transcriptional
regulation of developmental transitions; role of chromatin
remodeling in development
My lab is interested in understanding at the molecular
level the complex changes that occur when an organism switches
developmental programs. Specifically, we investigate the transition
from vegetative to reproductive development in the plant model
system Arabidopsis thaliana. This transition is not only vital,
important for species survival, but also a great example of a
master regulator reprogramming cell fate and has important implications
for plant breeding and biofuel production. Because of its importance,
many external signals (such as temperature and day length) as
well as internal cues input into the timing of reproductive development.
Upon perception of the required inductive signals cells at the
flanks of the shoot apical meristem will give rise to flowers
instead of leaves and secondary stems (Figure 1).
Figure 1 - Meristem identity switch
(A, B) The stem cell population of the shoot apical meristem
(SAM) gives rise to leaves at the flanks of the meristem
in
young seedlings. (C) At the onset of the reproductive phase,
the SAM enlarges and gives rise to leaves with axillary
meristems
(arrow). (D) The meristem identity switch results in formation
of the reproductive structures, the flowers (F) at the
flank of the SAM (arrowhead).
Interestingly, one master regulator, the plant specific LEAFY
(LFY) protein, is necessary and sufficient for the reproductive
transition in Arabidopsis.
An activatable LEAFY protein
We have previously demonstrated that the LEAFY protein acts as
a transcription factor in vivo using an activatable, biologically
functional chimeric protein consisting of LEAFY and the glucocorticoid
receptor hormone binding domain (LFY-GR) (Wagner et al., Science,
1999). Nuclear import and therefore activity of the fusion
protein
is dependent on steroid application without complicating side
effects on the plant (Figure 2).
Figure 2 - A system for post-translational activation
of LEAFY
A fusion protein between LEAFY (LFY) and the rat glucocorticoid
receptor hormone binding domain (GR) is retained in the cytoplasm
in absence of the synthetic steroid hormone dexamethasone
as determined by immunolocalization using an anti-LFY antibody.
Upon addition of hormone the LFY-GR protein enters the nucleus
and rescues a lfy null mutant flower (top) to a wild-type
flower, indicating that LFY-GR is biologically active.
Steroid activation of LFY-GR is used to identify
direct targets of LFY during the reproductive transition by assaying
genome-wide expression changes on microarrays.
We are complementing these studies with global binding analyses
using chromatin immunoprecipitation followed by hybridization
to a tiling array (ChIP on chip). Using these and
classical genetic tools, we are elucidating how the LFY master
regulator reprograms cell fate during development.
Chromatin remodeling and development
In a genetic screen we isolated an enhancer of a weak lfy mutant
(Figure 3), which we called SPLAYED (SYD) (Wagner and Meyerowitz,
Current Biology, 2002).
Figure 3 - splayed enhances floral homeotic defects
in lfy-5 mutants
The splayed (syd) mutant enhances the floral defects found
in the weak lfy-5 mutant (compare B to A). As in the lfy-6
null mutant flower (C), no floral organs with petal or stamen
identity are formed.
Cloning of SYD
revealed
that it is one of four Arabidopsis orthologs of the Snf2/BRM
ATPases (Figure 4). These proteins are central subunits of
large multi-protein
complexes that regulate stage- or tissue-specific transcription
in the context of chromatin. These enzymes remodel chromatin
to alter the accessibility of the DNA to transcription factors
or to the
transcriptional machinery by inducing non-covalent, local changes
in the DNA-nucleosome association in promoter regions. Since
most chromatin remodeling mutants in other higher eukaryotes
are embryonic lethal,
Arabidopsis provides a new entry point to genetic investigation
of this
process in multicellular organisms. Chromatin remodeling
complexes have intrinsic tumor suppressor activity and regulate
expression of other tumor suppressor, thus elucidating
the in vivo role of these ATPases is of utmost importance.
Figure 4 - Arabidopsis SWI/SNF ATPases
SYD is one of four Arabidopsis thaliana (At) SNF2/BRM-type
chromatin remodeling ATPases. These proteins are the
central
subunit of large (2MD) protein complexes which regulate transcription
by altering the DNA-histone octamer interaction in chromatin. The
red asterisk highlights Arabdopsis SYD and BRM.
Figure modified from Verbske and Richards, Current Opinion
in Plant Biology, 2001.
We are currently analyzing the role of chromatin remodeling
during Arabidopsis development and growth using molecular, biochemical,
genomic, genetic,
and reverse genetic approaches. Our goal is to identify the pathways
and transcription events controlled by SYD, as well as to uncover
potential
interactions
of SYD with other chromatin modifying complexes that might act
in concert with the putative SYD complex to control development
in Arabidopsis.
People