DNA-binding Transcriptional Regulators Research Articles

The Arabidopsis B‐type response regulator 18 homomerizes and positively regulates cytokinin responses

In higher plants, the two-component system (TCS) is a signaling mechanism based on a His-to-Asp phosphorelay. The Arabidopsis TCS involves three different types of proteins, namely the histidine kinases (AHKs), the histidine phosphotransfer proteins (AHPs) and the response regulators (ARRs). The ARRs comprise three different families, namely A, B and C types, according to their protein structure. While some members of the B-type family of ARRs have been studied extensively and reported to act as DNA-binding transcriptional regulators, very limited information is available for other B-type ARRs such as ARR18. In this study, we characterize in detail the molecular and functional properties of ARR18. ARR18 acts as a transcriptional regulator in plant cells and forms homodimers in planta as shown by FRET-FLIM studies. As demonstrated by mutational analysis, the aspartate at position 70 (D70) in the receiver domain of ARR18 acts as crucial phosphorylation site. The modification of D70 affects the response regulator's ability to homodimerize and to activate its target genes. Furthermore, physiological investigations of Arabidopsis lines ectopically expressing ARR18 introduce ARR18 as a new member within the cytokinin-regulated response pathway regulating root elongation.

Analysis of High Dimensionality Yeast Gene Expression Data Using Data Mining

Data Mining is the process of discovering interesting knowledge from large amounts of data stored either in databases, data warehouses, or other information repositories. From biological studies, the Yeast Proteome Database (YPD) is a model for the organization and presentation of genome-wide functional data. Accordingly, a yeast gene expression which is a unicellular DNA is selected which contains 6103 genes and the database combined with a number of related dataset to create a general dataset. DNA-binding transcriptional regulators interpret the genome’s regulatory code by binding to specific sequences to induce or repress gene expression. The gene products including RNA and protein are responsible for the development and functioning of all living membranes by 2 steps process, transcription and translation. Various transcription factors control gene transcription by binding to the promoter regions. Translation is the production of proteins from mRNA produced in transcription. In this study, out of the 169 transcription factors known to access yeast, we are considering those thought to be involved in the response of Hydrogen Peroxide (H2O2). They are 22 transcription factors. Each one is partitioned to 3 parts: TF with No H2O2, TF with Low H2O2 and TF with High H2O2. The aim of this paper was to enhance the effectiveness of the integration of hydrogen peroxide response data related to yeast gene expression data to obtain a protein response process model and to label a set of important genes related to this approach.

In silico cloning and characterization of the TGA (TGACG MOTIF-BINDING FACTOR) transcription factors subfamily in Carica papaya

The TGA transcription factors belong to the subfamily of bZIP group D that play a major role in disease resistance and development. Most of the TGA identified in Arabidopsis interact with the master regulator of SAR, NPR1 that controls the expression of PR genes. As a first approach to determine the possible involvement of these transcription factors in papaya defense, we characterized Arabidopsis TGA orthologs from the genome of Carica papaya cv. SunUp. Six orthologs CpTGA1 to CpTGA6, were identified. The predicted CpTGA proteins were highly similar to AtTGA sequences and probably share the same DNA binding properties and transcriptional regulation features. The protein sequences alignment evidenced the presence of conserved domains, characteristic of this group of transcription factors. The phylogeny showed that CpTGA evolved into three different subclades associated with defense and floral development. This is the first report of basal expression patterns assessed by RT-PCR, from the whole subfamily of CpTGA members in different tissues from papaya cv. Maradol mature plants. Overall, CpTGA1, CpTGA3 CpTGA6 and CpTGA4 showed a basal expression in all tissues tested; CpTGA2 expressed strongly in all tissues except in petioles while CpTGA5 expressed only in petals and to a lower extent in petioles. Although more detailed studies in anthers and other floral structures are required, we suggest that CpTGA5 might be tissue-specific, and it might be involved in papaya floral development. On the other hand, we report here for the first time, the expression of the whole family of CpTGA in response to salicylic acid (SA). The expression of CpTGA3, CpTGA4 and CpTGA6 increased in response to SA, what would suggest its involvement in the SAR response in papaya.

