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We isolated and characterized a novel K-Cl cotransporter, KCC3, from human placenta. The deduced protein contains 1,150 amino acids. KCC3 shares 75-76% identity at the amino acid level with human, pig, rat, and rabbit KCC1 and 67% identity with rat KCC2. KCC3 is 40 and 33% identical to two Caenorhabditis elegans K-Cl cotransporters and approximately 20% identical to other members of the cation-chloride cotransporter family (CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Cl cotransporter (NCC). Hydropathy analysis indicates a typical KCC topology with 12 transmembrane domains, a large extracellular loop between transmembrane domains 5 and 6 (unique to KCCs), and large NH(2) and COOH termini. KCC3 is predominantly expressed in kidney, heart, and brain, and is also expressed in skeletal muscle, placenta, lung, liver, and pancreas. KCC3 was localized to chromosome 15. KCC3 transiently expressed in human embryonic kidney (HEK)-293 cells fulfilled three criteria for increased expression of K-Cl cotransport: stimulation of cotransport by swelling, treatment with N-ethylmaleimide, or treatment with staurosporine.

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The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1␤. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBP␤ carboxylterminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity.The ETS family of transcription factors serves a variety of roles in development and differentiation (1). A highly conserved DNA-binding domain (DBD) 3 defines the family. Spi-1/PU.1 (Spi-1), one of the most diverse members, is critical for myeloid and B cell differentiation and function (2-6). It also regulates the expression of many effector genes, including IL1B, coding for the IL-1␤ protein (7). The Spi-1 molecule possesses a mutipartite transactivation domain (TAD) consisting of the amino half of the molecule that binds TBP, RB (8), and CBP/p300 (9) (Fig. 1a). The carboxyl end of the TAD contains a phosphorylatable PEST sequence that aids in the formation of a protein-protein interaction with transcription factors 11). Located at the COOH-terminal end of the protein is the ETS DBD. Besides DNA binding, this domain is also a tethering site for multiple transactivators, some of which occur at composite DNA-binding sites (reviewed in Ref. 12), and contains the previously coarsely mapped nuclear localization signal (NLS) (13). The structure of the Spi-1 DBD, like other ETS factors, consists of 85 conserved residues folded into a winged-helix-turn-helix (wHTH) conformation (14) (Fig. 1b). Two arginines, which are solventexposed in helix ␣3, are conserved in all ETS family members and form direct and indirect hydrogen bonds to a core DNA sequence of A/GGAA in Spi-1 (1, 7, 15). The Spi-1 DBD is 98% conserved at the amino acid level between mouse and human, suggesting stringent structural and functional requirements.Spi-1 plays a necessary role in the regulation of the IL1B promoter by cooperating functionally with C/EBP␤, a basic leucine zipper (bZIP) family member (16,17). This cooperativity may be derived, in part, by a physical interaction between the DBD of both molecules and the two composite DNA-binding sites in the IL1B promoter (Fig. 2a) (18). The downstream TATA-proximal site binds relatively well to Spi-1 but weakly to C/EBP␤ (7). C/EBP␤ DNA binding at this site appears to be supported by a protein interaction with the adjace...

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The immediate early (IE) proteins of human cytomegalovirus (hCMV) have diverse roles in directing viral and host cell transcription. Among these is the ability of IE2 to induce transcription of the IL1B gene that codes for IL-1β in monocytes. This function is partially explained by interaction between IE2 and the host cell transcription factor Spi-1/PU.1 (Spi-1). We now show that maximal IE2 function also depends on productive interactions localizing to two C/EBP sites on the IL1B promoter suggesting either bi-or tri-molecular interactions between IE2, Spi-1 and C/EBPβ at two different locations on the promoter. The IE2 interaction region on Spi-1 was previously mapped to the DNAbinding ETS domain and overlaps the region of Spi-1 that interacts with the transcription factor C/ EBPβ, a factor known to be critical for the induction of IL1B in response to Toll/IL-1 receptor (TIR) family signal transduction. The Spi-1 interacting region of IE2 maps to amino acids 315-328, a sequence that also interacts with the bZIP domain of C/EBPβ. An expression vector coding for amino acids 291-364 of IE2 can suppress LPS induction of a cotransfected IL1B enhancer-promoter fragment in a monocyte cell line. This inhibition is likely the result of competition between Spi-1 and C/EBPβ, thus blunting gene induction.