Marjorie L. Beggs

Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non‐ribosomal peptide synthetase genes

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell‐associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611 ) mutagenesis techniques were used to generate exochelin‐deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS‐reactive material excreted by the possible mutant strains with that of the wild‐type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell‐associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild‐type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb Pst I fragment and a 4.8 kb Pst I/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5′ region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non‐ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non‐ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.

Alterations in the TGFβ signaling pathway in myogenic progenitors with age

Myogenic progenitors in adult muscle are necessary for the repair, maintenance and hypertrophy of post‐mitotic muscle fibers. With age, fat deposition and fibrosis contribute to the decline in the integrity and functional capacity of muscles. In a previous study we reported increased accumulation of lipid in myogenic progenitors obtained from aged mice, accompanied by an up‐regulation of genes involved in adipogenic differentiation. The present study was designed to extend our understanding of how aging affects the fate and gene expression profile of myogenic progenitors. Affymetrix murine U74 Genechip analysis was performed using RNA extracted from myogenic progenitors isolated from adult (8‐month‐old) and aged (24‐month‐old) DBA/2JNIA mice. The cells from the aged animals exhibited major alterations in the expression level of many genes directly or indirectly involved with the TGFβ signaling pathway. Our data indicate that with age, myogenic progenitors acquire the paradoxical phenotype of being both TGFβ activated based on overexpression of TGFβ‐inducible genes, but resistant to the differentiation‐inhibiting effects of exogenous TGFβ. The overexpression of TGFβ‐regulated genes, such as connective tissue growth factor, may play a role in increasing fibrosis in aging muscle.

Induction of cell proliferation and survival genes by estradiol-repressed microRNAs in breast cancer cells

BackgroundIn estrogen responsive MCF-7 cells, estradiol (E2) binding to ERα leads to transcriptional regulation of genes involved in the control of cell proliferation and survival. MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression. The aim of this study was to explore whether miRNAs were involved in hormonally regulated expression of estrogen responsive genes.MethodsWestern blot and QPCR were used to determine the expression of estrogen responsive genes and miRNAs respectively. Target gene expression regulated by miRNAs was validated by luciferase reporter assays and transfection of miRNA mimics or inhibitors. Cell proliferation was evaluated by MTS assay.ResultsE2 significantly induced bcl-2, cyclin D1 and survivin expression by suppressing the levels of a panel of miRNAs (miR-16, miR-143, miR-203) in MCF-7 cells. MiRNA transfection and luciferase assay confirmed that bcl-2 was regulated by miR-16 and miR-143, cyclinD1 was modulated by miR-16. Importantly, survivin was found to be targeted by miR-16, miR-143, miR-203. The regulatory effect of E2 can be either abrogated by anti-estrogen ICI 182, 780 and raloxifene pretreatment, or impaired by ERα siRNA, indicating the regulation is dependent on ERα. In order to investigate the functional significance of these miRNAs in estrogen responsive cells, miRNAs mimics were transfected into MCF-7 cells. It revealed that overexpression of these miRNAs significantly inhibited E2-induced cell proliferation. Further study of the expression of the miRNAs indicated that miR-16, miR-143 and miR-203 were highly expressed in triple positive breast cancer tissues, suggesting a potential tumor suppressing effect of these miRNAs in ER positive breast cancer.ConclusionsThese results demonstrate that E2 induces bcl-2, cyclin D1 and survivin by orchestrating the coordinate downregulation of a panel of miRNAs. In turn, the miRNAs manifest growth suppressive effects and control cell proliferation in response to E2. This sheds a new insight into the integral post-transcriptional regulation of cell proliferation and survival genes by miRNAs, a potential therapeutic option for breast cancer.

Differences in the prevalence of IS6110 insertion sites in Mycobacterium tuberculosis strains: low and high copy number of IS6110

SETTING Mycobacterium tuberculosis (M. tuberculosis) isolates from various parts of the USA which have few copies of the insertion sequence IS6110. OBJECTIVES To characterize the sites of insertion of IS6110 among M. tuberculosis isolates that have one to six copies of the insertion sequence. DESIGN The mixed-linker polymerase chain reaction (ML-PCR) procedure was used to amplify the terminal repeats on the ends of IS6110 and adjacent flanking sequences. From the ML-PCR products, sequences flanking 14 copies of IS6110 in strains containing less than seven copies of the insertion were determined. Sequence information from the flanking deoxyribonucleic acid was used to construct flanking primers that can be used to indicate the presence of IS6110 at a particular site when paired with outbound IS6110 primers in a PCR. Over 200 strains of diverse origin were screened for the insertion of IS6110 at several distinct sites using this procedure. RESULTS The direct repeat (DR) locus has been described as a highly preferred site for insertion of IS6110 in strains of M. tuberculosis. Another highly preferred site of insertion of IS6100, DK1, is herein described. Insertions at DK1 are highly prevalent in M. tuberculosis strains harboring two to six copies of IS6110. The prevalence of insertions at this site decreases in strains with more than six copies of IS6110, even though the sequence itself is present in strains lacking a copy of IS6110 at this site. CONCLUSION In addition to the DR locus there are other conserved sites of insertion among M. tuberculosis strains. The data further suggest a separate lineage for the high copy and the low copy strains, and a possible sequential insertion of IS6110 in strains of M. tuberculosis with less than seven copies.

