Gene-Centered Regulatory Network Mapping - Caenorhabditis Elegans: Molecular Genetics and Development - page 275

The Y1H system uses a reporter gene readout in yeast to detect interactions
between a ''DNA bait'' and a ''protein prey'' (e.g., TF) ( Fig. 4 ). The first step in
Y1H assays involves the selection of the DNA bait. In most of the cases, this will be a
gene promoter or a small cis-regulatory element. Next, the DNA bait is cloned
upstream of two reporter genes, HIS3 and LacZ ( Fig. 4 ). Traditionally, this was done
by restriction enzyme/ligation-based methods ( Li and Herskowitz, 1993 ). However,
this is difficult to standardize and thus not amenable to the high-throughput settings
that are required for regulatory network studies. To enable high-throughput cloning
of DNA baits, we have combined the Y1H system with Gateway cloning, a recom-
bination-based method that is compatible with the Promoterome resource
( Deplancke et al., 2004 ). With this method, multiple DNA baits can be transferred
to the Y1H reporter Destination vectors simultaneously (e.g., in 96-well plates).
After cloning, the two DNA bait::reporter constructs are linearized and integrated
into the genome of a suitable yeast strain. DNA bait::HIS3 constructs are integrated
into a mutant HIS3 locus and plated on media lacking histidine. There is enough
background His3 expression conferred by the basal yeast promoter present in the
DNA bait::reporter constructs to enable growth on media lacking histidine. When
the same construct is used in a protein-DNA interaction assay, however, the media
are supplemented with 3-aminotriazole (3AT), a competitive inhibitor of the His3
enzyme. That way, the growth of the yeast depends on an increase in expression of
His3, conferred by an interacting AD-TF hybrid protein (see below and Fig. 4 ). DNA
bait::LacZ constructs contain a wild-type URA3 gene and are integrated into a
mutant URA3 locus, thereby rescuing the Ura3 deficiency when plated on media
lacking uracil. The DNA bait::reporter constructs do not carry a yeast origin of
replication and, therefore, the formation of colonies is strictly dependent on their
Fig. 4 Cartoon of yeast one-hybrid assays. AD - Gal4 transcription activation domain; TF - tran-
scription factor.