18. Real-time analyses of HLH transcription factors

2007 ◽  
Vol 148 (3) ◽  
pp. 347
Author(s):  
Osamu Hisatomi ◽  
Mari Kotoura ◽  
Daisuke Kitano ◽  
Koji Hasegawa ◽  
Tatsushi Goto ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Real Time ◽  
Hlh Transcription Factors

scholarly journals Enhancer priming enables fast and sustained transcriptional responses to Notch signaling

2018 ◽  
Author(s):  
Julia Falo-Sanjuan ◽  
Nicholas C Lammers ◽  
Hernan G Garcia ◽  
Sarah Bray
Keyword(s):  
Transcription Factors ◽  
Dynamic Response ◽  
Notch Signaling ◽  
Real Time ◽  
Single Cells ◽  
Transcriptional Responses ◽  
Sustained Activity ◽  
Developmental Signaling ◽  
Cellular Context ◽  
Nascent Transcripts

SummaryInformation from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2/MCP system to visualize nascent transcripts in single cells in Drosophila embryos we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.

46 EXPRESSION OF TRANSCRIPTION FACTORS SPECIFIC TO THE TROPHOBLAST LINEAGE IN MOUSE SOMATIC NUCLEAR TRANSFER EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 103
Author(s):  
T. Mitani ◽  
M. Nishiwaki ◽  
M. Anzai ◽  
H. Kato ◽  
Y. Hosoi ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Real Time ◽  
Real Time Pcr ◽  
Nuclear Transfer ◽  
Ectopic Expression ◽  
Expression Level ◽  
Pcr Analysis ◽  
Cell Stage ◽  
Cdx2 Expression ◽  
Morula Stage

Somatic cell nuclear transfer (SCNT) embryos can develop at relatively high rates during the preimplantation period; however, most of these fail after implantation. Development of extraembryonic tissue is indispensable for normal embryonic development. Hence, an abnormality of trophoblast development might be a significant factor in post-implantation lethality of SCNT embryos. A transcription factor, caudal-related homeobox 2 (Cdx2), appears to be involved in the segregation of ICM and trophectoderm (TE) in preimplantation embryos (Niwa et al. 2005 Cell 123, 917–929). Both Cdx2 and Oct3/4 are expressed in all cells at the morula stage, and then Cdx2 expression becomes restricted to the TE and Oct3/4 to the ICM as the blastocyst develops. Mouse embryos deficient in Cdx2 are able to develop to normal blastocysts but die soon after implantation, probably because of defects in the TE lineage. Moreover, dysplasia of the spongiotrophoblast layer might attribute to an abnormality of Tpbpa expression in mouse SCNT embryos (Wakisaka-Saito et al. 2006 Biochem. Biophys. Res. Commun. 349, 106–114). In this study, we examined the expression profiles of transcription factors implicated in trophoblast development in mouse SCNT embryos and intracytoplasmic sperm injection (ICSI) embryos by immunohistochemistry and real-time PCR analysis. SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998 Nature 394, 369–374). In brief, B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. After nuclear transfer, the oocytes were activated and cultured in KSOM to the morula and blastocyst stages. Immunohistochemical analysis demonstrated that in ICSI embryos Cdx2 was only partially expressed at the 8-cell stage but completely in early morulae. In contrast, in SCNT embryos, it was absent at the 8-cell stage and appeared partially at the early morula stage. Thereafter, Cdx2 expression became restricted to the TE cells in both the ICSI and the SCNT blastocysts. However, ectopic expression of Oct3/4 was observed in the TE cells of SCNT, but not in ICSI blastocysts. Real-time PCR analysis showed that at the 8-cell stage, Cdx2 was expressed in ICSI but not in SCNT embryos. In addition, the expression level of Cdx2 in SCNT embryos at the blastocyst stage was only half that in ICSI embryos (P < 0.05). However, there was no significant difference in expression level of Oct3/4 between ICSI and SCNT embryos. Eomesodermin (Eomes) is also implicated in trophoblast development and its expression depends on Cdx2, BMP4, and FGF4. In SCNT embryos, the expression level of Eomes was also only half that in ICSI embryos. These results indicate that the delayed expression of Cdx2 in SCNT embryos may lead to the ectopic expression of Oct3/4 in blastocysts and, along with the limited expression of Cdx2 and Eomes, may contribute to disorders in the function of the trophoblast lineage for normal placental development. This work was supported by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

