Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF.

The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCF- target sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growth-regulatory circuits, such as p19ARF, are likely to contribute to CTCF-induced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF.

CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S(604)KKEDS(609)S(610)DS(612)E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.

Identification of a novel recognition sequence for integrin alphaM beta2 within the gamma-chain of fibrinogen.

The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.