***************
Poster
Contents

Abstract

Introduction

Materials
& Methods

Results

Discussion
& Conclusion

References

Discussion
Board

INABIS '98 Home Page Your Poster Session Related Symposia & Posters Scientific Program Exhibitors' Foyer Personal Itinerary New Search

Isolation and Characterization of the 5'-Flanking Region of the Rat C/EBP alpha Gene.

Introduction

The CCAAT/enhancer-binding protein alpha (C/EBP alpha) belongs to a bZIP protein family of transcription factors (1). The bZIP proteins are characterized by a basic amino acid-rich DNA-binding region and a leucine zipper domain that is necessary for dimer formation (2). C/EBP alpha conforms functional homo- or heterodimers with another member of the C/EBP family and binds the DNA site (TT/GNNGC/TAAT/G) (3,4). This C/EBP alpha binding site exists in the promoter of some liver-enriched genes, e.g. albumin and aldolase B (4). Hence, this transcription factor plays important roles for expression of proteins related to liver functions.
C/EBP alpha is detected at high level in liver, white and brown adipocyte tissue,placenta, intestine, lung and myeloid cells (5,6,7,8). On the other hand, it is well known that the C/EBP alpha mRNA level is found abundantly in differentiated hepatoma cells, but scarcely in dedifferentiated hepatoma cells (9,10). Moreover primary culture of hepatocytes on collagen-coated dishes leads to a gradual decrease in C/EBP alpha protein level and DNA-binding activity. The addition of EHS (Engelbreth-Holm-Swarm) extra cellular matrix gel in the culture induces re-differentiation of the cultured hepatocytes and re-expression of C/EBP alpha, as well as remodeling the hepatocyte morphology and phenotype (11).
Thus C/EBP alpha was closely associated with hepatocyte differentiation and functions. However the regulation of C/EBP alpha gene expression is little known in differentiated hepatoma cells. In this study, we cloned and sequenced 5f-flanking region to elucidate the regulation of the rat C/EBP alpha gene.

Back to the top.


Materials and Methods

(1) Isolation and sequencing of the rat C/EBP alpha gene
A rat genomic library (105 colonies in cosmid vector) was screened with a rat C/EBP alpha cDNA as describe elswhere. Positive clones were further analyzed by Southern blot hybridization using the C/EBP alpha cDNA as a probe. 7.9-kilobase HindIII fragment was subcloned into the Bluescript II KS (-) vector, and used to generate a series of deletion constructs corresponding to various restriction enzyme sites. Sequencing of these subclones was performed by using a Thermo Sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham) on the automated sequencer.

(2) Northern blot analysis
Total RNA from rat liver or cultured cells was extracted using the guanidium thiocyanate/cesium chloride method. 10ug of total RNA was electrophoresed through a 1% agarose-6.7% formaldehyde gel and transferred to nylon membrane. The blots were hybridized according to previous report. The probe, a 1500bp NruI/NspI fragment from the HindIII-subclone, was labeled [-32P]dCTP by the random primer method (TaKaRa).

Back to the top.


Results

(1) Cloning and sequence analysis of 5f-upstream region of the rat C/EBP alpha gene
Previously, we showed decrease in C/EBP alpha gene expression associated with dedifferentiation of hepatoma cells. To study transcriptional regulation for the C/EBP alpha gene, we first isolated the rat C/EBP alpha gene from a genomic library using a 1.5-kilobase cDNA for rat C/EBP alpha as a probe. Three colonies were identified by the presence of a 7.9-kilobase HindIII fragment that contained the entire C/EBP alpha gene. And the complete sequence was determined in approximately 3-kilobase of the 5f-upstream region. As shown in Fig. 1, the putative binding sites for transcription factors were found in the 5f-flanking region of the C/EBP alpha gene. Binding sites for Sp1 and zinc finger protein are found in proximal upstream of the TATA box. The nucleotide sequence spanning from -195 to -173bp was well corresponded to the C/EBP binding site in the mouse promoter. The rat C/EBP alpha promoter possesses a CUP/AP-2 binding site, as well as the mouse and human promoters. A GT-repeat was mapped at 945bp upstream from the transcription start site, and C/EBP beta, HNF-3 beta and MyoD binding sites were also found at distal sites in the 5f-flanking region of the rat C/EBP alpha gene.
Fig. 1 Schematic representation of the DNA: protein interactions in the rat C/EBP alpha promoter.

