High-throughput SELEX SAGE method for quantitative modeling of transcription-factor binding sites.

The ability to determine the location and relative strength of all transcription-factor binding sites in a genome is important both for a comprehensive understanding of gene regulation and for effective promoter engineering in biotechnological applications. Here we present a bioinformatically driven experimental method to accurately define the DNA-binding sequence specificity of transcription factors. A generalized profile was used as a predictive quantitative model for binding sites, and its parameters were estimated from in vitro-selected ligands using standard hidden Markov model training algorithms. Computer simulations showed that several thousand low- to medium-affinity sequences are required to generate a profile of desired accuracy. To produce data on this scale, we applied high-throughput genomics methods to the biochemical problem addressed here. A method combining systematic evolution of ligands by exponential enrichment (SELEX) and serial analysis of gene expression (SAGE) protocols was coupled to an automated quality-controlled sequence extraction procedure based on Phred quality scores. This allowed the sequencing of a database of more than 10,000 potential DNA ligands for the CTF/NFI transcription factor. The resulting binding-site model defines the sequence specificity of this protein with a high degree of accuracy not achieved earlier and thereby makes it possible to identify previously unknown regulatory sequences in genomic DNA. A covariance analysis of the selected sites revealed non-independent base preferences at different nucleotide positions, providing insight into the binding mechanism.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Role of β-catenin and γ-catenin in haematopoiesis : Generation of floxed Delta like 4 gene targeted mice

Résumé La voie de signalisation de Wnt est extrêmement conservée au cours de l'évolution. Les protéines Wnt sont des molécules sécrétées qui se lient à la famille de récepteurs Frizzled. Cette interaction mène à la stabilisation de la protéine β-caténine, qui va s'accumuler dans le cytoplasme puis migrer dans le noyau où elle peut s'hétérodimériser avec les facteurs de transcription de la famille TCF/LEF. Il a été démontré que cette voie de signalisation joue un rôle important durant la lymphopoïèse et de récents résultats suggèrent un rôle clé de cette voie dans le renouvellement des Cellules Souches Hématopoïétique (CSH). Des études se basant sur un système de surexpression de protéines montrent clairement que la voie Wnt peut influencer l'hématopoïèse. Cependant, le rôle de la protéine β-caténine dans le système hématopoïétique n'a jamais été testé directement. Ce projet de thèse se propose d'étudier la fonction de la protéine β-caténine par sa délétion inductible via le système Cre-loxP. De façon surprenante, nous avons pu démontrer que les progéniteurs de la moelle osseuse, déficients en β-caténine, ne montrent aucune altération dans leur capacité à s'auto-renouveler et/ou à reconstituer toutes les lignées hématopoïétiques (myéloïde, érythroïde et lymphoïde) dans les souris-chimères. De plus, le développement, la survie des thymocytes ainsi que la prolifération des cellules T périphériques induite par un antigène, sont indépendants de β-caténine. Ces résultats suggèrent soit que la protéine β-caténine ne joue pas un rôle primordial dans le système hématopoiétique, soit que son absence pourrait être compensée par une autre protéine. Un candidat privilégié susceptible de se substituer à β-caténine, serait plakoglobine, aussi connu sous le nom de γ-caténine. En effet, ces deux protéines partagent de multiples caractéristiques structurelles. Afin de démontrer que la protéine γ-caténine peut compenser l'absence de β-caténine, nous avons généré des souris dans lesquelles, le système hématopoïétique est déficient pour ces deux protéines. Cette déficience combinée de β- caténine et γ-caténine ne perturbe pas la capacité des Cellules Souche Hématopoïétique-Long Terme (CSH-LT) de se renouveler, par contre elle agit sur un progéniteur précoce déjà différencié de la moelle osseuse. Ces résultats mettent en évidence que la protéine γ-caténine est capable de compenser l'absence de protéine β-caténine dans le système hématopoïétique. Par conséquent, ce travail contribue à une meilleure connaissance de la cascade Wnt dans l'hématopoïèse. Summary The canonical Wnt signal transduction pathway is a developmentally highly conserved. Wnts are secreted molecules which bind to the family of Frizzled receptors in a complex with the low density lipoprotein receptor related protein (LRP-5/6). This initial activation step leads to the stabilization and accumulation of β-catenin, first in the cytoplasm and subsequently in the nucleus where it forms heterodimers with TCF/LEF transcription factor family members. Wnt signalling has been shown to be important during early lymphopoiesis and has more recently, been suggested to be a key player in self-renewal of haematopoietic stem cells (HSCs). Although mostly gain of function studies indicate that components of the Wnt signalling pathway can influence the haematopoietic system, the role of β-catenin has never been directly investigated. The aim of this thesis project is to investigate the putatively critical role of β-catenin in vivo using the Cre-loxP mediated conditional loss of function approach. Surprisingly, β-catenin deficient bone marrow (BM) progenitors arc not impaired in their ability to self-renew and/or to reconstitute all haematopoietic lineages (myeloid, erythroid and lymphoid) in both mixed and straight bone marrow chimeras. In addition, both thymocyte development and survival, and antigen-induced proliferation of peripheral T cells are β- catenin independent. Our results do not necessarily exclude the possibility of an important function for β-catenin mediated Wnt signalling in the haematopoietic system, it rather raises the question that β-catenin is compensated for by another protein. A prime candidate that may take over the function of β-catenin in its absence, is the close relative plakoglobin, also know as γ-catenin. This protein shares multiple structural features with β-catenin. In order to investigate whether γ-catenin can compensate for the loss of β-catenin we have generated mice in which the haematopoietic compartment is deficient for both proteins. Combined deficiency of β-catenin and γ-catenin does not perturb Long Term-Haematopoietic Stem Cells (LT-HSC) self renewal, but affects an already lineage committed progenitor population within the BM. Our results demonstrate that y-catenin can indeed compensate for the loss of β-catenin within the haematopoietie system.

Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization.

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression.

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.

The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2.

In the eukaryotic cell cycle, there are major control points in late G2 to determine the timing of the initiation of mitosis, and in late G1, regulating entry into S phase. In yeasts, this latter control is called start. Traverse of the start control and progression to S phase is accompanied by an increase in the expression of some of the genes whose products are required for DNA synthesis. In Saccharomyces cerevisiae, the coordinate expression of these genes in late G1 is dependent on a cis-acting sequence element called the MluI cell cycle box (MCB). A transcription factor called DSC-1 binds these elements and mediates cell cycle regulated transcription, though it is unclear whether this is by cell cycle-dependent changes in its activity. A DSC-1-like factor has also been identified in the fission yeast S.pombe. This is composed of at least the products of the cdc10 and sct1/res1 genes, and binds to the promoters of genes whose expression increases prior to S phase. We demonstrate that p85cdc10 is a nuclear protein and that the activity of the S.pombe DSC-1 factor varies through the cell cycle; it is high in cells that have passed start, decreases at the time of anaphase, remains low during the pre-start phase of G1 and increases at the time of the next S phase. We also show that the reactivation in late G1 is dependent on the G1 form of p34cdc2.

Genomic Study of RNA Polymerase II and III SNAP(c)-Bound Promoters Reveals a Gene Transcribed by Both Enzymes and a Broad Use of Common Activators.

