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1.  Oncogenic LMO3 Collaborates with HEN2 to Enhance Neuroblastoma Cell Growth through Transactivation of Mash1 
PLoS ONE  2011;6(5):e19297.
Expression of Mash1 is dysregulated in human neuroblastoma. We have also reported that LMO3 (LIM-only protein 3) has an oncogenic potential in collaboration with neuronal transcription factor HEN2 in neuroblastoma. However, the precise molecular mechanisms of its transcriptional regulation remain elusive. Here we found that LMO3 forms a complex with HEN2 and acts as an upstream mediator for transcription of Mash1 in neuroblastoma. The high levels of LMO3 or Mash1 mRNA expression were significantly associated with poor prognosis in 100 primary neuroblastomas. The up-regulation of Mash1 remarkably accelerated the proliferation of SH-SY5Y neuroblastoma cells, while siRNA-mediated knockdown of LMO3 induced inhibition of growth of SH-SY5Y cells in association with a significant down-regulation of Mash1. Additionally, overexpression of both LMO3 and HEN2 induced expression of Mash1, suggesting that they might function as a transcriptional activator for Mash1. Luciferase reporter assay demonstrated that the co-expression of LMO3 and HEN2 attenuates HES1 (a negative regulator for Mash1)-dependent reduction of luciferase activity driven by the Mash1 promoter. Chromatin immunoprecipitation assay revealed that LMO3 and HEN2 reduce the amount of HES1 recruited onto putative HES1-binding sites and E-box within the Mash1 promoter. Furthermore, both LMO3 and HEN2 are physically associated with HES1 by immunoprecipitation assay. Thus, our present results suggest that a transcriptional complex of LMO3 and HEN2 may contribute to the genesis and malignant phenotype of neuroblastoma by inhibiting HES1 which suppresses the transactivation of Mash1.
PMCID: PMC3088666  PMID: 21573214
2.  LMO2 Is a Novel Predictive Marker for a Better Prognosis in Pancreatic Cancer1 
Neoplasia (New York, N.Y.)  2009;11(7):712-719.
LIM domain only 2 (LMO2) has been identified as a novel oncogene associated with carcinogenesis and better prognosis in several malignant tumors. We investigate the involvement of LMO2 in pancreatic cancer.
Experimental Design
We evaluated LMO2 expression in cultured cells, bulk tissues, and microdissected cells from pancreatic cancers by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry.
Of 164 pancreatic cancers, 98 (60%) were positive for LMO2 expression. LMO2 was more frequently detected in high-grade pancreatic intraepithelial neoplasia (PanIN) lesions (PanIN-2 and -3) than in low-grade PanIN lesions (PanIN-1A and -1B; P < .001) and was not detected in normal pancreatic ductal epithelium. The LMO2 messenger RNA levels were significantly higher in invasive ductal carcinoma cells than in normal pancreatic cells as evaluated by quantitative reverse transcription-polymerase chain reaction analyses of microdissected cells (P = .036). We also found higher incidence of LMO2 expression in histologic grade G1/G2 cancers than in grade G3 cancers (P < .001). The median survival time of LMO2-positive patients was significantly longer than that of LMO2-negative patients (P < .001), and multivariate analyses revealed that high LMO2 expression was an independent predictor of longer survival (risk ratio, 0.432, P < .001). Even among patients with a positive operative margin, LMO2-positive patients had a significant survival benefit compared with LMO2-negative patients. We further performed a large cohort study (n = 113) to examine the LMO2 messenger RNA levels in formalin-fixed paraffin-embedded samples and found similar results.
LMO2 is a promising marker for predicting a better prognosis in pancreatic cancer.
PMCID: PMC2697357  PMID: 19568416
3.  Decreased expression of LMO7 and its clinicopathological significance in human lung adenocarcinoma 
LIM-domain only protein 7 (LMO7) has been suggested to act as a tumor suppressor for murine lung adenocarcinoma, while its splice variant p100 LMO7/#16 is associated with invasion and metastasis of rat AH130W1 cells. However, the importance of LMO7 in human lung cancer is unknown. We investigated LMO7 protein expression by immunohistochemistry in tumor tissues obtained from 57 patients with adenocarcinoma of the lung using a rabbit anti-LMO7 antibody. Signals for LMO7 were localized to the apical surface of the bronchial epithelium and to the cell membranes of pneumocytes in non-cancerous pulmonary tissues, but were noted circumferentially around the plasma membrane of cancer cells in all 57 patients with adenocarcinoma. The LMO7-positive group (24 patients, 42%) showed equivocal to strong expression of LMO7 in more than 50% cancer cells, while the remaining 33 patients (58%) showed LMO7 expression in less than 50% of their cancer cells. The latter group had significantly more advanced disease than the LMO7-positive group with regard to T factor (p=0.011), nodal involvement (p=0.026) and p-stage (p=0.010; χ2 test). Multivariate analysis using a logistic regression model showed that LMO7 expression was independently associated with the T factor (p=0.041). Kaplan-Meier analysis showed that a poor prognosis was associated with low expression of LMO7 (p=0.036; log-rank test). Our findings are consistent with earlier observations and demonstrate that LMO7 is inversely correlated with the development and prognosis of human lung adenocarcinoma.
