Journal of Virology, March 2000, p. 2489-2501, Vol. 74, No. 6
Institute of Molecular and Cell Biology,
National University of Singapore, Singapore 117609, Republic of
Singapore
Received 1 October 1999/Accepted 15 December 1999
Two nuclear matrix attachment regions (MARs) bracket a 550-bp
segment of the long control region (LCR) containing the epithelial cell-specific enhancer and the E6 promoter of human papillomavirus type
16 (HPV-16). One of these MARs is located in the 5' third of the LCR
(5'-LCR-MAR); the other lies within the E6 gene (E6-MAR). To study
their function, we linked these MARs in various natural or artificial
permutations to a chimeric gene consisting of the HPV-16
enhancer-promoter segment and a reporter gene. In transient transfections of HeLa cells, the presence of either of these two MARs
strongly represses reporter gene expression. In contrast to this, but
similar to the published behavior of cellular MARs, reporter gene
expression is stimulated strongly by the E6-MAR and moderately by the
5'-LCR-MAR in stable transfectants of HeLa or C33A cells. To search for
binding sites of soluble nuclear proteins which may be responsible for
repression during transient transfections, we performed electrophoretic
mobility shift assays (EMSAs) of overlapping oligonucleotides that
represented all sequences of these two MARs. Both MARs contain multiple
sites for two strongly binding proteins and weak binding sites for
additional factors. The strongest complex, with at least five binding
sites in each MAR, is generated by the CCAAT displacement factor
(CDP)/Cut, as judged by biochemical purification, by EMSAs with
competing oligonucleotides and with anti-CDP/Cut oligonucleotides, and
by mutations. CDP/Cut, a repressor that is down-regulated during differentiation, apparently represses HPV-16 transcription in undifferentiated epithelials cells and in HeLa cells, which are rich in
CDP/Cut. In analogy to poorly understood mechanisms acting on cellular
MARs, activation after physical linkage to chromosomal DNA may result
from competition between the nuclear matrix and CDP/Cut. Our
observations show that cis-responsive elements that regulate the HPV-16 E6 promoter are tightly clustered over at least 1.3 kb and occur throughout the E6 gene. HPV-16 MARs are context dependent
transcriptional enhancers, and activated expression of HPV-16 oncogenes
dependent on chromosomal integration may positively select tumorigenic
cells during the multistep etiology of cervical cancer.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Nuclear Matrix Attachment Regions of Human
Papillomavirus Type 16 Repress or Activate the E6 Promoter,
Depending on the Physical State of the Viral DNA

and
*
Corresponding author. Mailing address: Institute of
Molecular and Cell Biology, National University of Singapore, 30 Medical Dr., Singapore 117609, Republic of Singapore. Phone:
65-778-8823. Fax: 65-779-1117. E-mail:
mcbhub{at}imcb.nus.edu.sg.
Present address: National Institutes of Health, Unit of Cell Cycle
Regulation, Bethesda, MD 20892-5431.
Present address: KuDOS Pharmaceuticals Ltd., Cambridge CB4
4GW, United Kingdom.
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