Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays

We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced–PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced–PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for ≈1,000 stress-induced and -repressed Arabidopsis genes.

γ-Secretase inhibitors repress thymocyte development

A major therapeutic target in the search for a cure to the devastating Alzheimer's disease is γ-secretase. This activity resides in a multiprotein enzyme complex responsible for the generation of Aβ42 peptides, precipitates of which are thought to cause the disease. γ-Secretase is also a critical component of the Notch signal transduction pathway; Notch signals regulate development and differentiation of adult self-renewing cells. This has led to the hypothesis that therapeutic inhibition of γ-secretase may interfere with Notch-related processes in adults, most alarmingly in hematopoiesis. Here, we show that application of γ-secretase inhibitors to fetal thymus organ cultures interferes with T cell development in a manner consistent with loss or reduction of Notch1 function. Progression from an immature CD4−/CD8− state to an intermediate CD4+/CD8+ double-positive state was repressed. Furthermore, treatment beginning later at the double-positive stage specifically inhibited CD8+ single-positive maturation but did not affect CD4+ single-positive cells. These results demonstrate that pharmacological γ-secretase inhibition recapitulates Notch1 loss in a vertebrate tissue and present a system in which rapid evaluation of γ-secretase-targeted pharmaceuticals for their ability to inhibit Notch activity can be performed in a relevant context.

Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding α-crystallin

Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis α-crystallin (acr) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

Developmental and Hormonal Regulation of Genes Coding for Proline-Rich Proteins in Female Inflorescences and Kernels of Maize1

The pattern of expression of two genes coding for proteins rich in proline, HyPRP (hybrid proline-rich protein) and HRGP (hydroxyproline-rich glycoprotein), has been studied in maize (Zea mays) embryos by RNA analysis and in situ hybridization. mRNA accumulation is high during the first 20 d after pollination, and disappears in the maturation stages of embryogenesis. The two genes are also expressed during the development of the pistillate spikelet and during the first stages of embryo development in adjacent but different tissues. HyPRP mRNA accumulates mainly in the scutellum and HRGP mRNA mainly in the embryo axis and the suspensor. The two genes appear to be under the control of different regulatory pathways during embryogenesis. We show that HyPRP is repressed by abscisic acid and stress treatments, with the exception of cold treatment. In contrast, HRGP is affected positively by specific stress treatments.

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.

Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNase I

Most methods for assessment of chromatin structure involve chemical or nuclease damage to DNA followed by analysis of distribution and susceptibility of cutting sites. The agents used generally do not permeate cells, making nuclear isolation mandatory. In vivo mapping strategies might allow detection of labile constituents and/or structures that are lost when chromatin is swollen in isolated nuclei at low ionic strengths. DNase I has been the most widely used enzyme to detect chromatin sites where DNA is active in transcription, replication or recombination. We have introduced the bovine DNase I gene into yeast under control of a galactose-responsive promoter. Expression of the nuclease leads to DNA degradation and cell death. Shorter exposure to the active enzyme allows mapping of chromatin structure in whole cells without isolation of nuclei. The validity and efficacy of the strategy are demonstrated by footprinting a labile repressor bound to its operator. Investigation of the inter-nucleosome linker regions in several types of repressed domains has revealed different degrees of protection in cells, relative to isolated nuclei.

A two-hybrid system for transactivator bait proteins

We describe a two-hybrid strategy for detection of interactions with transactivator proteins. This repressed transactivator (RTA) system employs the N-terminal repression domain of the yeast general repressor TUP1. TUP1-GAL80 fusion proteins, when coexpressed with GAL4, are shown to inhibit transcription of GAL4-dependent reporter genes. This effect requires the C-terminal 30 residues of GAL4, which are required for interaction with GAL80 in vitro. Furthermore, repression of GAL transcription by TUP1-GAL80 requires SRB10, demonstrating that the TUP1 repression domain, in the context of a two-hybrid interaction, functions by the same mechanism as endogenous TUP1. Using this strategy, we demonstrate interactions between the mammalian basic helix–loop–helix proteins MyoD and E12, and between c-Myc and Bin-1. We have also identified interacting clones from a TUP1-cDNA fusion expression library by using GAL4-VP16 as a bait fusion. These results demonstrate that RTA is generally applicable for identifying and characterizing interactions with transactivator proteins in vivo.

