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1.  Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts 
Background
Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4.
Methods
Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels.
Results
One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data.
Conclusions
These data are not only consistent with previous functional studies of KLF4’s role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4’s functions.
PMCID: PMC3165197  PMID: 21892412
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
2.  Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts 
Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions.
PMCID: PMC3165197  PMID: 21892412
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
3.  The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation1 
The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-κB pathway by blockade of IκB-α ubiquitination, a process catalyzed by the E3-SCFβ-TrCP ubiquitin ligase. The activity of this ubiquitin ligase is regulated via covalent modification of the Cullin-1 subunit by the ubiquitin-like protein NEDD8. We previously reported that interaction of viable commensal bacteria with mammalian intestinal epithelial cells resulted in a rapid and reversible generation of reactive oxygen species (ROS) that modulated neddylation of Cullin-1 and resulted in suppressive effects on the NF-κB pathway. Herein, we demonstrate that butyrate and other short chain fatty acids supplemented to model human intestinal epithelia in vitro and human tissue ex vivo results in loss of neddylated Cul-1 and show that physiological concentrations of butyrate modulate the ubiquitination and degradation of a target of the E3-SCFβ-TrCP ubiquitin ligase, the NF-κB inhibitor IκB-α. Mechanistically, we show that physiological concentrations of butyrate induces reactive oxygen species that transiently alters the intracellular redox balance and results in inactivation of the NEDD8-conjugating enzyme Ubc12 in a manner similar to effects mediated by viable bacteria. Because the normal flora produces significant amounts of butyrate and other short chain fatty acids, these data provide a functional link between a natural product of the intestinal normal flora and important epithelial inflammatory and proliferative signaling pathways.
PMCID: PMC2872164  PMID: 19109186
4.  The Probiotic Lactobacillus GG May Augment Intestinal Host Defense by Regulating Apoptosis and Promoting Cytoprotective Responses in the Developing Murine Gut 
Pediatric research  2008;64(5):511-516.
Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in preterm infants. Although its pathogenesis is poorly understood, inappropriate apoptosis of the mucosal epithelia has been implicated. Recent clinical trials have shown probiotics may reduce the incidence of NEC, and probiotics have been shown to suppress intestinal epithelial apoptosis in cultured cells. However, little is known about their mechanism of action in the developing intestine in vivo. Here, we confirm that the probiotic Lactobacillus rhamnosus GG (LGG) reduces chemically induced intestinal epithelial apoptosis in vitro. Furthermore, we report for the first time that LGG administered orally to live animals can reduce chemically induced epithelial apoptosis ex vivo, as measured by staining for active caspase 3 and TUNEL. Utilizing cDNA microarray analysis from the intestine of live, orally inoculated mice, we show that LGG upregulates of a battery of genes with known and likely cytoprotective effects. These studies indicate that probiotics such as LGG may augment intestinal host defenses in the developing intestine by stimulating anti-apoptotic and cytoprotective responses. Since apoptosis may be a precursor to NEC, understanding the mechanism behind probiotic modulation of apoptotic pathways may allow for development of more specifically targeted therapies or preventive strategies in the future.
doi:10.1203/PDR.0b013e3181827c0f
PMCID: PMC2694849  PMID: 18552706
Necrotizing enterocolitis; probiotics; Lactobacillus; apoptosis; intestinal epithelia
5.  Promoter Activation by Repositioning of RNA Polymerase▿  
Journal of Bacteriology  2008;190(9):3110-3117.
