Sci. Aging Knowl. Environ., 2 October 2002
Vol. 2002, Issue 39, p. pe15
[DOI: 10.1126/sageke.2002.39.pe15]

PERSPECTIVES

Setting a Trap for Aging-Related Genes in Drosophila

John Tower

The author is in the Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA. E-mail: jtower{at}usc.edu

http://sageke.sciencemag.org/cgi/content/full/sageke;2002/39/pe15

Key Words: enhancer trap • Drosophila • P element • gene expression

Introduction

In the October 2002 issue of the new journal Aging Cell, Seroude et al. report results of a study in which enhancer trapping was used to identify Drosophila genes whose expression changes with age. In this Perspective, I discuss the enhancer trap technique and the implications of the results of Seroude et al. with respect to aging in Drosophila.

P Elements and the Enhancer Trap Technique

The chromosome is a crowded place, chock full of genes and their transcriptional regulatory sequences. The latter include enhancer sequences that often act over considerable distances to control the tissue and temporal specificity of gene transcription. In addition to genes and enhancers, the genome of the fruit fly Drosophila melanogaster has various types of transposable elements, including those in the P family. P elements are widely used as versatile tools for manipulating the Drosophila genome.

For example, a reporter or transgene [such as {beta}-galactosidase ({beta}-Gal)] can be inserted into a P element, which is then integrated into the Drosophila genome at random via P-element transformation. For transformation, the P element (typically in a plasmid) is injected into embryos along with transposase, an enzyme that facilitates movement of the P element from the plasmid to a site in the genome of the embryo. Transcription of the transgene within the P element often is affected by regulatory DNA sequences near the integration site of the P element, a phenomenon called chromosomal position effects (PE). Although PE are usually a pain for anyone trying to study the transcriptional activity of a particular transgenic construct, they can be used to the researchers' advantage as a tricky way to detect the presence of enhancers with interesting regulatory properties. Using a transgene with a weak promoter maximizes its sensitivity to PE.

A transgene-containing P element can be readily mobilized to new positions in the genome to create individual Drosophila strains, each of which contains a single new insertion. At certain positions in the genome, the transgene will acquire intriguing patterns of expression due to the influence of enhancer(s) near the insertion site--a technique called "enhancer trapping" (1, 2).

Caveats of the Enhancer Trap Techique

A caveat of the enhancer trap technique is that without further experimentation, it is unclear whether the interesting changes in {beta}-Gal expression observed with the enhancer traps reflect changes in the expression patterns of actual genes near the insertion sites. Previous studies of changes in enhancer trap expression patterns during development have determined that the observed variations can sometimes reflect quite accurately the expression patterns of neighboring genes. However, enhancer traps can also exhibit only a subset of the expression pattern(s) of an adjacent gene or can sometimes exhibit novel expression patterns. One reason for this is that genes often have multiple enhancers, each of which controls expression in a particular tissue and/or at a particular time in the animal's life, and the enhancer trap may fall under the influence of only a subset of these regulatory elements. The net result of these events is that expression of the enhancer trap differs from that of the nearby gene. One way to confirm that expression of the nearby gene does indeed change with age as indicated by {beta}-Gal expression is by directly measuring the abundance of its RNA during aging. This can be achieved with Northern blots; quantitative reverse transcription-polymerase chain reaction; or, more recently, DNA microarrray technology.

Another consideration is whether the observed changes represent modulation of transcriptional activity or some other process. Because the RNA product of the enhancer trap is the same in each strain created, differences between strains in expression levels in the same tissue most likely reflect changes at the level of transcription of the enhancer trap. However, differences in expression patterns between strains, where expression is in different tissues, might include contributions from tissue-specific differences in RNA stability, translation, and {beta}-Gal protein stability.