Human prostate tumor growth in athymic mice: inhibition by androgens and stimulation by finasteride.

When the human prostate cancer cell line, LNCaP 104-S, the growth of which is stimulated by physiological levels of androgen, is cultured in androgen-depleted medium for > 100 passages, the cells, now called LNCaP 104-R2, are proliferatively repressed by low concentrations of androgens. LNCaP 104-R2 cells formed tumors in castrated male athymic nude mice. Testosterone propionate (TP) treatment prevented LNCaP 104-R2 tumor growth and caused regression of established tumors in these mice. Such a tumor-suppressive effect was not observed with tumors derived from LNCaP 104-S cells or androgen receptor-negative human prostate cancer PC-3 cells. 5 alpha-Dihydrotestosterone, but not 5 beta-dihydrotestosterone, 17 beta-estradiol, or medroxyprogesterone acetate, also inhibited LNCaP 104-R2 tumor growth. Removal of TP or implantation of finasteride, a 5 alpha-reductase inhibitor, in nude mice bearing TP implants resulted in the regrowth of LNCaP 104-R2 tumors. Within 1 week after TP implantation, LNCaP 104-R2 tumors exhibited massive necrosis with severe hemorrhage. Three weeks later, these tumors showed fibrosis with infiltration of chronic inflammatory cells and scattered carcinoma cells exhibiting degeneration. TP treatment of mice with LNCaP 104-R2 tumors reduced tumor androgen receptor and c-myc mRNA levels but increased prostate-specific antigen in serum- and prostate-specific antigen mRNA in tumors. Although androgen ablation has been the standard treatment for metastatic prostate cancer for > 50 years, our study shows that androgen supplementation therapy may be beneficial for treatment of certain types of human prostate cancer and that the use of 5 alpha-reductase inhibitors, such as finasteride or anti-androgens, in the general treatment of metastatic prostate cancer may require careful assessment.

Activation domain-mediated enhancement of activator binding to chromatin in mammalian cells.

DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.

Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation.

BCL-2-deficient T cells demonstrate accelerated cell cycle progression and increased apoptosis following activation. Increasing the levels of BCL-2 retarded the G0-->S transition, sustained the levels of cyclin-dependent kinase inhibitor p27Kip1, and repressed postactivation death. Proximal signal transduction events and immediate early gene transcription were unaffected. However, the transcription and synthesis of interleukin 2 and other delayed early cytokines were markedly attenuated by BCL-2. In contrast, a cysteine protease inhibitor that also blocks apoptosis had no substantial affect upon cytokine production. InterleUkin 2 expression requires several transcription factors of which nuclear translocation of NFAT (nuclear factor of activated T cells) and NFAT-mediated transactivation were impaired by BCL-2. Thus, select genetic aberrations in the apoptotic pathway reveal a cell autonomous coregulation of activation.

Identification of potential target genes for the neuron-restrictive silencer factor.

The neuron-restrictive silencer factor (NRSF) represses transcription of several neuronal genes in nonneuronal cells by binding to a 21-bp element called the neuron-restrictive silencer element (NRSE). We have performed data base searches with a composite NRSE to identify additional candidate NRSF target genes. Twenty-two more genes, 17 of which are expressed mainly in neurons, were found to contain NRSE-like sequences. Many of these putative NRSEs bound NRSF in vitro and repressed transcription in vivo. Most of the neuronal genes identified contribute to the basic structural or functional properties of neurons. However, two neuronal transcription factor genes contain NRSEs, suggesting that NRSF may repress neuronal differentiation both directly and indirectly. Functional NRSEs were also found in several nonneuronal genes, implying that NRSF may play a broader role than originally anticipated.

Association of yeast SIN1 with the tetratrico peptide repeats of CDC23.

