Gene-Centered Regulatory Network Mapping - Caenorhabditis Elegans: Molecular Genetics and Development - page 273

worms ( Friedlander et al., 2008; Kato et al., 2009 ). As with TFs, microRNAs can
also be grouped into families, based on their seed sequence, the part with which they
basepair with their target genes. It is not yet clear whether all C. elegans microRNAs
have been identified. Indeed, it may be that additional microRNAs will be uncovered
when the animal is exposed to particular conditions, in males or dauers, or when
sequencing techniques further improve to detect microRNAs of very low abundance.
3. Other Regulators
In addition to TFs and microRNAs, other RNA and protein molecules contribute
to differential gene regulation. These include RNA binding proteins, transcriptional
co-factors, and signaling molecules such as kinases and phosphatases, as well as
endogenous siRNAs and, perhaps, long non-coding RNAs. Systematic computa-
tional and experimental analyses will shed light on the number of molecules in each
class of regulators.
C. Delineating Gene Regulatory Network Edges
1. TF-Target Gene Interactions
Interactions between TFs and their target genes can be identified using two
conceptually different and highly complementary strategies. The first are TF-cen-
tered (protein-to-DNA); they start with a TF of interest and identify the genes with
which this factor interacts. The second are gene-centered (DNA-to-protein); they
start with a gene of interest and identify the TFs with which it interacts ( Fig. 3 ).
2. Transcription Factor-Centered Methods: ChIP
The most widely used TF-centered method is chromatin immunoprecipitation
(ChIP). In ChIP assays, an anti-TF antibody is used to precipitate TFs in vivo.
Briefly, worm extracts are first treated with formaldehyde to crosslink proteins to
proteins and proteins to DNA. After precipitation of the TF, associated DNA mole-
cules can be identified 1) by PCR using primer sets of interest ( Deplancke et al.,
2006a ); 2) by cloning and sequencing ( Oh et al., 2006 ); 3) using microarrays that tile
the entire C. elegans genome ( Tabuchi et al., 2011; Whittle et al., 2009 ); or more
recently 4) by deep sequencing (e.g., 454 or Solexa). Controls include a non-relevant
antibody and, if possible, mutant animals that do not express the TF of interest
( Walhout, 2011 ).
ChIP is a powerful method to identify TF-target gene interactions that occur
in vivo. However, it is mostly limited to TFs that are highly and/or broadly expressed
throughout the lifetime of the animal, and to TFs for which ChIP-grade antibodies
are available. It is, however, also feasible to use ChIP in transgenic animals that
overexpress an epitope-tagged TF. Although ChIP is usually the method of choice
when one is interested in one or a few TFs, it is less suitable when one is interested in