NCoA4 Containing Transcriptional Complexes Modulates Response of Myelomonocytic Leukemic Cell Line to Retinoids and VD3.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4238-4238
Author(s):  
Aurelie Baudet ◽  
Laurent Delva ◽  
Patrick Balaguer ◽  
David Piquemal ◽  
Jacques Marti ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Line ◽  
Real Time ◽  
Nuclear Receptors ◽  
Growth Arrest ◽  
Leukemic Cells ◽  
U937 Cells ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Transcriptional Complexes

Abstract Large scale analyses of transcriptome improve comprehension of complex processes such as differentiation or cell proliferation. SAGE libraries construction of the AML model U937 allowed the identification of new markers of myelomonocytic differentiation induced by retinoids and vitamin D3 (VD3) (Piquemal et al., 2002). Those molecules act through ligand-dependent transcription factors of the nuclear receptors family: RAR, RXR and VDR. Among differentially expressed members of transcriptional complexes, the most relevant was the co-regulator NCoA4 (Nuclear receptor Coactivator 4). This protein that modulates interactions between transcription factors, RNA polymerase II and chromatin remodeling factors, was initially described as a co-activator of the androgen receptor (AR). Its activity has been extended to receptors of estrogens (ER), peroxisome-proliferating activators (PPAR), retinoid X (RXR) and recently to VD3 receptor (VDR). Using real-time semi-quantitative PCR, we found that NCoA4 is specifically expressed during the monocytic differentiation of U937 but not during the granulocytic differentiation of NB4 cell lines. These results were confirmed by analysis on normal and in vitro-differentiated leukemia primary cells. Moreover, its early induction, within 6 hours of retinoids and VD3 treatment on U937 cells, suggests that its expression may be controlled by one or several nuclear receptors. Because of cross-talks between retinoids and VD3 pathways, we used NB4-LR2 cells in which RAR is knock-down. In this cell line, NCoA4 is expressed in a VDR-dependent fashion reinforcing the hypothesis that the coregulator is specifically involved in the VD3-monocytic differentiation of leukemic cells. Next, to explore the role of NCoA4, U937 cells were stably transfected to constitutively over-express the protein. The doubling time of this cell line (U-NCoA4) reaches to 48 hours against 24 hours in U937 cells. Concerning ligand-induced growth arrest, these cells are particularly sensitive to the RXR and VDR agonists while no significative difference was observed after treatment by the RAR agonist or by any combination. In addition, over-expression of NCoA4 induces a slow down of differentiation, as shown by expression of CD11b and CD14 myelomonocytic markers. Thus, in U-NCoA4, except for the RAR agonist, treatment for 72 hours corresponds to treatment for 48 hours levels of U937 cells. To conclude, in term of growth arrest, NCoA4 over-expressing cells are particularly sensitive to RXR and VDR agonists. Thus, natural ligands present in the culture medium might reduce or delay proliferation, inducing the same effect on differentiation. In order to have a large view of networks, transcriptome of U-NCoA4 was analyzed by real time PCR on Low Density Array composed of a hundred messengers extracted from U937 SAGE libraries. Analysis is currently in progress.

scholarly journals Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene.

