Biology Reference
In-Depth Information
section on a few transgenic strategies that have been used to great effect in
Drosophila
, including enhancer-reporter constructs and genetically encoded
miRNA sensors.
5.1. miRNA promoters “identified” by
P
elements
It is first worth commenting on the fact that
P
elements tend to insert in
active promoters. Although information on miRNA promoters and tran-
script models from microarray analysis following knockdown of nuclear
miRNA biogenesis factors (
Kadener
et al
., 2009b
), or from RNA-seq and
CAGE analyses (
Enderle
et al
., 2011
;
Graveley
et al
., 2011
), is growing, our
knowledge is far from complete. It is therefore humbling to realize that
P
elements were far ahead of humans in identifying not only miRNA genes
but also their promoters. In some cases, the miRNA promoter is located
quite distantly from the miRNA gene itself. For example, the
mir-278
hairpin is associated with a cluster of
P
elements located 45kb upstream,
which identifies its promoter (
Nairz
et al
., 2006
;
Teleman
et al
., 2006
).
Similarly, the
bantam
miRNA hairpin is associated with multiple
P
element
insertion clusters, which likely signify complexity of its transcriptional
control via multiple promoters; the most distal promoter is some 40kb
away from the
bantam
hairpin (
Graveley
et al
., 2011
;
Peng
et al
., 2009
).
Importantly,
P
elements that contain marker genes can report on the
transcriptional output of the promoter within which they are inserted
(
Fig. 8.4
A) through expression of the marker gene (e.g.,
bereft/mir-263a:
lacZ
;
Section 2.2
). This is particularly useful for miRNA genes, as it
provides a relatively stable readout of primary transcript expression of the
tagged miRNA locus.
5.2. miRNA enhancer:reporter transgenes
“Enhancer bashing” is a time-honored tradition in
Drosophila
, an exercise in
which one attempts to identify fragments of genomic DNA that can drive a
reporter gene (e.g.,
lacZ
,
GFP
,
DsRed
, etc.) in a pattern that recapitulates
known aspects of the endogenous transcript (
Fig. 8.4
A). An
in situ
hybridi-
zation survey of primary miRNA transcripts revealed a diversity of spatially
restricted patterns in
Drosophila
embryos (
Aboobaker
et al
., 2005
), and
relevant
cis
-regulatory modules (CRMs) for several embryonically
expressed miRNAs have been isolated, including for the
mir-309/3/286/
3/4/5/6
cluster in the early blastoderm embryo (
Biemar
et al
., 2005
), for
mir-1
in the mesoderm and muscles (
Biemar
et al
., 2005
;
Kwon
et al
., 2005
;
Sokol and Ambros, 2005
), and for
mir-124
in the central nervous system
(
Xu
et al
., 2008
). Similarly, a
mir-7
CRM located within the intron of its
host transcript
bancal
(
Li and Carthew, 2005
;
Li
et al
., 2009
) and a CRM
upstream of
mir-279
(
Cayirlioglu
et al
., 2008
) have proven useful to probe
