Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the transcriptional activation of a group of adipose-specific genes. As an approach to defining the mechanism responsible for activating the expression of these genes, we investigated the binding of nuclear factors to the promoters of two differentiation-induced genes, the 422(aP2) and stearoyl-CoA desaturase 1 (SCDl) genes. DNase I footprinting and gel retardation analysis identified two binding regions within the promoters of each gene that interact with nuclear factors present in differentiated 3T3-L1 adipocytes. One differentiation-induced nuclear factor interacts specifically with a single binding site in the promoter of each gene. Competition experiments showed that the interaction of this nuclear factor with the SCDl promoter was prevented specifically by a synthetic oligonucleotide corresponding to the site footprinted in the 422(aP2) promoter. Several lines of evidence indicate that the differentiation-induced nuclear factor is CCAAT/enhancer binding protein (C/EBP), a DNA-binding protein first isolated from rat liver. Bacterially expressed recombinant C/EBP binds to the same site at which the differentiation-specific nuclear factor interacts within the promoter of each gene. Northern analysis with RNA from 3T3-L1 cells shows that C/EBP mRNA abundance increases markedly during differentiation. Transient cotransfection studies using a C/EBP expression vector demonstrate that C/EBP can function as a trans-activator of both the 422(aP2) and SCDl gene promoters.

An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21 WAF1/Cip1 gene. We show that the gene encoding the gut-enriched Krü ppel-like factor (GKLF, KLF4) is concurrently induced with p21 WAF1/Cip1during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21 WAF1/Cip1 due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21, suggesting that GKLF may be involved in the induction of p21 Indeed, GKLF activates p21WAF1/Cip1 through a specific Sp1-like cis-element in the p21 WAF1/Cip1 proximal promoter. The same element is also required by p53 to activate the p21 WAF1/Cip1 promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21 WAF1/Cip1 promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21 WAF1/Cip1 promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21 WAF1/Cip1 is demonstrated by the ability of antisense Gklf oligonucleotides to block the production of p21 WAF1/Cip1 in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21 WAF1/Cip1 and may be part of a novel pathway by which cellular responses to stress are modulated.

We describe a postgenomic in silico approach for identifying genes that are likely to be essential and estimate their proportion in haploid genomes. With the knowledge of all sites eligible for mutagenesis and an experimentally determined partial list of nonessential genes from genome mutagenesis, a Bayesian statistical method provides reasonable predictions of essential genes with a subsaturation level of random mutagenesis. For mutagenesis, a transposon such as Himar1 is suitable as it inserts randomly into TA sites. All of the possible insertion sites may be determined a priori from the genome sequence and with this information, data on experimentally hit TA sites may be used to predict the proportion of genes that cannot be mutated. As a model, we used the Mycobacterium tuberculosis genome. Using the Himar1 transposon, we created a genetically defined collection of 1,425 insertion mutants. Based on our Bayesian statistical analysis using Markov chain Monte Carlo and the observed frequencies of transposon insertions in all of the genes, we estimated that the M. tuberculosis genome contains 35% (95% confidence interval, 28%-41%) essential genes. This analysis further revealed seven functional groups with high probabilities of being enriched in essential genes. The PE-PGRS (Pro-Glu polymorphic GC-rich repetitive sequence) family of genes, which are unique to mycobacteria, the polyketide͞ nonribosomal peptide synthase family, and mycolic and fatty acid biosynthesis gene families were disproportionately enriched in essential genes. At subsaturation levels of mutagenesis with a random transposon such as Himar1, this approach permits a statistical prediction of both the proportion and identities of essential genes of sequenced genomes.