Spo0A, a classical two-component-type response regulator in Bacillus subtilis, binds to a specific DNA sequence found in many promoters to repress or activate the transcription of over 100 genes. On the spoIIG promoter, one of the Spo0A binding sites, centered at position −40, overlaps a consensus −35 element that may also interact with region 4 of the sigma A (σA) subunit of RNA polymerase. Molecular modeling corroborated by genetic evidence led us to propose that the binding of Spo0A to this site repositions σA region 4 on the promoter. Therefore, we used a chemical nuclease, p-bromoacetamidobenzyl-EDTA-Fe, that was covalently tethered to a single cysteine in region 4 of σA to map the position of σA on the promoter. The results indicated that in the absence of Spo0A, σA region 4 of the RNA polymerase was located near the −35 element sequence centered at position −40. However, in the presence of Spo0A, σA region 4 was displaced downstream from the −35 element by 4 bp. These and other results support the model in which the binding of Spo0A to the spoIIG promoter stimulates promoter utilization by repositioning prebound RNA polymerase and stabilizing the repositioned RNA polymerase-promoter complex at a new position that aligns σA region 2 with the −10 region sequences of the promoter, thus facilitating open complex formation.
doi:10.1128/JB.00096-08
PMCID: PMC2347386  PMID: 18296515
6.  Toll-Like Receptor 5-Deficient Mice Have Dysregulated Intestinal Gene Expression and Nonspecific Resistance to Salmonella-Induced Typhoid-Like Disease▿ †  
Infection and Immunity  2008;76(3):1276-1281.
The recognition of flagellin by Toll-like receptor 5 (TLR5) is the dominant means by which model intestinal epithelia activate proinflammatory gene expression in response to Salmonella enterica. The role of the flagellin-TLR5 interaction in vivo has been addressed primarily via studies that use flagellar mutants. Such studies suggest that host recognition of flagellin promotes rapid neutrophil recruitment that protects the host from this pathogen. However, these works do not directly address the role of TLR5 and are subject to the caveat that flagellar mutations may broadly affect Salmonella gene expression. Thus, we examined the role of the flagellin-TLR5 interaction via the use of TLR5-deficient (TLR5KO) mice. We utilized both the traditional model of murine Salmonella infection, wherein low-dose oral infection of mice with Salmonella enterica subsp. enterica serovar Typhimurium results in systemic typhoid-like disease, and a more recently characterized model in which mice are pretreated with streptomycin to result in gut-restricted acute enteritis. In the enteritis model, TLR5KO mice had more severe gut pathology, thus “phenocopying” previous results obtained with Salmonella mutants. In contrast, TLR5KO mice were resistant to Salmonella-induced typhoid-like disease. However, such resistance was not specific for flagellated serovar Typhimurium, but rather, TLR5KO mice were also resistant to challenges by flagellin-deficient serovar Typhimurium. Such resistance associated with elevations in the microbiota was ablated by antibiotic pretreatment and correlated with basal elevations in intestinal host defense gene expression. All together, these results indicate that the resistance of TLR5KO mice to Salmonella-induced typhoid-like illness resulted from alterations in their basal phenotype rather than from the lack of TLR5 ligation during the infection per se.
doi:10.1128/IAI.01491-07
PMCID: PMC2258833  PMID: 18195036
7.  Deletion of TLR5 results in spontaneous colitis in mice 
The Journal of Clinical Investigation  2007;117(12):3909-3921.
Activation of TLRs by bacterial products results in rapid activation of genes encoding products designed to protect the host from perturbing microbes. In the intestine, which is colonized by a large and diverse population of commensal bacteria, TLR signaling may not function in a simple on/off mode. Here, we show that the flagellin receptor TLR5 has an essential and nonredundant role in protecting the gut from enteric microbes. Mice lacking TLR5 (TLR5KO mice) developed spontaneous colitis, as assessed by well-defined clinical, serologic, and histopathologic indicators of this disorder. Compared with WT littermates, TLR5KO mice that had not yet developed robust colitis exhibited decreased intestinal expression of TLR5-regulated host defense genes despite having an increased bacterial burden in the colon. In contrast, such TLR5KO mice displayed markedly increased colonic expression of hematopoietic-derived proinflammatory cytokines, suggesting that elevated levels of bacterial products may result in activation of other TLRs that drive colitis in TLR5KO mice. In accordance, deletion of TLR4 rescued the colitis of TLR5KO mice in that mice lacking both TLR4 and TLR5 also had elevated bacterial loads in the colon but lacked immunological, histopathological, and clinical evidence of colitis. That an engineered innate immune deficiency ultimately results in spontaneous intestinal inflammation supports the notion that an innate immune deficiency might underlie some instances of inflammatory bowel disease.
doi:10.1172/JCI33084
PMCID: PMC2075480  PMID: 18008007
8.  Spo0A-Dependent Activation of an Extended −10 Region Promoter in Bacillus subtilis 
Journal of Bacteriology  2006;188(4):1411-1418.
