Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase

In skeletal muscle, transcription of the gene encoding the mouse type Iα (RIα) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIα protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIα or RIIα fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIα subunits and requires the amino-terminal residues 1–81. Mutagenesis of Phe-54 to Ala in the full-length RIα–green fluorescent protein template abolishes localization, indicating that dimerization of RIα is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIα at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Iα homologue RCE with AKAPCE and for in vitro binding of RIα to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIα tethering at this site.

Linking osteopetrosis and pycnodysostosis: Regulation of cathepsin K expression by the microphthalmia transcription factor family

Various genetic conditions produce dysfunctional osteoclasts resulting in osteopetrosis or osteosclerosis. These include human pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K mutation, cathepsin K-deficient mice, and mitf mutant rodent strains. Cathepsin K is a highly expressed cysteine protease in osteoclasts that plays an essential role in the degradation of protein components of bone matrix. Cathepsin K also is expressed in a significant fraction of human breast cancers where it could contribute to tumor invasiveness. Mitf is a member of a helix–loop–helix transcription factor subfamily, which contains the potential dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative, but not recessive, mutations of mitf, produce osteopetrosis, suggesting a functional requirement for other family members. Mitf also has been found—and TFE3 has been suggested—to modulate age-dependent changes in osteoclast function. This study identifies cathepsin K as a transcriptional target of Mitf and TFE3 via three consensus elements in the cathepsin K promoter. Additionally, cathepsin K mRNA and protein were found to be deficient in mitf mutant osteoclasts, and overexpression of wild-type Mitf dramatically up-regulated expression of endogenous cathepsin K in cultured human osteoclasts. Cathepsin K promoter activity was disrupted by dominant negative, but not recessive, mouse alleles of mitf in a pattern that closely matches their osteopetrotic phenotypes. This relationship between cathepsin K and the Mitf family helps explain the phenotypic overlap of their corresponding deficiencies in pycnodysostosis and osteopetrosis and identifies likely regulators of cathepsin K expression in bone homeostasis and human malignancy.

Protein-coding genes are epigenetically regulated in Arabidopsis polyploids

The fate of redundant genes resulting from genome duplication is poorly understood. Previous studies indicated that ribosomal RNA genes from one parental origin are epigenetically silenced during interspecific hybridization or polyploidization. Regulatory mechanisms for protein-coding genes in polyploid genomes are unknown, partly because of difficulty in studying expression patterns of homologous genes. Here we apply amplified fragment length polymorphism (AFLP)–cDNA display to perform a genome-wide screen for orthologous genes silenced in Arabidopsis suecica, an allotetraploid derived from Arabidopsis thaliana and Cardaminopsis arenosa. We identified ten genes that are silenced from either A. thaliana or C. arenosa origin in A. suecica and located in four of the five A. thaliana chromosomes. These genes represent a variety of RNA and predicted proteins including four transcription factors such as TCP3. The silenced genes in the vicinity of TCP3 are hypermethylated and reactivated by blocking DNA methylation, suggesting epigenetic regulation is involved in the expression of orthologous genes in polyploid genomes. Compared with classic genetic mutations, epigenetic regulation may be advantageous for selection and adaptation of polyploid species during evolution and development.

Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B

Human cytomegalovirus (HCMV) infection alters the expression of many cellular genes, including IFN-stimulated genes (ISGs) [Zhu, H., Cong, J.-P., Mamtora, G., Gingeras, T. & Shenk, T. (1998) Proc. Natl. Acad. Sci. USA 95, 14470–14475]. By using high-density cDNA microarrays, we show that the HCMV-regulated gene expression profile in fibroblasts does not differ substantially from the response generated by IFN. Furthermore, we identified the specific viral component triggering this response as the envelope glycoprotein B (gB). Cells treated with gB, but not other herpesviral glycoproteins, exhibited the same transcriptional profile as HCMV-infected cells. Thus, the interaction of gB with its as yet unidentified cellular receptor is the principal mechanism by which HCMV alters cellular gene expression early during infection. These findings highlight a pioneering paradigm for the consequences of virus–receptor interactions.

Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: Population genetics, human serologic response, and gene transcription

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase–PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

Dopamine receptor regulating factor, DRRF: A zinc finger transcription factor

Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors. In vivo, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain.