Differentiation of 3T3-L1 preadipocytes into adipocytes is accompanied by increased expression of the nuclear protein C/EBP (CCAAT/enhancer binding protein) and by transcriptional activation of a group of adipose-specific genes. We report here the isolation of the murine C/EBP gene and the characterization of its promoter. Consistent with its proposed role in coordinating transcription during preadipocyte differentiation, an increase in the rate of transcription of the C/EBP gene precedes that of several adipose-specific genes whose promoters are transactivated by C/EBP. DNase I cleavage-inhibition patterns (footprinting) of the C/EBP gene promoter by nuclear factors from differentiated and undifferentiated 3T3-L1 cells identified two sites of differential factor binding. One site in the C/EBP gene promoter between nudeotides -252 and -239 binds a nuclear factor(s) present in preadipocytes that is lost or modified upon differentiation. Another site, between nucleotides -203 and -176, exhibits different but overlapping footprints by nuclear factors present in differentiated and undifferentiated cells. Gel retardation analysis with oligonucleotides corresponding to these sites revealed protein-oligonucleotide complexes containing these differentially expressed nuclear factors. The factor present in differentiated cells that binds at this site was identified as C/EBP (possibly in heterodimeric form with a homologous leucine-zipper protein), suggesting that C/EBP may regulate expression of its own gene.Differentiation of 3T3-L1 preadipocytes into adipocytes in culture is accompanied by a dramatic rise in the rate of transcription of adipose-specific genes (1, 2). The isolation of a number of these genes (3-5) and characterization of their respective promoters prompted a search for transcription factors that coordinately activate their expression. Recently, it was discovered (6-8) that the CCAAT/enhancer binding protein (C/EBP) exhibits many of the characteristics of such a transcriptional activator.C/EBP was first isolated from rat liver nuclei as a sequence-specific DNA binding protein with preference for binding to CCAAT boxes and to the enhancer regions of several viral promoters (7). High levels of expression of C/EBP were subsequently found to be limited to terminally differentiated cell types-e.g., hepatocytes and adipocytesthat play a central role in energy metabolism, particularly in the synthesis and mobilization of glycogen and fat (6, 9, 10). Most recently, C/EBP was identified as the differentiationinduced nuclear factor that binds specifically to homologous regulatory elements within the promoters of three genes that are coordinately expressed when 3T3-L1 preadipocytes differentiate into adipocytes (6, 11). It was further demonstrated that C/EBP can function as a trans-activator ofthe promoters of these genes (6,11,12) and that mutation of the C/EBP binding site obliterates trans-activation (6, 12).Given that C/EBP may play a pleiotropic role in coordinating gene expression in this system, it wa...

Krü ppel-like factor 4 (KLF4) is an epithelial cell-enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Previous studies show that constitutive expression of KLF4 inhibits DNA synthesis but the manner by which KLF4 exerts this effect is unclear. In the present study, we developed a system in which expression of KLF4 is controlled by a promoter that is induced upon treatment of cells containing the receptors for the insect hormone, ecdysone, with ponasterone A, an ecdysone analogue. The rate of proliferation of a stably transfected colon cancer cell line, RKO, was significantly decreased following addition of ponasterone A when compared with untreated cells. Flow cytometric analyses indicated that the inducible expression of KLF4 caused a block in the G 1 /S phase of the cell cycle. A similar block was observed when ecdysone receptor-containing RKO cells were infected with a replication-defective recombinant adenovirus containing an inducible KLF4 and treated with ponasterone A. Results of these studies provide evidence that the inhibitory effect of KLF4 on cell proliferation is mainly exerted at the G 1 /S boundary of the cell cycle.