Using the Enhancer Trap to Identify Changes in Gene Expression with Age

In their study, Seroude et al. used the Drosophila enhancer trap technique to search for genes whose expression is altered during aging (Fig. 1). First, the investigators generated a P element with a transgene that encoded the yeast transcription factor Gal4. They then mobilized the P-element construct to 180 different positions in the genome in hopes of trapping enhancers that would yield aging-specific patterns of expression. Each line was then crossed with a strain that carried a reporter construct in which the Gal4 DNA binding site, called the upstream activating sequence or UAS, was part of a promoter that controlled the expression of the adjacent lacZ gene, which encodes {beta}-Gal. In the newly created strains, the trapped enhancer drives the expression of Gal4 in some interesting (hopefully age-specific) pattern, and then Gal4 drives the expression of {beta}-Gal in the same pattern. This two-component system for driving expression of a gene such a lacZ is called the Gal4/UAS system (3). {beta}-Gal expression can then be easily detected as blue-staining tissue when fly sections are incubated with the appropriate substrate (X-Gal), or quantitated when fly extracts are incubated with the appropriate substrate (chlorophenol red-{beta}-D-galactopyranoside or CPRG) in a spectrophotometer cuvette.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. Scheme for monitoring transcriptional activity via an enhancer trap approach based on the GAL4-UAS system. A collection of GAL4 enhancer-trap lines referred to as DJ lines was generated by mobilizing a transposable element (P{GawB}). Each strain was crossed to a UAS-lacZ reporter and progeny reared at 25°C. Quantitative measurements of total lacZ activity were performed using homogenized whole flies. The spatio-temporal pattern of expression was assessed across life-span using X-Gal staining on cryo-sectioned adult flies. [Figure from the journal Aging Cell. http://www.aging-cell.com Reproduced by kind permission of Blackwell Publishing and the Anatomical Society of Great Britain and Ireland.]

 
Out of 180 lines examined, ~80% displayed some change in {beta}-Gal expression during aging. A variety of tissue-specific patterns of gene expression were observed, and these changes included increases in expression with age, decreases in expression with age, or both increases and decreases. Similar observations had been made in an earlier study of enhancer trap {beta}-Gal staining during aging in the Drosophila antenna (4). And in another related analysis, Pletcher et al. used the Affymetrix GeneChip to measure the changes in abundance of virtually all Drosophila RNAs during aging (5). A drawback of this approach is that whole flies, rather than fly sections, are assayed. Therefore, all the subtleties of tissue-specific expression patterns are lost, and one gets an average picture of gene expression over the whole fly. Even so, the GeneChip analysis confirms the up-regulation with age of at least one of the genes identified by Seroude et al., namely the peptidoglycan recognition protein LC (PGRP-LC) immune response gene.

The 180 enhancer trap lines assayed represent about 1.3% of the ~13,600 genes in the Drosophila genome. What might this sampling tell us about changes in the expression of the Drosophila genome with age? One thing to keep in mind is that P-element insertion is a semi-random process, with an apparent preference for genes that are expressed or in an open chromatin conformation in the dividing germline cells that give rise to sperm (6). Some genes cannot be mutated by P-element insertion and some are hotspots for insertion, yielding a somewhat less than random sampling of the genome (7). In most cases, expression of the enhancer traps decreased with age, and the authors speculate that this might reflect a global decrease in transcription and/or protein synthesis. Another related possibility is that a smaller fraction of the {beta}-Gal enzyme molecules are active in old flies, as has been observed for other enzymes in aging organisms (8, 9). If there is a global down-regulation at some level of gene expression, it just makes the lines with increased expression all the more striking.

Several lines were identified in which expression of the enhancer trap went up with age or went up and down in a characteristic pattern. In most cases, the nearby gene is expected to have increased transcription with age as well. However, as described above, there are mechanisms by which an enhancer trap could have increased expression with age even if the nearby gene does not. A possible precedent comes from studies of the gene encoding human blood coagulation factor IX. Promoter mapping in transgenic mice revealed that certain transcriptional control elements lost activity with age, whereas other transcriptional control elements apparently increased activity with age (10). The net result for the factor IX transgenes was maintenance of constant transcription with age. If a Drosophila gene had an analogous pattern of regulation, an enhancer trap insertion might fall under the influence of only one of the two types of elements. Of course, this scenario could be as interesting and informative as identifying a gene whose RNA levels change with age.