Expression of an Arabidopsis Aspartate Kinase/Homoserine Dehydrogenase Gene Is Metabolically Regulated by Photosynthesis-Related Signals but Not by Nitrogenous Compounds1

Although the control of carbon fixation and nitrogen assimilation has been studied in detail, relatively little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper we report our study of the metabolic regulation of expression of an Arabidopsis aspartate kinase/homoserine dehydrogenase (AK/HSD) gene, which encodes two linked key enzymes in the biosynthetic pathway of aspartate family amino acids. Northern blot analyses, as well as expression of chimeric AK/HSD-β-glucuronidase constructs, have shown that the expression of this gene is regulated by the photosynthesis-related metabolites sucrose and phosphate but not by nitrogenous compounds. In addition, analysis of AK/HSD promoter deletions suggested that a CTTGACTCTA sequence, resembling the binding site for the yeast GCN4 transcription factor, is likely to play a functional role in the expression of this gene. Nevertheless, longer promoter fragments, lacking the GCN4-like element, were still able to confer sugar inducibility, implying that the metabolic regulation of this gene is apparently obtained by multiple and redundant promoter sequences. The present and previous studies suggest that the conversion of aspartate into either the storage amino acid asparagine or aspartate family amino acids is subject to a coordinated, reciprocal metabolic control, and this biochemical branch point is a part of a larger, coordinated regulatory mechanism of nitrogen and carbon storage and utilization.

Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1

DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] is an orphan nuclear receptor that represses transcription by steroidogenic factor-1 (SF-1), a factor that regulates expression of multiple steroidogenic enzymes and other genes involved in reproduction. Mutations in the human DAX1 gene (also known as AHC) cause the X-linked syndrome AHC, a disorder that is associated with hypogonadotropic hypogonadism also. Characterization of Dax1-deficient male mice revealed primary testicular defects that included Leydig cell hyperplasia (LCH) and progressive degeneration of the germinal epithelium, leading to infertility. In this study, we investigated the effect of Dax1 disruption on the expression profile of various steroidogenic enzyme genes in Leydig cells isolated from Dax1-deficient male mice. Expression of the aromatase (Cyp19) gene, which encodes the enzyme that converts testosterone to estradiol, was increased significantly in the Leydig cells isolated from mutant mice, whereas the expression of other proteins (e.g., StAR and Cyp11a) was not altered. In in vitro transfection studies, DAX-1 repressed the SF-1-mediated transactivation of the Cyp19 promoter but did not inhibit the StAR or Cyp11a promoters. Elevated Cyp19 expression was accompanied by increased intratesticular levels of estradiol. Administration of tamoxifen, a selective estrogen-receptor modulator, restored fertility to the Dax1-deficient male mice and partially corrected LCH, suggesting that estrogen excess contributes to LCH and infertility. Based on these in vivo and in vitro analyses, aromatase seems to be a physiologic target of Dax-1 in Leydig cells, and increased Cyp19 expression may account, in part, for the infertility and LCH in Dax1-deficient mice.

CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F.

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B, and H1 is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified. Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp91-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.

Hepatocyte nuclear factor 6, a transcription factor that contains a novel type of homeodomain and a single cut domain.

Tissue-specific transcription is regulated in part by cell type-restricted proteins that bind to defined sequences in target genes. The DNA-binding domain of these proteins is often evolutionarily conserved. On this basis, liver-enriched transcription factors were classified into five families. We describe here the mammalian prototype of a sixth family, which we therefore call hepatocyte nuclear factor 6 (HNF-6). It activates the promoter of a gene involved in the control of glucose metabolism. HNF-6 contains two different DNA-binding domains. One of these corresponds to a novel type of homeodomain. The other is homologous to the Drosophila cut domain. A similar bipartite sequence is coded by the genome of Caenorhabditis elegans.

Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.

The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins.

ECA39, a conserved gene regulated by c-Myc in mice, is involved in G1/S cell cycle regulation in yeast.

