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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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