The transcriptional regulatory network of Corynebacterium jeikeium K411 and its interaction with metabolic routes contributing to human body odor formation

Lipophilic corynebacteria are involved in the generation of volatile odorous products in the process of human body odor formation by degrading skin lipids and specific odor precursors. Therefore, these bacteria represent appropriate model systems for the cosmetic industry to examine axillary malodor formation on the molecular level. To understand the transcriptional control of metabolic pathways involved in this process, the transcriptional regulatory network of the lipophilic axilla isolate Corynebacterium jeikeium K411 was reconstructed from the complete genome sequence. This bioinformatic approach detected a gene-regulatory repertoire of 83 candidate proteins, including 56 DNA-binding transcriptional regulators, nine two-component systems, nine sigma factors, and nine regulators with diverse physiological functions. Furthermore, a cross-genome comparison among selected corynebacterial species of the taxonomic cluster 3 revealed a common gene-regulatory repertoire of 44 transcriptional regulators, including the MarR-like regulator Jk0257, which is exclusively encoded in the genomes of this taxonomical subline. The current network reconstruction comprises 48 transcriptional regulators and 674 gene-regulatory interactions that were assigned to five interconnected functional modules. Most genes involved in lipid degradation are under the combined control of the global cAMP-sensing transcriptional regulator GlxR and the LuxR-family regulator RamA, probably reflecting the essential role of lipid degradation in C. jeikeium. This study provides the first genome-scale in silico analysis of the transcriptional regulation of metabolism in a lipophilic bacterium involved in the formation of human body odor.

Gene order and chromosome dynamics coordinate spatiotemporal gene expression during the bacterial growth cycle

In Escherichia coli crosstalk between DNA supercoiling, nucleoid-associated proteins and major RNA polymerase σ initiation factors regulates growth phase-dependent gene transcription. We show that the highly conserved spatial ordering of relevant genes along the chromosomal replichores largely corresponds both to their temporal expression patterns during growth and to an inferred gradient of DNA superhelical density from the origin to the terminus. Genes implicated in similar functions are related mainly in trans across the chromosomal replichores, whereas DNA-binding transcriptional regulators interact predominantly with targets in cis along the replichores. We also demonstrate that macrodomains (the individual structural partitions of the chromosome) are regulated differently. We infer that spatial and temporal variation of DNA superhelicity during the growth cycle coordinates oxygen and nutrient availability with global chromosome structure, thus providing a mechanistic insight into how the organization of a complete bacterial chromosome encodes a spatiotemporal program integrating DNA replication and global gene expression.

A Wide Extent of Inter-Strain Diversity in Virulent and Vaccine Strains of Alphaherpesviruses

Alphaherpesviruses are widespread in the human population, and include herpes simplex virus 1 (HSV-1) and 2, and varicella zoster virus (VZV). These viral pathogens cause epithelial lesions, and then infect the nervous system to cause lifelong latency, reactivation, and spread. A related veterinary herpesvirus, pseudorabies (PRV), causes similar disease in livestock that result in significant economic losses. Vaccines developed for VZV and PRV serve as useful models for the development of an HSV-1 vaccine. We present full genome sequence comparisons of the PRV vaccine strain Bartha, and two virulent PRV isolates, Kaplan and Becker. These genome sequences were determined by high-throughput sequencing and assembly, and present new insights into the attenuation of a mammalian alphaherpesvirus vaccine strain. We find many previously unknown coding differences between PRV Bartha and the virulent strains, including changes to the fusion proteins gH and gB, and over forty other viral proteins. Inter-strain variation in PRV protein sequences is much closer to levels previously observed for HSV-1 than for the highly stable VZV proteome. Almost 20% of the PRV genome contains tandem short sequence repeats (SSRs), a class of nucleic acids motifs whose length-variation has been associated with changes in DNA binding site efficiency, transcriptional regulation, and protein interactions. We find SSRs throughout the herpesvirus family, and provide the first global characterization of SSRs in viruses, both within and between strains. We find SSR length variation between different isolates of PRV and HSV-1, which may provide a new mechanism for phenotypic variation between strains. Finally, we detected a small number of polymorphic bases within each plaque-purified PRV strain, and we characterize the effect of passage and plaque-purification on these polymorphisms. These data add to growing evidence that even plaque-purified stocks of stable DNA viruses exhibit limited sequence heterogeneity, which likely seeds future strain evolution.

