PMID- 11222776
OWN - NLM
STAT- MEDLINE
DCOM- 20010503
LR  - 20190501
IS  - 1362-4962 (Electronic)
IS  - 0305-1048 (Print)
IS  - 0305-1048 (Linking)
VI  - 29
IP  - 5
DP  - 2001 Mar 1
TI  - Pituitary Ets-1 and GABP bind to the growth factor regulatory sites of the rat 
      prolactin promoter.
PG  - 1251-60
AB  - Ets factors play a critical role in oncogenic Ras- and growth factor-mediated 
      regulation of the proximal rat prolactin (rPRL) promoter in pituitary cells. The 
      rPRL promoter contains two key functional Ets binding sites (EBS): a composite 
      EBS/Pit-1 element located at -212 and an EBS that co-localizes with the basal 
      transcription element (BTE, or A-site) located at -96. Oncogenic Ras exclusively 
      signals to the -212 site, which we have named the Ras response element (RRE); 
      whereas the response of multiple growth factors (FGFs, EGF, IGF, insulin and TRH) 
      maps to both EBSs. Although Ets-1 and GA binding protein (GABP) have been 
      implicated in the Ras and insulin responses, respectively, the precise identity 
      of the pituitary Ets factors that specifically bind to the RRE and BTE sites 
      remains unknown. In order to identify the Ets factor(s) present in GH4 and GH3 
      nuclear extracts (GH4NE and GH3NE) that bind to the EBSs contained in the RRE and 
      BTE, we used EBS-RRE and BTE oligonucleotides in electrophoretic mobility shift 
      assays (EMSAs), antibody supershift assays, western blot analysis of partially 
      purified fractions and UV-crosslinking studies. EMSAs, using either the BTE or 
      EBS-RRE probes, identified a specific protein-DNA complex, designated complex A, 
      which contains an Ets factor as determined by oligonucleotide competition 
      studies. Using western blot analysis of GH3 nuclear proteins that bind to 
      heparin-Sepharose, we have shown that Ets-1 and GABP, which are MAP kinase 
      substrates, co-purify with complex A, and supershift analysis with specific 
      antisera revealed that complex A contains Ets-1, GABPalpha and GABPbeta1. In 
      addition, we show that recombinant full-length Ets-1 binds equivalently to BTE 
      and EBS-RRE probes, while recombinant GABPalpha/beta preferentially binds to the 
      BTE probe. Furthermore, comparing the DNA binding of GH4NE containing both Ets-1 
      and GABP and HeLa nuclear extracts devoid of Ets-1 but containing GABP, we were 
      able to show that the EBS-RRE preferentially binds Ets-1, while the BTE binds 
      both GABP and Ets-1. Finally, UV-crosslinking experiments with radiolabeled 
      EBS-RRE and BTE oligonucleotides showed that these probes specifically bind to a 
      protein of approximately 64 kDa, which is consistent with binding to Ets-1 (54 
      kDa) and/or the DNA binding subunit of GABP, GABPalpha (57 kDa). These studies 
      show that endogenous, pituitary-derived GABP and Ets-1 bind to the BTE, whereas 
      Ets-1 preferentially binds to the EBS-RRE. Taken together, these data provide 
      important insights into the mechanisms by which the combination of distinct Ets 
      members and EBSs transduce differential growth factor responses.
FAU - Schweppe, R E
AU  - Schweppe RE
AD  - Department of Biochemistry, Program in Molecular Biology, University of Colorado 
      Health Sciences Center, 4200 East Ninth Avenue, Box B-151, Denver, CO 80262, USA. 
      a.gutierrez-hartmann@uchsc.edu
FAU - Gutierrez-Hartmann, A
AU  - Gutierrez-Hartmann A
LA  - eng
GR  - R56 DK046868/DK/NIDDK NIH HHS/United States
GR  - P30 CA46934/CA/NCI NIH HHS/United States
GR  - DK46868/DK/NIDDK NIH HHS/United States
GR  - R01 DK046868/DK/NIDDK NIH HHS/United States
GR  - P30 CA046934/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Nucleic Acids Res
JT  - Nucleic acids research
JID - 0411011
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (ETS1 protein, human)
RN  - 0 (Ets1 protein, rat)
RN  - 0 (GA-Binding Protein Transcription Factor)
RN  - 0 (Oligonucleotides)
RN  - 0 (Proto-Oncogene Protein c-ets-1)
RN  - 0 (Proto-Oncogene Proteins)
RN  - 0 (Proto-Oncogene Proteins c-ets)
RN  - 0 (Transcription Factors)
RN  - 9002-62-4 (Prolactin)
SB  - IM
MH  - Animals
MH  - Binding Sites
MH  - Binding, Competitive
MH  - DNA-Binding Proteins/*metabolism
MH  - GA-Binding Protein Transcription Factor
MH  - HeLa Cells
MH  - Humans
MH  - Oligonucleotides/genetics/metabolism
MH  - Pituitary Neoplasms/metabolism/pathology
MH  - Prolactin/*genetics
MH  - Promoter Regions, Genetic/*genetics
MH  - Protein Binding/radiation effects
MH  - Proto-Oncogene Protein c-ets-1
MH  - Proto-Oncogene Proteins/*metabolism
MH  - Proto-Oncogene Proteins c-ets
MH  - Regulatory Sequences, Nucleic Acid
MH  - Transcription Factors/*metabolism
MH  - Tumor Cells, Cultured
MH  - Ultraviolet Rays
PMC - PMC29733
EDAT- 2001/02/27 10:00
MHDA- 2001/05/22 10:01
PMCR- 2001/03/01
CRDT- 2001/02/27 10:00
PHST- 2001/02/27 10:00 [pubmed]
PHST- 2001/05/22 10:01 [medline]
PHST- 2001/02/27 10:00 [entrez]
PHST- 2001/03/01 00:00 [pmc-release]
AID - gke223 [pii]
AID - 10.1093/nar/29.5.1251 [doi]
PST - ppublish
SO  - Nucleic Acids Res. 2001 Mar 1;29(5):1251-60. doi: 10.1093/nar/29.5.1251.