Transcriptional regulation of the murine k‐fgf gene

A Angie Rizzino, E. Rosfjord

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Embryonal carcinoma (EC) cells provide a useful model system for studying the roles of growth factors during early mammalian development. In 1988, we determined that EC cells express a member of the fibroblast growth factor (FGF) family that cannot be detected after EC cells undergo differentiation. Attempts to understand how differentiation regulates the production of FGFs led to the finding that EC cells express the fibroblast growth factor k‐FGF (FGF‐4), whereas there is a large decrease in the steady state levels of k‐FGF mRNA when EC cells differentiate. This suggested that transcription of the k‐fgf gene is repressed when EC cells differentiate. To investigate this possibility, we prepared a series of reporter gene constructs containing various regions of the murine k‐fgf gene. These constructs were transfected into two mouse EC cell lines and one mouse embryonic stem (ES) cell line. We determined that the mouse 5′ flanking region cannot support expression of the reporter gene. In both EC and ES cell lines, expression of the reporter gene is elevated greatly by the addition of a 316 bp region from the third exon of the murine k‐fgf gene. Sequence analysis of the 316 bp region identified one and possibly two conserved octamer binding motifs. These sequences are likely to be involved in regulation of the k‐fgf gene, because differentiation of EC cells is known to reduce the expression of octamer binding proteins, including Oct‐3. To test the possible role of octamer binding proteins, we examined the expression of our reporter gene constructs in F9‐differentiated cells and in PYS‐2 cells. In these cells, the expression of our k‐fgf/reporter gene constructs is very low. However, expression of the reporter gene is elevated significantly when these cells are cotransfected with a construct that contains an Oct‐3 cDNA under the control of a strong viral promoter. To test further the importance of the octamer motifs found in the 316 bp enhancer‐like region, we replaced the 316 bp region in our constructs with a smaller region (48 bp) that contains the downstream octamer motif and flanking sequences. Like the 316 bp region, this 48 bp region elevated the expression of the reporter gene, but it does so at a much lower level. Thus, it appears that the downstream octamer motif may be involved in the regulation of the k‐fgf gene, but that at least one other cis‐regulatory element present in the 316 bp enhancer is likely to be involved in the expression of the k‐fgf gene in EC cells. Lastly, we have identified two potentially important regulatory regions upstream of the transcription start site. One appears to regulate positively the expression of the murine k‐fgf gene in EC cells and in ES cells, and the other appears to regulate negatively the expression of the k‐fgf gene in these cells. © 1994 Wiley‐Liss, Inc.

Original languageEnglish (US)
Pages (from-to)106-111
Number of pages6
JournalMolecular Reproduction and Development
Volume39
Issue number1
DOIs
StatePublished - Sep 1994

Fingerprint

Embryonal Carcinoma Stem Cells
Reporter Genes
Genes
Fibroblast Growth Factors
Embryonic Stem Cells
Cell Line
Carrier Proteins
Gene Expression
5' Flanking Region
Nucleic Acid Regulatory Sequences
Transcription Initiation Site
Sequence Analysis
Cell Differentiation
Exons
Intercellular Signaling Peptides and Proteins
Complementary DNA

Keywords

  • Embryonal carcinoma cells
  • Fibroblast growth factor
  • Transcription

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology
  • Cell Biology

Cite this

Transcriptional regulation of the murine k‐fgf gene. / Rizzino, A Angie; Rosfjord, E.

In: Molecular Reproduction and Development, Vol. 39, No. 1, 09.1994, p. 106-111.