PMCID: PMC3440790  PMID: 22977619
LIM-domain only protein 7; immunohistochemistry; human lung adenocarcinoma
4.  Upstream Distal Regulatory Elements Contact the Lmo2 Promoter in Mouse Erythroid Cells 
PLoS ONE  2012;7(12):e52880.
The Lim domain only 2 (Lmo2) gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Several distal regulatory elements have been identified upstream of the Lmo2 gene in the human and mouse genomes that are capable of enhancing reporter gene expression in erythroid cells and may be responsible for the high level transcription of Lmo2 in the erythroid lineage. In this study we investigate how these elements regulate transcription of Lmo2 and whether or not they function cooperatively in the endogenous context. Chromosome conformation capture (3C) experiments show that chromatin-chromatin interactions exist between upstream regulatory elements and the Lmo2 promoter in erythroid cells but that these interactions are absent from kidney where Lmo2 is transcribed at twelve fold lower levels. Specifically, long range chromatin-chromatin interactions occur between the Lmo2 proximal promoter and two broad regions, 3–31 and 66–105 kb upstream of Lmo2, which we term the proximal and distal control regions for Lmo2 (pCR and dCR respectively). Each of these regions is bound by several transcription factors suggesting that multiple regulatory elements cooperate in regulating high level transcription of Lmo2 in erythroid cells. Binding of CTCF and cohesin which support chromatin loops at other loci were also found within the dCR and at the Lmo2 proximal promoter. Intergenic transcription occurs throughout the dCR in erythroid cells but not in kidney suggesting a role for these intergenic transcripts in regulating Lmo2, similar to the broad domain of intergenic transcription observed at the human β-globin locus control region. Our data supports a model in which the dCR functions through a chromatin looping mechanism to contact and enhance Lmo2 transcription specifically in erythroid cells. Furthermore, these chromatin loops are supported by the cohesin complex recruited to both CTCF and transcription factor bound regions.
PMCID: PMC3528669  PMID: 23285212
5.  Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance ▿  
Applied and Environmental Microbiology  2008;74(22):6848-6858.
Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.
PMCID: PMC2583506  PMID: 18806006
6.  A DNA-binding mutant of TAL1 cooperates with LMO2 to cause T cell leukemia in mice 
Oncogene  2010;30(10):1252-1260.
The most common translocation in childhood T-cell acute lymphoblastic leukemia (T-ALL) involves the LMO2 locus, resulting in ectopic expression of the LMO2 gene in human thymocytes. The LMO2 gene was also activated in patients with X-linked Severe Combined Immune Deficiency treated with gene therapy because of retroviral insertion in the LMO2 locus. The LMO2 insertions predisposed these children to T-ALL, yet how LMO2 contributes to T cell transformation remains unclear. The LIM (Lin 11, Isl-1, Mec-3) domain containing LMO2 protein regulates erythropoiesis as part of a large transcriptional complex consisting of LMO2, TAL1, E47, GATA1 and LDB1 that recognizes bipartite E-box-GATA1 sites on target genes. Similarly, a TAL1/E47/LMO2/LDB1 complex is observed in human T-ALL and Tal1 and Lmo2 expression in mice results in disease acceleration. To address the mechanism(s) of Tal1/Lmo2 synergy in leukemia, we generated Lmo2 transgenic mice and mated them with mice that express wild-type Tal1 or a DNA-binding mutant of TAL1. Tal1/Lmo2 and MutTAL1/Lmo2 bitransgenic mice exhibit perturbations in thymocyte development due to reduced E47/HEB transcriptional activity and develop leukemia with identical kinetics. These data demonstrate that the DNA-binding activity of Tal1 is not required to cooperate with Lmo2 to cause leukemia in mice and suggest that Lmo2 may cooperate with Tal1 to interfere with E47/HEB function(s).
PMCID: PMC3691994  PMID: 21057528
TAL1; Lmo2; E47; HEB; T cell acute lymphoblastic leukemia (T-ALL)
7.  Bivalent promoter marks and a latent enhancer may prime the leukaemia oncogene LMO1 for ectopic expression in T-cell leukaemia 
Leukemia  2013;27(6):1348-1357.
LMO1 is a transcriptional regulator and a T-acute lymphoblastic leukaemia (T-ALL) oncogene. Although first identified in association with a chromosomal translocation in T-ALL, the ectopic expression of LMO1 occurs far more frequently in the absence of any known mutation involving its locus. Given that LMO1 is barely expressed in any haematopoietic lineage, and activation of transcriptional drivers in leukaemic cells is not well described, we investigated the regulation of this gene in normal haematopoietic and leukaemic cells. We show that LMO1 has two promoters that drive reporter gene expression in transgenic mice to neural tissues known to express endogenous LMO1. The LMO1 promoters display bivalent histone marks in multiple blood lineages including T-cells, and a 3' flanking region at LMO1 +57 contains a transcriptional enhancer that is active in developing blood cells in transgenic mouse embryos. The LMO1 promoters become activated in T-ALL together with the 3' enhancer, which is bound in primary T-ALL cells by SCL/TAL1 and GATA3. Taken together, our results show that LMO1 is poised for expression in normal progenitors, where activation of SCL/TAL1 together with a breakdown of epigenetic repression of LMO1 regulatory elements induces ectopic LMO1 expression that contributes to the development and maintenance of T-ALL.
PMCID: PMC3677138  PMID: 23302769
LMO1; transcriptional regulation; T-ALL; bivalent chromatin
8.  SCL, LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells 
PLoS Genetics  2014;10(12):e1004768.
The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL.
Author Summary
Deciphering the initiating events in lymphoid leukemia is important for the development of new therapeutic strategies. In this manuscript, we define oncogenic reprogramming as the process through which non-self-renewing progenitors are converted into pre-leukemic stem cells with sustained self-renewal capacities. We provide strong genetic evidence that this step is rate-limiting in leukemogenesis and requires the activation of a self-renewal program by oncogenic transcription factors, as exemplified by SCL and LMO1. Furthermore, NOTCH1 is a pathway that drives cell fate in the thymus. We demonstrate that homeostatic NOTCH1 levels that are highest in specific thymocyte subsets determine their susceptibilities to oncogenic reprogramming by SCL and LMO1. Our data provide novel insight into the acquisition of self-renewal as a critical first step in lymphoid cell transformation, requiring the synergistic interaction of oncogenic transcription factors with a cellular context controlled by high physiological NOTCH1.
PMCID: PMC4270438  PMID: 25522233
9.  Human but Not Mouse Adipogenesis Is Critically Dependent on LMO3 
Cell Metabolism  2013;18(1):62-74.
Increased visceral fat is associated with a high risk of diabetes and metabolic syndrome and is in part caused by excessive glucocorticoids (GCs). However, the molecular mechanisms remain undefined. We now identify the GC-dependent gene LIM domain only 3 (LMO3) as being selectively upregulated in a depot-specific manner in human obese visceral adipose tissue, localizing primarily in the adipocyte fraction. Visceral LMO3 levels were tightly correlated with expression of 11β-hydroxysteroid dehydrogenase type-1 (HSD11B1), the enzyme responsible for local activation of GCs. In early human adipose stromal cell differentiation, GCs induced LMO3 via the GC receptor and a positive feedback mechanism involving 11βHSD1. No such induction was observed in murine adipogenesis. LMO3 overexpression promoted, while silencing of LMO3 suppressed, adipogenesis via regulation of the proadipogenic PPARγ axis. These results establish LMO3 as a regulator of human adipogenesis and could contribute a mechanism resulting in visceral-fat accumulation in obesity due to excess glucocorticoids.
•LMO3 is a target and amplifier of glucocorticoid action in human fat cells•LMO3 levels are selectively elevated in visceral adipose tissue of obese humans•LMO3 is required for human but not mouse adipocyte differentiation•LMO3 promotes adipogenesis by boosting a proadipogenic PPARγ program
PMCID: PMC3701325  PMID: 23823477
10.  GATA2 and Lmo2 control angiogenesis and lymphangiogenesis via direct transcriptional regulation of neuropilin-2 
Angiogenesis  2013;16(4):939-952.
GATA-binding protein 2 (GATA2) and LIM domain only 2 (Lmo2) form common transcription complexes during hematopoietic differentiation. Here we show that these two transcription factors also play a key role in endothelial cells (EC) and lymphatic EC (LEC) function. Primary EC and tumor-associated blood vessels expressed GATA2 and Lmo2. VEGF-induced sprouting angiogenesis in both differentiating embryonic stem cells (embryoid bodies) and primary EC increased GATA2 and Lmo2 levels. Conversely, silencing of GATA2 and Lmo2 expression in primary EC inhibited VEGF-induced angiogenic activity, including EC migration and sprouting in vitro, two key steps of angiogenesis in vivo. This inhibition of EC function was associated with downregulated expression of neuropilin-2 (NRP2), a co-receptor for VEGF, at the protein, mRNA and promoter levels. NRP2 overexpression partially rescued the impaired angiogenic sprouting in the GATA2/Lmo2 knockdown EC, confirming that GATA2 and Lmo2 mediated EC function, at least in part, by directly regulating NRP2 gene expression. Furthermore, it was found that primary LEC expressed GATA2 and Lmo2 as well. Silencing of GATA2 and Lmo2 expression in LEC inhibited VEGF-induced LEC sprouting, also in a NRP2-dependent manner. In conclusion, our results demonstrate that GATA2 and Lmo2 cooperatively regulate VEGF-induced angiogenesis and lymphangiogenesis via NRP2.
PMCID: PMC3793839  PMID: 23892628
GATA2; Lmo2; Neuropilin-2; Transcriptional Regulation; Angiogenesis; Lymphangiogenesis
11.  The Structure of an LIM-Only Protein 4 (LMO4) and Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Complex Reveals a Common Mode of Binding to LMO4 
PLoS ONE  2014;9(10):e109108.
LIM-domain only protein 4 (LMO4) is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1), with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1) and C-terminal binding protein interacting protein (CtIP/RBBP8). Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways.
PMCID: PMC4195752  PMID: 25310299
12.  The oncoprotein HBXIP activates transcriptional coregulatory protein LMO4 via Sp1 to promote proliferation of breast cancer cells 
Carcinogenesis  2013;34(4):927-935.
Hepatitis B X-interacting protein (HBXIP) is an important oncoprotein that plays critical role in the development of cancer. In this study, we report that HBXIP activates LIM-only protein 4 (LMO4), a transcriptional coregulatory protein, in promotion of cell proliferation. We observed that the messenger RNA (mRNA) expression levels of HBXIP were positively associated with those of LMO4 in clinical breast cancer tissues. We further identified that HBXIP upregulated LMO4 at the levels of promoter, mRNA and protein in MCF-7 and LM-MCF-7 breast cancer cell lines. The expression of cyclin D1 and cyclin E, downstream effectors of LMO4, could be upregulated by HBXIP through LMO4. Then, chromatin immunoprecipitation (ChIP) assay revealed that HBXIP was able to interact with the promoter region of LMO4. Electrophoretic mobility shift assay showed that HBXIP occupied the -237/-206 region of LMO4 promoter containing Sp1 binding element. The mutant of Sp1 binding site in the LMO4 promoter impeded the interaction of HBXIP with the promoter. Co-immunoprecipitation, ChIP and luciferase reporter gene assays showed that HBXIP activated LMO4 promoter through binding to Sp1. In function, flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays and animal transplantation assays demonstrated that HBXIP-enhanced cell proliferation of breast cancer through upregulating LMO4 in vitro and in vivo. Thus, we concluded that oncoprotein HBXIP is able to activate the transcriptional coregulatory protein LMO4 through transcription factor Sp1 in promotion of proliferation of breast cancer cells. HBXIP may serve as a driver gene to activate transcription in the development of cancer.
PMCID: PMC3616668  PMID: 23291272
13.  Lmo2 induces hematopoietic stem cell like features in T-cell progenitor cells prior to leukemia 
Stem cells (Dayton, Ohio)  2013;31(5):882-894.
LIM domain Only 2 (Lmo2) is frequently deregulated in sporadic and gene therapy-induced acute T-cell lymphoblastic leukemia (T-ALL) where its overexpression is an important initiating mutational event. In transgenic and retroviral mouse models, Lmo2 expression can be enforced in multiple hematopoietic lineages but leukemia only arises from T cells. These data suggest that Lmo2 confers clonal growth advantage in T-cell progenitors. We analyzed proliferation, differentiation, and cell death in CD2-Lmo2 transgenic thymic progenitor cells to understand the cellular effects of enforced Lmo2 expression. Most impressively, Lmo2 transgenic T-cell progenitor cells were blocked in differentiation, quiescent, and immortalized in vitro on OP9-DL1 stromal cells. These cellular effects were concordant with a transcriptional signature in Lmo2 transgenic T-cell progenitor cells that is also present in hematopoietic stem cells and Early T-cell Precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the hematopoietic stem cell. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL.
PMCID: PMC3652616  PMID: 23378057
T cells; hematopoiesis; stem cells; leukemia
14.  Two Novel Type II Restriction-Modification Systems Occupying Genomically Equivalent Locations on the Chromosomes of Listeria monocytogenes Strains 
Listeria monocytogenes is responsible for the potentially life-threatening food-borne disease listeriosis. One epidemic-associated clonal group of L. monocytogenes, epidemic clone I (ECI), harbors a Sau3AI-like restriction-modification (RM) system also present in the same genomic region in certain strains of other lineages. In this study, we identified and characterized two other, novel type II RM systems, LmoJ2 and LmoJ3, at this same locus. LmoJ2 and LmoJ3 appeared to recognize GCWGC (W = A or T) and GCNGC, respectively. Both RM systems consisted of genes with GC content below the genome average and were in the same genomic region in strains of different serotypes and lineages, suggesting site-specific horizontal gene transfer. Genomic DNA from the LmoJ2 and LmoJ3 strains grown at various temperatures (4 to 42°C) was resistant to digestion with restriction enzymes recognizing GCWGC or GCNGC, indicating that the methyltransferases were expressed under these conditions. Phages propagated in an LmoJ2-harboring strain exhibited moderately increased infectivity for this strain at 4 and 8°C but not at higher temperatures, while phages propagated in an LmoJ3 strain had dramatically increased infectivity for this strain at all temperatures. Among the sequenced Listeria phages, lytic phages possessed significantly fewer recognition sites for these RM systems than lysogenic phages, suggesting that in lytic phages sequence content evolved toward reduced susceptibility to such RM systems. The ability of LmoJ2 and LmoJ3 to protect against phages may affect the efficiency of phages as biocontrol agents for L. monocytogenes strains harboring these RM systems.
PMCID: PMC3318782  PMID: 22327591
15.  LMO4 is an essential mediator of ErbB2/HER2/Neu-induced breast cancer cell cycle progression 
Oncogene  2009;28(41):3608-3618.
ErbB2/HER2/Neu-overexpressing breast cancers are characterized by poor survival due to high proliferation and metastasis rates and identifying downstream targets of ErbB2 should facilitate developing novel therapies for this disease. Gene expression profiling revealed the transcriptional regulator LIM-only protein 4 [LMO4] is upregulated during ErbB2-induced mouse mammary gland tumorigenesis. While LMO4 is frequently overexpressed in breast cancer and LMO4-overexpressing mice develop mammary epithelial tumors, the mechanisms involved are unknown. Herein, we report that LMO4 is a downstream target of ErbB2 and PI3K in ErbB2-dependent breast cancer cells. Furthermore, LMO4 silencing reduces proliferation of these cells, inducing a G2/M arrest that was associated with decreased cullin-3, an E3-ubiquitin ligase component important for mitosis. Loss of LMO4 subsequently results in reduced Cyclin D1 and Cyclin E. Further supporting a role for LMO4 in modulating proliferation by regulating cullin-3 expression, we found that LMO4 expression oscillates throughout the cell cycle with maximum expression occurring during G2/M and these changes precede oscillations in cullin-3 levels. LMO4 levels are also highest in high grade/less differentiated breast cancers, which are characteristically highly proliferative. We conclude that LMO4 is a novel cell cycle regulator with a key role in mediating ErbB2-induced proliferation, a hallmark of ErbB2-positive disease.
PMCID: PMC2762490  PMID: 19648968
Breast Cancer; cullin-3; ErbB2; HER2; LMO4
16.  Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes 
PLoS Pathogens  2014;10(8):e1004301.
We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.
Author Summary
Listeria monocytogenes is ubiquitously present in the environment, highly adaptable and tolerant to various stresses. L. monocytogenes is also a foodborne pathogen associated with the largest foodborne outbreaks in recent US history. Signaling pathways involving the second messenger c-di-GMP play important roles in increased stress survival of proteobacteria and mycobacteria, yet roles of c-di-GMP signaling pathways in L. monocytogenes have remained unexplored. Here, we identified and systematically characterized functions of the proteins involved in c-di-GMP synthesis, degradation and sensing. We show that elevated c-di-GMP levels in L. monocytogenes result in synthesis of a previously unknown exopolysaccharide that promotes cell aggregation, inhibits motility in semi-solid media, and importantly, enhances bacterial tolerance to commonly used disinfectants as well as desiccation. These properties of the exopolysaccharide may increase listerial survival in food processing plants as well as on produce during transportation and storage. Elevated c-di-GMP levels also grossly diminish listerial invasiveness in enterocytes in vitro, and impair bacterial accumulation in selected mouse organs during oral infection.
PMCID: PMC4125290  PMID: 25101646
17.  LMO2 protein expression, LMO2 germline genetic variation, and overall survival in diffuse large B-cell lymphoma in the pre-rituximab era 
Leukemia & lymphoma  2012;53(6):1105-1112.
Both LMO2 mRNA and protein expression in diffuse large B-cell lymphoma (DLBCL) have been associated with superior survival; however, a role for germline genetic variation in LMO2 has not been previously reported. Immunohistochemistry (IHC) for LMO2 was conducted on tumor tissue from diagnostic biopsies, and 20 tag single nucleotide polymorphisms (SNPs) from LMO2 were genotyped from germline DNA. LMO2 IHC positivity was associated with superior survival (HR=0.55; 95% CI 0.31–0.97). Four LMO2 SNPs (rs10836127, rs941940, rs750781, rs1885524) were associated with survival after adjusting for LMO2 IHC and clinical factors (p<0.05), and one of these SNPs (rs941940) was also associated with IHC positivity (p=0.02). Compared to a model with clinical factors only (c-statistic=0.676), adding the 4 SNPs (c-statistic=0.751) or LMO2 IHC (c-statistic=0.691) increased the predictive ability of the model, while inclusion of all 3 factors (c-statistic=0.754) did not meaningfully add predictive ability above a model with clinical factors and the 4 SNPs. In conclusion, germline genetic variation in LMO2 was associated with DLBCL prognosis and provided slightly stronger predictive ability relative to LMO2 IHC status.
PMCID: PMC3575512  PMID: 22066713
Diffuse large B-cell lymphoma; LMO2; prognosis; single nucleotide polymorphisms
18.  Lmo Mutants Reveal a Novel Role for Circadian Pacemaker Neurons in Cocaine-Induced Behaviors 
PLoS Biology  2004;2(12):e408.
Drosophila has been developed recently as a model system to investigate the molecular and neural mechanisms underlying responses to drugs of abuse. Genetic screens for mutants with altered drug-induced behaviors thus provide an unbiased approach to define novel molecules involved in the process. We identified mutations in the Drosophila LIM-only (LMO) gene, encoding a regulator of LIM-homeodomain proteins, in a genetic screen for mutants with altered cocaine sensitivity. Reduced Lmo function increases behavioral responses to cocaine, while Lmo overexpression causes the opposite effect, reduced cocaine responsiveness. Expression of Lmo in the principal Drosophila circadian pacemaker cells, the PDF-expressing ventral lateral neurons (LNvs), is sufficient to confer normal cocaine sensitivity. Consistent with a role for Lmo in LNv function, Lmo mutants also show defects in circadian rhythms of behavior. However, the role for LNvs in modulating cocaine responses is separable from their role as pacemaker neurons: ablation or functional silencing of the LNvs reduces cocaine sensitivity, while loss of the principal circadian neurotransmitter PDF has no effect. Together, these results reveal a novel role for Lmo in modulating acute cocaine sensitivity and circadian locomotor rhythmicity, and add to growing evidence that these behaviors are regulated by shared molecular mechanisms. The finding that the degree of cocaine responsiveness is controlled by the Drosophila pacemaker neurons provides a neuroanatomical basis for this overlap. We propose that Lmo controls the responsiveness of LNvs to cocaine, which in turn regulate the flies' behavioral sensitivity to the drug.
Expression of the Drosophila LIM-only (LMO) gene in circadian pacemaker cells is required for the normal behavioral sensitivity of these animals to cocaine
PMCID: PMC529317  PMID: 15550987
19.  The LMO2 T-Cell Oncogene Is Activated via Chromosomal Translocations or Retroviral Insertion during Gene Therapy but Has No Mandatory Role in Normal T-Cell Development 
Molecular and Cellular Biology  2003;23(24):9003-9013.
The LMO2 gene encodes a LIM-only protein and is a target of chromosomal translocations in human T-cell leukemia. Recently, two X-SCID patients treated by gene therapy to rescue T-cell lymphopoiesis developed T-cell leukemias with retroviral insertion into the LMO2 gene causing clonal T-cell proliferation. In view of the specificity of LMO2 in T-cell tumorigenesis, we investigated a possible role for Lmo2 in T-lymphopoiesis, using conditional knockout of mouse Lmo2 with loxP-flanked Lmo2 and Cre recombinase alleles driven by the promoters of the lymphoid-specific genes Rag1, CD19, and Lck. While efficient deletion of Lmo2 was observed, even in the earliest detectable lymphoid cell progenitors of the bone marrow, there was no disturbance of lymphopoiesis in either T- or B-cell lineages, and in contrast to Lmo2 transgenic mice, there were normal distributions of CD4− CD8− thymocytes. We conclude that there is no mandatory role for LMO2 in lymphoid development, implying that its specific role in T-cell tumorigenesis results from a reprogramming of gene expression after enforced expression in T-cell precursors.
PMCID: PMC309712  PMID: 14645513
20.  Aberrant expression of LMO4 induces centrosome amplification and mitotic spindle abnormalities in breast cancer cells 
The Journal of pathology  2010;222(3):271-281.
The LIM-only protein, LMO4, is a transcriptional modulator overexpressed in breast cancer. It is oncogenic in murine mammary epithelium and required for G2/M progression of ErbB2-dependent cells as well as growth and invasion of other breast cancer cell types. However, the mechanisms underlying the oncogenic activity of LMO4 remain unclear. Herein, we show that LMO4 is expressed in all breast cancer subtypes examined and its expression level correlates with the degree of proliferation of such tumors. In addition, we have determined that LMO4 silencing induces G2/M arrest in cells from various breast cancer subtypes, suggesting LMO4 action in the cell cycle is not restricted to a single breast cancer subtype. This arrest was accompanied by increased cell death, amplification of centrosomes and formation of abnormal mitotic spindles. Consistent with its ability to positively and negatively regulate the formation of active transcription complexes, overexpression of LMO4 also resulted in an increase in centrosome number. Centrosome amplification has been shown to prolong the G2/M phase of the cell cycle and induce apoptosis, thus we conclude that supernumerary centrosomes mediate the G2/M arrest and cell death in LMO4-deficient cells. Furthermore, the correlation of centrosome amplification with genomic instability suggests that the impact of dysregulated LMO4 on the centrosome cycle may promote LMO4-induced tumor formation.
PMCID: PMC3600378  PMID: 20814902
Breast Cancer; LMO4; centrosome; cell cycle; proliferation
21.  Null Mutation of the Lmo4 Gene or a Combined Null Mutation of the Lmo1/Lmo3 Genes Causes Perinatal Lethality, and Lmo4 Controls Neural Tube Development in Mice 
Molecular and Cellular Biology  2004;24(5):2063-2073.
The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. We have carried out gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, we assessed the effect of compound Lmo1/Lmo3 null mutations. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. Our results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways.
PMCID: PMC350562  PMID: 14966285
22.  A novel transcript of the LMO2 gene, LMO2-c, is regulated by GATA-1 and PU.1 and encodes an antagonist of LMO2 
Ectopic expression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations or retroviral insertion, plays an important role in the onset of T-cell leukemias. Two transcripts of LMO2 gene (LMO2-a and LMO2-b) have been reported to encode a same 158-amino-acid protein. We have previously reported a novel transcript of human LMO2 gene (LMO2-c) encoding a 151-amino-acid protein, and defined its promoter region. In the present study, we investigated the regulation of the LMO2-c expression and the functions of LMO2-c. We found that LMO2-c expression is regulated by the cooperation of two essential hematopoietic transcription factors GATA-1 and PU.1 in various hematopoietic cell lines, suggesting an important functional role for LMO2-c in the hematopoietic system. More importantly, we demonstrated that LMO2-c acts as an antagonist of LMO2-a/b binding to its partners, therefore blocking the transactivation of LMO2-a/b on its target genes. These findings provide novel evidence to the functions of LMO2 gene in the hematopoietic system and leukemia.
PMCID: PMC2676384  PMID: 17361224
leukemia; LIM-only protein 2 (LMO2); GATA-1; PU.1; antagonist
23.  The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5′-UTR Variant 
PLoS Genetics  2014;10(10):e1004765.
Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized. Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (∼6-fold). Rapid amplification of 5′-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5′-untranslated regions (5′-UTR) of 28 and 234 nucleotides. The transcript containing the long 5′-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5′-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5′-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5′-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria. In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5′-UTR. These findings emphasize how 5′-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5′-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.
Author Summary
Listeria monocytogenes has evolved to adapt to numerous environments, including the intracellular niche of eukaryotic cells. Small RNAs (sRNA) play important regulatory roles in changing environments, and are thus predicted to modulate L. monocytogenes adaption to the intracellular lifestyle. This study shows how the regulatory activity of an sRNA on a defined target is restricted to bacteria in the intracellular infection phase. This regulation relies on a long (234-nucleotide) 5′-UTR that bears the sRNA-binding site present in a transcript variant that is upregulated by intracellular L. monocytogenes. The concomitant increase in both the target transcript containing the long 5′-UTR and the sRNA, which is postulated to facilitate opening of the Shine-Dalgarno site, culminates in markedly higher protein levels in intracellular bacteria. The limited amounts of both the target and the regulator in extracellular bacteria ensure that production of this bacterial protein is confined mainly to the host rather than the non-host environment.
PMCID: PMC4214639  PMID: 25356775
24.  TAL1 and LIM-Only Proteins Synergistically Induce Retinaldehyde Dehydrogenase 2 Expression in T-Cell Acute Lymphoblastic Leukemia by Acting as Cofactors for GATA3 
Molecular and Cellular Biology  1998;18(12):6939-6950.
Previously, we have shown that TAL1 and the LIM-only protein gene (LMO) are regularly coactivated in T-cell acute lymphoblastic leukemia (T-ALL). This observation is likely to relate to the findings that TAL1 and LMO are highly synergistic in T-cell tumorigenesis in double-transgenic mice. To understand the molecular mechanisms of functional synergy between TAL1 and LMO in tumorigenesis and transcriptional regulation, we tried to identify downstream target genes regulated by TAL1 and LMO by a subtractive PCR method. One of the isolated genes, that for retinaldehyde dehydrogenase 2 (RALDH2), was regularly expressed in most of the T-ALL cell lines that coexpressed TAL1 and LMO. Exogenously transfected TAL1 and LMO, but not either alone, induced RALDH2 expression in a T-ALL cell line, HPB-ALL, not expressing endogeneous TAL1 or LMO. The RALDH2 transcripts in T-ALL were, however, mostly initiated within the second intron. Promoter analysis revealed that a GATA site in a cryptic promoter in the second intron was essential and sufficient for the TAL1- and LMO-dependent transcriptional activation, and GATA3 binds to this site. In addition, forced expression of GATA3 potentiated the induction of RALDH2 by TAL1 and LMO, and these three factors formed a complex in vivo. Furthermore, a TAL1 mutant not binding to DNA also activated the transcription of RALDH2 in the presence of LMO and GATA3. Collectively, we have identified the RALDH2 gene as a first example of direct transcriptional target genes regulated by TAL1 and LMO in T-ALL. In this case, TAL1 and LMO act as cofactors for GATA3 to activate the transcription of RALDH2.
PMCID: PMC109277  PMID: 9819382
25.  Lmo4 in the Basolateral Complex of the Amygdala Modulates Fear Learning 
PLoS ONE  2012;7(4):e34559.
Pavlovian fear conditioning is an associative learning paradigm in which mice learn to associate a neutral conditioned stimulus with an aversive unconditioned stimulus. In this study, we demonstrate a novel role for the transcriptional regulator Lmo4 in fear learning. LMO4 is predominantly expressed in pyramidal projection neurons of the basolateral complex of the amygdala (BLC). Mice heterozygous for a genetrap insertion in the Lmo4 locus (Lmo4gt/+), which express 50% less Lmo4 than their wild type (WT) counterparts display enhanced freezing to both the context and the cue in which they received the aversive stimulus. Small-hairpin RNA-mediated knockdown of Lmo4 in the BLC, but not the dentate gyrus region of the hippocampus recapitulated this enhanced conditioning phenotype, suggesting an adult- and brain region-specific role for Lmo4 in fear learning. Immunohistochemical analyses revealed an increase in the number of c-Fos positive puncta in the BLC of Lmo4gt/+ mice in comparison to their WT counterparts after fear conditioning. Lastly, we measured anxiety-like behavior in Lmo4gt/+ mice and in mice with BLC-specific downregulation of Lmo4 using the elevated plus maze, open field, and light/dark box tests. Global or BLC-specific knockdown of Lmo4 did not significantly affect anxiety-like behavior. These results suggest a selective role for LMO4 in the BLC in modulating learned but not unlearned fear.
PMCID: PMC3317997  PMID: 22509321

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