Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos.

We have analyzed the structure and the regulation of Krox-20, a mouse zinc finger-encoding gene which is transiently activated following serum stimulation of quiescent fibroblast cells in culture. The gene is localized on chromosome 10, band B5, in the mouse, and the homologous human gene also maps to chromosome 10 (region q21.1 to q22.1). Alternative splicing of the 5'-most intron of the Krox-20 gene gives rise to mRNAs encoding putative zinc finger proteins with different N termini. The first exon contains a sequence element with strong similarity to the c-fos proto-oncogene serum response element (SRE). This element can functionally substitute for the c-fos SRE, and it binds the same nuclear protein. It is probably responsible for the serum induction of Krox-20, possibly in combination with a weaker SRE located in the 5'-flanking region of the gene. Our findings suggest that c-fos, Krox-20, and a number of immediate-early serum response genes are coregulated and that the SRE and its cognate protein are essential components of this regulatory pathway.     

A novel plant nuclear gene encoding chloroplast ribosomal protein S9 has a transit peptide related to that of rice chloroplast ribosomal protein L12

We have cloned a novel nuclear gene for a ribosomal protein of rice and Arabidopsis that is like the bacterial ribosomal protein S9. To determine the subcellular localization of the gene product, we fused the N-terminal region and green fluorescent protein and expressed it transiently in rice seedlings. Localized fluorescence was detectable only in chloroplasts, indicating that this nuclear gene encodes chloroplast ribosomal protein S9. The N-terminal region of rice ribosomal protein S9 was found to have a high sequence similarity to the transit peptide region of the rice chloroplast ribosomal protein L12, suggesting that these transit peptides have a common lineage.     

Isolation of a novel interleukin-1-inducible nuclear protein bearing ankyrin-repeat motifs

We isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other IkappaB family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.11. Northern blot analyses revealed that INAP was not expressed in any examined tissues without stimulation, but INAP expression was rapidly and transiently induced by IL-1 although not by tumor necrosis factor alpha nor by phorbol 12-myristate 13-acetate in OP9 cells. Immunoblots with anti-INAP-specific antibody demonstrated that INAP was rapidly and specifically produced by IL-1 stimulation and was predominantly localized in the nucleus. Immunofluorescence stainings showed that the INAP newly synthesized by IL-1 stimulation was promptly translocated into the nucleus, and FLAG-tagged INAP forcibly expressed in NIH/3T3 cells was also specifically localized in the nucleus. The possible interaction of INAP with RelA/p65, NF-kappaB1/p50, NF-kappaB2/p52, C/EBPbeta, and retinoid X receptor was examined, but we could detect none of these interactions in the nuclear extracts of IL-1-stimulated cells. Unlike Bcl-3 and other IkappaB family members, INAP may play a unique role in IL-1-induced specific gene expression and/or signal transduction in the nucleus.     

Structure and regulation of the envoplakin gene

Envoplakin, a member of the plakin family of proteins, is a component of desmosomes and the epidermal cornified envelope. To understand how envoplakin expression is regulated, we have analyzed the structure of the mouse envoplakin gene and characterized the promoters of both the human and mouse genes. The mouse gene consists of 22 exons and maps to chromosome 11E1, syntenic to the location of the human gene on 17q25. The exon-intron structure of the mouse envoplakin gene is common to all members of the plakin family: the N-terminal protein domain is encoded by 21 small exons, and the central rod domain and the C-terminal globular domain are coded by a single large exon. The C terminus shows the highest sequence conservation between mouse and human envoplakins and between envoplakin and the other family members. The N terminus is also conserved, with sequence homology extending to Drosophila Kakapo. A region between nucleotides -101 and 288 was necessary for promoter activity in transiently transfected primary keratinocytes. This region is highly conserved between the human and mouse genes and contains at least two different positively acting elements identified by site-directed mutagenesis and electrophoretic mobility shift assays. Mutation of a GC box binding Sp1 and Sp3 proteins or a combined E box and Krüppel-like element interacting with unidentified nuclear proteins virtually abolished promoter activity. 600 base pairs of the mouse upstream sequence was sufficient to drive expression of a beta-galactosidase reporter gene in the suprabasal layers of epidermis, esophagus, and forestomach of transgenic mice. Thus, we have identified a regulatory region in the envoplakin gene that can account for the expression pattern of the endogenous protein in stratified squamous epithelia.     

Evidence for a novel cardiac-enriched retinoid X receptor partner.

