Functional characterization of the murine Tnk1 promoter.
Tnk1/Kos1 is a non-receptor protein tyrosine kinase found to be a tumor suppressor. It negatively regulates cell growth by indirectly suppressing Ras activity. We identified and characterized the critical cis-elements required for Tnk1/Kos1's promoter activity. Results indicate that the murine Tnk1 promoter lacks a conventional TATA, CAAT or initiator element (Inr) but contains multiple transcription start sites. Transcription is initiated by a TATA-like element composed of an AT rich sequence at -30 (30 bp upstream) from the major transcription start site and an Inr-like element that overlaps the multiple start sites. Deletion analysis of the m-Tnk1 promoter reveals the presence of both positive (-25 to -151) and negative (-151 to -1201) regulatory regions. The three GC boxes which bind Sp1 and Sp3 with high affinity, an AP2 site (that overlaps with an AML1 site) and a MED1 site comprise the necessary cis-elements of the proximal promoter required for both constitutive and inducible Tnk1/Kos1 expression. Importantly, results reveal that cellular stress reverses the repression of Tnk1/Kos1 and induces its expression through increased high affinity interactions between nuclear proteins Sp1, Sp3, AP2 and MED1 for the m-Tnk1 promoter. These findings provide a mechanism by which the m-Tnk1 promoter can be dynamically regulated during normal growth.
Hoare
Sarasija
S
Department of Internal Medicine, University of Florida Shands Cancer Center, P.O. Box No: 103633, Gainesville, FL 32610, USA.
Hoare
Kishalay
K
Reinhard
Mary K
MK
Flagg
Tammy O
TO
May
William Stratford
WS
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D056913:Mediator Complex Subunit 1; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D050656:Transcription Factor AP-2 / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site; D015533:Transcriptional Activation
C538856:Med1 protein, mouse; D056913:Mediator Complex Subunit 1; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D011505:Protein-Tyrosine Kinases; C475466:Kos1 protein, mouse