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Fig. 6. Brdt protein is involved in the regulation of expression of histone
H1t. (A) Histone H1t expression is upregulated in
Brdt
BD1/BD1
mutant testes. Real-time reverse transcription (RT)-PCR was used to quantify
changes in the levels of expression of selected genes from control and
BrdtBD1/BD1
mutant testes. Among the genes examined for expression was Brdt
itself, using primers specifically designed such that they would not recognize
the mutant transcripts. As predicted, no normal Brdt mRNA was
detected (first bar). The expression levels are presented as relative levels
of the wild-type expression and the wild-type expression levels are set as
one. The results, corrected with GAPDH expression, are means±s.e.m. of
at least three experiments in three pairs of animals. (B) Immunoblot of
H1t protein in wild-type and
BrdtBD1/BD1
mutant testes. H1t protein level was elevated in the mutant testis as compared
with control. ß-tubulin was used as a loading control. (C)
Immunostaining of testicular sections with anti-H1t antibody, showing that H1t
was expressed in pachytene spermatocytes and spermatids and that H1t protein
level was elevated in the mutant testis. (D) Chromatin
immunoprecipitation (ChIP) assay showing that Brdt protein binds the histone
H1t promoter. Two pairs of PCR primers corresponding to the histone
H1t promoter proximal and distal regions were used. Total testicular
cells were prepared from wild-type and
BrdtBD1/BD1
mutant testis and used for ChIP experiments with anti-Brdt C-terminal
antibody. The cartoon in the upper panel shows the positions of the primers in
the H1t promoter. I, input chromatin; C, Brdt peptide blocked control
Brdt antibody; E, Brdt unblocked antibody experimental.
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