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REGULATION OF HOMEOTIC GENE FUNCTION IN DROSOPHILA
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James A. Kennison, PhD, Head,
Section on Drosophila Gene Regulation Mark Mortin, PhD, Staff
Scientist Helen Green, PhD, Postdoctoral
Fellow Monica T. Cooper, BA, Senior
Research Technician Der-Hwa Huang, PhD, Guest
Researchera Gabriel Band, Summer Student |
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Our
goal is to understand how genes control cell fates during development. The homeotic
genes in Drosophila specify segmental identities at both the embryonic
and adult stages, encoding 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. Such 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 Cooper, Kennison Assays
in transgenes in Drosophila have previously identified cis-acting
transcriptional regulatory elements from the homeotic genes. In particular,
they 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
both defining the structure of the cis-regulatory elements and
identifying trans-acting factors that bind to them, the functions of
the regulatory elements within the context 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.
Such chromosomal rearrangements identified an imaginal leg enhancer about 35
kb upstream of the Sex combs reduced promoter. The imaginal leg
enhancer can activate transcription not only of the Sex combs reduced
promoter that is 35 kb distant but also that of the Sex combs reduced
promoter on the homologous chromosome. This trans-activation
phenomenon was first observed for the homeotic gene Ultrabithorax and
named transvection. While the imaginal leg enhancer can activate
transcription of both the Sex combs reduced promoter 35 kb distant and
on the homologous chromosome, it does not activate transcription from the ftz
gene promoter, which is about half-way between the enhancer and the Sex
combs reduced promoter. 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, the elements interact
with elements on the homologous chromosome and cause derepression of its
wild-type Sex combs reduced gene. To
validate our model, we characterized a transposable element insertional
mutation isolated 50 years ago that has highly unusual genetic properties.
The transposable element is inserted about 150 kb upstream of the Sex comb
reduced promoter; we believe that the unusual genetic properties of this
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 used an
unlinked genetic suppressor of Springer to show that the unusual
genetic properties are indeed attributable to the Springer insertion
and have identified a 400–base pair region of the Springer
retrotransposon that functions as a homeotic repression element in a
transgene assay. We have also begun to test DNA fragments from the Sex
combs reduced gene in transgene assays to identify other cis-regulatory
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. 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
repressors and activators of homeotic genes Mortin, Green, Cooper,
Band, Kennison; in collaboration with Kassis, Vázquez, Zurita The
initial domains of homeotic gene repression are set by the segmentation
proteins, which also divide the embryo into segments. Maintenance of
repression requires proteins encoded by the Polycomb group genes. We have
identified a number of homeotic repressors, including the Su(z)12 and Mi-2
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 have generated
approximately 500 new lethal mutants that die very late in development (after
the formation of the adult cuticle during pupation). Among these new mutants
are six with homeotic phenotypes. Genetic
studies have identified the trithorax group of genes that are 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 newly identified at least two dozen trithorax group
genes. Two such genes (skuld and kohtalo) encode subunits of
the mediator coactivator complex, which is highly conserved between Drosophila
and man, but only about a third of the subunits are conserved between yeast
and man. We have also 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 characterized mutations that interact with mutations
in the Brahma complex and 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
in that 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 are sumoylated. The genetic
interactions with tonalli suggest that sumoylation may play an
important role in regulating the function of the Brahma chromatin-remodeling
complex. We have also isolated a number of new mutations that show very
strong genetic interactions with brahma, osa, taranis,
and tonalli mutations. We are currently mapping these mutations to
identify new genes required for homeotic gene regulation. 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. Kennison JA. Introduction to Trx-G and Pc-G genes. Methods
Enzymol 2004;377:61-70. aLeft the
laboratory in 2003. COLLABORATORS Judy A. Kassis, PhD, Laboratory of
Molecular Genetics, NICHD, Martha Vázquez, PhD, Instituto de
Biotecnología, UNAM, Mario Zurita, PhD, Instituto de
Biotecnología, UNAM,
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