We have isolated and analyzed human CTCF cDNA clones and show here that the ubiquitously expressed 11-zinc-finger factor CTCF is an exceptionally highly conserved protein displaying 93% identity between avian and human amino acid sequences. It binds specifically to regulatory sequences in the promoter-proximal regions of chicken, mouse, and human c-myc oncogenes. CTCF contains two transcription repressor domains transferable to a heterologous DNA binding domain. One CTCF binding site, conserved in mouse and human c-myc genes, is found immediately downstream of the major P2 promoter at a sequence which maps precisely within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse and human cells show that CTCF is the predominant factor binding to this sequence. Mutational analysis of the P2-proximal CTCF binding site and transient-cotransfection experiments demonstrate that CTCF is a transcriptional repressor of the human c-myc gene. Although there is 100% sequence identity in the DNA binding domains of the avian and human CTCF proteins, the regulatory sequences recognized by CTCF in chicken and human c-myc promoters are clearly diverged. Mutating the contact nucleotides confirms that CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are absent in the chicken c-myc CTCF binding site. Moreover, proteolytic-protection assays indicate that several more CTCF Zn fingers are involved in contacting the human CTCF binding site than the chicken site. Gel shift assays utilizing successively deleted Zn finger domains indicate that CTCF Zn fingers 2 to 7 are involved in binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF binding to the human promoter. This flexibility in Zn finger usage reveals CTCF to be a unique "multivalent" transcriptional factor and provides the first feasible explanation of how certain homologous genes (i.e., c-myc) of different vertebrate species are regulated by the same factor and maintain similar expression patterns despite significant promoter sequence divergence.

Gamma interferon (IFN-␥) induces transcription of a distinct set of genes by activating STAT1, one member of a family of latent cytoplasmic transcription factors that are activated via phosphorylation on tyrosine residues (6). The IFN-␥ receptor (IFN-␥R) lacks intrinsic tyrosine kinase activity, but on ligand binding, receptor multimerization results in reciprocal activation of JAK1 and JAK2, two Janus tyrosine kinases noncovalently attached to the IFN-␥R ␣ and ␤ chains (2, 20, 22). Phosphorylation of the tyrosine residue on IFN-␥R␣ by JAK molecules creates a docking site for the Src homology 2 domain of STAT1, which is then phosphorylated on tyrosine and ultimately forms a homodimeric transcription factor that translocates to the nucleus (6, 21). The factors that govern the traffic of cytoplasmic STAT molecules to the docking site on the IFN-␥R are unknown. We have recently found that the activation of STAT1 in response to IFN-␥ is suppressed in hematopoietic cells from children with Fanconi anemia of type C (FA-C) and in mice nullizygous at the FA-C locus. However, in the ground state (uninduced by IFN), IFN response factor 1 (IRF-1) is expressed at high levels in mutant FA-C cells (35), suggesting that a non-STAT1 pathway is involved in constitutive activation of IRF-1 in FA cells. In addition, complementation of the defect by retrovirus mediated transfer of normal FANCC cDNA reconstitutes the normal STAT1 response (10,38).Linkage of FANCC function with that of STAT1 provided us with an opportunity to test whether the relationship of these two molecules was direct or indirect. We report herein results of experiments in which the assembly of the fully activated IFN-␥R complex, including STAT1, JAK1, and JAK2, was examined in isogenic murine and human FA-C cells. We report that in IFN-␥-stimulated FA-C cells, phosphorylation of JAK1, JAK2, and IFN-␥R␣ occurs normally, but STAT1 does not dock at the receptor ␣ chain. In FA-C cells nuclear STAT1 is reduced, and IFN fails to induce STAT1-specific DNA-binding complexes and expression of IRF-1. Expression of the normal FANCC cDNA in mutant cells results in normal STAT1 docking and phosphorylation as well as normal induction of nuclear STAT1-DNA complex and normal induction of IRF-1. We also find that a variety of cytokines and hematopoietic growth factors stimulate the association of STAT1 with glutathione S-transferase (GST) fusion proteins encoding the normal FANCC but not a naturally occurring inactivating mutant FANCC (L554P) and that the association occurs rapidly and prior to STAT1 phosphorylation on Y 701 . Coimmunoprecipitation experiments confirmed the IFN-inducible association of

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5–E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16–32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

Patients with the genomic instability syndrome Fanconi anemia (FA) commonly develop progressive bone marrow (BM) failure and have a high risk of cancer. Certain manifestations of the disease suggest that the FA immune system is dysfunctional and may contribute to the pathogenesis of both BM failure and malignancies. In this study, we have investigated inflammation and innate immunity in FA hemopoietic cells using mice deficient in Fanconi complementation group C gene (Fancc). We demonstrate that Fancc-deficient mice exhibit enhanced inflammatory response and are hypersensitive to LPS-induced septic shock as a result of hemopoietic suppression. This exacerbated inflammatory phenotype is intrinsic to the hemopoietic system and can be corrected by the re-expression of a wild-type FANCC gene, suggesting a potential role of the FANCC protein in innate immunity. LPS-mediated hemopoietic suppression requires two major inflammatory agents, TNF-α and reactive oxygen species. In addition, LPS-induced excessive accumulation of reactive oxygen species in Fancc−/− BM cells overactivates the stress kinase p38 and requires prolonged activation of the JNK. Our data implicate a role of inflammation in pathogenesis of FA and BM failure diseases in general.

Hematopoietic cells bearing inactivating mutations of Fanconi anemia group C (FANCC) are excessively apoptotic and demonstrate hypersensitivity not only to cross-linking agents but also to interferon gamma (IFN-gamma) and tumor necrosis factor-alpha. Seeking essential signaling pathways for this phenotype, this study quantified constitutive and induced RNA-dependent protein kinase (PKR) activation in Fanconi anemia cells of the C complementation group (FA-C). PKR was constitutively phosphorylated and exhibited an increased binding affinity for double-stranded RNA (dsRNA) in FANCC(-/-) cells. FANCC(-/-) cells were hypersensitive to both dsRNA and the combination of dsRNA and IFN-gamma in that these agents induced a higher fraction of apoptosis in FANCC(-/-) cells than in normal cells. Overexpression of wild-type PKR-sensitized FANCC(-/-) cells to apoptosis induced by IFN-gamma and dsRNA. Conversely, inhibition of PKR function by enforced expression of a dominant-negative inhibitory mutant of PKR (PKRDelta6) substantially reduced the IFN and dsRNA hypersensitivity of FANCC(-/-) cells. Two PKR target molecules, IkappaB-alpha and IRF-1, were not differentially activated in FANCC(-/-) cells, but enforced expression of a nonphosphorylatable form of eukaryotic translation initiation factor-2alpha reversed the PKR-mediated block of messenger RNA translation and partially abrogated the PKR-mediated apoptosis in FANCC(-/-) cells. Because no evidence was found of a PKR/FANCC complex in normal cells, it was concluded that an essential function of FANCC is to suppress, indirectly, the activity of PKR and that FANCC inactivation results in IFN hypersensitivity, at least in part, because this function of FANCC is abrogated.