Biology Reference
In-Depth Information
2. False Positives
As with Y2H assays, there are two types of false positives with Y1H assays:
technical false positives that cannot be reproduced in the same assay and biological
false positives that represent genuine Y1H/Y2H interactions that nonetheless do not
occur in vivo. To keep the rate of technical false positives low several issues need to
be taken into consideration. First, it is best to only consider interactions that score
positively for both Y1H reporters, that is, that induce growth on media lacking
histidine and containing 3AT and that are bluer than an ''AD only'' control.
Second, it is important to make sure that the TF retrieved is in frame (only relevant
to cDNA library screens). Third, all Y1H interactions need to be retested in fresh
DNA bait cells (i.e., from a frozen stock that has not been used in the screen itself),
either by gap-repair (
Walhout and Vidal, 2001
) or by directly transforming an AD-
TF clone. This is necessary because baits can mutate in yeast and give rise to a colony
with an apparent interaction phenotype that is not reproducible (
Walhout and Vidal,
1999
). Fourth, it is absolutely critical to integrate DNA bait::reporter constructs into
the yeast genome. We have tried to perform the assay with replicating plasmids, but
the background expression was highly variable, probably due to different plasmid
copy numbers. Finally, it is important to note that interactions obtained with highly
auto-active DNA baits are more difficult to assess and may be less specific. We have
developed an interaction scoring scheme to assess the results obtained from Y1H
library screens (
Vermeirssen et al., 2007a
).
Biological false positives are more challenging to assess. First, the genome itself is
the same in every cell and thus, when a TF is expressed in any given cell one may
expect the interaction to occur. However, the nucleosome occupancy likely varies in
different cell types and this may prevent interactions from occurring in vivo. The
integration of the DNA baits into the yeast genome ensures that they are incorporated
into chromatin and, thus, Y1H assays are not based on interactions with naked DNA.
However, it could be that the integration of the DNA baits in yeast only partially
recapitulates the chromatin state in any C. elegans cell in vivo. In Y1H assays, we can
find multiple members of a TF family binding to a particular DNA bait. This could
be because these members have very similar DNA binding specificities and that this
does not reflect in vivo functionality. However, we, and others, have found that
multiple members of a TF family can bind the same DNA targets in vivo and can
function redundantly (
Hollenhorst et al., 2007; Ow et al., 2008
). For instance,
multiple TFs with a FLYWCH DNA binding domain were found to interact with
microRNA promoters in Y1H assays and to redundantly repress microRNA expres-
sion in the early C. elegans embryo (
Ow et al., 2008
). It is also important to note that
not all TF-DNA interactions lead to a regulatory consequence. For instance, ChIP
has identified numerous interactions that do not have an apparent biological function
(
Li et al., 2008
). This should be taken into account when physical interactions are
being assessed by regulatory assays such as target gene expression in TF mutants or
by TF knockdown with RNAi. Finally, different validation assays each have their
own rate of false negatives,
that
is,
they cannot detect every single genuine
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