Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering

Telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA at the ends of most eukaryotic chromosomes. Human telomerase is composed of an RNA moiety (hTR) and a protein catalytic subunit (hTERT) [1]. In humans, telomerase activity is absent or greatly reduced in most somatic cells but present in germ and stem cells, and highly reactivated in the majority of human cancers [2,3]. Several reports suggest that hTERT protein levels reflect the amount of nuclear telomerase activity [4,5]. The hTERT gene appears to be controlled transcriptionally, though various post-transcriptional mechanisms have been suggested to be involved in hTERT regulation. Several lines of evidence suggest that the regulation of hTERT transcription involves both repressive and activating transcription mechanisms [6,7]. However, the hTERT transcriptional regulatory system is highly complex and largely unknown. Therefore, the identification of hTERT repressor(s) is an important step in understanding how telomerase is controlled during development, aging and tumorigenesis.We previously reported that the introduction of a normal human chromosome 3 restored cellular senescence in two immortal renal cell carcinoma cell lines, RCC23 and KC12, using microcell-mediated chromosome transfer (MMCT) [8,9]. This inactivation of immortal growth by human chromosome 3 transfer was attributed to the loss of telomerase activity due to the transcriptional repression of the hTERT gene [9,10]. This work suggested that human chromosome 3 contains a locus responsible for hTERT repression.Here we report a unique approach using MMCT to introduce truncated chromosome 3 regions into cells forcibly expressing hTERT to identify an hTERT repressor(s). By the transfer of each truncated human chromosome 3, we successfully identified a 7-Mb region within 3p21.3 containing an hTERT repressor(s). We present this approach as a useful tool for the identification and further analysis of hTERT repressor(s).To deter