Enhancer activity of human papillomavirus type 11 in mouse embryonal carcinoma cells

Kasinadhuni, Satyanarayana (1994) Enhancer activity of human papillomavirus type 11 in mouse embryonal carcinoma cells. Masters thesis, Memorial University of Newfoundland.

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Abstract

The activity of minimal constitutive enhancer (MCE (nucleotides 7657-7870) of human papillomavirus 11 was studied in undifferentiated and retinoic acid-differentiated P19 mouse embryonal carcinoma cells. The enhancer was active in RA-differentiated cells but not in undifferentiated cells. The in vivo activity was correlated with in vitro DNA-protein interactions by DNaseI protection assay. Four protected regions (I-IV) were detected with RA-differentiated nuclear extracts,, while protection was observed with undifferentiated nuclear extracts for the regions III and IV. To further study the functional importance of the protected regions, deletion mutant enhancers were tested for their in vivo activity. Mutant enhancers were active like wild type in RA-differentiated but not in undifferentiated cells. Deletion of protected region I and a 9 base pair sequence of the imperfect NF1 palindrome of protected region II of HPV11 MCE displayed activity close to that of wild type MCE. This suggests that the major constitutive enhancer activity is exhibited by the minimal sequences of MCE (nucleotide 7785-7870) in P19 RA-differentiated cells. The mutant enhancer (nucleotide 7811-7870) which lost both protected regions I and II showed only 50% of the wild type activity. This indicates that sequences displaying major constitutive activity of HPV11 MCE are present in the protected region II, although functional synergism appears to be important for full activity of the enhancer. The role of entire protected region II by itself including the 9 base pair sequence which was deleted in the minimal enhancer sequences, was examined for its in vivo activity. The protected region II alone showed substantial in vivo activity in RA-differentiated cells which was 90-fold higher than the residual activity observed for undifferentiated cells. The in vivo activity of protected region II in RA-differentiated-dependent manner was correlated with in vitro DNA-protein interactions. The protected region II showed an extra DNA-protein complex with RA-differentiated nuclear extracts in the gel retardation assay. The formation of this complex was competed by the imperfect NF1 palindromic sequences but not by the NF1 half-site sequence of protected region II. This suggests that certain factor(s) in RA-differentiated cells specifically binds the imperfect NF1 palindrome. The DNA-protein interaction was specific for HPV11 imperfect NF1 palindrome as the complex formation was not abolished either by polyoma JC virus perfect NF1 palindrome or its mutant sequence. This suggests the existence of family of NF1 proteins in P19 RA-differentiated cells that show different binding specificities for the NF1 binding sequences of imperfect and perfect palindromic nature. As the extra RA-differentiated DNA-protein complex was specific for the imperfect NF1 palindrome, UV cross-linking was performed to determine the number of proteins interacting with it. At least six proteins were found to interact with the imperfect NF1 palindrome and three of them were specific for RA-differentiated nuclear extracts. The exact nature and binding sites of these three proteins for the functional importance of protected region II in RA-differentiated cells require further investigation.

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