Increased levels of the repressor coup-TFII and coup-TF association with histone deacetylases contribute to reduced MHC class I transcription in tumorigenic adenovirus type 12 transformed cells / Denis A. Smirnov.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Smirnov, Denis A.

Contributor:

Ricciardi, Robert P., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Biology.

Biology--Penn dissertations.

Biology.

Academic Dissertations as Topic.

Medical Subjects:

Biology.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Biology.

Biology--Penn dissertations.

Physical Description:

xi, 145 pages : illustrations (some color) ; 29 cm

Production:

2000.

Summary:

In tumorigenic Ad12-transformed cells, compared to non-turmorigenic Ad5-transformed cells, transcription of the MHC class I antigens is down-regulated as a result of the strong binding activity of COUP-TF repressor to the R2 site and lack of binding of NF-kappaB activator to the R1 site within the class I enhancer. To further investigate how single viral gene Ad12 E1A mediates transcriptional repression of MHC class I, it was important to establish which member of the family of COUP-TF repressors (COUP-TFI or COUP-TFII) is responsible for differential binding to the R2 element. Here, I show that COUP-TFII, but not COUP-TFI, is expressed in Ad-transformed cells. Because hypophosphorylation of NF-kappaB in Ad12-transformed cells prevents it from binding to the class I enhancer, it was essential to determine whether the higher COUP-TFII binding activity to the R2 site, observed in Ad12-transformed cells, is due to higher levels of COUP-TFII expression or to modification of COUP-TFII protein. Here, I demonstrate that up-regulation of COUP-TFII transcription is responsible for strong COUP-TF binding activity in Ad12-transformed cells. These results suggest that Ad12 E1A-mediated mechanisms, responsible for the differential binding activities of COUP-TFII and NF-kappaB to the class I enhancer, are dissimilar.

The mechanism by which COUP-TFII represses transcription has yet to be elucidated. Here, I show that COUP-TFII represses transcription through its association with a histone deacetylase. Using reciprocal binding assays, it was determined that COUP-TFII interacts with histone deacetylase (HDAC1) through COUP-TFII C-terminal repression domain. Moreover, a histone deacetylase enzymatic activity was found to be associated with COUP-TFII in Ad12-transformed cells. Transfection experiments further revealed that exogenous histone deacetylase facilitates transcriptional repression by COUP-TFs. Also, supershift assays suggest that the transcriptional corepressor N-CoR and the histone deacetylase HDAC1 are both part of the COUP-TFII complex bound to the MHC class I enhancer R2 site. Finally, I provide evidence that the inhibition of histone deacetylases relieves the repression of MHC class I expression in Ad12-transformed cells. Taken together, these results support the notion that the deacetylation of histones, mediated through COUP-TFII binding to the R2 site, serves to down-regulate MHC class I transcription in Ad12-transformed cells.

Finally, fundamental changes underlying differential tumorigenicity observed between Ad5- and Ad12-transformed cells still remain to be explored in greater details. Using cDNA microarray technology, global gene expression profiles were generated for Ad5 and Ad12-transformed cells. Here, I describe the initial comparison of these gene expression profiles.

Notes:

Supervisor: Robert P. Ricciardi.

Thesis (Ph.D. in Biology) -- University of Pennsylvania, 2000.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 9965570.

OCLC:

244971185