Association analysis of PARP1 polymorphisms with Parkinson’s disease

Alpha-synuclein accumulation in intracellular inclusions, oxidative stress and microglia-mediated inflammation in the substantia nigra are crucial events in the pathogenesis of Parkinson’s disease (PD). Poly (ADP-ribose) polymerase-1 (PARP1), a DNA-binding enzyme and transcriptional regulator, plays an important role in modulating the cellular response to oxidative stress, inflammatory stimuli, and in apoptotic cell death. Inhibition of PARP1 results in significant neuroprotection in PD animal models; moreover PARP1 has a physiological role in the regulation of alpha-synuclein expression. A previous study had demonstrated that variants located within the PARP1 gene promoter reduce the risk of PD and delay the disease age at onset. In light of these data, we carried out an association study to investigate whether variability within this gene is associated with PD risk and disease age at onset in an Italian cohort composed of 600 PD patients and 592 healthy controls. To this purpose, we used a comprehensive tag SNP approach spanning the entire gene and the upstream and downstream regions. We did not detect any significant association of the PARP1 gene with PD either at genotypic or haplotypic level; none of the 11 genotyped SNPs was significantly associated with PD age at onset. We conclude that, despite previous evidence, PARP1 is not a susceptibility gene for PD in our population.

Structure of the leukemia oncogene LMO2: implications for the assembly of a hematopoietic transcription factor complex

AbstractThe LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.

Intracellular Yersinia pestis expresses general stress response and tellurite resistance proteins in mouse macrophages

Yersinia pestis inoculated subcutaneously via fleabite or experimental injection in natural rodent hosts multiply initially in macrophage phagolysosomes. Survival and multiplication of Y. pestis in this acidic low [Ca 2+] and [Mg 2+] environment likely necessitates compensatory mechanisms involving expression of specific proteins compared to those expressed during extracellular growth. A proteomics approach was used to identify these proteins using mouse macrophage RAW264.7 cells infected with Y. pestis strain KIM6-2053.1+ for 8 h. Intracellular Y. pestis protein samples were prepared by detergent lysis of infected RAW264.7 cells, isolation of intracellular Y. pestis by differential centrifugation, and sonication of isolated Y. pestis. Protein samples were similarly prepared from Y. pestis grown extracellularly in tissue culture media. Two intracellular and extracellular Y. pestis protein samples were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared in silico identifying 12 protein spots present in both intracellular samples but absent in extracellularly grown Y. pestis. Mass spectrometry analysis of these identified nine proteins at a high level of confidence in the Y. pestis genome: superoxide dismutase-A (sodA), inorganic pyrophosphatase, autonomous glycyl radical cofactor GrcA, molecular chaperone DnaK, serine endoprotease GsrA, global DNA-binding transcriptional dual regulator H-NS, urease subunit gamma UreA, and tellurite resistance proteins TerD and TerE. These results support the involvement of various general stress response regulators of Y. pestis during the intracellular parasitism of host macrophages as well as identification of UreA, TerD and TerE with as yet unknown roles in the process of intracellular survival of Y. pestis.