1993 ◽  
Vol 13 (4) ◽  
pp. 2342-2353 ◽  
Author(s):  
M Therrien ◽  
J Drouin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Proteins ◽  
Specific Activity ◽  
Pituitary Cells ◽  
Immunoglobulin Gene ◽  
Helix Loop Helix ◽  
E Box ◽  
E Boxes ◽  
Hlh Transcription Factors

Pro-opiomelanocortin (POMC)-expressing cells appear to be the first pituitary cells committed to hormone production. In this work, we have identified an element of the POMC promoter which confers cell-specific activity. This element did not exhibit any activity on its own and required at least one other element of the promoter to manifest its cell-specific activity. Fine mutagenesis of this element indicated that a CANNTG motif is responsible for activity. This E-box motif is typical of binding sites for helix-loop-helix (HLH) transcription factors; however, the POMC cell-specific E box cannot be replaced by other E boxes like the kappa E2 site of the immunoglobulin gene or a muscle-specific E box. Similar E boxes which are present in the insulin gene promoter were shown to contribute to the pancreatic specificity of the insulin promoter. However, E-box-binding proteins found in nuclear extracts from POMC-expressing AtT-20 cells and from insulin-expressing cells have different electrophoretic mobilities. The AtT-20 proteins were named CUTE (for corticotroph upstream transcription element-binding) proteins, and they were not found in any other cells. CUTE proteins have DNA-binding properties characteristic of HLH transcription factors. Overexpression of the dominant negative HLH protein Id or of the ubiquitous positive HLH factor rat Pan-2 decreased or augmented POMC promoter activity, respectively. These observations are consistent with the hypothesis that CUTE factors might be heterodimers. This hypothesis was further supported by antibody shift experiments and by abrogation of DNA binding in the presence of bacterially expressed Id protein. Thus, the cell-specific CUTE proteins and their binding site in the POMC promoter appear to be important determinants for cell specificity of this promoter. The requirement for HLH factors in POMC transcription also presents the possibility that these factors are involved in differentiation of pituitary cells, in analogy with the role of HLH factors in muscle development.

201. Homeobox gene DLX3 regulates forskolin induced trophoblast differentiation

2008 ◽  
Vol 20 (9) ◽  
pp. 1
Author(s):  
A. Chui ◽  
B. Kalionis ◽  
S. Brennecke ◽  
P. Murthi
Keyword(s):  
Transcription Factors ◽  
Real Time ◽  
Fetal Growth ◽  
Real Time Pcr ◽  
Fetal Growth Restriction ◽  
Homeobox Gene ◽  
Trophoblast Cell ◽  
Trophoblast Differentiation ◽  
Human Trophoblast ◽  
Β Hcg

Trophoblast cells carry out important functions required for the development of the normal placenta. Disruption of these functions is associated with significant pregnancy disorders such as fetal growth restriction and pre-eclampsia. Transcription factors regulate trophoblast functions. We are interested in one such class of transcription factors known as homeobox genes. The homeobox gene Distal-less 3 (DLX3) plays a vital role in the development of the mouse placenta (Morasso, Grinberg et al. 1999) and increased levels of DLX3 have been found in placentae affected by human fetal growth restriction (Murthi and Chui, unpubl. data). However, the function of DLX3 in the human placenta is not well established. Here, we investigated whether DLX3 regulates trophoblast differentiation using a plasmid construct to overexpress DLX3 in the human trophoblast cell line, BeWo. Real-time PCR showed a significant increase in DLX3 mRNA (3.1 ± 0.1 v. 1.0 ± 0.2 control, P < 0.05, n = 3), as well as the mRNA of two known markers of differentiation 3-β-hydroxysteroid dehydrogenase (3β-HSD) (8.1 ± 1.8 v. 1.2 ± 0.1 control, P < 0.05, n = 3) and β-human chorionic gonadotropin (β-hCG) (54.9 ± 0.9 v. 49.2 ± 1.6 control, P < 0.05, n = 3). Furthermore, forskolin mediated trophoblast differentiation was verified in BeWo cells. Following forskolin induction, real-time PCR showed a significant increase in the expression of DLX3 mRNA (12.9 ± 1.2 v. 3.8 ± 0.9 control, P < 0.05, n = 4), as well as a significant increase in the mRNA expression of the differentiation markers previously tested, 3β-HSD (28.3 ± 2.4 v. 1.0 ± 0.08 control, P < 0.05, n = 4) and β-hCG (2.3 ± 1.9 v. 30.9 ± 0.08 control, P < 0.001, n = 3). The expression of an additional differentiation marker, syncytin, was also significantly increased (4.0 ± 1.9 v. 1.0 ± 0.08 control, P < 0.05, n = 4). Thus, we have shown that DLX3 is a regulator of human trophoblast cell differentiation, and that forskolin acts through DLX3 to induce trophoblast differentiation. (1) Morasso, M. I., A. Grinberg et al. (1999).

