A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter

Jill E. Slansky, Yue Li, William G. Kaelin, Peggy J. Farnham

Research output: Contribution to journalArticle

248 Citations (Scopus)

Abstract

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

Original languageEnglish (US)
Pages (from-to)1610-1618
Number of pages9
JournalMolecular and Cellular Biology
Volume13
Issue number3
StatePublished - Mar 1993
Externally publishedYes

Fingerprint

Tetrahydrofolate Dehydrogenase
Messenger RNA
Growth
Proteins
Serum
S Phase
G1 Phase
Cell Cycle
E2F Transcription Factors
NIH 3T3 Cells
Oncogene Proteins
Nucleotides
Cell Proliferation

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter. / Slansky, Jill E.; Li, Yue; Kaelin, William G.; Farnham, Peggy J.

In: Molecular and Cellular Biology, Vol. 13, No. 3, 03.1993, p. 1610-1618.

Research output: Contribution to journalArticle

Slansky, Jill E. ; Li, Yue ; Kaelin, William G. ; Farnham, Peggy J. / A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter. In: Molecular and Cellular Biology. 1993 ; Vol. 13, No. 3. pp. 1610-1618.
@article{a21dc6829f874352bd3c8d3877e10ae3,
title = "A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter",
abstract = "Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.",
author = "Slansky, {Jill E.} and Yue Li and Kaelin, {William G.} and Farnham, {Peggy J.}",
year = "1993",
month = "3",
language = "English (US)",
volume = "13",
pages = "1610--1618",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "3",

}

TY - JOUR

T1 - A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter

AU - Slansky, Jill E.

AU - Li, Yue

AU - Kaelin, William G.

AU - Farnham, Peggy J.

PY - 1993/3

Y1 - 1993/3

N2 - Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

AB - Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

UR - http://www.scopus.com/inward/record.url?scp=0027403342&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027403342&partnerID=8YFLogxK

M3 - Article

C2 - 8441401

AN - SCOPUS:0027403342

VL - 13

SP - 1610

EP - 1618

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 3

ER -