We previously cloned ZNF74, a developmentally expressed zinc finger gene commonly deleted in DiGeorge syndrome. Here, the intron/exon organization of the human gene and the functional properties of the expressed protein are presented. This zinc finger gene from the transcription factor IIIA/Kruppel family contains three exons. A truncated Kruppel-associated box (KRAB) located at the N terminus of the predicted 64-kDa zinc finger protein is encoded by exon 2. The remainder of the protein including the zinc finger domain as well as the 3-untranslated region (UTR) is encoded by exon 3. Both 5-UTR (exon 1) and 3-UTR contain repetitive Alu elements. In vitro translation of a cDNA encoding the entire ZNF74 coding region produced a 63-kDa protein as determined on sodium dodecyl sulfate-polyacrylamide gel. A bacterially expressed fusion protein shown to bind tightly to 65 zinc was used to test the nucleic acid binding properties of ZNF74. By RNA binding assays, ZNF74 was found to bind specifically to poly(U) and poly(G) RNA homopolymers. The restricted binding to these homopolymers and not to poly(A) and poly(C) suggested that ZNF74 displays RNA sequence preferences. RNA binding was mediated by the zinc finger domain. Immunofluorescence studies on transfected cells revealed ZNF74 nuclear localization. The labeling pattern observed in the nuclei clearly excluded the nucleoli. The zinc finger region lacks a classical nuclear localization signal but was found to be responsible for nuclear targeting. Subcellular and in situ sequential fractionations further showed that ZNF74 is associated with the nuclear matrix. The RNA binding properties of this protein and its tight association with the nuclear matrix, a subnuclear compartment involved in DNA replication as well as RNA synthesis and processing, suggest a role for ZNF74 in RNA metabolism.

Transcriptional intermediary factor 1 (TIF1) ␣ and KAP-1/TIF1␤, two members of the TIF1 family of nuclear cofactors, are ubiquitous co-regulators of nuclear receptors and KRAB motif-containing zinc finger transcription factors, respectively. Despite the functional evidence suggesting a role for TIF1 proteins as modulators of transcription, the study of their interactions with transcriptional machineries in physiologically relevant systems has been difficult. Here, we have developed a bioluminescence resonance energy transfer (BRET) biophysical approach to study protein-protein interactions in the nuclear compartment of living mammalian cells. We report that TIF1␣ and KAP-1 form homo-and heterooligomers in intact mammalian cells. BRET titration experiments indicate that both homo-and hetero-oligomers occur with relatively high affinity suggesting that they could co-exist in cells. Furthermore, we demonstrate that KAP-1 but not TIF1␣ interacts with the KRAB multifinger ZNF74 in the nuclear matrix. Splice variants and point mutants of ZNF74 that lack transcriptional activity were found not to interact with KAP-1 confirming the physiological importance of this interaction in living cells. The interaction of ZNF74 with KAP-1 did not prevent KAP-1 homomerization indicating that the oligomers most likely represent the transcriptionally active species. Furthermore, the detection of ternary ZNF74⅐KAP-1⅐TIF1␣ complexes suggests the existence of cross-talk between KAP-1-interacting KRAB proteins and TIF1␣-interacting nuclear receptors. In addition to providing new insights into the molecular interactions involved in the transcriptional activities of these proteins, this study shows that BRET can be advantageously used as a non-transcriptionbased oligomerization detection system to study the interaction of transcriptionally active proteins, including nuclear matrix proteins, in living cells.

We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome. ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys 2 -His 2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with premRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing.Zinc finger proteins of the TFIIIA/Kruppel type belong to the largest known family of transcription factors (1, 2). These proteins are characterized by Cys 2 -His 2 zinc finger motifs often repeated in tandem that fold around zinc ions and function as nucleic acid binding domains (3-6). About one-third of mammalian Cys 2 -His 2 zinc finger proteins contain a conserved domain of approximately 75 amino acids called KRAB (Kruppelassociated box) (7). The KRAB domain, located at the N terminus of Cys 2 -His 2 multifinger proteins, can confer strong distance-independent transcriptional repression of both activated and basal RNA polymerase II promoter activity (8 -14). Since they encode a repression motif and a potential DNA binding domain, members from the large KRAB/Cys 2 -His 2 protein family are presently thought to function as transcriptional regulators of gene expression.We previously cloned ZNF74, a gene that encodes a KRAB/ Cys 2 -His 2 protein (15). ZNF74 lies a few kilobases proximal to a polymorphic CA repeat (D22S264) (16) that was shown to be a distal marker for 22q11.2 deletions associated with increased susceptibility to schizophrenia (17). ZNF74 is also one of the few genes found hemizygously deleted in the majority of patients with the DiGeorge syndrome, a microdeletion disorder associated with a wide variety of congenital malformations including cardiac defects, thymic hypoplasia, and hypocalcemia (18 -20). To date, however, both the role of embryologically expressed ZNF74 in the DiGeorge syndrome (19) and its biochemical and cellular functions remain unclear.Although the presence of a KRAB motif and of 1...