Regulation of the potential tumorsuppressor gene mad1 by G-CSF

The human Mad1 protein, 35kD, is a 220 amino acid polypeptide with a short half-life (t1/2=15-30min), whose gene is localized on chromosome 2p12-13. The expression of Mad1 is tightly regulated and its synthesis can be modulated in response to diverse signals that control cell behavior. One example is that granulocyte colony-stimulating factor (G-CSF) that drives differentiation of granulocytic cells induces mad1 expression. In general, Mad1 is well expressed in a variety of cells undergoing differentiating and in non-cycling terminally differentiated cells. It has been shown that Mad1 interferes with cell cycle progression, drives cell differentiation, prevents apoptosis and blocks transformation. Mad1 can regulate different aspects of cell behavior by modulating gene expression. At the molecular level, Mad1 acts as a repressive transcription factor through Mad1/Max heterodimers by binding to 5’-CACGTG E-box DNA site and by recruiting histone deacetylase-containing corepressor complexes. However, little is known about the regulatory mechanisms that control the expression of the mad1 gene. The aims of the study were to investigate the transcriptional regulation of the mad1 gene. In particular, we would like to: 1) identify the key regulatory elements of the mad1 promoter that mediate the response to G-CSF; 2) identify transcription factors controlling the expression of the mad1 gene; 3) investigate possible cooperations between the transcription factors defined; and 4) define the signal transduction pathways, which are relevant for G-CSF-dependent induction of mad1 gene expression in hematopoietic cells. In the study presented here, the following observtions were made: First, in U937 promyelocytic cell, G-CSF-induced expression of the mad1 gene was demonstrated by real time PCR. This induction was sensitive to inhibition of MAP-kinases. Second, the -184 to 247 mad1 promoter fragment, containing part of the homology region, was identified as functional promoter. Althougth this fragment lacks a TATA-box it overlaps with a CpG-island that contains GC-boxes. In response to G-CSF, the region from -1282 to -796 reduces activation, whereas the region from -184 to -59 contains G-CSF responsive elements. It appears that for this two CCAAT-boxes are critical. The GC-boxes are important for mad1 promoter’s activity, they are probably not directly relevant for the activation of the promoter in response to G-CSF/G-CSFR. In addition, a possible cooperativity exists between CCAAT- and GC-boxes in the regulation of the human mad1 promoter. Third, both C/EBP and Sp proteins regulate the activity of the mad1 promoter in response to G-CSF through CCAAT- and GC-boxes respectively. While Sp proteins bind to the mad1 promoter constitutively, C/EBP proteins, especially C/EBPbeta, appear to play major roles in the induction of the mad1 gene in response to G-CSF. Fourth, STAT3 is implicated in the regulation of mad1 expression in an indirect way, suggesting a new cooperative mechanism with C/EBP. Finally, regarding signal transduction pathways, the data presented here demonstrate that four tyrosine residues (Y704, Y729, Y744 and Y764) within the cytoplasmic domain of the G-CSFR orchestrate the induction of mad1 expression. Both the JAK/STAT and the RAS/RAF/MAPK pathways are involved in the activation and the final signals may converge on the transcription factor C/EBPbetawhich interacts with STAT3, supporting a CEBPbeta-dependent model of the induction of mad1 expression in response to G-CSF.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Jiang, Kan

Contributors dc:contributor

• Lüscher, Bernhard

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• Genexpression
• Zelldifferenzierung
• Signaltransduktion
• G-CSF
• Gene expression
• differentiation
• signal transduction
• mad1

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-122486%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:60796