Impaired Wnt Signaling in Embryonal Rhabdomyosarcoma Cells from p53/c-fos Double Mutant Mice

Rhabdomyosarcoma is a primitive neoplasm with a poorly understood etiology that exhibits features of fetal skeletal muscle. It represents the most frequent malignant soft tissue sarcoma affecting the pediatric population and is often treated very aggressively. Embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma constitute the two major subtypes and exhibit different molecular features. We investigated one potential molecular basis for ERMS by using cells derived from tumors produced in p53(-/-)/c-fos(-/-) mice. This model closely recapitulates the timing, location, molecular markers, and histology seen in human ERMS. A combined chromatin immunoprecipitation/promoter microarray approach was used to identify promoters bound by the c-Jun-containing AP-1 complex in the tumor-derived cells that lacked c-Fos. Identification of the Wnt2 gene and its overexpression in ERMS cells was confirmed in human rhabdomyosarcoma cell lines and prompted further analysis of the Wnt signaling pathway. Contrary to our expectations, the canonical Wnt/β-catenin signaling pathway was down-regulated in ERMS cells compared with normal myoblasts, and activating this pathway promoted myogenic differentiation. Furthermore, the identification of both survivin and sfrp2 through promoter and expression analyses suggested that increased resistance to apoptosis was associated with the inhibition of the Wnt signaling pathway. These results suggest that altered AP-1 activity that leads to the down-regulation of the Wnt pathway may contribute to the inhibition of myogenic differentiation and resistance to apoptosis in ERMS cases.

Transcriptome Profiling of Peripheral Blood Cells Identifies Potential Biomarkers for Doxorubicin Cardiotoxicity in a Rat Model

Aims Doxorubicin (DOX), a widely used anticancer agent, can cause an unpredictable cardiac toxicity which remains a major limitation in cancer chemotherapy. There is a need for noninvasive, sensitive and specific biomarkers which will allow identifying patients at risk for DOX-induced cardiotoxicity to prevent permanent cardiac damage. The aim of this study was to investigate whether the expression of specific genes in the peripheral blood can be used as surrogate marker(s) for DOX-induced cardiotoxicity. Methods/Results Rats were treated with a single dose of DOX similar to one single dose that is often administered in humans. The cardiac and peripheral blood mononuclear cells (PBMCs) genome-wide expression profiling were examined using Illumina microarrays. The results showed 4,409 differentially regulated genes (DRG) in the hearts and 4,120 DRG in PBMC. Of these 2411 genes were similarly DRG (SDRG) in both the heart and PBMC. Pathway analysis of the three datasets of DRG using Gene Ontology (GO) enrichment analysis and Ingenuity Pathways Analysis (IPA) showed that most of the genes in these datasets fell into pathways related to oxidative stress response and protein ubiquination. IPA search for potential eligible biomarkers for cardiovascular disease within the SDRG list revealed 188 molecules. Conclusions We report the first in-depth comparison of DOX-induced global gene expression profiles of hearts and PBMCs. The high similarity between the gene expression profiles of the heart and PBMC induced by DOX indicates that the PBMC transcriptome may serve as a surrogate marker of DOX-induced cardiotoxicity. Future directions of this research will include analysis of PBMC expression profiles of cancer patients treated with DOX-based chemotherapy to identify the cardiotoxicity risk, predict DOX-treatment response and ultimately to allow individualized anti-cancer therapy.

Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling

Changes in gene expression associated with skeletal muscle atrophy due to aging are distinct from those due to disuse, suggesting that the response of old muscle to inactivity may be altered. The goal of this study was to identify changes in muscle gene expression that may contribute to loss of adaptability of old muscle. Muscle atrophy was induced in young adult (6-mo) and old (32-mo) male Brown Norway/F344 rats by 2 wk of hindlimb suspension (HS), and soleus muscles were analyzed by cDNA microarrays. Overall, similar changes in gene expression with HS were observed in young and old muscles for genes encoding proteins involved in protein folding (heat shock proteins), muscle structure, and contraction, extracellular matrix, and nucleic acid binding. More genes encoding transport and receptor proteins were differentially expressed in the soleus muscle from young rats, while in soleus muscle from old rats more genes that encoded ribosomal proteins were upregulated. The gene encoding the cold-shock protein RNA-binding motif protein-3 (RBM3) was induced most highly with HS in muscle from old rats, verified by real-time RT-PCR, while no difference with age was observed. The cold-inducible RNA-binding protein (Cirp) gene was also overexpressed with HS, whereas cold-shock protein Y-box-binding protein-1 was not. A time course analysis of RBM3 mRNA abundance during HS showed that upregulation occurred after apoptotic nuclei and markers of protein degradation increased. We conclude that a cold-shock response may be part of a compensatory mechanism in muscles undergoing atrophy to preserve remaining muscle mass and that RBM3 may be a therapeutic target to prevent muscle loss.

Evidence of the Presence of IS1245 and IS1311 or Closely Related Insertion Elements in Nontuberculous Mycobacteria outside of the Mycobacterium avium Complex

ABSTRACT A PCR assay based on the simultaneous detection of IS1245 and IS1311 was developed and used to determine the host range of these insertion elements. Specific PCR products were observed in Mycobacterium malmoense, Mycobacterium scrofulaceum, and Mycobacterium nonchromogenicum, indicating that IS1245 and IS1311 are not limited to the Mycobacterium avium complex.

Functional relationships between genes associated with differentiation potential of aged myogenic progenitors

Aging is accompanied by considerable heterogeneity with possible co-expression of differentiation pathways. The present study investigates the interplay between crucial myogenic, adipogenic, and Wnt-related genes orchestrating aged myogenic progenitor differentiation (AMPD) using clonal gene expression profiling in conjunction with Bayesian structure learning (BSL) techniques. The expression of three myogenic regulatory factor genes (Myogenin, Myf-5, MyoD1), four genes involved in regulating adipogenic potential (C/EBPα, DDIT3, FoxC2, PPARγ), and two genes in the Wnt signaling pathway (Lrp5, Wnt5a) known to influence both differentiation programs were determined across 34 clones by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Three control genes were used for normalization of the clonal expression data (18S, GAPDH, and B2M). Constraint-based BSL techniques, namely (a) PC Algorithm, (b) Grow-shrink (GS) algorithm, and (c) Incremental Association Markov Blanket (IAMB) were used to model the functional relationships (FRs) in the form of acyclic networks from the clonal expression profiles. A novel resampling approach that obviates the need for a user-defined confidence threshold is proposed to identify statistically significant FRs at small sample sizes. Interestingly, the resulting acyclic network consisted of FRs corresponding to myogenic, adipogenic, Wnt-related genes and their interaction. A significant number of these FRs were robust to normalization across the three house-keeping genes and the choice of the BSL technique. The results presented elucidate the delicate balance between differentiation pathways (i.e., myogenic as well as adipogenic) and possible cross-talk between pathways in AMPD.

A stress-activated, p38 mitogen-activated protein kinase-ATF/CREB pathway regulates posttranscriptional, sequence-dependent decay of target RNAs.

ABSTRACT Broadly conserved, mitogen-activated/stress-activated protein kinases (MAPK/SAPK) of the p38 family regulate multiple cellular processes. They transduce signals via dimeric, basic leucine zipper (bZIP) transcription factors of the ATF/CREB family (such as Atf2, Fos, and Jun) to regulate the transcription of target genes. We report additional mechanisms for gene regulation by such pathways exerted through RNA stability controls. The Spc1 (Sty1/Phh1) kinase-regulated Atf1-Pcr1 (Mts1-Mts2) heterodimer of the fission yeast Schizosaccharomyces pombe controls the stress-induced, posttranscriptional stability and decay of sets of target RNAs. Whole transcriptome RNA sequencing data revealed that decay is associated nonrandomly with transcripts that contain an M26 sequence motif. Moreover, the ablation of an M26 sequence motif in a target mRNA is sufficient to block its stress-induced loss. Conversely, engineered M26 motifs can render a stable mRNA into one that is targeted for decay. This stress-activated RNA decay (SARD) provides a mechanism for reducing the expression of target genes without shutting off transcription itself. Thus, a single p38-ATF/CREB signal transduction pathway can coordinately induce (promote transcription and RNA stability) and repress (promote RNA decay) transcript levels for distinct sets of genes, as is required for developmental decisions in response to stress and other stimuli.