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Fig. 6. Juxtaposition of EED and BMI1 complexes at Hox target loci.
(A) A schematic representation of the Hoxc8 upstream region is
shown. The blue arrow depicts the Hoxc8 transcription start site, and
the green lines demark two upstream regions identified by ChIP located
immediately upstream (a) and 1.5 kb upstream (b) of the Hoxc8
transcription start site. Red asterisks indicate putative YY1-binding sites.
(B) ChIP using antibodies against EED, BMI1, YY1 and trimethylated
H3-K27 detected the proteins at the two upstream regions (a and b) of the
Hoxc8 locus. While dimethylated H3-K27 localized to region b of the
Hoxc8 locus, results for region a were variable and, hence,
inconclusive. EED, BMI1, YY1 and trimethylated H3-K27 were also detected 1.5
kb upstream of the Hoxa5 transcription start site. ChIP using an
antibody against Fpn1 served as a negative control. (C) ChIP detected
differential binding of EED and BMI1 binding to Hox regulatory regions in
dissected anterior and posterior regions of E12.5 trunk. In all cases, input
encompassed 1% of the chromatin used for ChIP, and mock ChIP without antibody
served as additional negative controls. As a negative control, EED and BMI1
did not associate with the ß-actin promoter.
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