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1.
BMC Mol Biol ; 19(1): 10, 2018 08 01.
Article in English | MEDLINE | ID: mdl-30068312

ABSTRACT

BACKGROUND: Brucella melitensis bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity and economic loss worldwide. The majority of experiments on the transcriptional responses of Brucella to conditions inside the host have been performed following invasion of cultured mammalian cells, and do not address gene expression patterns during long-term infection. RESULTS: Here, we examine the application of the previously developed coincidence cloning methodology to recover and characterize B. melitensis RNA from the supramammary lymph node of experimentally-infected goats. Using coincidence cloning, we successfully recovered Brucella RNA from supramammary lymph nodes of B. melitensis-infected goats at both short-term (4 weeks) and long-term (38 weeks) infection time points. Amplified nucleic acid levels were sufficient for analysis of Brucella gene expression patterns by RNA-sequencing, providing evidence of metabolic activity in both the short-term and the long-term samples. We developed a workflow for the use of sequence polymorphism analysis to confirm recovery of the inoculated strain in the recovered reads, and utilized clustering analysis to demonstrate a distinct transcriptional profile present in samples recovered in long-term infection. In this first look at B. melitensis gene expression patterns in vivo, the subset of Brucella genes that was highly upregulated in long-term as compared to short-term infection included genes linked to roles in murine infection, such as genes involved in proline utilization and signal transduction. Finally, we demonstrated the challenges of qPCR validation of samples with very low ratios of pathogen:host RNA, as is the case during in vivo brucellosis, and alternatively characterized intermediate products of the coincidence cloning reaction. CONCLUSIONS: Overall, this study provides the first example of recovery plus characterization of B. melitensis RNA from in vivo lymph node infection, and demonstrates that the coincidence cloning technique is a useful tool for characterizing in vivo transcriptional changes in Brucella species. Genes upregulated in long-term infection in this data set, including many genes not previously demonstrated to be virulence factors in mice or macrophage experiments, are candidates of future interest for potential roles in Brucella persistence in natural host systems.


Subject(s)
Brucella melitensis/genetics , Cloning, Molecular/methods , Gene Expression Profiling/methods , Lymph Nodes/microbiology , RNA, Bacterial/genetics , Animals , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Goats , Polymorphism, Single Nucleotide , Sequence Analysis, RNA/methods
2.
J Mol Biol ; 392(2): 452-64, 2009 Sep 18.
Article in English | MEDLINE | ID: mdl-19616560

ABSTRACT

Pax5 (paired box binding factor 5) is a critical regulator of transcription and lineage commitment in B lymphocytes. In B cells, mb-1 (Ig-alpha/immunoglobulin-associated alpha) promoter transcription is activated by Pax5 through its recruitment of E74-like transforming sequence (Ets) family proteins to a composite site, the P5-EBS (Pax5-Ets binding site). Previously, X-ray crystallographic analysis revealed a network of contacts between the DNA-binding domains of Pax5 and Ets-1 while bound to the P5-EBS. Here, we report that Pax5 assembles these ternary complexes via highly cooperative interactions that overcome the autoinhibition of Ets-1. Using recombinant proteins, we calculated K(d(app)) values for the binding of Pax5, Ets-1, and GA-binding proteins, separately or together, to the P5-EBS. By itself, Pax5 binds the P5-EBS with high affinity (K(d) approximately equal 2 nM). Ets-1(331-440) bound the P5-EBS by itself with low affinity (K(d)=136 nM). However, autoinhibited Ets-1(280-440) alone does not bind detectably to the suboptimal sequences of the P5-EBS. Recruitment of Ets-1(331-440) or Ets-1(280-440) resulted in highly efficient ternary complex assembly with Pax5. Pax5 counteracts autoinhibition and increases binding of Ets-1 of the mb-1 promoter by >1000-fold. Mutation of Pax5 Gln22 to alanine (Q22A) enhances promoter binding by Pax5; however, Q22A greatly reduces recruitment of Ets-1(331-440) and Ets-1(280-440) by Pax5 (8.9- or >300-fold, respectively). Thus, Gln22 of Pax5 is essential for overcoming Ets-1 autoinhibition. Pax5 wild type and Q22A each recruited GA-binding protein alpha/beta1 to the mb-1 promoter with similar affinities, but recruitment was less efficient than that of Ets-1 (reduced by approximately 8-fold). Our results suggest a mechanism that allows Pax5 to overcome autoinhibition of Ets-1 DNA binding. In summary, these data illustrate requirements for partnerships between Ets proteins and Pax5.