The c-myc oncogene has been shown to play a role in cell proliferation and apoptosis. The realization that myc oncogenes may control the level of expression of other genes has opened the field to search for genetic targets for Myc regulation. Recently, using a subtraction/coexpression strategy, a murine genetic target for Myc regulation, called EC439, was isolated. To further characterize the ECA39 gene, we set out to determine the evolutionary conservation of its regulatory and coding sequences. We describe the human, nematode, and budding yeast homologs of the mouse ECA39 gene. Identities between the mouse ECA39 protein and the human, nematode, or yeast proteins are 79%, 52%, and 49%, respectively. Interestingly, the recognition site for Myc binding, located 3' to the start site of transcription in the mouse gene, is also conserved in the human homolog. This regulatory element is missing in the ECA39 homologs from nematode or yeast, which also lack the regulator c-myc. To understand the function of ECA39, we deleted the gene from the yeast genome. Disruption of ECA39 which is a recessive mutation that leads to a marked alteration in the cell cycle. Mutant haploids and homozygous diploids have a faster growth rate than isogenic wild-type strains. Fluorescence-activated cell sorter analyses indicate that the mutation shortens the G1 stage in the cell cycle. Moreover, mutant strains show higher rates of UV-induced mutations. The results suggest that the product of ECA39 is involved in the regulation of G1 to S transition.

The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression.

The ob gene product, leptin, apparently exclusively expressed in adipose tissue, is a signaling factor regulating body weight homeostasis and energy balance. ob gene expression is increased in obese rodents and regulated by feeding, insulin, and glucocorticoids, which supports the concept that ob gene expression is under hormonal control, which is expected for a key factor controlling body weight homeostasis and energy balance. In humans, ob mRNA expression is increased in gross obesity; however, the effects of the above factors on human ob expression are unknown. We describe the structure of the human ob gene and initial functional analysis of its promoter. The human ob gene's three exons cover approximately 15 kb of genomic DNA. The entire coding region is contained in exons 2 and 3, which are separated by a 2-kb intron. The first small 30-bp untranslated exon is located >10.5 kb upstream of the initiator ATG codon. Three kilobases of DNA upstream of the transcription start site has been cloned and characterized. Only 217 bp of 5' sequence are required for basal adipose tissue-specific expression of the ob gene as well as enhanced expression by C/EBPalpha. Mutation of the single C/EBPalpha site in this region abolished inducibility of the promoter by C/EBPalpha in cotransfection assays. The gene structure will facilitate our analysis of ob mutations in human obesity, whereas knowledge of sequence elements and factors regulating ob gene expression should be of major importance in the prevention and treatment of obesity.

SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis.

Cell-specific activation of the transcription factor sigma F during sporulation in Bacillus subtilis is controlled by a regulatory pathway involving the proteins SpoIIE, SpoIIAA, and SpoIIAB. SpoIIAB is an antagonist of sigma F, and SpoIIAA, which is capable of overcoming SpoIIAB-mediated inhibition of sigma F, is an antagonist of SpoIIAB. SpoIIAA is, in turn, negatively regulated by SpoIIAB, which phosphorylates SpoIIAA on serine 58. SpoIIAA is also positively regulated by SpoIIE, which dephosphorylates SpoIIAA-P, the phosphorylated form of SpoIIAA. Here, isoelectric focusing and Western blot analysis were used to examine the phosphorylation state of SpoIIAA in vivo. SpoIIAA was found to be largely in the phosphorylated state during sporulation in wild-type cells but a significant portion of the protein that was unphosphorylated could also be detected. Consistent with the idea that SpoIIE governs dephosphorylation of SpoIIAA-P, SpoIIAA was entirely in the phosphorylated state in spoIIE mutant cells. Conversely, overexpression of spoIIE led to an increase in the ratio of unphosphorylated SpoIIAA to SpoIIAA-P and caused inappropriate activation of sigma F in the predivisional sporangium. We also show that a mutant form of SpoIIAA (SpoIIAA-S58T) in which serine 58 was replaced with threonine was present exclusively as SpoIIAA-P, a finding that confirms previous biochemical evidence that the mutant protein is an effective substrate for the SpoIIAB kinase but that SpoIIAA-S58T-P cannot be dephosphorylated by SpoIIE. We conclude that SpoIIE plays a crucial role in controlling the phosphorylation state of SpoIIAA during sporulation and thus in governing the cell-specific activation of sigma F.