partner of MEF2C, and not other B cell specific transcription factors such as E12, E47, or PAX5. Genome-wide survey of MEF2C and EBF1 binding sites via chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in a proB cell line revealed that these two sequence-specific transcription factors co-occupy the promoters and intragenic regions of many B cell specific genes such as Il7ra, Myb, Foxo1, Ets1, Ebf1 itself, and Pou2af1. Regulatory regions of Il7ra and Foxo1 derived from the ChIP-seq data, as well as an artificial enhancer containing trimerized MEF2C and EBF1 binding sites, were examined in luciferase reporter assays and found to be sufficient to drive transcription from a minimal reporter in 293T cells. Further, this activation was co-dependent on MEF2C and EBF1 expression. The functional relevance of MEF2C binding was further supported by gene expression analyses of MEF2C-regulated B lineage genes in Mx1-Cre Mef2c KO mice compared to WT mice. Consistent with ChIP-seq and luciferase reporter assays, Myb, Ebf1, Il7ra, and Foxo1 all had significantly decreased expression levels in MEF2C-null HSPCs as well as B lineage progenitor cells, compared to sex-matched littermate control mice. Interestingly, myeloid gene expression in Mef2c-KO mice was increased compared to WT control. MEF2C ChIP-seq in a murine HSPC line revealed that it binds myeloid lineage gene targets that are not regulated by MEF2C in proB cells. These results suggest that MEF2C can repress myeloid gene expression in HSPCs. To elucidate the mechanism of this functional switch, we tested the requirement for MAPK pathways to phosphorylate and activate MEF2C at three previously identified residues in order to drive B cell differentiation. Inhibition of p38 MAPK (p38i), but not ERK1/2/5, decreased the potential of HSPCs to differentiate into B220+CD19+ B cells cultured with cytokines that drive this particular lineage fate. Instead, p38i-treated progenitor cells gave rise to more myeloid cells. 65% of this phenotype was rescued by over-expressing a phosphomimetic mutant of MEF2C that can bypass p38 inhibition. Furthermore, MEF2C is known to bind class II HDAC proteins to repress gene expression, providing a possible mechanism for its repression of myeloid transcription program. Supporting this mechanism, phosphomimetic and HDAC-binding double mutant of MEF2C can rescue p38 inhibition phenotype almost 100%. Taken together, this study elucidated the molecular mechanisms of a key lymphoid-specific lineage fate determinant, MEF2C. We discovered that p38 MAPK converts MEF2C from a transcriptional repressor to an activator by phosphorylation in B cell specification, which can be applied to understanding other cell differentiation processes regulated by this important stress-induced signaling pathway. Furthermore, we identified MEF2C’s binding and co-activating partner EBF1, several novel B cell specific targets that it activates in proB cells, and a novel myeloid transcription program that it represses in hematopoietic progenitors. Therefore, these results expanded our understanding of the intricate transcription network that regulates B cell differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Epigenomics of Neural Cells: REST-Induced Down- and Upregulation of Gene Expression in a Two-Clone PC12 Cell Model

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Jose M. Garcia-Manteiga ◽  
Silvia Bonfiglio ◽  
Maria Luisa Malosio ◽  
Dejan Lazarevic ◽  
Elia Stupka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Pc12 Cell ◽  
Cell Model ◽  
Synaptic Function ◽  
Neural Cells ◽  
Strong Decrease ◽  
Upregulated Genes

Cell epigenomics depends on the marks released by transcription factors operating via the assembly of complexes that induce focal changes of DNA and histone structure. Among these factors is REST, a repressor that, via its strong decrease, governs both neuronal and neural cell differentiation and specificity. REST operation on thousands of possible genes can occur directly or via indirect mechanisms including repression of other factors. In previous studies of gene down- and upregulation, processes had been only partially investigated in neural cells. PC12 are well-known neural cells sharing properties with neurons. In the widely used PC12 populations, low-REST cells coexist with few, spontaneous high-REST PC12 cells. High- and low-REST PC12 clones were employed to investigate the role and the mechanisms of the repressor action. Among 15,500 expressed genes we identified 1,770 target and nontarget, REST-dependent genes. Functionally, these genes were found to operate in many pathways, from synaptic function to extracellular matrix. Mechanistically, downregulated genes were predominantly repressed directly by REST; upregulated genes were mostly governed indirectly. Among other factors, Polycomb complexes cooperated with REST for downregulation, and Smad3 and Myod1 participated in upregulation. In conclusion, we have highlighted that PC12 clones are a useful model to investigate REST, opening opportunities to development of epigenomic investigation.

