title: The functional mapping of long-range transcription control elements of the HOX11 proto-oncogene.
abstract: Mapping of transcriptional control elements normally depends on the generation of a series of deletion mutants. The consequences of particular deletions are then functionally assessed by their ability to alter gene expression. The information derived from such investigations provides a general regulatory profile of the gene of interest, as well as generating a focus for future experiments. Due to the limitations of conventional DNA cloning methods, it has previously not been possible to use such an approach to rapidly assess the role of long-range regulatory elements that frequently lie further than 20 kb away from the coding region. In order to identify regulatory elements of the proto-oncogene HOX11 that may be mutated in a subset of childhood T-cell acute lymphoblastic leukaemia specimens, we generated nested deletions from a P1 artificial chromosome (PAC). This clone contained 95 kilobases (kb) of the HOX11 locus at 10q24; including 63 kb of 5' regulatory DNA. The deletion series was produced by the use of a recombination based cloning system and clones were subsequently transfected into mammalian cells. We have identified several long-range regulatory elements that mediate transcriptional control of HOX11. This approach is simple, rapid, and inexpensive. Furthermore, it generates multiple deletion clones in a single experiment. This novel approach opens up a new avenue for investigating long-range transcription control. Additionally, by allowing analysis of these elements in the natural context of large integrants the approach does not require the use of artificial extrachromosomal elements. This methodology can be applied to any gene cloned into a PAC or BAC vector and could also be useful in identifying appropriately sized deletion mutants for functional testing in transgenic models.
authors.affiliation: Division of Children's Leukaemia and Cancer Research, Telethon Institute for Child Health Research and Centre for Child Health Research, The University of Western Australia, West Perth, WA 6872, Australia. rachaelb@ichr.uwa.edu.au
authors.forename: Rachael L
authors.initials: RL
authors.lastname: Brake
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Chatterjee
authors.forename: Ursula R
authors.initials: UR
authors.lastname: Kees
authors.forename: Paul M
authors.initials: PM
authors.lastname: Watt
mesh_terms: D045744:Cell Line, Tumor; D002874:Chromosome Mapping; D022222:Chromosomes, Artificial, Human; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D005434:Flow Cytometry; D015973:Gene Expression Regulation, Leukemic; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D015513:Oncogene Proteins / Q000235:genetics*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
chemical_list: D017931:DNA Primers; D004251:DNA Transposable Elements; D018398:Homeodomain Proteins; C000715470:MAS1 protein, human; D015513:Oncogene Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; C071250:TLX1 protein, human; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases