Mga Expression Research Articles

In vivo CRISPR screens identify Mga as an immunotherapy target in triple-negative breast cancer

Immune evasion is not only critical for tumor initiation and progression, but also determines the efficacy of immunotherapies. Through iterative in vivo CRISPR screens with seven syngeneic tumor models, we identified core and context-dependent immune evasion pathways across cancer types. This valuable high-confidence dataset is available for the further understanding of tumor intrinsic immunomodulators, which may lead to the discovery of effective anticancer therapeutic targets. With a focus on triple-negative breast cancer (TNBC), we found that Mga knock-out significantly enhances antitumor immunity and inhibits tumor growth. Transcriptomics and single-cell RNA sequencing analyses revealed that Mga influences various immune-related pathways in the tumor microenvironment. Our findings suggest that Mga may play a role in modulating the tumor immune landscape, though the precise mechanisms require further investigation. Interestingly, we observed that low MGA expression in breast cancer patients correlates with a favorable prognosis, particularly in those with active interferon-γ signaling. These observations provide insights into tumor immune escape mechanisms and suggest that further exploration of MGA's function could potentially lead to effective therapeutic strategies in TNBC.

DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes

DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (ΔRSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the ΔRSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the ΔRSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the ΔRSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence.

Postimplantation Mga expression and embryonic lethality of two gene-trap alleles

BackgroundThe dual-specificity T-box/basic helix-loop-helix leucine zipper transcription factor MGA is part of the MAX-interacting network of proteins. In the mouse, MGA is necessary for the survival of the pluripotent epiblast cells of the peri-implantation embryo and a null, gene-trap allele MgaGt results in embryonic lethality shortly after implantation. We have used this allele to document expression of Mga in postimplantation embryos and also investigated a second, hypomorphic gene-trap allele, MgaInv. ResultsCompound heterozygotes, MgaGt/MgaInv, die prior to midgestation. The extraembryonic portion of the embryos appears to develop relatively normally while the embryonic portion, including the pluripotent cells of the epiblast, is severely retarded by E7.5. Mga expression is initially limited to the pluripotent inner cell mass of the blastocyst and epiblast, but during organogenesis it is widely expressed, notably in the central nervous system and sensory organs, reproductive and excretory systems, heart, somites and limbs. ConclusionsWidespread yet specific areas of expression of Mga during organogenesis raise the possibility that the transcription factor may play roles in controlling proliferation and potency in the progenitor cell populations of different organ systems. Documentation of these patterns sets the stage for the investigation of specific progenitor cell types.

The Regulatory Small RNA MarS Supports Virulence of Streptococcus pyogenes

Small regulatory RNAs (sRNAs) play a role in the control of bacterial virulence gene expression. In this study, we investigated an sRNA that was identified in Streptococcus pyogenes (group A Streptococcus, GAS) but is conserved throughout various streptococci. In a deletion strain, expression of mga, the gene encoding the multiple virulence gene regulator, was reduced. Accordingly, transcript and proteome analyses revealed decreased expression of several Mga-activated genes. Therefore, and because the sRNA was shown to interact with the 5′ UTR of the mga transcript in a gel-shift assay, we designated it MarS for mga-activating regulatory sRNA. Down-regulation of important virulence factors, including the antiphagocytic M-protein, led to increased susceptibility of the deletion strain to phagocytosis and reduced adherence to human keratinocytes. In a mouse infection model, the marS deletion mutant showed reduced dissemination to the liver, kidney, and spleen. Additionally, deletion of marS led to increased tolerance towards oxidative stress. Our in vitro and in vivo results indicate a modulating effect of MarS on virulence gene expression and on the pathogenic potential of GAS.

Abstract 806: MGA is a potential tumor suppressor in acute myeloid leukemia

Abstract MGA is an incompletely studied gene with a high mutation frequency in MLL-PTD AML (9%) and in core bind factor AML (8%). This gene encodes a MAX-interacting protein and is believed to act as a transcription factor that suppresses MYC binding to its target. By in silico analysis, we found that MGA is expressed in normal myeloid hematopoietic cells and AML, and the expression level is comparable with TET2 or DNMT3A. Further data mining of TCGA revealed a high frequency of inactivating mutations of the MGA gene in a variety of cancers such as various adenocarcinomas. To interrogate functionally its role in leukemogenesis, lentiviral constructs containing either shRNA or CRISPR-sgRNA targeted to different regions of the MGA gene were generated. MGA expressing AML cell line EOL-1 was silenced by shRNA or CRISPER system. Silencing was confirmed by western blot (shRNA) and Sanger Sequencing (sgRNA). An increase of methylcellulose colony number (~30%) was observed in MGA silenced cell lines. Control EOL-1 cells or EOL-1 cells silenced with MGA CRISPR sgRNAs were injected into both flanks of NSG mice, and tumor masses were harvested 21 days after injection. Silencing of MGA by CRISPR-sgRNA consistently enhanced in vivo xenograft cell growth. In addition, western blot analyses revealed silencing of MGA in EOL-1 cells increased protein levels of Cyclin E1 and phos-RB (S807 phosphorylation inhibits the ability of RB to target protein allowing cell cycle progression), indicative of a proliferative advantage conferred by the silencing of MGA. MGA may be a potential regulator of the MYC pathway. We, therefore, examined whether silencing of MGA alters MYC transcriptional activity. Luciferase reporter assay was carried out in 293FT cells stabilized with either scramble or shRNA- targeting MGA. Luciferase activities were measured 48 h after transfection of cells with MYC activity reporter pMyc4ElbLuc and normalized to the corresponding co-transfected Renilla luciferase activity. A fourfold increase in luciferase activity was observed in MGA silenced cells when compared with non- targeting shRNA controls. Furthermore, Kaplan–Meier survival analysis was performed in the TCGA-AML patients by comparison of cases with highest versus lowest expression of MGA. P-values were calculated by log-rank test. MGA expression data and patient survival data were retrieved from TCGA-AML patients RNA seq, or microarray (70 AML patients). The MGA expression ‘high’ and ‘low’ groups were defined by 15% higher than the median or 15% lower than the median, respectively. AML patients with lower levels of MGA in their leukemic samples had a worse outcome compared with those whose leukemic cells expressed higher levels of MGA. Collectively, our results suggest that MGA may function as a potential tumor-suppressor in AML. Citation Format: Qiaoyang Sun, Lingwen Ding, Kar-Tong Tan, Wenwen Chien, Xinyi Loh, Jinfen Xiao, Anand Mayakonda, Dechen Lin, Yanyi Jiang, Henry Yang, Sigal Gery, H. Phillip Koeffler. MGA is a potential tumor suppressor in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2017-806

A Naturally Occurring Single Amino Acid Replacement in Multiple Gene Regulator of Group A Streptococcus Significantly Increases Virulence

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation within a species; however, few investigations demonstrate how naturally occurring SNPs may increase strain virulence. We recently used group A Streptococcus as a model pathogen to study bacteria strain genotype-patient disease phenotype relationships. Whole-genome sequencing of approximately 800 serotype M59 group A Streptococcus strains, recovered during an outbreak of severe invasive infections across North America, identified a disproportionate number of SNPs in the gene encoding multiple gene regulator of group A Streptococcus (mga). Herein, we report results of studies designed to test the hypothesis that the most commonly occurring SNP, encoding a replacement of arginine for histidine at codon 201 of Mga (H201R), significantly increases virulence. Whole transcriptome analysis revealed that the H201R replacement significantly increased expression of mga and 54 other genes, including many proven virulence factors. Compared to the wild-type strain, a H201R isogenic mutant strain caused significantly larger skin lesions in mice. Serial quantitative bacterial culture and noninvasive magnetic resonance imaging also demonstrated that the isogenic H201R strain was significantly more virulent in a nonhuman primate model of joint infection. These findings show that the H201R replacement in Mga increases the virulence of M59 group A Streptococcus and provide new insight to how a naturally occurring SNP in bacteria contributes to human disease phenotypes.

Impact of the Streptococcus pyogenes Mga regulator on human matrix protein binding and interaction with eukaryotic cells

The Streptococcus pyogenes (group A streptococci, GAS) stand-alone Mga regulator has been shown to positively control surface-expressed virulence factors like the antiphagocytic M protein during exponential growth phase and thus, was implicated to contribute to the acute infection process. In the present study, we generated mga mutants as well as mga promoter – luciferase reporter fusions in weakly and strongly encapsulated serotype M2 and M49 GAS strains. Employing the luc reporter fusions, we showed that the complex growth medium THY-broth decreased mga expression and identified albumin as one component responsible for this effect. Fibrinogen and c U50980omponents of the complex DMEM cell culture medium induced the mga transcription rate. The attachment of mga mutants to immobilized human matrix proteins (collagen type I, fibronectin, keratin, laminin) and serum proteins (albumin, fibrinogen) was consistently reduced. Changing the Mn 2+ or Ca 2+ growth medium concentrations did not affect the fibronectin/collagen binding of M49 GAS wild-type and mga mutant strains. Medium supplementation with the oxidative stressor paraquat or anaerobic growth on THY-agar led to a relatively increased human matrix protein binding of the mga mutant. Opposite to their matrix protein-binding behaviour, the M2 and M49 mga mutants displayed an increased attachment and internalization rate for eukaryotic cells. The host cell viability was considerably reduced after prolonged exposure to mga mutants. By generating and testing corresponding M protein gene ( emm) mutants, features of the eukaryotic cell interaction could not be associated to the Mga – M protein regulatory axis. In conclusion, the present results support the postulated central role of Mga regulation for GAS host colonization and acute infection stages.

TrxR, a New CovR-Repressed Response Regulator That Activates the Mga Virulence Regulon in Group A Streptococcus

Coordinate regulation of virulence factors by the group A streptococcus (GAS) Streptococcus pyogenes is important in this pathogen's ability to cause disease. To further elucidate the regulatory network in this human pathogen, the CovR-repressed two-component system (TCS) trxSR was chosen for further analysis based on its homology to a virulence-related TCS in Streptococcus pneumoniae. In a murine skin infection model, an insertion mutation in the response regulator gene, trxR, led to a significant reduction in lesion size, lesion severity, and lethality. Curing the trxR mutation restored virulence comparable to the wild-type strain. The trxSR operon was defined in vivo, and CovR was found to directly repress its promoter in vitro. DNA microarray analysis established that TrxR activates transcription of Mga-regulated virulence genes, which may explain the virulence attenuation of the trxR mutant. This regulation appears to occur by activation of the mga promoter, Pmga, as demonstrated by analysis of a luciferase reporter fusion. Complementation of the trxR mutant with trxR on a plasmid restored expression of Mga regulon genes and restored virulence in the mouse model to wild-type levels. TrxR is the first TCS shown to regulate Mga expression. Because it is CovR repressed, TrxR defines a new pathway by which CovR can influence Mga to affect pathogenesis in the GAS.

The Mga virulence regulon: infection where the grass is greener

Co-ordinate regulation of virulence gene expression in response to different host environments is central to the success of the group A streptococcus (GAS, Streptococcus pyogenes) as an important human pathogen. Mga represents a ubiquitous stand-alone virulence regulator that controls genes (Mga regulon) whose products are necessary for adherence, internalization and host immune evasion. Mga highly activates a core set of virulence genes, including its own gene, by directly binding to their promoters. Yet, Mga also influences expression of over 10% of the GAS genome, primarily genes and operons involved in metabolism and sugar utilization. Expression of the Mga regulon is influenced by conditions that signify favourable growth conditions, presumably allowing GAS to take advantage of promising new niches in the host. The ability of Mga to respond to growth signals clearly involves regulation of mga expression via global regulatory networks such as RALPs, Rgg/RopB and the catabolite control protein CcpA. However, the presence of predicted PTS regulatory domains (PRDs) within Mga suggests an intriguing model whereby phosphorylation of Mga by the PTS phosphorelay might link growth and sugar utilization with virulence in GAS. As Mga homologues have been found in several important Gram-positive pathogens, the Mga regulon could provide a valuable paradigm for increasing our understanding of global virulence networks in bacteria.

Role of Streptococcus pyogenes two-component response regulators in the temporal control of Mga and the Mga-regulated virulence gene emm.

We examined the role of Streptococcus pyogenes two-component response regulators (SptR) in expression of Mga and the Mga-regulated gene emm. Both serotype M6 and serotype M1 mutants in 12 of the 13 identified sptR genes exhibited levels of emm transcripts and Mga protein comparable to those of the wild type during exponential and stationary phases of growth. Thus, temporal control of these virulence genes does not require Spt response regulators.

AmrA encodes a putative membrane protein necessary for maximal exponential phase expression of the Mga virulence regulon in Streptococcus pyogenes

The transcriptional regulator Mga activates a regulon of virulence genes important for colonization and immune evasion in GAS. Using transposon mutagenesis of a serotype M6 group A streptococcus (GAS) reporter strain KSM148, we have identified an open reading frame (ORF) designated amrA that is required for maximal activation of the Mga regulon during exponential phase. A deletion in amrA, but not in the downstream transcriptionally linked ORF Spy0798, was able to reproduce the phenotype seen in the transposon mutants. Northern analysis for mga and emm transcripts, as well as Western analysis of Mga, confirmed a reduction in mga expression leading to a decrease in transcription of the Mga-regulated emm in the amrA deletion and transposon mutants. Furthermore, both the amrA deletion mutant and an original transposon mutant could be complemented using amrA expressed from a nisin-inducible expression system. As amrA is strongly conserved across the sequenced streptococcal M types, and inactivation of amrA in an M3 serotype also resulted in reduction of emm transcripts, the role of amrA does not appear to be serotype specific. Although the specific function of AmrA is unknown, its putative membrane localization and homology to transporters involved in cell wall synthesis suggest a link between growth and virulence gene expression in GAS.

Two DNA-binding domains of Mga are required for virulence gene activation in the group A streptococcus.

Mga is a DNA-binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm), C5a peptidase (scpA) and Mga (mga). To determine the functionality of four potential helix-turn-helix DNA-binding motifs (HTH1-HTH4) identified within the amino-terminus of Mga, alanine substitutions were introduced within each domain in a MBP-Mga fusion allele and purified proteins were assayed for binding to Mga-specific promoter fragments (Pmga, PscpA and Pemm) in vitro. Although HTH-1 and HTH-2 mutations showed wild type DNA-binding activity, an altered HTH-3 domain resulted in reduced binding to the three promoters and an HTH-4 mutant was devoid of detectable binding activity. Plasmid-encoded expression of the HTH-3 and HTH-4 alleles from a constitutive promoter (Pspac) in the mga-deleted GAS strain JRS519 demonstrated that Mga-regulated emm expression correlated directly to the DNA-binding activity observed for each mutant protein in vitro. Single-copy expression of HTH-3 and HTH-4 from their native Pmga resulted in a dramatic reduction in autoregulated mga expression in both mutant strains. Thus, Mga appears to contain two DNA-binding domains (HTH-3 and HTH-4) that are required for direct activation of the Mga virulence regulon in vivo.

Role of mga in growth phase regulation of virulence genes of the group A streptococcus.

To determine whether growth phase affects the expression of mga and other virulence-associated genes in the group A streptococcus (GAS), total RNA was isolated from the serotype M6 GAS strain JRS4 at different phases of growth and transcript levels were quantitated by hybridization with radiolabeled DNA probes. Expression of mga (which encodes a multiple gene regulator) and the Mga-regulated genes emm (which encodes M protein) and scpA (which encodes a complement C5a peptidase) was found to be maximal in exponential phase and shut off as the bacteria entered stationary phase, while the housekeeping genes recA and rpsL showed constant transcript levels over the same period of growth. Expression of mga from a foreign phage promoter in a mga-deleted GAS strain (JRS519) altered the wild-type growth phase-dependent transcription profile seen for emm and scpA, as well as for mga. Therefore, the temporal control of mga expression requires its upstream promoter region, and the subsequent growth phase regulation of emm and scpA is Mga dependent. A number of putative virulence genes in JRS4 were shown not to require Mga for their expression, although several exhibited growth phase-dependent regulation that was similar to mga, i.e., slo (which encodes streptolysin O) and plr (encoding the plasmin receptor/glyceraldehyde-3-phosphate dehydrogenase). Still others showed a markedly different pattern of expression (the genes for the superantigen toxins MF and SpeC). These results suggest the existence of complex levels of global regulation sensitive to growth phase that directly control the expression of virulence genes and mga in GAS.