scholarly journals VirtualCytometry: a webserver for evaluating immune cell differentiation using single-cell RNA sequencing data

2019 ◽  
Vol 36 (2) ◽  
pp. 546-551 ◽  
Author(s):  
Kyungsoo Kim ◽  
Sunmo Yang ◽  
Sang-Jun Ha ◽  
Insuk Lee
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Signaling Molecules ◽  
Single Cell Rna Sequencing ◽  
Functional States

Abstract Motivation The immune system has diverse types of cells that are differentiated or activated via various signaling pathways and transcriptional regulation upon challenging conditions. Immunophenotyping by flow and mass cytometry are the major approaches for identifying key signaling molecules and transcription factors directing the transition between the functional states of immune cells. However, few proteins can be evaluated by flow cytometry in a single experiment, preventing researchers from obtaining a comprehensive picture of the molecular programs involved in immune cell differentiation. Recent advances in single-cell RNA sequencing (scRNA-seq) have enabled unbiased genome-wide quantification of gene expression in individual cells on a large scale, providing a new and versatile analytical pipeline for studying immune cell differentiation. Results We present VirtualCytometry, a web-based computational pipeline for evaluating immune cell differentiation by exploiting cell-to-cell variation in gene expression with scRNA-seq data. Differentiating cells often show a continuous spectrum of cellular states rather than distinct populations. VirtualCytometry enables the identification of cellular subsets for different functional states of differentiation based on the expression of marker genes. Case studies have highlighted the usefulness of this subset analysis strategy for discovering signaling molecules and transcription factors for human T-cell exhaustion, a state of T-cell dysfunction, in tumor and mouse dendritic cells activated by pathogens. With more than 226 scRNA-seq datasets precompiled from public repositories covering diverse mouse and human immune cell types in normal and disease tissues, VirtualCytometry is a useful resource for the molecular dissection of immune cell differentiation. Availability and implementation www.grnpedia.org/cytometry

The MDS-Associated Gene DLK Modulates Gene Expression in Human Myeloid Cells by Altering the Activity of Multiple Transcription Factors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3428-3428
Author(s):  
Liang Li ◽  
Rushabh Modi ◽  
Xiwei Wu ◽  
Stephen J. Forman ◽  
Ravi Bhatia
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Linear Model ◽  
Expression Profiles ◽  
Myeloid Cells ◽  
Gene Expression Profiles ◽  
Differentially Expressed ◽  
Differentiation And Proliferation ◽  
And Control

