The eukaryotic cell nucleus is highly structured into subdomains of specialized function that include compartments of high and low transcriptional capacity, the euchromatin and heterochromatin (reviewed in (1
). The most transcriptionally inactive compartment, constitutive heterochromatin, consists typically of repetitive DNAs and specialized, highly compact chromatin concentrated near centromeres or telomeres (5
). Although repetitive DNAs retain their historic label as ‘junk’ DNA, they play an important role in chromosomal segregation as an essential structural component of the centromere (7
). In addition, the heterochromatic chromosomal domains repress the activity of nearby genes (2
). This indicates an indirect role for repetitive DNA in transcriptional regulation but it remains unknown if repetitive DNAs can somehow directly affect transcription.
A number of transcription factors concentrate at heterochromatic regions (9
). Where examined, the concentration of transcription factors at heterochromatin depends upon retention of their DNA binding domain (14
). This suggests that direct DNA binding, possibly to the repetitive DNAs, may target those transcription factors to the heterochromatin. Indeed, some transcription factors can bind specific repetitive elements in vitro
). The correlation between repetitive DNA binding and targeting to heterochromatin is strengthened by a report that treatment of a preadipocyte cell line with growth hormone enhances targeting of the transcription factor CCAAT/Enhancer Binding Protein beta (C/EBPβ2
to heterochromatin and the ability of that factor to bind a repetitive DNA in vitro
). Furthermore, Drosophila embryos undergo homeotic transformations when treated with polyamide drugs that bind a repetitive DNA element to which the GAGA factor binds (20
). However, establishing if there is a causal or coincidental relationship between repetitive DNA binding, heterochromatic targeting and biologic response of a site-specific transcription factor has been difficult to address experimentally (1
). In the current study, we identify methods to preferentially block the binding of a transcription factor to repetitive DNA, and then follow the effects of that intervention on promoter binding and activation.
CCAAT/enhancer binding protein alpha (C/EBPα) is a transcription factor important to the differentiation and transcriptional regulation of a number of cell types (25
). In addition to binding and activating a large number of gene promoters, C/EBPα also is capable of binding in vitro
to the mouse major α-satellite repetitive DNA (11
). The major α-satellite DNA repeat (referred to hereafter as α-satellite DNA) is prevalent at murine pericentromeric heterochromatin (29
). Endogenous C/EBPα expressed upon differentiation of mouse 3T3-L1 cells into adipocytes concentrates strongly at the pericentromeric heterochromatin (11
), possibly through its binding to α-satellite DNA. The concentration of C/EBPα at pericentromeric heterochromatin is an intrinsic property of C/EBPα as C/EBPα ectopically expressed in undifferentiated 3T3-L1 pre-adipocyte cells and in pituitary progenitor cells also concentrates there (31
). The ability to mimic the intranuclear localization with ectopically expressed proteins enabled us to manipulate C/EBPα and establish that the conserved bZIP domain of C/EBPα was necessary and sufficient for ectopically expressed C/EBPα to target to pericentromeric heterochromatin (21
). We also showed previously that the concentration of C/EBPα in transcriptionally inactive heterochromatin is highly regulated by the pituitary-specific transcription factor Pit-1 which translocates C/EBPα from a pericentromeric to a non-pericentromeric distribution in the pituitary progenitor cell model (31
Here, we provide experimental evidence that specific binding of C/EBPα binding to α-satellite DNA sequesters C/EBPα in the pericentromeric heterochromatin of the cell nucleus. Furthermore, the regulated translocation of C/EBPα away from the pericentromeric heterochromatin (31
) correlated with a loss of C/EBPα binding to α-satellite DNA, enhanced promoter binding and enhanced promoter activation. This indicates that transcription factor binding to repetitive DNA can act as a direct negative regulator of transcriptional activity. The impact of repetitive DNA binding on the activity of other transcription factors remains unknown but this report suggests that it is a facet of transcription regulation that should be considered for some factors.