SNAP(c) is one of a few basal transcription factors used by both RNA polymerase (pol) II and pol III. To define the set of active SNAP(c)-dependent promoters in human cells, we have localized genome-wide four SNAP(c) subunits, GTF2B (TFIIB), BRF2, pol II, and pol III. Among some seventy loci occupied by SNAP(c) and other factors, including pol II snRNA genes, pol III genes with type 3 promoters, and a few un-annotated loci, most are primarily occupied by either pol II and GTF2B, or pol III and BRF2. A notable exception is the RPPH1 gene, which is occupied by significant amounts of both polymerases. We show that the large majority of SNAP(c)-dependent promoters recruit POU2F1 and/or ZNF143 on their enhancer region, and a subset also recruits GABP, a factor newly implicated in SNAP(c)-dependent transcription. These activators associate with pol II and III promoters in G1 slightly before the polymerase, and ZNF143 is required for efficient transcription initiation complex assembly. The results characterize a set of genes with unique properties and establish that polymerase specificity is not absolute in vivo.

The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.

Proteins secreted from adipose tissue are increasingly recognized to play an important role in the regulation of glucose metabolism. However, much less is known about their effect on lipid metabolism. The fasting-induced adipose factor (FIAF/angiopoietin-like protein 4/peroxisome proliferator-activated receptor gamma angiopoietin-related protein) was previously identified as a target of hypolipidemic fibrate drugs and insulin-sensitizing thiazolidinediones. Using transgenic mice that mildly overexpress FIAF in peripheral tissues we show that FIAF is an extremely powerful regulator of lipid metabolism and adiposity. FIAF overexpression caused a 50% reduction in adipose tissue weight, partly by stimulating fatty acid oxidation and uncoupling in fat. In addition, FIAF overexpression increased plasma levels of triglycerides, free fatty acids, glycerol, total cholesterol, and high density lipoprotein (HDL)-cholesterol. Functional tests indicated that FIAF overexpression severely impaired plasma triglyceride clearance but had no effect on very low density lipoprotein production. The effects of FIAF overexpression were amplified by a high fat diet, resulting in markedly elevated plasma and liver triglycerides, plasma free fatty acids, and plasma glycerol levels, and impaired glucose tolerance in FIAF transgenic mice fed a high fat diet. Remarkably, in mice the full-length form of FIAF was physically associated with HDL, whereas truncated FIAF was associated with low density lipoprotein. In human both full-length and truncated FIAF were associated with HDL. The composite data suggest that via physical association with plasma lipoproteins, FIAF acts as a powerful signal from fat and other tissues to prevent fat storage and stimulate fat mobilization. Our data indicate that disturbances in FIAF signaling might be involved in dyslipidemia.

Mutations in the LHX2 gene are not a frequent cause of micro/anophthalmia.

Purpose: Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. A few genes (orthodenticle homeobox 2 [OTX2], retina and anterior neural fold homeobox [RAX], SRY-box 2 [SOX2], CEH10 homeodomain-containing homolog [CHX10], and growth differentiation factor 6 [GDF6]) have been implicated mainly in isolated micro/anophthalmia but causative mutations of these genes explain less than a quarter of these developmental defects. The essential role of the LIM homeobox 2 (LHX2) transcription factor in early eye development has recently been documented. We postulated that mutations in this gene could lead to micro/anophthalmia, and thus performed molecular screening of its sequence in patients having micro/anophthalmia. Methods: Seventy patients having non-syndromic forms of colobomatous microphthalmia (n=25), isolated microphthalmia (n=18), or anophthalmia (n=17), and syndromic forms of micro/anophthalmia (n=10) were included in this study after negative molecular screening for OTX2, RAX, SOX2, and CHX10 mutations. Mutation screening of LHX2 was performed by direct sequencing of the coding sequences and intron/exon boundaries. Results: Two heterozygous variants of unknown significance (c.128C > G [p.Pro43Arg]; c.776C > A [p.Pro259Gln]) were identified in LHX2 among the 70 patients. These variations were not identified in a panel of 100 control patients of mixed origins. The variation c.776C > A (p.Pro259Gln) was considered as non pathogenic by in silico analysis, while the variation c.128C > G (p.Pro43Arg) considered as deleterious by in silico analysis and was inherited from the asymptomatic father. Conclusions: Mutations in LHX2 do not represent a frequent cause of micro/anophthalmia.