At the onset of endospore formation in Bacillus subtilis the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, Pskf, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of Pskf provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate the promoter directly, since Spo0A is required for Pskf activity in a ΔabrB strain. Here we investigate the mechanism of Pskf activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the −10 region of the promoter was found to be similar to those of extended −10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended −10 region, as well as other base pairs in the −10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position −9 of Pskf produced a promoter that was active constitutively in both ΔabrB and Δspo0A ΔabrB strains. Therefore, the base pair at position −9 of Pskf makes its activity dependent on Spo0A binding, and the extended −10 region motif of the promoter contributes to its high level of activity.
doi:10.1128/JB.188.4.1411-1418.2006
PMCID: PMC1367231  PMID: 16452424
10.  α-Helix E of Spo0A Is Required for σA- but Not for σH-Dependent Promoter Activation in Bacillus subtilis 
Journal of Bacteriology  2004;186(4):1078-1083.
At the onset of endospore formation in Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two types of promoters. The first type includes the spoIIG and spoIIE promoters, which are used by σA-RNA polymerase, whereas the second type includes the spoIIA promoter, which is used by RNA polymerase containing the secondary sigma factor σH. Previous genetic analyses have identified specific amino acids in α-helix E of Spo0A that are important for activation of Spo0A-dependent, σA-dependent promoters. However, these amino acids are not required for activation of the σH-dependent spoIIA promoter. We now report the effects of additional single-amino-acid substitutions and the effects of deletions in α-helix E. The effects of alanine substitutions revealed one new position (239) in Spo0A that appears to be specifically required for activation of the σA-dependent promoters. Based on the effects of a deletion mutation, we suggest that α-helix E in Spo0A is not directly involved in interaction with σH-RNA polymerase.
doi:10.1128/JB.186.4.1078-1083.2004
PMCID: PMC344211  PMID: 14762002
11.  Surfaces of Spo0A and RNA Polymerase Sigma Factor A That Interact at the spoIIG Promoter in Bacillus subtilis 
Journal of Bacteriology  2004;186(1):200-206.
In Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two classes of promoters, those used by RNA polymerase containing the primary sigma factor, σA (e.g., spoIIG), and those used by RNA polymerase containing the secondary sigma factor, σH (e.g., spoIIA). Several single amino acid substitutions in region 4 of σA define positions in σA that are specifically required for Spo0A-dependent promoter activation. Similarly, several single amino acid substitutions in Spo0A define positions in Spo0A that are required for σA-dependent promoter activation but not for other functions of Spo0A. It is unknown whether these amino acids in Spo0A interact directly with those in region 4 of σA or whether they interact with another subunit of RNA polymerase to effect promoter activation. Here we report the identification of a new amino acid in region 4 of σA, arginine at position 355 (R355), that is involved in Spo0A-dependent promoter activation. To further investigate the role of R355, we used the coordinates of Spo0A and sigma region 4, each in complex with DNA, to build a model for the interaction of σA and Spo0A at the spoIIG promoter. We tested the model by examining the effects of amino acid substitutions in the putative interacting surfaces of these molecules. As predicted by the model, we found genetic evidence for interaction of R355 of σA with glutamine at position 221 of Spo0A. These results appear to define the surfaces of Spo0A and σA that directly interact during activation of the spoIIG promoter.
doi:10.1128/JB.186.1.200-206.2004
PMCID: PMC303461  PMID: 14679239

Results 1-11 (11)