Almost nothing is known about the sequences involved in transcription initiation of protein-coding genes in the parasite Leishmania. We describe here the transcriptional analysis of chromosome 1 (chr1) from Leishmania major Friedlin (LmjF) which encodes the first 29 genes on one DNA strand, and the remaining 50 on the opposite strand. Strand-specific nuclear run-on assays showed that a low level of nonspecific transcription probably takes place over the entire chromosome, but an approximately 10-fold higher level of coding strand-specific RNA polymerase II (Pol II)-mediated transcription initiates within the strand-switch region. 5' RACE studies localized the initiation sites to a <100 bp region. Transfection studies support the presence of a bidirectional promoter within the strand-switch region, but suggest that other factors are also involved in Pol II transcription. Thus, while in most eukaryotes each gene possesses its own promoter, a single region seems to drive the expression of the entire chr1 in LmjF.

Genome projects involving Leishmania and other trypanosomatids have revealed that most genes in these organisms are organized into large clusters of genes on the same DNA strand. We have previously shown that transcription of the entire Leishmania major Friedlin (LmjF) chromosome 1 (chr1) initiates bidirectionally between two divergent gene clusters. Here, we analyze transcription of LmjF chr3, which contains two convergent clusters of 67 and 30 genes, separated by a tRNA gene, with a single divergent protein-coding gene located close to the "left" telomere. Nuclear run-on analyses indicate that specific transcription of chr3 initiates bidirectionally between the single subtelomeric gene and the adjacent 67-gene cluster, close to the "right" telomere upstream of the 30-gene cluster, and upstream of the tRNA gene. Transcription on both strands terminates within the tRNA-gene region. Transient-transfection studies support the role of the tRNA-gene region as a transcription terminator for RNA polymerase II (Pol II) and Pol III, and also for Pol I.Leishmania is a protozoan parasite (order Kinetoplastida) which alternates life-forms between an intracellular amastigote stage residing in vertebrate macrophages and an extracellular promastigote stage living in the digestive tract of sandflies. The numerous human-infective Leishmania species cause a spectrum of disease ranging from asymptomatic to lethal, resulting in widespread human suffering and death, as well as considerable economic loss (35).Leishmania, as well as other members of the Trypanosomatidae family, possesses unusual mechanisms of gene expression, such as polycistronic transcription (13,19) and RNA editing of the mitochondrial transcripts (37). In these organisms, the mature nuclear mRNAs are generated from primary transcripts by trans-splicing, a process that adds a capped 39-nucleotide (nt) miniexon or splice leader (SL) to the 5Ј termini of the mRNAs (27). The steady-state levels of most of the mature mRNAs appear to be regulated posttranscriptionally by mechanisms that involve their 3Ј untranslated region sequences (23). Promoters for RNA polymerase I (Pol I) have been extensively characterized in trypanosomatids (31,44,46), as have some Pol III promoters (3,26). However, little is known about the sequences that drive the expression of protein-coding genes by Pol II.The Leishmania haploid genome content is ϳ34 Mb, consisting of 36 chromosomes which range in size from 0.3 to 2.5 Mb (40). The Leishmania Genome Network was established with the support of the World Health Organization to map and sequence the genome of Leishmania major Friedlin (LmjF), the reference strain of the project. The sequence of chromosome 1 (chr1), the smallest in the parasite, revealed the presence of 79 putative genes, the first 29 of which are in a cluster on the "bottom" DNA strand, while the remaining 50 are in a cluster on the "top" strand (21). Importantly, nuclear run-on analysis of chr1 showed that specific transcription, leading to the production of stable transcripts, ...

The ends of human chromosomes (telomeres) lose up to 200 bp of DNA per cell division. Chromosomal shortening ultimately leads to senescence and death in normal cells. Many human carcinoma lines are immortal in vitro, suggesting that these cells have a mechanism for maintaining the ends of their chromosomes. Telomerase is a ribonucleoprotein complex that synthesizes telomeric DNA onto chromosomes using its RNA component as template. Telomerase activity is found in most tumor cells, but is absent from normal cells. Little is known about how normal human cells repress telomerase (hTERT) gene expression. Mice carrying an E2F-1 null mutation develop a variety of malignant tumors, suggesting that this transcription factor has a tumor suppressor function. To determine mechanisms by which E2F-1 suppresses tumor formation, we examined the role of this transcription factor in regulation of the hTERT promoter in human cells. We identified two putative E2F-1-binding sites proximal to the transcriptional start site of the hTERT promoter. Mutation of these sites produced dramatic increases in promoter activity. Overexpression of E2F-1 but not a mutant E2F-1 repressed hTERT promoter activity in reporter gene assays. This repression was abolished by mutation of the E2F-1-binding sites in the hTERT promoter. Human cancer cell lines stably overexpressing E2F-1 exhibited decreased hTERT mRNA expression and telomerase activity. We conclude that E2F-1 has an atypical function as a transcriptional repressor of the hTERT gene in human cells.

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most frequent cancer worldwide. Because HNSCC is largely acquired by environmental carcinogen exposure rather than through germ line mutations, there are no known familial forms of the disease in humans nor are there inbred rodent strains prone to spontaneous head and neck tumors. Transgenic animals with inactivation of tumor suppressor genes commonly mutated in human cases of HNSCC provide attractive models for studying the pathogenesis of head and neck cancer. p53 is the most frequently inactivated tumor suppressor gene in HNSCC. We used a chemical induction protocol in mice heterozygous for the p53 gene to evaluate how p53 inactivation contributed to head and neck carcinogenesis the mouse model. Metastatic squamous cell carcinomas developed in 100% of animals. Histopathologically, the tumors ranged from well to poorly differentiated and showed many molecular features of human HNSCC. Mice carrying only one p53 allele developed tumors with significantly reduced latency compared with wild-type controls (average, 18 versus 22 weeks). Metastatic cancer cells showed complete loss of p53 expression when compared with primary tumors. Transcriptional profiling showed not only distinct genetic differences between primary and metastatic tumors, but also when cancers from heterozygous null and wild-type animals were compared. Our results provide novel insights into the molecular genetics of tumor progression in head and neck cancer. (Mol Cancer Res 2007;5(4):351 -62)