Regulation of Homeotic Gene Function in Drosophila

James A. Kennison, PhD, Head, Section on Drosophila Gene Regulation
Mark Mortin, PhD, Staff Scientist
Monica T. Cooper, BA, Senior Research Technician
Nader Jameel, BS, Postbaccalaureate Fellow¹

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 provide powerful assays for identifying 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 human. 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

Kennison, Cooper, Jameel

Assays in transgenes in Drosophila previously identified cis-acting transcriptional regulatory elements from the homeotic genes. The assays 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 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 remain poorly understood. We 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 rearrangements identified an imaginal leg enhancer about 35 kb upstream of the Sex combs reduced promoter. This enhancer is not only able to 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. 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. The 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 tested 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). We are using targeted gene replacement to delete these clusters of elements in the endogenous gene to assay their role in transcriptional regulation.

Trans-acting activators and repressors of homeotic genes

Kennison, Mortin, Cooper; in collaboration with Vázquez, Zurita, Huang, Dorsett, Brock, Honda

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, have identified two dozen mutants with homeotic phenotypes. These mutants identify genes required for expression or function of the homeotic genes (Kennison, Methods Enzymol 2004;377:61).

Reduced function of the trithorax group genes mimics loss of function of the homeotic genes. We 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 human. To understand further the function of the Brahma complex, we have been isolating and characterizing mutations that interact with brahma. One of the 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 for rendering the homeotic promoter accessible to interactions with proteins bound to distant regulatory elements. The characterization of two other brahma-interacting genes, verthandi and Pearl, suggests that two protein complexes known to be important for chromosome segregation during mitosis also play important roles in transcriptional regulation. We have shown that verthandi encodes the RAD21 subunit of the cohesin complex. The cohesin complex is required for proper segregation of sister chromatids during anaphase. We have also shown that Pearl encodes one of the two gamma-tubulin isoforms in Drosophila. During the characterization of Pearl, we 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. Our results demonstrate that gamma-tubulin and microtubule organizer complexes are required not only for chromosome segregation during mitosis but may also play a role in transcriptional regulation.

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;27:5486-5498.
Hallson G, Syrzycka M, Beck SA, Kennison JA, Dorsett D, Page SL, Hunter SM, Keall R, Warren WD, Brock HW, Sinclair DAR, Honda BM. The Drosophila cohesin subunit Rad21 is a trithorax group (trxG) protein. Proc Natl Acad Sci USA 2008;105:12405-12410.
Vázquez M, Cooper MT, Zurita M, Kennison JA. γTub23C interacts genetically with Brahma chromatin-remodeling complexes in Drosophila melanogaster. Genetics 2008;180:835-843.

¹Left the laboratory in May 2008.

Collaborators

Hugh W. Brock, PhD, University of British Columbia, Vancouver, Canada
Dale Dorsett, PhD, Saint Louis University School of Medicine, St. Louis, MO
Barry M. Honda, PhD, Simon Fraser University, Burnaby, Canada
Der-Hwa Huang, PhD, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC
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.