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The seven Mycobacterium tuberculosis whiB-like genes encode small proteins postulated to be transcriptional regulators. A systematic real-time reverse transcription-PCR analysis following exposure to antibiotics and a variety of growth and in vitro stress conditions indicates differential, and in some cases dramatic, transcription modulations for the different M. tuberculosis whiB family members. This information together with biochemical analyses of the whiB1 to whiB7 gene products will be important for understanding the biology of this novel family of proteins in mycobacteria and related actinomycetes.Upon infection, Mycobacterium tuberculosis has the ability to adapt to many different environments within the host organism. Tubercle bacilli are able to avoid immune system detection and persist inside the host for decades. Many of the conditions to which the bacteria are exposed, such as the acidic environment within the phagolysosomal compartment, are harsh (14). In the lung, there is a recruitment of activated macrophages to the infection site, and these, along with other immune cells, contain the infection by forming a tuberculous granuloma (49). M. tuberculosis is adept at surviving within the hypoxic and fatty acid-rich granulomatous environment (8), a facet critical to its ability to persist despite immune pressure (25,28). Moreover, tubercle bacilli can survive for long periods of exposure to low-nutrient conditions, such as may occur within a granuloma (4, 34), as well as temperature and oxidative stress which may act to disrupt the M. tuberculosis cell membrane. The bacterium also has a remarkable tolerance to a wide range of antibiotics (12,26). To survive, the organism must sense and respond to exogenous stress conditions. Hence, differential expression of transcriptional regulators, which control sets of genes that respond to environmental stimuli, is an important mechanism of stress survival.A family of genes which may be key transcriptional regulators in M. tuberculosis is the whiB gene family. The whiB-like genes are exclusive to the actinomycetes, such as Mycobacterium and Streptomyces spp., and are absent from all other organisms studied thus far (9, 44). whiB was identified in Streptomyces coelicolor as an essential gene for sporulation of aerial hyphae (9), and early sporulation genes were identified by morphological studies of a collection of mutants unable to form normal gray spore pigment (17). Because their aerial mycelium remained white upon prolonged incubation, these mutants were designated whi mutants. S. coelicolor whiB encodes an 87-amino-acid polypeptide with attributes suggesting that it may be a DNA binding protein (9). S. coelicolor WhiB and its related homologues, including S. coelicolor WhiD, each contain four cysteine residues, a feature common in metalcoordinating DNA binding proteins (33). Indeed, a recent study has found that Sc WhiD binds a [4Fe-4S] cluster under anaerobic conditions similar to the situation with Escherichia coli DNA binding regulators SoxR and Fnr (6,10,...

The Mycobacterium tuberculosis alternate sigma factor, SigF, is expressed during stationary growth phase and under stress conditions in vitro. To better understand the function of SigF we studied the phenotype of the M. tuberculosis ⌬sigF mutant in vivo during mouse infection, tested the mutant as a vaccine in rabbits, and evaluated the mutant's microarray expression profile in comparison with the wild type. In mice the growth rates of the ⌬sigF mutant and wild-type strains were nearly identical during the first 8 weeks after infection. At 8 weeks, the ⌬sigF mutant persisted in the lung, while the wild type continued growing through 20 weeks. Histopathological analysis showed that both wild-type and mutant strains had similar degrees of interstitial and granulomatous inflammation during the first 12 weeks of infection. However, from 12 to 20 weeks the mutant strain showed smaller and fewer lesions and less inflammation in the lungs and spleen. Intradermal vaccination of rabbits with the M. tuberculosis ⌬sigF strain, followed by aerosol challenge, resulted in fewer tubercles than did intradermal M. bovis BCG vaccination. Complete genomic microarray analysis revealed that 187 genes were relatively underexpressed in the absence of SigF in early stationary phase, 277 in late stationary phase, and only 38 genes in exponential growth phase. Numerous regulatory genes and those involved in cell envelope synthesis were down-regulated in the absence of SigF; moreover, the ⌬sigF mutant strain lacked neutral red staining, suggesting a reduction in the expression of envelope-associated sulfolipids. Examination of 5-untranslated sequences among the downregulated genes revealed multiple instances of a putative SigF consensus recognition sequence: GGTTTCX 18 GGGTAT. These results indicate that in the mouse the M. tuberculosis ⌬sigF mutant strain persists in the lung but at lower bacterial burdens than wild type and is attenuated by histopathologic assessment. Microarray analysis has identified SigF-dependent genes and a putative SigF consensus recognition site.