James A. Kennison, PhD, Head, Section on Drosophila Gene Regulation
Mark Mortin, PhD, Staff Scientist
Monica T. Cooper, BA, Senior Research Technician
Amer Jameel, BS, Postbaccalaureate Fellow ¹
Nader Jameel, BS, Postbaccalaureate Fellow

Our goal is to understand how genes control cell fates during development. The homeotic genes in Drosophila specify cell identities at both the embryonic and adult stages. They encode homeodomain-containing transcription factors that control cell fates by regulating the transcription of downstream target genes. The homeotic genes are expressed in precise spatial patterns that are crucial for the proper determination of cell fates. Both loss of expression and ectopic expression in the wrong tissues lead to changes in cell fates. These changes provide powerful assays to identify the trans-acting factors that regulate the homeotic genes and the cis-acting sequences through which they act. Both the homeotic genes and the trans-acting factors that regulate them are conserved between Drosophila and man. In addition to many conserved developmental genes, at least half of the disease and cancer-causing genes in man are conserved in Drosophila, making Drosophila an important model system for the study of human development and disease.

Cis-acting sequences for transcriptional regulation of the Sex combs reduced homeotic gene

Kennison, Cooper, Jameel A, Jameel N

Cis-acting transcriptional regulatory elements from the homeotic genes have been previously identified by assays in transgenes in Drosophila. These assays have identified tissue-specific enhancer elements as well as cis-regulatory elements that are required for the maintenance of activation or repression throughout development. While these transgenic assays have been important in both defining the structure of the cis-regulatory elements and identifying trans-acting factors that bind to them, their functions within the contexts of the endogenous genes are still not well understood. We have used a large number of existing chromosomal rearrangements in the Sex combs reduced homeotic gene to investigate the functions of the cis-acting elements within the endogenous gene. These chromosomal rearrangements identified an imaginal leg enhancer about 35 kb upstream of the Sex combs reduced promoter. The imaginal leg enhancer not only can activate transcription of the Sex combs reduced promoter that is 35 kb distant, but it can also activate transcription of the Sex combs reduced promoter on the homologous chromosome. This trans-activation was first observed for the homeotic gene Ultrabithorax and named transvection. Although the imaginal leg enhancer can activate transcription in all three pairs of legs, it is normally silenced in the second and third pairs of legs. Such silencing requires the Polycomb group proteins. We are currently trying to identify the cis-regulatory DNA sequences in the Sex combs reduced gene that are required for transcriptional activation in the first leg and for Polycomb group silencing in the second and third legs. Characterization of the chromosomal rearrangements also revealed that two genetic elements about 70 kb apart in the Sex combs reduced gene must be in cis to maintain proper repression. When not physically linked to each other, these elements interact with elements on the homologous chromosome and cause derepression of its wild-type Sex combs reduced gene. We are testing DNA fragments from the Sex combs reduced gene in transgenic assays to identify endogenous cis-regulatory elements that could interact. We have identified two fragments of DNA from the regions that include the regulatory elements required for the maintenance of silencing because they strongly promote pairing-sensitive silencing (an assay for interaction of cis-regulatory DNA fragments). We are currently using targeted gene replacement to delete both these elements in the endogenous gene to assay their role in transcriptional regulation.

Trans-acting activators and repressors of homeotic genes

Kennison, Mortin, Green, ² Cooper; in collaboration with Badenhorst, Huang, Hursh, Vázquez, Zurita

The initial domains of homeotic gene repression are set by the segmentation proteins, which also divide the embryo into segments. Genetic studies have identified the trithorax group of genes that are required for expression or function of the homeotic genes, including the maintenance of transcriptional activation. Maintenance of transcriptional repression requires the proteins encoded by the Polycomb group genes. To identify new trithorax group activators and Polycomb group repressors, we are screening for new mutations that mimic the following phenotypes: loss-of-function or ectopic expression of the homeotic genes. We have generated over 4,000 lethal mutants, and, among those that die very late in development (after the formation of the adult cuticle during pupation), we have identified two dozen mutants with homeotic phenotypes. The mutations identify several new genes that are required for expression or function of the Sex combs reduced homeotic gene. We have recovered three alleles of the rhinoceros gene, which encodes the Drosophila homologue of a mammalian transcriptional co-activator that interacts with the von Hippel-Lindau tumor suppressor protein. We have also recovered seven alleles of a novel gene that is required for both function of the Sex combs reduced gene and melanotic tumor suppression. We have localized this novel gene to a small genomic region of about 80kb and are currently working to identify the transcription unit with which it corresponds.

Reduced function of the trithorax group genes mimics loss of function of the homeotic genes. We had previously identified several trithorax group genes that encode subunits of chromatin-remodeling complexes. The brahma, moira, and osa genes encode subunits of the Brahma chromatin-remodeling complex, which is conserved from yeast (the SWI/SNF and RSC complexes) to man (the BRG1 and HBRM complexes). To understand further the function of the Brahma complex, we have been characterizing mutations that interact with mutations in the Brahma complex. We have shown that one of these new genes, Pearl, encodes one of the two gamma-tubulin isoforms in Drosophila. During the characterization of Pearl, we also identified a recessive suppressor mutation that rescues the zygotic lethality associated with loss of gamma-tubulin function. This recessive suppressor mutation is in a gene that encodes a protein required for formation of the gamma-tubulin ring complex.

Regulation of nuclear import and export of homeodomain proteins

Mortin, Kennison; in collaboration with Bi, Hursh, Rong

The prospero homeodomain protein in Drosophila is required for patterning the embryonic nervous system. The subcellular localization of prospero is affected by both nuclear import and export. Both DNA-binding and nuclear export require the prospero homeodomain. We identified the caliban protein by its interaction with the prospero homeodomain. The human homologue of caliban has been implicated in colon and lung cancer. We have shown that both Drosophila and human caliban are bipartite mediators of Exportin-dependent nuclear export in cultured cells. The carboxyl terminus of caliban binds to the prospero homeodomain while the amino terminus binds to Exportin. Both interactions are required for the nuclear export of the prospero homeodomain. While noncancerous human lung cells have functional human caliban, human lung carcinoma cell lines do not. Expression of Drosophila caliban in human lung cancer cells reduced cell invasiveness and the cells' ability to form colonies on soft agar. We have deleted the Drosophila caliban gene and shown that it is important for melanotic tumor suppression following irradiation of developing larvae.

¹ Left the laboratory in 2006.
² Helen Green, PhD, former Postdoctoral Fellow

COLLABORATORS

Paul Badenhorst, PhD, Institute of Biomedical Research, University of Birmingham, Edgbaston, UK
Xiaolin Bi, PhD, Laboratory of Molecular Cell Biology, NCI, Bethesda, MD
Der-Hwa Huang, PhD, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC
Deborah A. Hursh, PhD, Division of Cellular and Gene Therapies, CBER, FDA, Bethesda, MD
Yikong Rong, PhD, Laboratory of Molecular Cell Biology, NCI, Bethesda, MD
Martha Vázquez, PhD, Instituto de Biotecnología, UNAM, Cuernavaca, Mexico
Mario Zurita, PhD, Instituto de Biotecnología, UNAM, Cuernavaca, Mexico

For further information, contact Jim_Kennison@nih.gov.