Solution NMR structure of the plasmid‐encoded fimbriae regulatory protein PefI from Salmonella enterica serovar Typhimurium

The surfaces of many bacteria feature pili or fimbriae, proteinaceous filaments that play an integral role in the adhesion of bacteria to host cells and, hence, in their pathogenicity.1,2 These extracellular structures are key virulence factors and potential targets for antibacterial drug and vaccine development. In certain Salmonella enterobacteria strains, one such class of fimbriae, the virulence plasmid-encoded fimbriae encoded by the pef operon,3,4 has been shown to be important for adhesion to murine small intestine and fluid accumulation,5 as well as biofilm formation on eukaryotic cell surfaces.6 Transcription of the pef operon, induced under acidic conditions, is modulated by the PefI regulatory protein.7 PefI represses plasmid-encoded fimbrial protein production by activating the leucine-responsive regulatory protein (Lrp) mediated inhibition of DNA methylation within the pef promoter region.7 PefI expression also inhibits transcription of flagellar proteins and consequently cell motility.8 Here we present the solution NMR structure of the 70-residue PefI transcription regulator from Salmonella enterica serovar Typhimurium LT2 [UniProtKB/TrEMBL ID, Q04822_SALTY; NESG ID, StR82; hereafter referred to as stPefI], a member of the FaeA-like protein domain family (Pfam identifier, PF04703). The sequence alignment of stPefI with its functional homolog in Escherichia coli, PapI, is shown in Fig. 1(A). We demonstrate that the structure of stPefI adopts a winged helix-turn-helix motif,9,10 consistent with its role as a DNA-binding transcriptional regulator. Moreover, in spite of their relatively low sequence identity (29%), the structure of stPefI is highly similar to that of E. coli PapI,11 which activates the expression of pyelonephritis-associated pili.12,13 Fig. 1 (A) Structure-based sequence alignment of stPefI and E. coli PapI (ecPapI). The sequence numbering for stPefI and the secondary structural elements found in its solution NMR structure (PDB ID, 2JT1) are shown above the alignment; t, turn and l, loop. ... Materials and Methods Isotopically enriched samples of stPefI were cloned, expressed, and purified, and the sample buffer was optimized for NMR studies following standard protocols of the NESG consortium;14,15 see Supplementary Material for a complete description of the methods used in this work. Briefly, samples of [U-13C,15N]- and [U-5%-13C,100%-15N]-stPefI for NMR spectroscopy were concentrated by ultracentrifugation to 0.5 to 0.7 mM in 95% H2O / 5% 2H2O solution containing 20 mM ammonium acetate, 450 mM NaCl, 10 mM DTT, 5 mM CaCl2 at pH 5.5. Analytical gel filtration chromatography, static light scattering (Supplementary Fig. S1) and one-dimensional 15N T1 and T2 relaxation data (Supplementary Fig. S2) demonstrate that the protein is monomeric under the conditions used in the NMR studies. All NMR data for resonance assignment and structure determination were collected at 20 °C on Bruker AVANCE 600 and 800 MHz spectrometers equipped with conventional 5-mm TXI probes, and a Varian INOVA 600 MHz instrument with a 5-mm HCN cold probe, and referenced to internal DSS (2,2-dimethyl-2-silapentane-5-sulfonic acid). Complete 1H, 13C, and 15N resonance assignments for stPefI were determined using conventional triple resonance NMR methods, with automated backbone assignment made by AutoAssign 2.4.0,16 followed by manual side chain assignment. Resonance assignments were validated using the Assignment Validation Suite (AVS) software package17 and deposited in the BioMagResDB (BMRB accession number, 15386). The solution NMR structure of stPefI was calculated using CYANA 2.1,18,19 and the 20 structures with lowest target function out of 100 in the final cycle were further refined by restrained molecular dynamics in explicit water using CNS 1.2.20,21 Structural statistics and global structure quality factors were computed using the PSVS 1.4 suite of structure quality assessment programs.22 The global goodness-of-fit of the final structure ensemble with the NOESY peak list data was determined using the RPF analysis program.23 The final ensemble of 20 models (excluding the C-terminal His6) were deposited into the Protein Data Bank (PDB ID, 2JT1). All structure figures were rendered using PyMOL (www.pymol.org).