Subject(s)
DNA/metabolism , PAX5 Transcription Factor/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Amino Acid Substitution/genetics , Electrophoretic Mobility Shift Assay , Humans , Kinetics , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutant Proteins/metabolism , PAX5 Transcription Factor/genetics , Promoter Regions, Genetic , Protein Binding
3.
J Biol Chem ; 280(3): 1982-91, 2005 Jan 21.
Article in English | MEDLINE | ID: mdl-15539407

ABSTRACT

Increased protein-tyrosine kinase activity is a prognostic indicator of decreased disease-free survival in patients with advanced breast tumors. Breast tumor kinase (Brk) is a soluble protein-tyrosine kinase overexpressed in the majority of breast cancers and also in normal skin and gut epithelium, but not in normal breast epithelial cells. Herein, we show that Brk interacts with protein kinase B/Akt, a serine/threonine kinase involved in cell growth and survival. Epidermal growth factor (EGF) treatment of human keratinocytes or Brk-transfected COS-1 cells leads to the dissociation of the Brk.Akt complex, whereas a constitutively active Brk mutant containing a point mutation at Tyr-447 (YF-Brk) failed to dissociate from Akt upon EGF treatment. In addition, Brk.Akt dissociation was blocked by the inhibition of phosphatidylinositol 3-kinase. Similar to ectopic Brk, endogenous Brk in T47D breast cancer cells was less phosphorylated upon EGF treatment, but it remained constitutively associated with Akt in the presence of EGF. Overexpression of wild-type (wt)-Brk, kinase-inactive (KM)-Brk, or YF-Brk increased the Tyr phosphorylation of multiple signaling molecules including EGF receptor. However, only wt- and YF-Brk, but not KM-Brk, induced phosphorylation of Akt and inhibited the kinase activity of Akt in unstimulated cells. Similarly, overexpression of wt- or YF-, but not KM-Brk, blocked the phosphorylation of the forkhead transcription factor, a downstream Akt target. These results suggest that Brk may function as a signaling molecule whose kinase activity normally limits the activity of Akt in unstimulated cells. Additionally, these results suggest that in breast cancer cells Brk behaves similarly to a constitutively active Brk mutant (YF-Brk) and associates with tyrosine-phosphorylated proteins in deregulated signaling complexes. Together these data provide clues to the possible proto-oncogenic and oncogenic functions of Brk.


Subject(s)
Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Antigen-Antibody Complex/metabolism , COS Cells , Cell Line, Tumor , Humans , Neoplasm Proteins , Protein Binding , Proto-Oncogene Proteins c-akt
4.
Nucleic Acids Res ; 31(19): 5483-9, 2003 Oct 01.
Article in English | MEDLINE | ID: mdl-14500810

ABSTRACT

Pax-5, a member of the paired domain family of transcription factors, is a key regulator of B lymphocyte-specific transcription and differentiation. A major target of Pax-5-mediated activation is the mb-1 gene, which encodes the essential transmembrane signaling protein Ig-alpha. Pax-5 recruits three members of the Ets family of transcription factors: Ets-1, Fli-1 and GABPalpha (with GABPbeta1), to assemble ternary complexes on the mb-1 promoter in vitro. Using the Pax-5:Ets-1:DNA crystal structure as a guide, we defined amino acid requirements for transcriptional activation of endogenous mb-1 genes using a novel cell-based assay. Mutations in the beta-hairpin/beta-turn of the DNA-binding domain of Pax-5 demonstrated its importance for DNA sequence recognition and activation of mb-1 transcription. Mutations of amino acids contacting Ets-1 in the crystal structure reduced or blocked mb-1 promoter activation. One of these mutations, Q22A, resulted in greatly reduced mb-1 gene transcript levels, concurrent with the loss of its ability to recruit Fli-1 to bind the promoter in vitro. In contrast, the mutation had no effect on recruitment of the related Ets protein GABPalpha (with GABPbeta1). These data further define requirements for Pax-5 function in vivo and reveal the complexity of interactions required for cooperative partnerships between transcription factors.