(2) Expression of C/EBP alpha mRNA in differentiated and dedifferentiated hepatoma cells
To study whether the expression of C/EBP alpha mRNA is related to the differentiation of hepatoma cell lines, we examined the level of mRNA coding C/EBP alpha in different hepatoma cells. The pattern of C/EBP alpha mRNA expression in hepatoma cells is shown in Fig. 2. C/EBP alpha transcript was observed in normal rat liver. Further more, it was detected in the fully differentiated Reuber cell line at a significant level, while scarcely in the dedifferentiated variant AH66. Thus the pattern of C/EBP alpha expression suggested to correlation with the differentiation state of hepatoma cell lines.
Fig. 2 C/EBP alpha mRNA level in differentiated and dedifferentiated hepatoma cells.
Northern blot analysis of the indicated cell line was performed as described in Materials and Methods.
Back to the top.


Discussion and Conclusion

C/EBP alpha has been suggested to play an important role as a key factor in the switching of proliferation and differentiation, mainly based on work in adipocyte- and hepatocyte- derived cell lines (11, 12). Recent study has demonstrated that the transduction of CUP/AP-2 in 3T3-L1 preadipocyte trans-inhibits the C/EBP alpha gene expression (13). However, the factor regulating the C/EBP alpha gene is little known in the hepatocyte during its differentiation. In this report, we have isolated and sequenced the 5f-flanking region of the rat C/EBP alpha gene. We found putative binding sites for C/EBP beta, HNF-3 beta and MyoD in the upstream region of the gene. Rana et al. reported previously that C/EBP alpha was transactivated by C/EBP beta that bound at -190 to -170 in C/EBP alpha gene (14). However, C/EBP beta binding site of the rat C/EBP alpha gene was additionally located in distal region. HNF-3 beta is one of the liver-enriched transcription factors, and first appears in liver development (15). Therefore HNF-3 beta seems to be associated with the regulation of C/EBP alpha expression in the hepatocyte differentiation. MyoD, which induces the differentiation of muscle cells (16), may participate with the down-regulation of the C/EBP alpha gene.
The expression of C/EBP alpha gene has been shown to be downregulated in hepatoma cells. The phenotypic difference between Reuber and AH66 seems to be caused by the amount of C/EBP alpha gene expression. To understand the correlation between the C/EBP alpha expression and the dedifferentiation of hepatoma cells, the fine regulatory mechanism of expression C/EBP alpha gene is necessary further study using differentiated and dedifferentiated hepatoma cells.

Back to the top.


References

  1. Vinson, C. R., Sigler, P. B. and McKnight, S. L. (1989) Science 246: 911-916.
  2. Landschulz, W. H., Johnson, P. F. and McKnight, S. L. (1988) Science 240: 1759-1764.
  3. Cao, Z., Umek, R. M. and McKnight, S. L. (1991) Genes Dev. 5: 1538-1552.
  4. McKnight, S. L., Lane, M. D. and Gluecksohn-Waelsch, S. (1989) Genes Dev. 3: 2021-2024.
  5. Xanthopoulos, K. G., Mirkovitch, J., Decker, T., Kuo, C. F. and Darnell Jr., J. E. (1989) Proc. Natl. Acad. Sci. USA 86:4117-4121.
  6. Williams, S. C., Cantwell, C. A. and Johnson, P. F. (1991) Genes Dev. 5: 1553-1567.
  7. Birkenmeier, E. H., Gwynn, B., Howard, S., Jerry, J., Gordon, J. I., Landschulz, W. H. and McKnight, S. L. (1989) Genes Dev. 3: 1146-1156.
  8. Scott, L. M., Civin, C. I., Routh, P. and Friedman, A. D. (1992) Blood 80: 1725-1735.
  9. Cereghini, S., Yaniv, M. and Cortese, R. (1990) EMBO J. 9: 2257-2263.
  10. Herbst, R. S., Nielsch, U., Sladek, F., Lai, E., Babiss, L. E. and Darnell Jr, J. E. (1991) New Biol. 3: 289-296.
  11. Runge, D. Runge, D. M., Bowen, W. C., Locker, J. and Michalopoulos, G. K. (1997) Biol. Chem. 378: 873-881.
  12. Umek, R. M., Friedman, A. D. and McKnight, S. L. (1991) Science 251: 288-292.
  13. Jiang, M-S., Tang, Q-Q., McLenithan, J., Geiman, D., Shillinglow, W., Henzel, W. and Lane, M. D. (1998) Proc. Natl. Acad. Sci. USA 95: 3467-3471.
  14. Rana, B., Xie, Y., Mischoulon, D., Bucher, N. L. and Farmer, S. R. (1995) J. Biol. Chem. 270: 18123-18132.
  15. Cereghini, S. (1996) FASEB J. 10: 267-282.
  16. Weintraub, H. (1993) Cell 75: 1241-1244.

Back to the top.


| Discussion Board | Previous Page | Your Poster Session |
(C) 1998 Author(s) Hold Copyright