Genome-wide
analysis of the role of PcG and trxG proteins
(Bernd
Schüttengruber, Mythily Ganapathi, Benjamin Leblanc)
Identification
of downstream targets for
regulatory proteins has been a major challenge in the past decades.
Regulatory
factors often bind in vitro
to specific DNA sequences but in vivo
binding
is found only at a subset of all consensus sites in the genome. Even
more
dramatic is the case of chromatin associated factors
that
associate
to specific targets in vivo
but do not bind to specific DNA sequences
in
vitro as bacterially purified proteins. In vivo cross-linking of
proteins
to their target DNA is the method of choice to identify protein targets
in
the cell nucleus. Our lab has identified PcG target genes during development. In a first
study (Negre, N.et al. (2006). PLoS Biol 4, e170), we
coupled
ChIP with
hybridization
on
a DNA
microarray that contains 10 Mb of the X-chromosome (ChIP on chip).
We then extended these studies using
genome-wide oligonucleotide arrays, with the aim to map the
distribution of trxG and PcG proteins, as well as their associated
chromatin marks: trimethylation of Lysine 27 of histone H3
(H3K27me3, a PcG mark)
and trimethylation
of
Lysine 4 of histone H3
(H3K4me3, a trxG mark). Finally, we have mapped the binding of several
proteins that were previously reported to recruit PcG proteins to PREs.
In the right hand side figure we show an example of the results,
showing one PcG protein, Polyhomeotic (PH) and the two histone marks on
a target locus containing three Hox genes.
The results we have obtained are
surprising
in
many ways. First, the majority of PcG recruiter binding sites are
associated with H3K4me3 and not with PcG binding, suggesting that
recruiter proteins have a dual function in activation
as
well as silencing. One major discriminant between activation and
silencing is the strong binding of Pleiohomeotic (PHO) to silenced
regions, while its homolog Pleiohomeotic-like (PHOL) binds
preferentially to active promoters. In addition, the C-terminal
fragment of TRX (TRX-C) showed high affinity to PcG binding sites,
while the N-terminal fragment (TRX-N) bound mainly to active promoter
regions trimethylated on H3K4.
These results indicate that DNA binding proteins serve as platforms to
assist PcG and trxG binding. Furthermore, several DNA sequence features
discriminate between PcG and TRX-N bound regions,
indicating
that underlying DNA sequence contains critical information
to drive PREs and TREs towards silencing or activation. These data have
been published (Schuettengruber,
B.,
Ganapathi,
M.et al (2009). PloS
Biol,
7(1): e1000013). Moreover, we
provide these data in a genome browser, which also collects data from
previously published work. All these data are also freely available for
download.
In the future, will extend these studies by mapping the distribution of
other proteins, by analysing other developmental stages and tissues in
different Drosophila species
and
by analyzing the effect of mutations on chromatin landscapes. These
studies will be combined
with expression analysis to describe
transcriptional changes in cells mutated for PcG/trxG genes, with the
goal to understand the PcG/trxG regulatory
circuitry.
Last update:
13/03/2012