Research output: Contribution to journalArticle

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abstract = "Embryonal carcinoma (EC) cells provide a useful model system for studying the roles of growth factors during early mammalian development. In 1988, we determined that EC cells express a member of the fibroblast growth factor (FGF) family that cannot be detected after EC cells undergo differentiation. Attempts to understand how differentiation regulates the production of FGFs led to the finding that EC cells express the fibroblast growth factor k‐FGF (FGF‐4), whereas there is a large decrease in the steady state levels of k‐FGF mRNA when EC cells differentiate. This suggested that transcription of the k‐fgf gene is repressed when EC cells differentiate. To investigate this possibility, we prepared a series of reporter gene constructs containing various regions of the murine k‐fgf gene. These constructs were transfected into two mouse EC cell lines and one mouse embryonic stem (ES) cell line. We determined that the mouse 5′ flanking region cannot support expression of the reporter gene. In both EC and ES cell lines, expression of the reporter gene is elevated greatly by the addition of a 316 bp region from the third exon of the murine k‐fgf gene. Sequence analysis of the 316 bp region identified one and possibly two conserved octamer binding motifs. These sequences are likely to be involved in regulation of the k‐fgf gene, because differentiation of EC cells is known to reduce the expression of octamer binding proteins, including Oct‐3. To test the possible role of octamer binding proteins, we examined the expression of our reporter gene constructs in F9‐differentiated cells and in PYS‐2 cells. In these cells, the expression of our k‐fgf/reporter gene constructs is very low. However, expression of the reporter gene is elevated significantly when these cells are cotransfected with a construct that contains an Oct‐3 cDNA under the control of a strong viral promoter. To test further the importance of the octamer motifs found in the 316 bp enhancer‐like region, we replaced the 316 bp region in our constructs with a smaller region (48 bp) that contains the downstream octamer motif and flanking sequences. Like the 316 bp region, this 48 bp region elevated the expression of the reporter gene, but it does so at a much lower level. Thus, it appears that the downstream octamer motif may be involved in the regulation of the k‐fgf gene, but that at least one other cis‐regulatory element present in the 316 bp enhancer is likely to be involved in the expression of the k‐fgf gene in EC cells. Lastly, we have identified two potentially important regulatory regions upstream of the transcription start site. One appears to regulate positively the expression of the murine k‐fgf gene in EC cells and in ES cells, and the other appears to regulate negatively the expression of the k‐fgf gene in these cells. {\circledC} 1994 Wiley‐Liss, Inc.",
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AB - Embryonal carcinoma (EC) cells provide a useful model system for studying the roles of growth factors during early mammalian development. In 1988, we determined that EC cells express a member of the fibroblast growth factor (FGF) family that cannot be detected after EC cells undergo differentiation. Attempts to understand how differentiation regulates the production of FGFs led to the finding that EC cells express the fibroblast growth factor k‐FGF (FGF‐4), whereas there is a large decrease in the steady state levels of k‐FGF mRNA when EC cells differentiate. This suggested that transcription of the k‐fgf gene is repressed when EC cells differentiate. To investigate this possibility, we prepared a series of reporter gene constructs containing various regions of the murine k‐fgf gene. These constructs were transfected into two mouse EC cell lines and one mouse embryonic stem (ES) cell line. We determined that the mouse 5′ flanking region cannot support expression of the reporter gene. In both EC and ES cell lines, expression of the reporter gene is elevated greatly by the addition of a 316 bp region from the third exon of the murine k‐fgf gene. Sequence analysis of the 316 bp region identified one and possibly two conserved octamer binding motifs. These sequences are likely to be involved in regulation of the k‐fgf gene, because differentiation of EC cells is known to reduce the expression of octamer binding proteins, including Oct‐3. To test the possible role of octamer binding proteins, we examined the expression of our reporter gene constructs in F9‐differentiated cells and in PYS‐2 cells. In these cells, the expression of our k‐fgf/reporter gene constructs is very low. However, expression of the reporter gene is elevated significantly when these cells are cotransfected with a construct that contains an Oct‐3 cDNA under the control of a strong viral promoter. To test further the importance of the octamer motifs found in the 316 bp enhancer‐like region, we replaced the 316 bp region in our constructs with a smaller region (48 bp) that contains the downstream octamer motif and flanking sequences. Like the 316 bp region, this 48 bp region elevated the expression of the reporter gene, but it does so at a much lower level. Thus, it appears that the downstream octamer motif may be involved in the regulation of the k‐fgf gene, but that at least one other cis‐regulatory element present in the 316 bp enhancer is likely to be involved in the expression of the k‐fgf gene in EC cells. Lastly, we have identified two potentially important regulatory regions upstream of the transcription start site. One appears to regulate positively the expression of the murine k‐fgf gene in EC cells and in ES cells, and the other appears to regulate negatively the expression of the k‐fgf gene in these cells. © 1994 Wiley‐Liss, Inc.

KW - Embryonal carcinoma cells

KW - Fibroblast growth factor

KW - Transcription

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