Recent studies indicate that retinoid-mediated pathways play a pivotal role in cardiac morphogenesis and function. To identify proteins that serve as interacting partners of the retinoid X receptor alpha (RXRalpha) in heart, DNA-protein binding studies were performed with an RXR-responsive element (NRRE-1) derived from the medium chain acyl-CoA dehydrogenase gene promoter and nuclear protein extracts prepared from adult rat heart. NRRE-1 is a pleiotropic RXR-responsive element comprised of three potential recognition sites for class II members of the nuclear receptor superfamily. Gel mobility shift assays performed with an NRRE-1 probe in the absence or presence of bacterially overproduced RXRalpha and nuclear protein extracts prepared from adult rat heart, liver, or brain identified a cardiac-specific, RXR-dependent DNA-protein interaction. The NRRE-1-RXR.cardiac-enriched RXR-interacting protein (CERIP) complex exhibited a distinct mobility compared with NRRE-1-RXR.peroxisome proliferator-activated receptor, NRRE-1-RXR.retinoic acid receptor, or NRRE-1-RXR.thyroid receptor complexes. Mutational analysis demonstrated that two of the three potential binding half-sites of NRRE-1 (an everted repeat separated by an 8-base pair spacer) are required for the NRRE-1-RXR. CERIP interaction. Gel mobility shift assays demonstrated that CERIP interacted with RXRalpha and RXRgamma but not with RXRbeta, indicating a receptor subtypespecific binding preference and suggesting an RXR AB region-dependent interaction. The RXR.CERIP complex did not form on NRRE-1 when a mutant GST-RXRalpha fusion protein lacking the NH(2)-terminal AB region (but containing the receptor dimerization domain) of RXRalpha was added in place of the full-length RXRalpha, confirming a role for the AB region in the RXR. CERIP interaction. DNA-protein cross-linking studies demonstrated that CERIP is a DNA-binding protein of approximately 110 kDa. These results provide evidence for the existence of a cardiac-enriched DNA-binding protein that interacts with RXRalpha via the AB region and suggest a mechanism whereby cardiac retinoid signaling is controlled in an RXR subtype-specific manner.     

Hairless contains a novel nuclear matrix targeting signal and associates with histone deacetylase 3 in nuclear speckles

Hair follicle cycling is a highly regulated and dynamic cellular process consisting of phases of growth, regression, and quiescence. The hairless (hr) gene encodes a nuclear factor that is highly expressed in the skin, where it appears to be an essential regulator during the regression in the catagen hair follicle. In hairless mice, as well as humans with congenital atrichia, the absence of hr protein initiates a premature and abnormal catagen due to defects in the signaling required for hair follicle remodeling. Here, we report that hr protein is a nuclear protein that is tightly associated with the nuclear matrix scaffold. Using a series of deletion constructs of the mouse hr gene, we monitored the sub-cellular localization of the recombinant protein by in situ immunolocalization and biochemical fractionation after nuclear matrix extraction of transiently transfected cells. We identified a novel nuclear matrix-targeting signal (NMTS) in the hr protein and mapped the domain to amino acid residues 111-186 of the mouse hr sequence. Furthermore, we provide evidence that this region not only mediates the interaction of hr with components of the nuclear architecture, but also specifies the sub-nuclear location of the hr protein to nuclear domains containing deacetylase activity. The N-terminal region directs hr to a speckled nuclear pattern that co-localizes with the histone deacetylase 3 (HDAC), but not with HDAC1 or HDAC7. Based on our findings, we propose that hr protein is part of a specific multi-protein repressor complex and that hr may be involved in chromatin remodeling.     

Identification of amino acid sequence in the hinge region of human vitamin D receptor that transfers a cytosolic protein to the nucleus

The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D(3) was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the subcellular distribution of the fusion proteins was examined. GFP-tagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout the cells. 10(-8) M 1,25-dihydroxyvitamin D(3) promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus.     

Phorbol ester, forskolin, and serum induction of a human colon nuclear hormone receptor gene related to the NUR 77/NGFI-B genes.

Two closely related members (mouse NUR 77 and rat NGFI-B) of the serum-inducible "early intermediate" gene family are nuclear hormone receptors containing zinc fingers of the cys2-cys2 type. This paper describes the complementary DNA cloning of the human equivalent of the NUR 77/NGFI-B genes isolated from LS-180 colon adenocarcinoma cells and named the ST-59 gene. ST-59 RNA expression was shown to be rapidly and transiently induced by fetal calf serum. To a lesser extent, epidermal growth factor could induce ST-59 RNA expression, but nerve growth factor, insulin-like growth factor, and fibroblast growth factor were ineffective. ST-59 receptor induction by serum was greatly amplified by cycloheximide and could be detected in actively growing LS-180 cells. The serum induction of RNA expression in these cells could be augmented by treatment with phorbol esters (10(-5) M), forskolin (10(-5) M), and 8-bromo cyclic AMP (4 x 10(-3) M). These results suggest that at least two signal pathways (protein kinase C and protein kinase A) participate in the ST-59 gene mRNA induction.     

Intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells

The nucleocapsid (N) protein of several members within the order Nidovirales localizes to the nucleolus during infection and after transfection of cells with N genes. However, confocal microscopy of N protein localization in Vero cells infected with the severe acute respiratory syndrome coronavirus (SARS-CoV) or transfected with the SARS-CoV N gene failed to show the presence of N in the nucleoplasm or nucleolus. Amino acids 369 to 389, which contain putative nuclear localization signal (NLS) and nucleolar localization signal motifs, failed to restore nuclear localization to an NLS-minus mutant Rev protein. These data indicate that nuclear localization is not a conserved property among all nidoviruses.