Subject(s)
Antigens, CD/genetics , DNA-Binding Proteins/metabolism , Receptors, Antigen, B-Cell/genetics , Transcription Factors/metabolism , Transcriptional Activation , Animals , Antigens, CD/biosynthesis , CD79 Antigens , Cell Line , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , GA-Binding Protein Transcription Factor , Macromolecular Substances , Models, Molecular , Mutation , PAX5 Transcription Factor , Protein Structure, Secondary , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Protein c-fli-1 , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-ets , Receptors, Antigen, B-Cell/biosynthesis , Trans-Activators/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics
5.
Mol Cell Biol ; 23(6): 1946-60, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12612069

ABSTRACT

Methylation of cytosine in CpG dinucleotides promotes transcriptional repression in mammals by blocking transcription factor binding and recruiting methyl-binding proteins that initiate chromatin remodeling. Here, we use a novel cell-based system to show that retrovirally expressed Pax-5 protein activates endogenous early B-cell-specific mb-1 genes in plasmacytoma cells, but only when the promoter is hypomethylated. CpG methylation does not directly affect binding of the promoter by Pax-5. Instead, methylation of an adjacent CpG interferes with assembly of ternary complexes comprising Pax-5 and Ets proteins. In electrophoretic mobility shift assays, recruitment of Ets-1 is blocked by methylation of the Ets site (5'CCGGAG) on the antisense strand. In transfection assays, selective methylation of a single CpG within the Pax-5-dependent Ets site greatly reduces mb-1 promoter activity. Prior demethylation of the endogenous mb-1 promoter is required for its activation by Pax-5 in transduced cells. Although B-lineage cells have only unmethylated mb-1 genes and do not modulate methylation of the mb-1 promoter during development, other tissues feature high percentages of methylated alleles. Together, these studies demonstrate a novel DNA methylation-dependent mechanism for regulating transcriptional activity through the inhibition of DNA-dependent protein-protein interactions.


Subject(s)
Antigens, CD/genetics , DNA-Binding Proteins/physiology , Promoter Regions, Genetic/genetics , Receptors, Antigen, B-Cell/genetics , Transcription Factors/physiology , Transcription, Genetic/genetics , Animals , B-Lymphocytes/metabolism , Binding Sites , Bone Marrow Cells/metabolism , CD79 Antigens , Cell Lineage , CpG Islands , DNA Methylation , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Humans , Macromolecular Substances , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , PAX5 Transcription Factor , Plasma Cells/metabolism , Plasmacytoma/pathology , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-ets , Recombinant Fusion Proteins/physiology , Specific Pathogen-Free Organisms , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection , Tumor Cells, Cultured
6.
Mol Cell Biol ; 22(24): 8539-51, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12446773

ABSTRACT

Previous studies have suggested that the early-B-cell-specific mb-1(Igalpha) promoter is regulated by EBF and Pax-5. Here, we used in vivo footprinting assays to detect occupation of binding sites in endogenous mb-1 promoters at various stages of B-cell differentiation. In addition to EBF and Pax-5 binding sites, we detected occupancy of a consensus binding site for E2A proteins (E box) in pre-B cells. EBF and E box sites are crucial for promoter function in transfected pre-B cells, and EBF and E2A proteins synergistically activated the promoter in transfected HeLa cells. Other data suggest that EBF and E box sites are less important for promoter function at later stages of differentiation, whereas binding sites for Pax-5 (and its Ets ternary complex partners) are required for promoter function in all mb-1-expressing cells. Using DNA microarrays, we found that expression of endogenous mb-1 transcripts correlates most closely with EBF expression and negatively with Id1, an inhibitor of E2A protein function, further linking regulation of the mb-1 gene with EBF and E2A. Together, our studies demonstrate the complexity of factors regulating tissue-specific transcription and support the concept that EBF, E2A, and Pax-5 cooperate to activate target genes in early B-cell development.


Subject(s)
Antigens, CD/metabolism , B-Lymphocytes/physiology , DNA-Binding Proteins/metabolism , Promoter Regions, Genetic , Receptors, Antigen, B-Cell/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Antigens, CD/genetics , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Binding Sites , CD79 Antigens , Cell Line , DNA Footprinting , DNA-Binding Proteins/genetics , Gene Expression Regulation , Genes, Reporter , Helix-Loop-Helix Motifs , Humans , Macromolecular Substances , Mice , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , PAX5 Transcription Factor , Protein Binding , Receptors, Antigen, B-Cell/genetics , Recombinant Fusion Proteins/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Transcription, Genetic
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