Revelations and Implications

This brings us to the first of several exciting revelations in the Seroude et al. paper: the intricate variety of dynamic tissue-specific patterns observed. The insertions and their molecular characterization should provide an entree into a wealth of aging-regulated transcriptional control elements. The dynamic patterns indicate that tissues go through a characteristic series of states or steps in the adult aging process. Studying the sequences and factors that regulate this aging-specific transcriptional regulation should tell us a great deal about the nature of the aging process in different tissues. A precedent comes from the study of Drosophila heat shock protein (hsp) genes. A subset of the hsp's, including hsp70 and hsp22, have been found to be induced at the RNA and protein levels in tissue-specific patterns during aging (11-13). Studies of the hsp transcriptional regulatory sequences in transgenic flies revealed that heat shock response elements, or HSEs, were required for induction during aging. The enhancer traps identified by Seroude et al. do not appear to be responding to HSEs, and therefore should help investigators to identify additional transcriptional regulatory elements that respond to aging.

Another exciting implication of the results of Seroude et al. is the identification of candidate biomarkers of aging. Currently, the best and virtually only metric of aging rate is life-span. Life-span is problematic as an assay because it takes a long time to measure, and, once the animal is dead, it limits the types of experiments you can do with it. A biomarker of aging that could be scored earlier in the life-span of the fly would have numerous applications and expedite experiments. If such biomarkers were conserved in humans, they would likely have medical applications. The expression of several of the enhancer traps identified by Seroude et al. was found to scale with life-span as altered by temperature. In cold-blooded invertebrates such as Drosophila, cooler culture temperatures reduce metabolic activity and lengthen life-span, whereas warmer temperatures increase metabolic activity and shorten life-span (14). For some of the enhancer traps, the time course of expression was changed in proportion to the change in life-span, suggesting that their expression is a function of physiological age. This is in agreement with what has previously been observed for enhancer trap expression in the antenna (4) and for hsp70 and hsp22 expression (11, 12). To determine the value of these enhancer traps as potential biomarkers, further characterization should include the effect on expression of other interventions that alter life-span, such as dietary restriction, single gene mutations, and drugs. One current limitation of the enhancer traps and hsp's as biomarkers is that the reporter used, lacZ, can only be scored in a sacrificed fly, so it is not yet possible to determine whether expression can actually predict life-span. However, the ideal technology now exists: the green fluorescent protein (GFP) and its other-color relatives, which can be visualized in live flies. Fig. 2 shows transgenic flies that carry a construct that allows GFP to be expressed specifically in eye tissue (15), and experiments in my laboratory show that this expression can readily be quantitated from captured images (16). The combination of GFP reporters with the panel of enhancer trap lines characterized by Seroude et al. should take us one step closer to finding the elusive biomarkers of Drosophila aging.



View larger version (88K):
[in this window]
[in a new window]
 
Fig. 2. GFP expression visualized in live Drosophila. Young adult Drosophila transgenic for an eyeless enhancer-GFP reporter construct (15) were anesthetized with CO2 gas and photographed with a Leica MZFLIII fluorescence dissecting microscope.

 
An equally stirring implication of the work described herein is the possibility that the enhancer traps may lead to the identification of genes with a role in regulating aging and life-span. All other things being equal, a set of genes whose expression is regulated or altered during aging is more likely to have aging-related functions than genes whose expression is not altered during aging. An example that illustrates this maxim comes from yeast, in which the first gene identified to affect yeast replicative life-span, LAG1, was identified initially on the basis of its differential expression during yeast aging (17). The PGRP-LC gene identified by Seroude et al. is a promising candidate for a gene with an important function during aging: PGRP-LC is a peptidoglycan recognition protein required for the induction of antibacterial peptide genes in response to infection (18). The PGRP family of genes, as well as numerous other immune-response genes, are induced during aging (5), suggesting that aging may be associated with an increased pathogen load.

Finally, these enhancer trap lines have an immediate practical value in studies of aging in Drosophila: They constitute a means by which to drive the expression of transgenes in a variety of tissue-specific patterns during adult aging. Instead of driving expression of the lacZ reporter, the same system can be used to drive expression of any gene of interest. For example, this Gal4/UAS system has been used to show that expression of human Cu/Zn superoxide dismutase (Cu/ZnSOD) preferentially in adult motor neurons can extend Drosophila life-span (19). The enhancer trap patterns characterized by Seroude et al. increase our arsenal of tissue-specific patterns that can be generated to study the function of genes such as SOD and others during the aging process.