Temperature-responsive sensing regulates biocontrol factor expression in Pseudomonas fluorescens CHA0.

In the plant-beneficial, root-colonizing strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively regulates the synthesis of biocontrol factors (mostly antifungal secondary metabolites) and contributes to oxidative stress response via the stress sigma factor RpoS. The backbone of this pathway consists of the GacS/GacA two-component system, which activates the expression of three small regulatory RNAs (RsmX, RsmY, RsmZ) and thereby counters translational repression exerted by the RsmA and RsmE proteins on target mRNAs encoding biocontrol factors. We found that the expression of typical biocontrol factors, that is, antibiotic compounds and hydrogen cyanide (involving the phlA and hcnA genes), was significantly lower at 35 degrees C than at 30 degrees C. The expression of the rpoS gene was affected in parallel. This temperature control depended on RetS, a sensor kinase acting as an antagonist of the GacS/GacA system. An additional sensor kinase, LadS, which activated the GacS/GacA system, apparently did not contribute to thermosensitivity. Mutations in gacS or gacA were epistatic to (that is, they overruled) mutations in retS or ladS for expression of the small RNAs RsmXYZ. These data are consistent with a model according to which RetS-GacS and LadS-GacS interactions shape the output of the Gac/Rsm pathway and the environmental temperature influences the RetS-GacS interaction in P. fluorescens CHA0.

Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

A next step in adeno-associated virus-mediated gene therapy for neurological diseases: regulation and targeting.

Recombinant adeno-associated virus (rAAV) vectors mediating long term transgene expression are excellent gene therapy tools for chronic neurological diseases. While rAAV2 was the first serotype tested in the clinics, more efficient vectors derived from the rh10 serotype are currently being evaluated and other serotypes are likely to be tested in the near future. In addition, aside from the currently used stereotaxy-guided intraparenchymal delivery, new techniques for global brain transduction (by intravenous or intra-cerebrospinal injections) are very promising. Various strategies for therapeutic gene delivery to the central nervous system have been explored in human clinical trials in the past decade. Canavan disease, a genetic disease caused by an enzymatic deficiency, was the first to be approved. Three gene transfer paradigms for Parkinson's disease have been explored: converting L-dopa into dopamine through AADC gene delivery in the putamen; synthesizing GABA through GAD gene delivery in the overactive subthalamic nucleus and providing neurotrophic support through neurturin gene delivery in the nigro-striatal pathway. These pioneer clinical trials demonstrated the safety and tolerability of rAAV delivery in the human brain at moderate doses. Therapeutic effects however, were modest, emphasizing the need for higher doses of the therapeutic transgene product which could be achieved using more efficient vectors or expression cassettes. This will require re-addressing pharmacological aspects, with attention to which cases require either localized and cell-type specific expression or efficient brain-wide transgene expression, and when it is necessary to modulate or terminate the administration of transgene product. The ongoing development of targeted and regulated rAAV vectors is described.

Protective effects of maternal nutritional supplementation with lactoferrin on growth and brain metabolism.

Background:Intrauterine growth restriction (IUGR) is a major risk factor for both perinatal and long-term morbidity. Bovine lactoferrin (bLf) is a major milk glycoprotein considered as a pleiotropic functional nutrient. The impact of maternal supplementation with bLf on IUGR-induced sequelae, including inadequate growth and altered cerebral development, remains unknown.Methods:IUGR was induced through maternal dexamethasone infusion (100 μg/kg during last gestational week) in rats. Maternal supplementation with bLf (0.85% in food pellet) was provided during both gestation and lactation. Pup growth was monitored, and Pup brain metabolism and gene expression were studied using in vivo (1)H NMR spectroscopy, quantitative PCR, and microarray in the hippocampus at postnatal day (PND)7.Results:Maternal bLf supplementation did not change gestational weight but increased the birth body weight of control pups (4%) with no effect on the IUGR pups. Maternal bLf supplementation allowed IUGR pups to recover a normalized weight at PND21 (weaning) improving catch-up growth. Significantly altered levels of brain metabolites (γ-aminobutyric acid, glutamate, N-acetylaspartate, and N-acetylaspartylglutamate) and transcripts (brain-derived neurotrophic factor (BDNF), divalent metal transporter 1 (DMT-1), and glutamate receptors) in IUGR pups were normalized with maternal bLf supplementation.Conclusion:Our data suggest that maternal bLf supplementation is a beneficial nutritional intervention able to revert some of the IUGR-induced sequelae, including brain hippocampal changes.

