First published online 10 May 2006
doi: 10.1242/dev.02405
Development 133, 2371-2381 (2006)
Published by The Company of Biologists 2006
Distinct roles of Polycomb group gene products in transcriptionally repressed and active domains of Hoxb8
Yu-ichi Fujimura1,*,
Kyo-ichi Isono1,*,
Miguel Vidal2,
Mitsuhiro Endoh1,
Hiroshi Kajita1,
Yoko Mizutani-Koseki1,
Yoshihiro Takihara3,
Maarten van Lohuizen4,
Arie Otte5,
Thomas Jenuwein6,
Jacqueline Deschamps7 and
Haruhiko Koseki1,
1 RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku,
Yokohama 230-0045, Japan.
2 Centro de Investigaciones Biologicas, Department of Developmental and Cell
Biology, Ramiro de Maeztu 9, 28040 Madrid, Spain.
3 Department of Stem Cell Biology, Research Institute for Radiation Biology and
Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553,
Japan.
4 Division of Molecular Genetics, The Netherlands Cancer Institute, 1066CX
Amsterdam, The Netherlands.
5 Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan
406, 1098 SM Amsterdam, The Netherlands.
6 Research Institute of Molecular Pathology, The Vienna Biocenter, Dr Bohrgasse
7, A-1030 Vienna, Austria.
7 Hubrecht Laboratory, Uppsalalaan 8 3584CT Utrecht, The Netherlands.
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Fig. 1. Comparison of PcG associations with Hoxb8 genomic surrounding
at 12.5 dpc between anterior and posterior tissues. (A) Embryonic
tissues used in this study. Wild-type embryos at 12.5 dpc were dissected as
illustrated and anterior (A) and posterior (P) tissues (paraxial mesoderm plus
neuroectoderm) were subjected to the ChIP analyses. (B) Comparative
analysis of Rnf2 association to regions 2, 14 and D1 between anterior and
posterior tissues. The chromatin fraction purified from A or P tissue was
subjected to the immunoprecipitation with anti-Rnf2 antibody. Amounts of
genomic DNA immunoprecipitated by anti-Rnf2 (A+ or P+) were quantified by
comparing with serially diluted genomic DNA isolated from the original
chromatin fractions designated as `Input' (see Fig. S2A in the supplementary
material) and equivalent amounts of immunoprecipitated DNA to that of `Input'
DNA loaded into lane 1 were subjected to PCR reactions. Usually 10 to 20 ng of
genomic DNA was used. Immunoprecipitated DNA was also serially diluted.
Mock-immunoprecipitated DNA (A- and P-) derived from the same volume of the
chromatin fraction as used for anti-Rnf2 immunoprecipitation were subjected to
the PCR. The adam34 locus was used as a negative control. (C)
Comparative analysis of Phc1 association to regions 2 and 14 between anterior
and posterior tissues. (D) Comparative analyses for association of Cbx2
and Ring1 to regions 2 and 14. Amounts of genomic DNA (A+ and P+)
immunoprecipitated by anti-Cbx2 and anti-Ring1 antibodies subjected to PCR
were equivalent to that of `Input' DNA loaded into lane 1.
Mock-immunoprecipitated DNA (A- and P-) derived from the same volume of the
chromatin fraction as used for anti-Cbx2 and -Ring1 immunoprecipitation were
subjected to the PCR. (E) Rnf110 association to regions 2 and 14. All
experiments were conducted as described using anti-Rnf110 antibody. (F)
Schematic comparisons of Rnf2, Ring1, Phc1, Cbx2 and Rnf110 association to the
Hoxb8 genomic surrounding between anterior and posterior tissues.
Genomic organization around Hoxb8 gene is shown at the top. Exonic
regions are indicated by black boxes and the exon numbers are numerically
shown in the boxes. Positions of known cis-acting regulatory elements are
represented by overlying bold bars indicated as BH1100, KA and DE
(Charité et al., 1995 ;
Vogels et al., 1993;
Oosterveen et al., 2003).
Putative promotor regions are indicated by folded arrows. The genomic regions
examined by PCR using specific primer pairs listed in
Table 1 are shown by bars and
numerically indicated. The relative quantity of each genomic region in
immunoprecipitated genomic DNA from anterior and posterior tissues was
estimated by referring to `Input' DNA isolated from the initial lysates and
enrichment values against the `Input', and are represented by the black and
gray bars, respectively. Genomic regions left unexamined are covered by boxes
crossed with a diagonal line.
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Fig. 2. Comparison of H3-K9 acetylation, H3-K4 methylation and H3-K27
trimethylation in Hoxb8 genomic surrounding at 12.5 dpc between
anterior and posterior tissues. (A) Comparative analysis of H3-K9
acetylation at regions 2, 7, 8 and 9. (Left) Whole-cell lysates (WCE) prepared
from anterior and posterior parts of 12.5 dpc embryos were subjected to ChIP
analyses by using anti-acetylated H3-K9. Amounts of immunoprecipitated genomic
DNA (A+ and P+) by anti-acetylated H3-K9 subjected to PCR were equivalent to
that of `Input' DNA loaded in lane 1. Mock-immunoprecipitated DNA (A- and P-)
derived from the same volume of the chromatin fraction as used for
anti-acetylated H3-K9 immunoprecipitation were subjected to the PCR. (Right)
Schematic comparison of H3-K9 acetylation between anterior and posterior
tissues. The relative quantity of each genomic region in immunoprecipitated
genomic DNA from anterior and posterior tissues was estimated by referring to
`Input' DNA isolated from the initial lysates and enrichment values against
the initial lysate are represented by the black and gray bars, respectively.
