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The visual responses of vertebrates are sensitive to the overall composition of retinal interneurons including amacrine cells, which tune the activity of the retinal circuitry. The expression of is regulated by multiple cis-DNA elements including the intronic α-enhancer, which is active in GABAergic amacrine cell subsets. Here, we report that the transforming growth factor ß1-induced transcript 1 protein (Tgfb1i1) interacts with the LIM domain transcription factors Lhx3 and Isl1 to inhibit the α-enhancer in the post-natal mouse retina. mice show elevated α-enhancer activity leading to overproduction of Pax6ΔPD isoform that supports the GABAergic amacrine cell fate maintenance. Consequently, the mouse retinas show a sustained light response, which becomes more transient in mice with the auto-stimulation-defective mutation. Together, we show the antagonistic regulation of the α-enhancer activity by Pax6 and the LIM protein complex is necessary for the establishment of an inner retinal circuitry, which controls visual adaptation.
Squamous cell carcinoma of the head and neck (HNSCC) accounts for more than 300,000 deaths worldwide per year as a consequence of tumor cell invasion of adjacent structures or metastasis. LIM-only protein 4 (LMO4) and LIM-domain binding protein 1 (LDB1), two directly interacting transcriptional adaptors that have important roles in normal epithelial cell differentiation, have been associated with increased metastasis, decreased differentiation, and shortened survival in carcinoma of the breast. Here, we implicate two LDB1-binding proteins, single-stranded binding protein 2 (SSBP2) and 3 (SSBP3), in controlling LMO4 and LDB1 protein abundance in HNSCC and in regulating specific tumor cell functions in this disease. First, we found that the relative abundance of LMO4, LDB1, and the two SSBPs correlated very significantly in a panel of human HNSCC cell lines. Second, expression of these proteins in tumor primaries and lymph nodes involved by metastasis were concordant in 3 of 3 sets of tissue. Third, using a Matrigel invasion and organotypic reconstruct assay, CRISPR/Cas9-mediated deletion of LDB1 in the VU-SCC-1729 cell line, which is highly invasive of basement membrane and cellular monolayers, reduced tumor cell invasiveness and migration, as well as proliferation on tissue culture plastic. Finally, inactivation of the LDB1 gene in these cells decreased growth and vascularization of xenografted human tumor cells in vivo. These data show that LMO4, LDB1, and SSBP2 and/or SSBP3 regulate metastasis, proliferation, and angiogenesis in HNSCC and provide the first evidence that SSBPs control LMO4 and LDB1 protein abundance in a cancer context.
LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R(320)LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Nuclear LASP-1 (LIM and SH3 protein-1) has a direct correlation with overall survival of breast cancer patients. In this study, immunohistochemical analysis of a human breast TMA showed that LASP-1 is absent in normal human breast epithelium but the expression increases with malignancy and is highly nuclear in aggressive breast cancer. We investigated whether the chemokines and growth factors present in the tumor microenvironment could trigger nuclear translocation of LASP-1.Treatment of human breast cancer cells with CXCL12, EGF and HRG, and HMEC-CXCR2 cells with CXCL8 facilitated nuclear shuttling of LASP-1. Data from the biochemical analysis of the nuclear and cytosolic fractions further confirmed the nuclear translocation of LASP-1 upon chemokine and growth factor treatment. CXCL12-dependent nuclear import of LASP-1 could be blocked by CXCR4 antagonist, AMD-3100. Knock down of LASP-1 resulted in alterations in gene expression leading to an increased level of cell-junction and extracellular matrix proteins and an altered cytokine secretory profile. Three-dimensional cultures of human breast cancer cells on Matrigel revealed an altered colony growth, morphology and arborization pattern in LASP-1 knockdown cells. Functional analysis of the LASP-1 knockdown cells revealed increased adhesion to collagen IV and decreased invasion through the Matrigel. Proteomic analysis of immunoprecipitates of LASP-1 and subsequent validation approaches revealed that LASP-1 associated with the epigenetic machinery especially UHRF1, DNMT1, G9a and the transcription factor Snail1. Interestingly, LASP-1 associated with UHRF1, G9a, Snail1 and di- and tri-methylated histoneH3 in a CXCL12-dependent manner based on immunoprecipitation and proximity ligation assays. LASP-1 also directly bound to Snail1 which may stabilize Snail1. Thus, nuclear LASP-1 appears to functionally serve as a hub for the epigenetic machinery.
PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN.
© 2015. Published by The Company of Biologists Ltd.
