Biology Reference
In-Depth Information
Excellent examples of how expression analyses have facilitated identification
of the genetic networks controlling diverse biological processes include aging
(
Budovskaya et al., 2008; Murphy et al., 2003
), development (
Baugh et al., 2009
),
and innate immunity (
Styer et al., 2008
). Illustrating its utility in the aging field,
Budovskaya et al. (2008)
recently discovered a development-related transcrip-
tional circuit that guides the aging process. By comparing DNA microarray
profiles, they identified a common set of 1254 genes that showed age-dependent
expression changes (both upregulated and downregulated) during normal aging.
This pattern of gene expression was found to be shared with dauer larvae (devel-
opmentally arrested worms whose life spans are ten times longer than normal
worms), and longevity mutants displaying either extended or shortened life spans.
For example, genes that show increased expression with age tend to have
increased expression in dauer larvae and long-lifespan mutants, but show
decreased expression in short-lifespan mutants. To search for transcription factors
that regulate these age-dependent expression changes, they analyzed the upstream
regulatory regions of these 1254 genes and identified a common consensus motif
recognized by a GATA transcription factor, elt-3, in 602 of them. The importance
of elt-3 was validated by showing that RNAi depletion of elt-3 activity resulted in
decreased expression of 12 representative GATA-site-containing age-regulated
genes. The expression of elt-3 itself over the normal life span was negatively
regulated by two other GATA transcription factors, elt-5 and elt-6 (
Fig. 5
), which
were previously known to function with elt-3 to regulate hypodermis differenti-
ation in embryos (
Gilleard et al., 1999; Gilleard and McGhee, 2001
). Consistent
with a role for these transcription factors in regulating longevity, loss of elt-3
suppressed the long-lifespan phenotype of a longevity mutant of daf-2 (encoding
a C. elegans insulin/IGF receptor), whereas elt-5 or elt-6 promoted aging as
reduction in activity of either elt-5 or elt-6 caused lifespan extension. Thus, using
a combination of transcriptional profiling, cis-regulatory analysis, and reverse
genetic approaches for validation, this study identified a development-related
transcriptional circuit consisting of three GATA transcription factors and revealed
its novel role in regulating aging late in life.
3. Protein Interaction Screens
Many genetic interactions are realized in the form of direct protein-protein
interactions. Analyzing interactions among proteins not only provides insight into
the function of their corresponding genes, but also helps unravel the genetic topology
of pathways and networks regulating biological processes. To acquire interaction
information between proteins in C. elegans, two strategies are often employed: yeast
two-hybrid (Y2H) screens (
Fields and Song, 1989
) and affinity-based protein iso-
lation coupled with mass spectrometry (MS) analysis. In a typical Y2H assay, one
protein is used as a bait and fused with the DNA-binding domain of a transcription
factor, whereas the other protein functions as a prey and is fused with the activating
domain of the transcription factor. These two fusion proteins are introduced into the
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