Genetic polymorphisms of the main transcription factors for adiponectin gene promoter in regulation of adiponectin levels: association analysis in three European cohorts.

Adiponectin serum concentrations are an important biomarker in cardiovascular epidemiology with heritability etimates of 30-70%. However, known genetic variants in the adiponectin gene locus (ADIPOQ) account for only 2%-8% of its variance. As transcription factors are thought to play an under-acknowledged role in carrying functional variants, we hypothesized that genetic polymorphisms in genes coding for the main transcription factors for the ADIPOQ promoter influence adiponectin levels. Single nucleotide polymorphisms (SNPs) at these genes were selected based on the haplotype block structure and previously published evidence to be associated with adiponectin levels. We performed association analyses of the 24 selected SNPs at forkhead box O1 (FOXO1), sterol-regulatory-element-binding transcription factor 1 (SREBF1), sirtuin 1 (SIRT1), peroxisome-proliferator-activated receptor gamma (PPARG) and transcription factor activating enhancer binding protein 2 beta (TFAP2B) gene loci with adiponectin levels in three different European cohorts: SAPHIR (n = 1742), KORA F3 (n = 1636) and CoLaus (n = 5355). In each study population, the association of SNPs with adiponectin levels on log-scale was tested using linear regression adjusted for age, sex and body mass index, applying both an additive and a recessive genetic model. A pooled effect size was obtained by meta-analysis assuming a fixed effects model. We applied a significance threshold of 0.0033 accounting for the multiple testing situation. A significant association was only found for variants within SREBF1 applying an additive genetic model (smallest p-value for rs1889018 on log(adiponectin) = 0.002, β on original scale = -0.217 µg/ml), explaining ∼0.4% of variation of adiponectin levels. Recessive genetic models or haplotype analyses of the FOXO1, SREBF1, SIRT1, TFAPB2B genes or sex-stratified analyses did not reveal additional information on the regulation of adiponectin levels. The role of genetic variations at the SREBF1 gene in regulating adiponectin needs further investigation by functional studies.

Programming of marginal zone B-cell fate by basic Kruppel-like factor (BKLF/KLF3).

Splenic marginal zone (MZ) B cells are a lineage distinct from follicular and peritoneal B1 B cells. They are located next to the marginal sinus where blood is released. Here they pick up antigens and shuttle the load onto follicular dendritic cells inside the follicle. On activation, MZ B cells rapidly differentiate into plasmablasts secreting antibodies, thereby mediating humoral immune responses against blood-borne type 2 T-independent antigens. As Krüppel-like factors are implicated in cell differentiation/function in various tissues, we studied the function of basic Krüppel-like factor (BKLF/KLF3) in B cells. Whereas B-cell development in the bone marrow of KLF3-transgenic mice was unaffected, MZ B-cell numbers in spleen were increased considerably. As revealed in chimeric mice, this occurred cell autonomously, increasing both MZ and peritoneal B1 B-cell subsets. Comparing KLF3-transgenic and nontransgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-regulated/down-regulated on normal MZ B-cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor-receptor signaling, indicating that KLF3 may complement alternative nuclear factor-κB signaling. Thus, KLF3 is a driving force toward MZ B-cell maturation.