We show here that CTCF physically interacts with the Smad2/3/4 complex. This interaction brings the Smad complex to CTCF-binding sites that generally map to linker regions between positioned nucleosomes. Most importantly, because CTCF-binding sites are generally sensitive to CpG methylation (32
), the CTCF link provides an epigenetic dimension to TGFβ signaling.
The selectivity of interaction of Smad3, but not Smad2 or Smad4, with CTCF (–) is of interest. This selective interaction requires the MH1 domain of Smad3 (A) and suggests that because Smad3 binds to CTCF via its DNA-binding domain, the interaction may preclude the possibility of concomitant Smad3 binding to DNA and to CTCF (B). In addition, the requirement for Smad4 for the recruitment of Smad2 and Smad3 to the H19 ICR (A) suggests that Smad4 may stabilize the nuclear Smad complex, which is necessary for its proper association to chromatin via CTCF (see also the model of B). Because Smad3 may be engaged with binding to CTCF by means of its MH1 domain and to other Smads via its phosphorylated MH2 domain, Smad4 may provide an additional binding site to DNA via its own available MH1 domain. The functional role of the Smad2 MH1 domain that fails to associate with either CTCF or DNA remains open for future investigation. However, a nuclear complex with the spliced isoform Smad2Δex3 is capable of providing a second CTCF-binding arm to the multimeric complex (B).
FIGURE 10. The CTCF-Smad complex. A, schematic model of the interacting protein domains between Smad3 and CTCF. The Smad3 MH1 and MH2 domains and the CTCF N- and C-terminal and 11 zinc finger domains are indicated. An arrow points to the interaction between MH1 (more ...)
The interaction between CTCF and Smads (–) and the dependence of CTCF-binding sites for the recruitment of Smads to the maternal H19
ICR allele strongly suggest that Smads do not bind to sequences within the H19
ICR but rather are recruited via CTCF. There is a single precedence for common recruitment of CTCF and Smad proteins to the regulatory region of the APP
) gene (23
). APP expression is induced by TGFβ signaling, and CTCF seems to participate in this regulatory mechanism. However, this study has not defined whether Smads bind directly to the APP
enhancer or whether they are recruited via binding to CTCF or other common interacting components. In contrast, we were unable to establish robust regulation of well established gene and cell responses to TGFβ in various cell types by CTCF ( and ). The examples of APP
gene regulation and our evidence on PAI-1
gene expression () do leave open the possibility that CTCF may be involved in regulation of the transcriptional output of a subset of TGFβ-responsive genes.
Moreover, TGFβ signaling did not modify the repressed status of the maternal Igf2
alleles (), which depends on the CTCF-binding sites within the H19
). We thus conclude that TGFβ signaling targets the paternal Igf2
allele independent of the maternal H19
ICR allele. Furthermore, the presence of Smad3 cannot play an essential role in the parent of origin-specific insulator function associated with the H19
ICR. This argument is based on previous work that established that the H19
ICR insulator function is maintained in human choriocarcinoma cells such as JEG-3 (29
), despite the fact that these tumor cells suffer a complete deletion of the Smad3
). Moreover, there is to our knowledge no other indication in the literature that the maternal-specific repression of the Igf2
locus depends on exogenous TGFβ.
Although these data render the functionality of the CTCF-Smad2/3/4 complex enigmatic, we note that CTCF has been identified bound to several thousand sites in the human, mouse, and fly genomes (34
). Even though most of these sites map at intergenic regions and at chromatin boundaries between transcriptionally silent and active chromatin, a number of the CTCF-binding sites reside in proximity to gene promoters/enhancers. It is thus possible that TGFβ signaling modulates the function of CTCF-binding sites in a context-dependent manner.
Furthermore, because CTCF has been implicated as a master weaver of the genome (5
), TGFβ signaling might also modulate the organization of large chromatin domains via the formation of chromatin loops and bridges (4
). This option is attractive given the ability of the Smad complex to interact with other proteins to potentially stabilize interactions between chromatin fibers involving CTCF.
In conclusion, this study provides the first evidence that CTCF recruits Smad proteins to its binding sites and that this recruitment can be epigenetically controlled. Such cross-talk can be achieved by the domain-specific molecular interaction between CTCF and Smads that we demonstrate here. This work opens the possibility that the functional consequence of such a molecular interaction may mediate control of long range chromatin associations by a major developmental signaling pathway such as TGFβ.