Helen Green, PhD, Postdoctoral Fellow
Monica T. Cooper, Senior Research Technician
Der-Hwa Huang, PhD, Guest Researcher

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 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 required for transcriptional regulation of the Sex combs reduced homeotic gene

Kennison, Cooper

Assays in transgenes in Drosophila have previously identified cis-acting transcriptional regulatory elements from the homeotic genes. The assays have identified both tissue-specific enhancer elements as well as cis-regulatory elements that are required for the maintenance of activation or repression throughout development. While these transgene assays have been important in defining the structure of the cis-regulatory elements and in identifying trans-acting factors that bind to them, their functions within the context of 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. This imaginal leg enhancer can activate transcription not only of the Sex combs reduced promoter that is 35 kb distant but also of the Sex combs reduced promoter on the homologous chromosome. This trans-activation 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 50 years ago and has highly 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 retrotransposon. We have used an unlinked genetic suppressor of Springer to show that the unusual genetic properties are actually attributable to the Springer insertion. We have identified a 400 base pair region of the Springer retrotransposon that functions as a homeotic repression element in a transgene assay. 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.

Kennison JA, Southworth JW. Transvection in Drosophila. Adv Genet 2002;46:399-420. Southworth JW, Kennison JA. Transvection and silencing of the Sex combs reduced homeotic gene of Drosophila melanogaster. Genetics 2002;161:733-746.

Trans-acting activators of homeotic genes

Kennison, Green, Cooper; in collaboration with Vázquez, Zurita 
Genetic studies have identified the trithorax group of genes as required for expression or function of the 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. We have identified at least two dozen trithorax group genes, most of which were not previously known. Two of the genes (skuld and kohtalo) encode subunits of the mediator coactivator complex. This 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. For example, as part of these studies, we have isolated and characterized mutations in the Asf1 histone chaperone as well as in the Drosophila homolog of the ELL RNA polymerase II transcriptional elongation factor [the Su(Tpl) gene]. We have recently identified two other genes (taranis and tonalli) that show genetic interactions with Brahma complex mutations. Both taranis and tonalli encode multiple protein isoforms. Some of the tonalli protein isoforms are particularly interesting, as they include an SP-RING finger domain that may function as a SUMO E3 ligase. SUMO is a small protein that is conjugated to target proteins to alter their function, cellular localization, or stability. The SUMO E3 ligases specify which target proteins get sumoylated. The genetic interactions with tonalli suggest that sumoylation may play an important role in regulating the function of the Brahma chromatin-remodeling complex.

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.

Gutiérrez L, Zurita M, Kennison JA, Vázquez M. The Drosophila trithorax group gene tonalli (tna)

interacts genetically with the Brahma remodeling complex and encodes an SP-RING finger protein. Development 2003;130:343-354.

Moshkin YM, Armstrong JA, Maeda RK, Tamkun JW, Verrijzer CP, Kennison JA, Karch F. Histone

chaperone ASF1 cooperates with the Brahma chromatin-remodelling machinery. Genes Dev 2002;16:2621-2626.

Trans-acting repressors of homeotic genes

Kennison; in collaboration with Kassis

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. We have identified a number of homeotic repressors, including the Su(z)12, Mi-2, and Deaf-1 genes. To identify new Polycomb group repressors, we are screening for new mutations that either interact genetically with Polycomb mutations or mimic the homeotic phenotypes of Polycomb group mutations. We are conducting the screens in collaboration with Judy Kassis and the Section on Gene Expression, also within the Laboratory of Molecular Genetics.

Birve A, Sengupta AK, Beuchle D, Larsson J, Kennison JA, Rasmusson-Lestander A, Muller J.

Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants. Development 2001;128:3371-3379.

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.

COLLABORATORS

Judy A. Kassis, PhD, Laboratory of Molecular Genetics, NICHD, Bethesda MD 
Martha Vázquez, PhD, Instituto de Biotecnologîa, UNAM, Cuernavaca, Mexico 
Mario Zurita, PhD, Instituto de Biotecnologîa, UNAM, Cuernavaca, Mexico 

For further information, contact kennisoj@exchange.nih.gov