NFAT, immunity and cancer: a transcription factor comes of age

Nuclear factor of activated T cells (NFAT) was first identified more than two decades ago as a major stimulation-responsive DNA-binding factor and transcriptional regulator in T cells. It is now clear that NFAT proteins have important functions in other cells of the immune system and regulate numerous developmental programmes in vertebrates. Dysregulation of these programmes can lead to malignant growth and cancer. This Review focuses on recent advances in our understanding of the transcriptional functions of NFAT proteins in the immune system and provides new insights into their potential roles in cancer development.

MeCP2 in the nucleus accumbens contributes to neural and behavioral responses to psychostimulants

MeCP2 is a methyl-DNA binding transcriptional regulator that contributes to the development and function of CNS synapses; however the requirement for MeCP2 in stimulus-regulated behavioral plasticity is not fully understood. Here we show that acute viral manipulation of MeCP2 expression in the Nucleus Accumbens (NAc) bidirectionally modulates amphetamine (AMPH)-induced conditioned place preference. Hypomorphic Mecp2 mutant mice have an increased number of NAc GABAergic synapses and show deficient AMPH-induced structural plasticity of NAc dendritic spines. Furthermore these mice show deficient plasticity of striatal immediate early gene inducibility following repeated AMPH administration. Intriguingly, psychostimulants induce phosphorylation of MeCP2 at Ser421, a site that regulates MeCP2's function as a repressor. Phosphorylation is selectively induced in GABAergic interneurons of the NAc, and its extent strongly predicts the degree of behavioral sensitization. These data reveal novel roles for MeCP2 in both mesolimbocortical circuit development and in the regulation of psychostimulant-induced behaviors.

Highlights of This Issue

Matrix metalloproteinases (MMP) have been implicated in multiple stages of cancer metastasis. The molecular basis underlying their regulation and associated cellular responses during metastatic extravasation has not been established. In this study, Shen and colleagues present evidence that breast cancer cell adhesion to lung microvascular endothelial cells induces MMP-2 activation, which increases endothelial permeability and tumor cell transmigration. They have further identified cancer cell–derived TIMP-2 as a major determinant of endothelial MMP-2 activity in the presence of MMP-14. Their findings suggest a cooperative mechanism between metastatic tumor cells and endothelial cells in regulating MMP-2 activation and tumor cell transmigration.WT1 encodes a tumor suppressor that is necessary for the proper development of multiple organs and is frequently inactivated in a subset of cancers. To understand the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a threeamino acid (KTS) insertion, Wells and colleagues undertook a genome-wide chromatin immunoprecipitation and cloning analysis to identify candidate transcriptional target genes. Their results suggest that this WT1 isoform encodes a DNA-binding transcriptional regulator that functionally links the key planar cell polarity gene SCRIBBLE to regulation of renal development and tumorigenesis.Replication Protein A (RPA) is essential for cellular DNA metabolism. Mutation of a conserved leucine residue in the DNAbinding site of RPA causes genomic instability and a high cancer rate in heterozygous mice. Hass and coworkers have used functional assays in cells and in vitro to determine the molecular defect of the homologous human RPA mutation. This mutation produces a stable nonfunctional complex. These results suggest having one functional and one nonfunctional RPA allele is not sufficient to maintain long-term genomic integrity. This study provides insights into the genetic causes of cancer.Aberrant promoter DNA methylation silences critical regulatory genes in human cancers and is potentially reversible through treatment with DNA demethylating agents. Although these agents induce clinical responses in leukemias, their effects are relatively transient, thus requiring extensive maintenance therapy. To understand the molecular mechanisms that control DNA remethylation, Kagey and colleagues characterized epigenetic alterations that accompany DNA demethylation and subsequent remethylation. Distinct locus-specific differences in the kinetics of DNA remethylation were observed and correlated with the ability to attain and subsequently maintain RNA polymerase II occupancy. A better understanding of the epigenetic events leading to resilencing of tumor suppressor genes may help to identify additional targets for therapeutic intervention.

