Does CTCF mediate between nuclear organization and gene expression?

Abstract

The multifunctional zinc-finger protein CCCTC-binding factor (CTCF) is a very strong candidate for the role of coordinating the expression level of coding sequences with their three-dimensional position in the nucleus, apparently responding to a "code" in the DNA itself. Dynamic interactions between chromatin fibers in the context of nuclear architecture have been implicated in various aspects of genome functions. However, the molecular basis of these interactions still remains elusive and is a subject of intense debate. Here we discuss the nature of CTCF-DNA interactions, the CTCF-binding specificity to its binding sites and the relationship between CTCF and chromatin, and we examine data linking CTCF with gene regulation in the three-dimensional nuclear space. We discuss why these features render CTCF a very strong candidate for the role and propose a unifying model, the "CTCF code," explaining the mechanistic basis of how the information encrypted in DNA may be interpreted by CTCF into diverse nuclear functions.

Figure 1.

Structure of CTCF and its known modifications and interacting partners. A: Schematic representation of CTCF. It is composed of three structurally distinct domains: the N-terminal domain (N), 11 ZFs and a C-terminal domain (C). Post-translational modifications of CTCF are shown in the upper part of the figure, with the domain that is modified indicated. B: Proteins that interact with CTCF through the known domain, with domain of interaction indicated. C: Other proteins interacting with CTCF. D: CTCF interaction with another CTCF molecule via ZF and C-terminal domains. (For more details in panels B, C, see Table 2).

Figure 2.

CTCF binding to CTCF target sites. A: Characteristic examples of DNA logos of CTCF ‘‘consensus’’ motifs identified via genome-wide screens. DNA logos of the consensus motifs identified in screening for binding sequences of cohesin subunits, Rad21 and Scc1, are shown at the bottom. The size of each base is indicative of the probability of its presence at a particular position within a binding site. Nucleotides conserved between species are boxed; the invariant core sequence is indicated (‘‘n’’ signifies any nucleotide). B: Different combinations of ZFs are employed depending on the primary sequence of the CTS. Several characteristic examples are shown. The panel on the left summarizes which ZFs of CTCF are involved in binding to the CTS shown on the right. Filled boxes indicate fingers that are essential for binding, lighter boxes indicate fingers less important for binding and empty boxes are fingers dispensable for binding. The panel on the right represents the sequences of the CTSs; contact guanine nucleotides are shown in red. The mutated guanine nucleotide in Xist C(—43)G mutation is shown in lower case in the third example. The CTSs are aligned with the consensus motif,⁽¹⁷⁾ on plus and minus DNA strands; the invariant nucleotides are highlighted in gray. A match with the invariant core is indicated in the column in the middle. In human c-myc-A CTS, the mutation of ‘‘G’’ outside the consensus (indicated by the asterisk) leads to complete loss of CTCF binding. C: The effect of mutations in the CTCF ZF3 and ZF7 on CTS recognition. The range of CTSs recognized by CTCF carrying tumor-specific mutations of individual ZFs⁽⁴⁸⁾ in gel retardation experiments is shown. Filled boxes indicate strong binding, lighter boxes indicate partial loss of binding, and empty boxes indicate no binding. The mutations are: K344E, R339W, and H345R in ZF3 and R448Q in ZF7.

Figure 3.

The CTCF code. Formation of diverse CTCF-protein complexes at different CTCF target sites (CTSs). The CTSs (CTS-1 and CTS-2) are recognized (‘‘read’’) by CTCF (blue line with ZFs shown as bars, and N- and C-terminal domains as an extension of the line). The red bars indicate the ZFs contacting the CTSs, and blue bars the ZFs that do not contact the CTSs. DNA is shown as a black line. Out of numerous possibilities we depict three general cases. In the first (i), CTCF uses different ZFs to contact two different CTSs. CTCF molecules bound at the two sites make direct contact with each other via, e.g., C-terminal ZF domain interactions (see Ref.⁽⁴⁹⁾). This brings together two CTSs. In the second example (ii), CTS-1 and CTS-2 are brought together by protein partners, which contact CTCF directly or indirectly via protein-protein bridges (green, red, and orange symbols in the image). Cohesin may act to stabilize the interactions in cases (i) and (ii), as demonstrated in Ref.⁽⁹⁸⁾ In the third example (iii), CTCF may directly (see, e.g., Ref.⁽⁹²⁾) or indirectly make contact with another DNA-binding trans-acting factor to bridge a CTS with a non-CTS. In this model, use of different ZFs at different CTSs results in different surfaces on CTCF being available for inter-action with protein partners. The resulting combination of the CTCF-CTSs-protein complexes specifies a particular nuclear function. According to the model, the underlying CTS determines the pattern of ZF utilization and hence the choice of protein partner in combina-tion with post-translational modifications to dictate the functional outcome, as indicated in the figure.