October 2, 2002
  1. H. J. Bellen, P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev . 3, 1288-1300 (1989).
  2. C. J. O'Kane, W. J. Gehring, Detection in situ of genomic regulatory elements in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 84, 9123-9127 (1987).[Abstract/Free Full Text]
  3. A. H. Brand, N. Perrimon, Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415 (1993).[Abstract]
  4. S. L. Helfand, K. J. Blake, B. Rogina, M. D. Stracks, A. Centurion, B. Naprta, Temporal patterns of gene expression in the antenna of the adult Drosophila melanogaster. Genetics 140, 549-555 (1995).[Abstract/Free Full Text]
  5. S. D. Pletcher, S. J. Macdonald, R. Marguerie, U. Certa, S. C. Stearns, D. B. Goldstein, L. Partridge, Genome-wide transcript profiles in aging and calorically restricted Drosophila melanogaster. Curr. Biol. 12, 712-723 (2002).[CrossRef][Medline]
  6. M. Bownes, Preferential insertion of P elements into genes expressed in the germ-line of Drosophila melanogaster. Mol. Gen. Genet. 222, 457-460 (1990).[CrossRef][Medline]
  7. A. C. Spradling, D. M. Stern, I. Kiss, J. Roote, T. Laverty, G. M. Rubin, Gene disruptions using P transposable elements: An integral component of the Drosophila genome project. Proc. Natl. Acad. Sci. U.S.A. 92, 10824-10830 (1995).[Abstract/Free Full Text]
  8. E. R. Stadtman, Protein oxidation and aging. Science 257, 1220-1224 (1992).[Abstract/Free Full Text]
  9. M. Rothstein, The formation of altered enzymes in aging animals. Mech. Ageing Dev. 9, 197-202 (1979).[CrossRef][Medline]
  10. S. Kurachi, Y. Deyashiki, J. Takeshita, K. Kurachi, Genetic mechanisms of age regulation of human blood coagulation factor IX. Science 285, 739-744 (1999).[Abstract/Free Full Text]
  11. J. C. Wheeler, E. T. Bieschke, J. Tower, Muscle-specific expression of Drosophila hsp70 in response to aging and oxidative stress. Proc. Natl. Acad. Sci. U.S.A. 92, 10408-10412 (1995).[Abstract/Free Full Text]
  12. V. King, J. Tower, Aging-specific expression of Drosophila hsp22. Dev. Biol. 207, 107-118 (1999).[CrossRef][Medline]
  13. J. C. Wheeler, V. King, J. Tower, Sequence requirements for upregulated expression of Drosophila hsp70 transgenes during aging. Neurobiol. Aging 20, 545-553 (1999).[CrossRef][Medline]
  14. J. Miquel, P. R. Lundgren, K. G. Bensch, H. Atlan, Effects of temperature on the life span, vitality and fine structure of Drosophila melanogaster. Mech. Ageing Dev. 5, 347-370 (1976).[CrossRef][Medline]
  15. A. J. Berghammer, M. Klingler, E. A. Wimmer, A universal marker for transgenic insects. Nature 402, 370-371 (1999).[CrossRef][Medline]
  16. J. Yang, J. Tower, unpublished data.
  17. N. P. D'mello, A. M. Childress, D. S. Franklin, S. P. Kale, C. Pinswasdi, S. M. Jazwinski, Cloning and characterization of LAG1, a longevity-assurance gene in yeast. J. Biol. Chem. 269, 15451-15459 (1994).[Abstract/Free Full Text]
  18. K.-M. Choe, T. Werner, S. Stoven, D. Hultmark, K. V. Anderson, Requirement for a peptidoglycan recognition protein (PGRP) in relish activation and antibacterial immune responses in Drosophila. Science 296, 359-362 (2002).[Abstract/Free Full Text]
  19. T. L. Parkes, A. J. Elia, D. Dickson, A. J. Hilliker, J. P. Phillips, G. L. Boulianne, Extension of Drosophila lifespan by overexpression of human SOD1 in motor neurons. Nat. Genet. 19, 171-174 (1998).[CrossRef][Medline]
Citation: J. Tower, Setting a Trap for Aging-Related Genes in Drosophila. Science's SAGE KE (2 October 2002), http://sageke.sciencemag.org/cgi/content/full/sageke;2002/39/pe15








Science of Aging Knowledge Environment. ISSN 1539-6150