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J. Biol. Chem., Vol. 277, Issue 20, 17765-17774, May 17, 2002
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From the Department of Molecular and Cell Biology, University of
Texas at Dallas, Richardson, Texas 75080
ALF (TFIIA The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF452125.
Regulation of ALF Gene Expression in Somatic and Male Germ Line
Tissues Involves Partial and Site-specific Patterns of Methylation*
/
-like factor) is a germ
cell-specific counterpart of the large (/) subunit of general
transcription factor TFIIA. Here we isolated homologous GC-rich
promoters from the mouse and human ALF genes and used promoter deletion
analysis to identify sequences active in COS-7 and 293 cells.
Further, bisulfite sequence analysis of the mouse ALF promoter
showed that all 21 CpG dinucleotides between 
179 and +207 were
partially methylated in five somatic tissues, brain, heart, liver,
lung, and muscle, and in epididymal spermatozoa from adult mice. In contrast, DNA from prepubertal mouse testis and from purified spermatocytes were unmethylated except at C+19G and
C+170G. We also found that ALF expression correlates
with a strong promoter-proximal DNase I-hypersensitive site present in
nuclei from testis but not from liver. Finally we show that in
vitro methylation of the ALF promoter inhibits activity and that
5-aza-2'-deoxycytidine treatment reactivates the endogenous ALF gene in
a panel of seven different mouse and human somatic cell lines. Overall
the results show that silencing in somatic cells is
methylation-dependent and reversible and that a unique
CpG-specific methylation pattern at the ALF promoter precedes
expression in pachytene spermatocytes. This pattern is transient as
remethylation of the ALF promoter in haploid germ cell DNA has occurred
by the time spermatozoa are present in the epididymis.
*
This work was supported by grants from the American Cancer
Society and The Welch Foundation (to J. D.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular and
Cell Biology, University of Texas at Dallas, 2601 N. Floyd Rd.,
Richardson, TX 75080. Tel.: 972-883-6882; Fax: 972-883-2409; E-mail:
dejong@utdallas.edu.
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