LIM domain only-2 (LMO2) overexpression in T cells induces leukemia but the molecular mechanism remains to be elucidated. In hematopoietic stem and progenitor cells, Lmo2 is part of a protein complex comprised of class II basic helix loop helix proteins, Tal1and Lyl1. The latter transcription factors heterodimerize with E2A proteins like E47 and Heb to bind E boxes. LMO2 and TAL1 or LYL1 cooperate to induce T-ALL in mouse models, and are concordantly expressed in human T-ALL. Furthermore, LMO2 cooperates with the loss of E2A suggesting that LMO2 functions by creating a deficiency of E2A. In this study, we tested this hypothesis in Lmo2-induced T-ALL cell lines. We transduced these lines with an E47/estrogen receptor fusion construct that could be forced to homodimerize with 4-hydroxytamoxifen. We discovered that forced homodimerization induced growth arrest in 2 of the 4 lines tested. The lines sensitive to E47 homodimerization accumulated in G1 and had reduced S phase entry. We analyzed the transcriptome of a resistant and a sensitive line to discern the E47 targets responsible for the cellular effects. Our results suggest that E47 has diverse effects in T-ALL but that functional deficiency of E47 is not a universal feature of Lmo2-induced T-ALL.
Copyright © 2014 Elsevier Ltd. All rights reserved.
In this study, we present a remarkable clonal cell line, 32080, derived from a CD2-Lmo2- transgenic T-cell leukemia with differentiation arrest at the transition from the intermediate single positive to double positive stages of T-cell development. We observed that 32080 cells had a striking variegated pattern in CD4 expression. There was cell-to-cell variability, with some cells expressing no CD4 and others expressing high CD4. The two populations were isogenic and yet differed in their rates of apoptosis and sensitivity to glucocorticoid. We sorted the 32080 line for CD4-positive or CD4-negative cells and observed them in culture. After 1 week, both sorted populations showed variegated CD4 expression, like the parental line, showing that the two populations could interconvert. We determined that cell replication was necessary to transit from CD4(+) to CD4(-) and CD4(-) to CD4(+). Lmo2 knockdown decreased CD4 expression, while inhibition of intracellular NOTCH1 or histone deacetylase activity induced CD4 expression. Enforced expression of RUNX1 repressed CD4 expression. We analyzed the CD4 locus by Histone 3 chromatin immunoprecipitation and found silencing marks in the CD4(-) cells and activating marks in the CD4(+) population. The 32080 cell line is a striking model of intermediate single positive to double positive T-cell plasticity and invokes a novel mechanism for LMO2's oncogenic functions.
Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.
The LMO2 oncogene is deregulated in the majority of human T-cell leukemia cases and in most gene therapy-induced T-cell leukemias. We made transgenic mice with enforced expression of Lmo2 in T-cells by the CD2 promoter/enhancer. These transgenic mice developed highly penetrant T-ALL by two distinct patterns of gene expression: one in which there was concordant activation of Lyl1, Hhex, and Mycn or alternatively, with Notch1 target gene activation. Most strikingly, this gene expression clustering was conserved in human Early T-cell Precursor ALL (ETP-ALL), where LMO2, HHEX, LYL1, and MYCN were most highly expressed. We discovered that HHEX is a direct transcriptional target of LMO2 consistent with its concordant gene expression. Furthermore, conditional inactivation of Hhex in CD2-Lmo2 transgenic mice markedly attenuated T-ALL development, demonstrating that Hhex is a crucial mediator of Lmo2's oncogenic function. The CD2-Lmo2 transgenic mice offer mechanistic insight into concordant oncogene expression and provide a model for the highly treatment-resistant ETP-ALL subtype.
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 (HSCs) and early T-cell precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the HSC. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL.
Copyright © 2013 AlphaMed Press.
Ldb1 and Ldb2 are coregulators that mediate Lin11-Isl1-Mec3 (LIM)-homeodomain (HD) and LIM-only transcription factor-driven gene regulation. Although both Ldb1 and Ldb2 mRNA were produced in the developing and adult pancreas, immunohistochemical analysis illustrated a broad Ldb1 protein expression pattern during early pancreatogenesis, which subsequently became enriched in islet and ductal cells perinatally. The islet-enriched pattern of Ldb1 was similar to pan-endocrine cell-expressed Islet-1 (Isl1), which was demonstrated in this study to be the primary LIM-HD transcription factor in developing and adult islet cells. Endocrine cell-specific removal of Ldb1 during mouse development resulted in a severe reduction of hormone⁺ cell numbers (i.e., α, β, and δ) and overt postnatal hyperglycemia, reminiscent of the phenotype described for the Isl1 conditional mutant. In contrast, neither endocrine cell development nor function was affected in the pancreas of Ldb2(-/-) mice. Gene expression and chromatin immunoprecipitation (ChIP) analyses demonstrated that many important Isl1-activated genes were coregulated by Ldb1, including MafA, Arx, insulin, and Glp1r. However, some genes (i.e., Hb9 and Glut2) only appeared to be impacted by Ldb1 during development. These findings establish Ldb1 as a critical transcriptional coregulator during islet α-, β-, and δ-cell development through Isl1-dependent and potentially Isl1-independent control.