Abstract Delta-Like 1 (DLK) is an EGF-like transmembrane protein, which is overexpressed in myelodysplastic syndrome (MDS) CD34+ cells. We have previously shown that ectopic DLK expression inhibits HL-60 cell differentiation and proliferation through intracellular domain interactions. To further investigate mechanisms underlying DLK effects on myeloid cell differentiation and proliferation, we compared gene expression profiles of DLK expressing and control HL-60 cells, with or without differentiating induction with ATRA, using Affymetrix HG-U133A arrays. Gene expression data was analyzed using affy and limma (linear model of microarray analysis) packages in the open-source BioConductor project (v 1.6). Raw data were processed using robust multi-chip average (RMA) algorithm, a linear model fit to each gene, and the following comparisons were made: (a) effects of DLK expression in unstimulated cells, (b) effects of DLK expression in ATRA exposed cells, (c) effects of ATRA induction on R1 cells, (d) effects of ATRA induction on DLK+ cells, and (e) differences in the response of DLK+ vs. control cells to ATRA. Adjusted P values and log odds of differential expression (B statistic, 50% probability when B=0) were calculated. B values > 0 were considered statistically significant. 523 genes were differentially expressed between unstimulated control and DLK+ cells, 343 genes were differentially expressed between control and DLK+ cells after ATRA stimulation, and 204 genes were common to the two sets. 802 genes were differentially expressed after ATRA stimulation in control cells, 742 genes in DLK+ cells, with 550 genes common to the two sets. 13 genes were differentially expressed when ATRA responses of control and DLK+ cells were compared. Gene ontology (GO) analyses indicated that "Biological processes" significantly affected by DLK overexpression included signal transduction, cell cycle, proliferation, cell death, protein metabolism and enzyme cascades, and "Molecular functions" most affected included nucleotide/DNA binding and protein kinase activity. These observations are consistent with observed cellular effects of DLK. Using MotifRegressor software, we performed promoter analysis correlating common transcription factor-binding motifs with expression profiles of genes differentially expressed between DLK+ and control cells. We identified the transcription factors (TF) PBX, GATA-1, c-Myc: Max, HIF-1, DEC1, Hand1, Lmo2, NKX25, GKLF and AP-1 as being potentially involved in DLK-mediated changes in gene expression. The observed patterns of differential gene expression were consistent with altered activities of these TF. Electrophoresis mobility shift assays (EMSA) indicated increased PBX and reduced HIF-1 and GATA-1 activities in DLK+ cells. Interestingly, Hand1, c-Myc: Max and Dec1 are basic Helix-loop-Helix (b-HLH) factors with E box binding sites, which are known to associate and form regulatory complexes with other TF. TF such as GATA-1, GLKF and Lmo2, also identified in our analysis, are known to be associated with such complexes. In conclusion, gene expression profiles of DLK expressing human myeloid cells are consistent with observed alterations in cell proliferation and differentiation. We have identified TF that may act individually and/or in concert to induce the observed changes in gene expression in DLK+ cells. Further evaluation of their role of these TF in mediating DLK effects and in abnormal hematopoietic cell growth in MDS is warranted.

Analysis of an approach to oviduct-specific expression of modified chicken lysozyme genes

2005 ◽  
Vol 83 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Mamiko Shimizu ◽  
Jan K Losos ◽  
Ann M. Verrinder Gibbins
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Gene Targeting ◽  
Stop Codon ◽  
Specific Gene ◽  
Specific Expression ◽  
Endogenous Gene ◽  
Lysozyme Gene ◽  
Tubular Gland ◽  
Chicken Lysozyme

The –2.7 kb enhancer (E) element of the chicken lysozyme gene domain appears to govern expression of the gene in macrophages but not in oviduct tubular gland cells, the only other site of lysozyme expression. The ultimate goal of our research was to determine whether lysozyme domain variants could be developed that would mainly be expressed in the oviduct so that transgenic birds could be produced that would deposit exogenous protein in the egg white. Accordingly, precise mutations were made by poxvirus-mediated gene targeting in FEF/PU.1 and CCAAT/enhancer-binding protein (C/EBP) transcription factor binding sites in the –2.7 kb E of cloned copies of a specific lysozyme gene variant that includes a hydrophobic pentapeptide tail encoding sequence inserted immediately prior to the stop codon. This variant contains the entire lysozyme domain and is cloned in a λ bacteriophage vector (λDIILys-HT); the novel tail sequence enables distinction in cell-based expression systems between transcripts of the variant and those of the endogenous gene. These various lysozyme domain mutants, in bacteriophage vector form, were tested for expression in cultured chicken blastodermal cells cotransfected with plasmids encoding the transcription factors C/EBP and v-Myb. In the absence of these plasmids, barely detectable levels of endogenous lysozyme gene transcription resulted in the blasto dermal cells. In the presence of the plasmids, however, transcripts of the endogenous gene could be detected as well as varying levels (as evaluated by quantitative real-time PCR) of transcripts of all of the lysozyme domain mutants. These results are discussed in the context of the known role and occurrence of various transcription factors involved in gene expression in differentiating macrophage cells. The ultimate test of expression of the variants in macrophages vs. oviduct cells will be to use them to produce transgenic birds.Key words: lysozyme, macrophage-specific gene expression, poxvirus-mediated gene targeting.

scholarly journals Redundant Regulation of T Cell Differentiation and TCRα Gene Expression by the Transcription Factors LEF-1 and TCF-1

Immunity ◽  
1998 ◽  
Vol 8 (1) ◽  
pp. 11-20 ◽  
Author(s):  
Ross M Okamura ◽  
Mikael Sigvardsson ◽  
Juan Galceran ◽  
Sjef Verbeek ◽  
Hans Clevers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
T Cell ◽  
T Cell Differentiation
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