Abstract
BackgroundCCCTC binding factor (CTCF) is a highly conserved zinc finger protein, which is involved in chromatin organization, local histone modifications, and RNA polymerase II-mediated gene transcription. CTCF may act by binding tightly to DNA and recruiting other proteins to mediate its various functions in the nucleus. To further explore the role of this essential factor, we used a mass spectrometry-based approach to screen for novel CTCF-interacting partners.ResultsUsing biotinylated CTCF as bait, we identified upstream binding factor (UBF) and multiple other components of the RNA polymerase I complex as potential CTCF-interacting partners. Interestingly, CTCFL, the testis-specific paralog of CTCF, also binds UBF. The interaction between CTCF(L) and UBF is direct, and requires the zinc finger domain of CTCF(L) and the high mobility group (HMG)-box 1 and dimerization domain of UBF. Because UBF is involved in RNA polymerase I-mediated ribosomal (r)RNA transcription, we analyzed CTCF binding to the rDNA repeat. We found that CTCF bound to a site upstream of the rDNA spacer promoter and preferred non-methylated over methylated rDNA. DNA binding by CTCF in turn stimulated binding of UBF. Absence of CTCF in cultured cells resulted in decreased association of UBF with rDNA and in nucleolar fusion. Furthermore, lack of CTCF led to reduced binding of RNA polymerase I and variant histone H2A.Z near the rDNA spacer promoter, a loss of specific histone modifications, and diminished transcription of non-coding RNA from the spacer promoter.ConclusionsUBF is the first common interaction partner of CTCF and CTCFL, suggesting a role for these proteins in chromatin organization of the rDNA repeats. We propose that CTCF affects RNA polymerase I-mediated events globally by controlling nucleolar number, and locally by regulating chromatin at the rDNA spacer promoter, similar to RNA polymerase II promoters. CTCF may load UBF onto rDNA, thereby forming part of a network that maintains rDNA genes poised for transcription.
Highlights
CCCTC binding factor (CTCF) is a highly conserved zinc finger protein, which is involved in chromatin organization, local histone modifications, and RNA polymerase II-mediated gene transcription
As CTCF often binds DNA in a methylation-sensitive manner, we tested whether the in vitro methylation of these two sites in R30 affected CTCF binding. We found that this was the case to some extent, as CTCF bound the non-methylated R30 probe slightly more efficiently, and this probe was a better competitor than methylated R30
CTCF maintains specific histone marks at the spacer promoter Given the role of CTCF in epigenetic chromatin remodeling near its binding sites, we examined the distribution of specific histone marks across the rDNA regulatory region in the presence and absence of CTCF
Summary
CCCTC binding factor (CTCF) is a highly conserved zinc finger protein, which is involved in chromatin organization, local histone modifications, and RNA polymerase II-mediated gene transcription. We have shown that loss of a CTCF-binding site affects chromatin looping and local histone modifications in the mouse b-globin locus, without significantly perturbing transcription [10]. These data indicate that CTCF is able to regulate the balance between active and repressive chromatin modifications near its binding sites, with different outcomes in terms of transcription. CTCF may control epigenetic modifications by binding to the chromatin remodeling factor CHD8 [11]
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