REGULATION OF HOMEOTIC GENE FUNCTION IN DROSOPHILA
, Head, Section on Drosophila Gene Regulation
Mark Mortin, PhD, Staff Scientist
Monica T. Cooper, BA, Senior Research Technician
Nader Jameel, BS, Postbaccalaureate Fellow
Alexander Conant, Summer Student
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 fate. Both loss of expression and ectopic expression in the wrong tissues lead to changes in cell fate. These changes in cell fate provide powerful assays for identifying the trans-acting factors that regulate the homeotic genes and the cis-acting sequences through which the genes act. Both the homeotic genes and the trans-acting factors 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 a particularly important model system for the study of human development and disease.
Cis-acting sequences for transcriptional regulation of the Sex combs reduced homeotic gene
Assays in transgenes in Drosophila have previously identified cis-acting transcriptional regulatory elements from homeotic genes. Specifically, the assays have identified both tissue-specific enhancer elements and cis-regulatory elements that are required for the maintenance of activation or repression throughout development. While the transgenic assays have been important in defining the structure of the cis-regulatory elements and identifying trans-acting factors that bind to them, their functions within the context of the endogenous genes are still not well understood. We have used several existing chromosomal rearrangements in the Sex combs reduced homeotic gene to investigate the functions of the cis-acting elements within the endogenous gene. The rearrangements identified an imaginal leg enhancer about 35 kb upstream of the Sex combs reduced promoter. The enhancer can activate transcription of both the Sex combs reduced promoter that is 35 kb distant and the Sex combs reduced promoter on the homologous chromosome. Even though the imaginal leg enhancer can activate transcription in all three pairs of legs, it is normally silenced in the second and third pairs and 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, the two 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. From the regions that include the regulatory elements required for the maintenance of silencing, we have identified two clusters of DNA fragments that promote pairing-sensitive silencing (an assay for interaction of cis-regulatory DNA fragments). To assay the regulatory elements’ role in transcriptional regulation, we are using targeted gene replacement to delete both in the endogenous gene.
Trans-acting activators and repressors of homeotic genes
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 phenotypes’ loss of function or ectopic expression of the homeotic genes. We have generated over 4,000 lethal mutants and, among those that die late in development, identified two dozen with homeotic phenotypes. These mutants 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 Pop2, which encodes a subunit of an mRNA deadenylase. Pop2 is required for both function of the Sex combs reduced gene and melanotic tumor suppression.
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 to man. To understand further the function of the Brahma complex, we have been characterizing genes that interact with brahma. One of these interacting genes, female-sterile homeotic, encodes a bromodomain protein that interacts with regulatory elements near the Ultrabithorax homeotic gene promoter. The interaction appears to be important in rendering the homeotic promoter accessible to interactions with proteins bound to distant regulatory elements. We have shown that another of the brahma-interacting 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.
Chang YL, King B, Lin SC, Kennison JA, Huang DH. A double-bromodomain protein, FSH-S, activates the homeotic gene Ultrabithorax through a critical promoter-proximal region. Mol Cell Biol 2007;27:5486-98.
Stultz BG, Jackson DG, Mortin MA, Yang X, Beachy PA, Hursh DA. Transcriptional activation by extradenticle in the Drosophila visceral mesoderm. Dev Biol 2006;290:482-94.
Regulation of nuclear import and export of homeodomain proteins
The prospero homeodomain protein in Drosophila is required for patterning the embryonic nervous system. Both nuclear import and export affect the subcellular localization of prospero while both DNA binding and nuclear export require the prospero homeodomain. We identified the protein encoded by caliban through 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, and the amino terminus binds to Exportin. Both interactions are required for the nuclear export of the prospero homeodomain. Non-cancerous human lung cells have functional caliban, but 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. The expressions of several genes, including interleukin-8, are higher in caliban-expressing cells. We have deleted the Drosophila caliban gene by homologous gene replacement. Even though flies lacking the caliban protein are viable and fertile, they develop more slowly and are more susceptible to tumor formation following irradiation.
Bi X, Jones T, Abbasi F, Lee H, Stultz B, Hursh DA, Mortin MA. Drosophila caliban, a nuclear export mediator, can function as a tumor suppressor in human lung cancer cells. Oncogene 2005;24:8229-39.
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.