NFκB-Mediated Up-Regulation of Transcription Factors Related to More Primitive State of Hematopoietic Progenitor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1246-1246
Author(s):  
Rodrigo A. Panepucci ◽  
Lucila H.B. Oliveira ◽  
Dalila L. Zanette ◽  
Greice A. Molfetta ◽  
Rita C.V. Carrara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Progenitor Cells ◽  
Real Time ◽  
Quantitative Pcr ◽  
Expression Profiles ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Primitive State

Abstract We have previously shown that a distinctive feature of umbilical cord blood (UCB) CD34+ hematopoietic progenitor cells (HSPC) as compared to bone marrow (BM) CD34+ is a higher expression of transcription targets and components of the nuclear factor kappa B (NF-κB) pathway. NFKB2 and RELB are sub-units of the transcription factor (TF) that specifically mediates the constitutive NF-κB signaling pathway and their increased levels could be related with the primitive state of the newborn’s HSPC. However, BM and UCB CD34+ HSPC differ in their sub-population compositions, and a higher proportion of more primitive cells among the CD34+ cells could account for those differences. CD133 is a surface marker expressed on a more primitive sub-population of CD34+ cells that are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells. We used flow cytometry, oligonucleotide microarray gene expression profiling and real time quantitative PCR to better characterize immunomagnetically sorted CD34+ and CD133+ HSPC derived from BM and UCB. We found that UCB CD34+ cells contain a larger proportion of CD133+ cells (around 70%), differing from BM CD34+ cells (around 30%). Cluster analysis of the expression profiles, encompassing 10.000 genes, showed that UCB CD133+ are more similar to UCB CD34+ than to BM CD133+ cells. Furthermore, a statistically significant higher expression of NFKB2 and RELB was demonstrated by quantitative PCR on UCB CD133+ HSPC, compared to BM. Overall this indicates that despite distinct compositions of the cells from UCB or BM, UCB HSPC display intrinsic molecular differences related to their ontological age. The comparison of the gene expression profiles of the CD133+ with the CD34+ populations revealed the higher expression of many well known factors related to more primitive HSPC and hemangioblasts. In fact, TFs such as RUNX1/AML1, GATA3, USF1, TAL1/SCL, HOXA9 and HOXB4 were all present at higher levels in CD133+ HSPC. In an attempt to identify a key TF that could be responsible for the expression of these important factors, we carried a promoter analysis for the set of highly expressed TF found in the CD133 cells. A frequency of TF binding sites significantly higher than the expected was observed for the NF-κB TFs, including potential NF-κB targets such as RUNX1, GATA3 and USF1. Measurements of GATA3, NFKB2 and RELB expression by real-time PCR showed a higher expression of the three genes in CD133+ samples (both from BM and UCB), as well as a correlation of the expression levels of NFkB2 and RELB with one another and with GATA3 (Sperman’s correlation), indicating that GATA3 could be, in fact, regulated by NF-κB. To further test this hypothesis, we used interference RNA (RNAi) against NFKB2 in HSPC. Levels of NFKB2, GATA3 and RELB (a known target of NFKB2/RELB dimmers) were down-modulated, in comparison with cells transfected with control RNAi. Taken together, our data indicates that constitutive NF-κB signaling may act up-regulating transcription factors related to a more primitive state of HSPC.

Sign in / Sign up

Export Citation Format

Share Document