(B) Comparative analysis of di- and trimethylation of H3-K4 at the
region 2, 7, 8 and 9. Representative results (left) and schematic summary
(right) are shown. (C) Comparative analysis of trimethylation of H3-K27
at the region 2, 3 and 8. Representative results (left) and schematic summary
(right) are shown.
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Fig. 4. De-repression of Hox genes in Rnf2-/- MEFs and ES
cells. (A) Conditional depletion of Rnf2 lead to de-repression of
Hoxb8 in MEFs derived from the cranial part of
Rnf2fl/fl 9.5 dpc embryos. (Top) Infection of
Cre-expressing adenovirus vector to MEFs derived from
Rnf2fl/fl embryos (fl/fl) depleted the Rnf2 gene products,
whereas the wild-type (+/+) MEFs were unaffected. Lamin B was used as a
control. (Bottom) The expression of Hoxb8 was induced by infection of
Cre-expressing adenovirus vector in Rnf2fl/fl MEFs
(fl/fl), but not in the wild type (+/+). (B)
Rnf2-/- (-/-) ES cells were derived from
Rnf2fl/fl (fl/fl) ES cells by transient overexpression of
Cre-recombinase. Rnf2 was re-expressed by transfecting
Rnf2-/- ES cells with a construct expressing Myc-tagged
Rnf2 (tr). The expression of endogenous and transfected Rnf2 was examined by
using anti-Rnf2 (left) and -Myc (middle) antibodies. CBB staining was used as
a loading control (right). (C) The expression of Hox cluster genes in
Rnf2fl/fl (fl/fl), Rnf2-/- (-/-) and
Rnf2 transfected (tr) ES cells was compared by RT-PCR. The quantity of
synthesized cDNA from respective cells was equalized by comparing the relative
amounts of ß-actin transcripts. (D) The expression of Phc1 and
Cbx2 gene products was reduced in Rnf2-/- ES cells (-/-)
in comparison with the wild type (fl/fl), whereas the expression of RYBP
(another Rnf2-binding protein that is not found in hPRC-H complex or class 1
PcG proteins) was not altered. (E) Rnf2 association and H3-K27
trimethylation at Hox promoter regions were compared between
Rnf2fl/fl and Rnf2-/- ES cells. For
the `Input', genomic DNA extracted from the original whole cell lysate
equivalent to the 1/40 volume of that used for the ChIP analysis was subjected
to the PCR. Hprt was used as a negative control.
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Fig. 5. De-repression of Hox genes in Suz12-/- ES cells
correlates with reduction of Rnf2 association to Hox genomic regions.
(A) The association of Suz12, Eed, Rnf2 and H3-K27 trimethylation at
the Hox promoter regions in the wild-type and suz12-/- ES
cells. Whole-cell lysates prepared from approximately the same number of wild
type (+/+) and suz12-/- (-/-) ES cells were subjected to
ChIP analyses using anti-Suz12, -Eed, -trimethylated H3-K27 and -Rnf2
antibodies. For the `Input', genomic DNA extracted from the original whole
cell lysate equivalent to the 1/40 volume of that used for the ChIP analysis
was subjected to the PCR. Hprt was used as a control. (B) The
expression of Hox cluster genes in the wild type (+/+) and
suz12-/- (-/-) ES cells was compared by RT-PCR.
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Fig. 6. Decreased H3-K9 acetylation at the first exonic region of Hoxb8
in the posterior tissues of
Rnf110-/-Bmi1-/- and
Phc1-/-Phc2-/- embryos at 9.5 dpc.
(A) Degree of H3-K9 acetylation in the anterior (A) and posterior (P)
regions were compared in
Rnf110+/-Bmi1+/- and
Rnf110-/-Bmi1-/- embryos. The
ß-actin promoter was used as a positive control. (B) Degree of
H3-K9 acetylation in the anterior (A) and posterior (P) regions were compared
in Phc1-/-Phc2+/-,
Phc1+/-Phc2-/- and
Phc1-/-Phc2-/- embryos. The
ß-actin promoter was used as a positive control. In this study, the
negative control ChIPs (A- and P-) were performed with rabbit IgG.
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© The Company of Biologists Ltd 2006
13-somite stage were dissected into
three pieces at the boundary between somites 9 and 10, and at the newly
generated somite boundary. WCE prepared from cranial region up to somite 9 (A)
and posterior unsegmented region (P) were subjected to ChIP analyses.
(D) Embryos at 9.5 dpc (about 25 somites) were bisected into anterior
(A) and posterior (P) pieces at the level of somite 9/10 boundary and
respective WCE were subjected to ChIP analyses. (E) Embryos at 10.5 dpc
(