The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sin1 mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two-hybrid system to identify proteins that interact with SIN1 in vivo. Here we demonstrate that CDC23, a protein known to be involved in sister chromatid separation during mitosis, is able to directly interact with SIN1. Furthermore, using recombinant molecules in vitro, we show that the N terminal of SIN1 is sufficient to bind a portion of CDC23 consisting solely of tetratrico peptide repeats. Earlier experiments identified the C-terminal domain of SIN1 to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1. Taken together with the results presented here, we suggest that SIN1 is a chromatin protein having at least a dual function: The N terminal of SIN1 interacts with the tetratrico peptide repeat domains of CDC23, a protein involved in chromosome segregation, whereas the C terminal of SIN1 binds proteins involved in transcriptional regulation.

Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus.

Using genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affinity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controlled by Tc. Cultured rat mesangial cells were cotransfected with a regulator plasmid and a response plasmid that introduces a beta-galactosidase gene. In vitro, stable double-transfectant MtTAG cells exhibited no beta-galactosidase activity in the presence of Tc. However, following withdrawal of Tc from culture media, expression of beta-galactosidase was induced within 24 h. When Tc was again added, the expression was rapidly resuppressed. Low concentrations of Tc were sufficient to maintain the silent state of tTA-dependent promoter. MtTAG cells were then transferred into the rat glomeruli via renal artery injection. In the isolated chimeric glomeruli, expression of beta-galactosidase was induced ex vivo in the absence of Tc, whereas it was repressed in its presence. When Tc-pretreated MtTAG cells were transferred into the glomeruli of untreated rats, beta-galactosidase expression was induced in vivo within 3 days. Oral administration of Tc dramatically suppressed this induction. These data demonstrate the feasibility of using mesangial cell vectors combined with the Tc regulatory system for site-specific in vivo control of exogenous gene expression in the glomerulus.

Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes.

Lack of leptin (ob) protein causes obesity in mice. The leptin gene product is important for normal regulation of appetite and metabolic rate and is produced exclusively by adipocytes. Leptin mRNA was induced during the adipose conversion of 3T3-L1 cells, which are useful for studying adipocyte differentiation and function under controlled conditions. We studied leptin regulation by antidiabetic thiazolidinedione compounds, which are ligands for the adipocyte-specific nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) that regulates the transcription of other adipocyte-specific genes. Remarkably, leptin gene expression was dramatically repressed within a few hours after thiazolidinedione treatment. The ED50 for inhibition of leptin expression by the thiazolidinedione BRL49653 was between 5 and 50 nM, similar to its Kd for binding to PPARgamma. The relatively weak, nonthiazolidinedione PPAR activator WY 14,643 also inhibited leptin expression, but was approximately 1000 times less potent than BRL49653. These results indicate that antidiabetic thiazolidinediones down-regulate leptin gene expression with potencies that correlate with their abilities to bind and activate PPARgamma.

Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation.

Genetic code differences prevent expression of nuclear genes within Saccharomyces cerevisiae mitochondria. To bridge this gap a synthetic gene, ARG8m, designed to specify an arginine biosynthetic enzyme when expressed inside mitochondria, has been inserted into yeast mtDNA in place of the COX3 structural gene. This mitochondrial cox3::ARG8m gene fully complements a nuclear arg8 deletion at the level of cell growth, and it is dependent for expression upon nuclear genes that encode subunits of the COX3 mRNA-specific translational activator. Thus, cox3::ARG8m serves as a mitochondrial reporter gene. Measurement of cox3::ARG8m expression at the levels of steady-state protein and enzymatic activity reveals that glucose repression operates within mitochondria. The levels of this reporter vary among strains whose nuclear genotypes lead to under- and overexpression of translational activator subunits, in particular Pet494p, indicating that mRNA-specific translational activation is a rate-limiting step in this organellar system. Whereas the steady-state level of cox3::ARG8m mRNA was also glucose repressed in an otherwise wild-type strain, absence of translational activation led to essentially repressed mRNA levels even under derepressing growth conditions. Thus, the mRNA is stabilized by translational activation, and variation in its level may be largely due to modulation of translation.