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REGULATION OF HOMEOTIC GENES IN DROSOPHILA
     
James A. Kennison, Ph.D., Head, Section on Drosophila Gene Regulation
Helen Green, Ph.D., Postdoctoral Fellow
Monica T. Cooper, Senior Research Technician
Jeffrey W. Southworth, Senior Research Techniciana
Daniel Segal, Ph.D., Guest Researcherb
James A. Kennison's photograph
 

Our goal is to understand the regulation of homeotic gene function in Drosophila. The homeotic genes specify segmental identities in Drosophila 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 segmental identities. Both loss of expression and ectopic expression in the wrong tissues lead to changes in segmental identities. These changes in identity provide a powerful assay 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 extremely important model system for the study of human development and disease.

Cis-Acting Sequences Required for Transcriptional Regulation of the Sex combs reduced Homeotic Gene
Kennison, Cooper, Southworth
Assays in transgenes in Drosophila have identified cis-acting transcriptional regulatory elements from homeotic genes. The 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 the transgene 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 contexts of the endogenous genes are not yet 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. The chromosomal rearrange-ments identified an imaginal leg enhancer about 35 kb upstream of the Sex combs reduced promoter. The imaginal leg enhancer can not only 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. The trans-activation phenomenon was first observed for the homeotic gene Ultrabithorax and named transvection. 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. To validate our model, we have characterized a transposable element insertional mutation that was isolated 49 years ago and has unusual genetic properties. The transposable element is inserted about 150 kb upstream of the Sex combs reduced promoter, and we believe that the unusual genetic properties of the insertion derive from its ability to mimic the endogenous genetic elements required for transcriptional repression. We have identified the transposable element as the Drosophila Springer retrotrans-poson and have used an unlinked genetic suppressor of Springer to show that the unusual genetic properties are actually attributable to the Springer insertion. We are currently trying to identify the region of the Springer retrotransposon that is able to mimic the endogenous Sex combs reduced repression elements. We believe that comparisons between the Springer sequences and the sequences of the endogenous elements should reveal target sites that interact with the trans-acting factors.

Trans-Acting Activators of Homeotic Genes
Kennison, Green, Cooper, Segal; in collaboration with Eissenberg, Shilatifard, Christensen, Karch, Tamkun, Verrijzer
Genetic studies have identified the trithorax group of genes required for expression or function of homeotic genes. Reduced function of the trithorax group genes mimics loss of function of the homeotic genes. Many of the trithorax group genes have been shown to be required for the maintenance of transcription of the homeotic genes during development. Many trithorax group proteins are subunits of chromatin-remodeling or transcriptional coactivator complexes. Our section has identified at least two dozen trithorax group genes, most of which were not previously identified. Two of the genes that we identified (skuld and kohtalo) encode subunits of the mediator coactivator complex. The complex is highly conserved between Drosophila and man, but only about a third of the subunits are conserved between yeast and man. We have identified several other 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. As part of these studies, we have recently isolated and characterized mutations in the Asf1 histone chaperone. We have shown that Asf1 is important for the structure of heterochromatin and that it interacts both functionally and physically with the Brahma chromatin-remodeling complex. We have also isolated and characterized mutations in Su(Tpl), which encodes the Drosophila homolog of the ELL RNA polymerase II transcriptional elongation factor. We have shown that Su(Tpl) is required for the transcription of multiple developmental genes, including the Sex combs reduced homeotic gene. Su(Tpl) is within the first intron of the Mi-2 gene, which is required for transcriptional repression of the homeotic genes. The regulation of Su(Tpl) and Mi-2 should prove interesting in that both are transcribed from the same DNA strand and in the same cells. We have isolated insertions of a transposon that differentially interfere with the functions of the two genes. The insertional mutations are at the same site in the DNA and differ only in the structure of the transposon.

Trans-Acting Repressors of Homeotic Genes
Kennison; in collaboration with Müller, Rasmuson-Lestander, McGinnis
The initial domains of homeotic gene repression are set the by the segmentation proteins, which also divide the embryo into segments. Maintenance of repression requires the proteins encoded by the Polycomb group genes. The switch from the initiation to the maintenance of repression involves the recruitment of a chromatin-remodeling complex that includes the brahma- and kismet-related protein Mi-2 and a histone deacetylase subunit. In addition, maintenance requires the Polycomb complex, which also has a histone deacetylase subunit. In collaboration with J. Müller and A. Rasmuson-Lestander, we have identified and characterized a new Polycomb group gene, Su(z)12. Su(z)12 encodes a zinc-finger protein that is required both maternally and zygotically for the maintenance of homeotic gene repression. In collaboration with W. McGinnis, we have also isolated and characterized mutations in the Deaf-1 gene. The DEAF-1 protein was originally identified as a factor that bound to a cis-regulatory enhancer element from the Deformed homeotic gene. Although Deaf-1 is essential for embryonic development, its precise role in transcription is still not understood.
 

SELECTED PUBLICATIONS

  1. Birve A, Sengupta AK, Beuchle D, Larsson J, Kennison JA, Rasmuson-Lestander A, Müller J. Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants. Development. 2001;128:3371-3379.

  2. Eissenberg JC, Ma J, Gerber MA, Christensen A, Kennison JA, Shilatifard A. dELL is an essential RNA polymerase II elongation factor with a general role in development. Proc Natl Acad Sci USA. 2002;99:9894-9899.

  3. Kennison JA, Southworth JW. Transvection in Drosophila. Adv Genet. 2002;46:399-420.

  4. Moshkin YM, Armstrong JA, Maeda RK, Tamkun JW, Verrijzer CP, Kennison JA, Karch F. Link of histone chaperone ASF1 to chromatin-remodelling machinery. Genes Dev. 2002;16:2621-2626.

  5. Schulze S, Sinclair DAR, Silva E, Fitzpatrick KA, Singh M, Lloyd VK, Morin KA, Kim J, Holm DG, Kennison JA, Honda BM. Essential genes in proximal 3L heterochromatin of Drosophila melanogaster. Molec Gen Genet. 2001;264:782-789.

  6. Southworth JW, Kennison JA. Transvection and silencing of the Sex combs reduced homeotic gene of Drosophila melanogaster. Genetics. 2002;161:733-746.

  7. Veraksa A, Li X, Kennison J, McGinnis W. DEAF-1 function is essential for the early embryonic development of Drosophila. Genesis. 2002;33:67-76.

aLeft NIH in 2000
bLeft NIH in 2002

Collaborators

Alan Christensen, Ph.D., University of Nebraska, Lincoln, NE
Joel C. Eissenberg, Ph.D., St. Louis University School of Medicine, St. Louis, MO
François Karch, Ph.D., University of Geneva, Geneva, Switzerland
William McGinnis, Ph.D., University of California, San Diego, La Jolla, CA
Jürg Müller, Ph.D., EMBL, Heidelberg, Germany
Asa Rasmuson-Lestander, Ph.D., Umea University, Umea, Sweden
Ali Shilatifard, Ph.D., St. Louis University School of Medicine, St. Louis, MO
John W. Tamkun, Ph.D., University of California, Santa Cruz, CA
Peter Verrijzer, Ph.D., Leiden University Medical Centre, Leiden, The Netherlands