Esophageal Squamous Cell Dysplasia and Delayed Differentiation With Deletion of Krüppel-Like Factor 4 in Murine Esophagus

Krüppel-like factor 4 (Klf; previously known a gut-enriched Krüppel-like factor) is a DNA-binding transcriptional regulator highly expressed in skin and gastrointestinal epithelia, specifically in regions of cellular differentiation. Homozygous null mice for Klf4 die shortly after birth from skin defects, precluding their analysis at later stages. The aim of this study was to analyze the function of Klf4 in keratinocyte biology and epithelial homeostasis in the adult by focusing on the squamous lined esophagus. By using the ED-L2 promoter of Epstein-Barr virus to drive Cre, we obtained tissue-specific ablation of Klf4 in the squamous epithelia of the tongue, esophagus, and forestomach. Mice with loss of Klf4 in esophageal epithelia survived to adulthood, bypassing the early lethality. Tissue-specific Klf4 knockout mice had increased basal cell proliferation and a delay in cellular maturation; these mice developed epithelial hypertrophy and subsequent dysplasia by 6 months of age. Moreover, loss of Klf4 in vivo was associated with increased expression of the pro-proliferative Klf5, and Klf4 down-regulated Klf5 both transcriptionally and posttranscriptionally. By using gene expression profiling, we also showed decreased expression of critical late-stage differentiation factors and identified alterations of several genes important in cellular differentiation. Klf4 is essential for squamous epithelial differentiation in vivo and interacts with Klf5 to maintain normal epithelial homeostasis.

The Zur regulon of Corynebacterium glutamicum ATCC 13032

BackgroundZinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators.ResultsThe cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays.ConclusionWhole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum.

Fhl5/Act, a CREM-binding transcriptional activator required for normal sperm maturation and morphology, is not essential for testicular gene expression

BackgroundThe LIM domain protein Fhl5 was previously found to interact with CREM, a DNA binding transcriptional regulator necessary for spermiogenesis in mammals. Co-transfection experiments using heterologous promoter constructs indicated a role for Fhl5 in transcriptional up-regulation of CREM-dependent testicular genes. Male mice lacking Fhl5 were reported to be fertile but displayed partially abnormal sperm maturation and morphology.MethodsTo identify Fhl5 testicular target genes we carried out two whole-genome expression profiling experiments using high-density oligonucleotide microarrays and total testis samples from Fhl5 wild-type versus homozygous mutant mice first in different and then in isogenic strain backgrounds.ResultsWeak signal differences were detected in non-isogenic samples but no statistically significant expression changes were observed when isogenic Fhl5 mutant and wild-type samples were compared.ConclusionThe outcome of these experiments suggests that testicular expression profiling is extremely sensitive to the genetic background and that Fhl5 is not essential for testicular gene expression to a level detected by microarray-based measurements. This might be due to redundant function of the related and similarly expressed protein Fhl4.

Genetic and biochemical analysis of the interaction of Bacillus subtilis CodY with branched-chain amino acids.

Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

Krüppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor β-mediated, Not by Retinoic Acid Receptor α-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells

Proliferation inhibition of vascular smooth muscle cells (VSMCs) is governed by the activity of a transcription factor network. Krüppel-like factor 4 (Klf4), retinoic acid receptor (RAR alpha), and platelet-derived growth factor receptor (PDGFR) are expressed in VSMCs and are components of such a network. However, the relationship among them in the regulation of VSMC proliferation remains unknown. Here, we investigated the mechanisms whereby Klf4 mediates the growth inhibitory effects in VSMCs through RAR alpha and PDGFR beta. We demonstrated that Klf4 directly binds to the 5' regulatory region of RAR alpha, down-regulates RAR alpha expression, and specifically inhibits RAR alpha-mediated phosphatidylinositol 3-kinase (PI3K) and ERK signaling in cultured VSMCs induced by the synthetic retinoid Am80. Of particular interest, Klf4 inhibits RAR alpha and PDGFR beta expression while blocking PI3K and ERK signaling induced by Am80 and PDGF-BB, respectively. The anti-proliferative effects of Klf4 on neointimal formation depend largely on PDGFR-mediated PI3K signaling without involvement of the RAR alpha-activated signaling pathways. These findings provide a novel mechanism for signal suppression and growth inhibitory effects of Klf4 in VSMCs. Moreover, the results of this study suggest that Klf4 is one of the key mediators of retinoid actions in VSMCs.

Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations

Rett syndrome (RTT) is an X chromosome-linked neurodevelopmental disorder associated with the characteristic neuropathology of dendritic spines common in diseases presenting with mental retardation (MR). Here, we present the first quantitative analyses of dendritic spine density in postmortem brain tissue from female RTT individuals, which revealed that hippocampal CA1 pyramidal neurons have lower spine density than age-matched non-MR female control individuals. The majority of RTT individuals carry mutations in MECP2, the gene coding for a methylated DNA-binding transcriptional regulator. While altered synaptic transmission and plasticity has been demonstrated in Mecp2-deficient mouse models of RTT, observations regarding dendritic spine density and morphology have produced varied results. We investigated the consequences of MeCP2 dysfunction on dendritic spine structure by overexpressing (∼twofold) MeCP2-GFP constructs encoding either the wildtype (WT) protein, or missense mutations commonly found in RTT individuals. Pyramidal neurons within hippocampal slice cultures transfected with either WT or mutant MECP2 (either R106W or T158M) showed a significant reduction in total spine density after 48 h of expression. Interestingly, spine density in neurons expressing WT MECP2 for 96 h was comparable to that in control neurons, while neurons expressing mutant MECP2 continued to have lower spine density than controls after 96 h of expression. Knockdown of endogenous Mecp2 with a specific small hairpin interference RNA (shRNA) also reduced dendritic spine density, but only after 96 h of expression. On the other hand, the consequences of manipulating MeCP2 levels for dendritic complexity in CA3 pyramidal neurons were only minor. Together, these results demonstrate reduced dendritic spine density in hippocampal pyramidal neurons from RTT patients, a distinct dendritic phenotype also found in neurons expressing RTT-associated MECP2 mutations or after shRNA-mediated endogenous Mecp2 knockdown, suggesting that this phenotype represent a cell-autonomous consequence of MeCP2 dysfunction.

The LacI/GalR family transcriptional regulator UriR negatively controls uridine utilization of Corynebacterium glutamicum by binding to catabolite-responsive element (cre)-like sequences

The Cg1547 protein of Corynebacterium glutamicum ATCC 13032 is a member of the LacI/GalR family of DNA-binding transcriptional regulators. A defined deletion in the cg1547 gene, now designated uriR (uridine utilization regulator), resulted in the mutant strain C. glutamicum KB1547. Comparison of gene expression levels in C. glutamicum KB1547 and the wild-type strain revealed enhanced expression of the uriR operon genes cg1546 (ribokinase), cg1545 (uridine transporter) and cg1543 (uridine-preferring nucleoside hydrolase). Gene expression of the uriR operon was stimulated by the presence of either uridine or ribose. Growth assays with C. glutamicum mutants showed that functional Cg1543 and Cg1545 proteins are essential for the utilization of uridine as the sole carbon source. Transcriptional regulation of the uriR operon is mediated by a 29 bp palindromic sequence composed of two catabolite-responsive element (cre)-like sequences and located in between the mapped -10 promoter region and the start codon of uriR. A similar cre sequence was detected in the upstream region of rbsK2 (cg2554), coding for a second ribokinase in C. glutamicum ATCC 13032. DNA band-shift assays with a streptavidin-tagged UriR protein and labelled oligonucleotides including the cre-like sequences of uriR and rbsK2 demonstrated the specific binding of the purified regulator in vitro. Whole-genome DNA microarray hybridizations comparing the gene expression in C. glutamicum KB1547 with that of the wild-type strain revealed that UriR is a pathway-specific repressor of genes involved in uridine utilization in C. glutamicum.