The Four and a Half LIM Domain Protein 2 (FHL2) Interacts with CALM and Is Highly Expressed in AML with Complex Aberrant Karyotpes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4337-4337 ◽  
Author(s):  
Zlatana Pasalic ◽  
Belay Tizazu ◽  
Leticia Archangelo ◽  
Alexandre Krause ◽  
Greif Philipp ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fusion Protein ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Luciferase Reporter ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domain Protein ◽  
Domain Protein ◽  
Two Hybrid

Abstract The balanced chromosomal translocation t(10;11)(p13;q14) results in the CALM/AF10 fusion gene. This translocation is found in acute myeloid leukemia (AML), T-cell acute lymphoblastic leukaemia (T-ALL) and malignant lymphoma. The CALM/AF10 fusion gene has recently been shown to cause an aggressive biphenotypic leukemia in a murine bone marrow transplant model. The CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) gene product is a clathrin assembly protein which plays a role in clathrin mediated endocytosis and trans Golgi network trafficking. AF10 is a putative transcription factor most likely involved in processes related to chromatin organization and has polycomb group gene like properties. To learn more about the function of the CALM/AF10 fusion protein, we searched for protein interaction partners of CALM. In a yeast two hybrid screen the four and a half LIM domain protein FHL2 was identified as putative CALM interacting partner. The CALM FHL2 interaction was confirmed by co-transformation assay in yeast and by GST-pulldown experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294 to 335 of CALM using the yeast two hybrid assay. In co-localization studies with transiently expressed fluorescent protein tagged CALM and FHL2, both proteins showed cytoplasmatic localization. Expression analysis (Affymetrix based) in different AML subtypes showed a significantly higher expression of FHL2 in AML with complex aberrant karyotypes compared to AML with normal karyotypes or balanced chromosomal translocations like the t(8;21), inv(16) or t(15;17). FHL2, which is also known as DRAL (downregulated in rhabdomyosarcoma LIM protein), is a TP53 responsive gene known to interact with numerous proteins in both the nucleus and the cytoplasm and can function as a transcriptional cofactor. Known FHL2 interactors include TP53, BRCA1, PLZF (promyelocytic leukemia zinc finger protein), the proto-oncogene SKI1 and beta-catenin. High expression of FHL2 in breast cancer has recently been shown to be associated with an adverse prognosis. CALM has been shown to shuttle between the nucleus and the cytoplasm because inhibition of CREM-mediated nuclear export by leptomycin B leads to the accumulation of CALM in the nucleus. Reporter gene assays using a GAL4-DNA binding domain CALM fusion protein and a GAL4 responsive luciferase reporter were able to demonstrate a transcriptional activation function of CALM. We are currently investigation the effect of FHL2 co-expression on this aspect of the CALM function. It is thus conceivable that FHL2 is playing an important role in CALM/AF10-mediated leukemogenesis by tethering the CALM/AF10 fusion protein to various nuclear transcription factor complexes.

scholarly journals The LIM domain protein nTRIP6 acts as a co-repressor for the transcription factor MEF2C in myoblasts

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Denise Kemler ◽  
Oliver Dahley ◽  
Sven Roßwag ◽  
Margarethe Litfin ◽  
Olivier Kassel
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Regulatory Function ◽  
Myoblast Differentiation ◽  
Lim Domain ◽  
Lim Domain Protein ◽  
Domain Protein ◽  
Two Hybrid

Abstract The transcription factor Myocyte enhancer factor 2C (MEF2C) plays a key role in the late differentiation of skeletal muscle progenitor cells, the so-called myoblasts. During myoblast differentiation, both MEF2C expression and transcriptional activity are regulated. We have reported that nTRIP6, the nuclear isoform of the focal adhesion LIM domain protein TRIP6, acts as an adaptor transcriptional co-activator for several transcription factors. It interacts with the promoter-bound transcription factors and consequently mediates the recruitment of other co-activators. Based on a described interaction between MEF2C and TRIP6 in a yeast-two-hybrid screen, we hypothesised a co-regulatory function of nTRIP6 for MEF2C. In proliferating myoblasts, nTRIP6 interacted with MEF2C and was recruited together with MEF2C to the MEF2-binding regions of the MEF2C target genes Myom2, Mb, Tnni2 and Des. Silencing nTRIP6 or preventing its interaction with MEF2C increased MEF2C transcriptional activity and increased the expression of these MEF2C target genes. Thus, nTRIP6 acts as a co-repressor for MEF2C. Mechanistically, nTRIP6 mediated the recruitment of the class IIa histone deacetylase HDAC5 to the MEF2C-bound promoters. In conclusion, our results unravel a transcriptional co-repressor function for nTRIP6. This adaptor co-regulator can thus exert either co-activator or co-repressor functions, depending on the transcription factor it interacts with.

scholarly journals Erratum: Corrigendum: The LIM domain protein nTRIP6 acts as a co-repressor for the transcription factor MEF2C in myoblasts

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Denise Kemler ◽  
Oliver Dahley ◽  
Sven Roßwag ◽  
Margarethe Litfin ◽  
Olivier Kassel
Keyword(s):  
Transcription Factor ◽  
Lim Domain ◽  
Lim Domain Protein ◽  
Domain Protein

LIM-Domain Protein Ajuba Is a Required Co-Factor for Gfi1-Induced Epigenetic Switch Regulating Runx2 Repression in Multiple Myeloma-Exposed Pre-Osteoblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4216-4216
Author(s):  
Juraj Adamik ◽  
Jixin Ding ◽  
Wei Zhao ◽  
Peng Zhang ◽  
Quanhong Sun ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Bone Lesions ◽  
Lim Domain ◽  
Lim Domain Protein ◽  
Histone Modifiers ◽  
Domain Protein

Abstract Multiple myeloma (MM) causes osteolytic bone lesions that rarely heal even after therapeutic remission. We reported that the MM-induced pro- inflammatory bone marrow microenvironment causes upregulation of the transcriptional repressor Gfi1 in bone marrow stromal cells (BMSC) via TNFα. Gfi1 represses the key osteoblast (OB) differentiation factor Runx2, resulting in impaired BMSC differentiation into OB. In this study, we explored the molecular mechanisms involved in Gfi1-mediated repression of Runx2 and the role of histone modifiers and possibly of cofactors, which co-operate with the MM-induced Gfi1 binding to epigenetically repress Runx2. The Runx2 promoter in MC4 pre-OB co-cultured with 5TGM1 MM cells (48 h) had reduced activating acetylation (H3K9ac) levels and enhanced heterochromatic methylation (H3K27me3), consistent with decreased mRNA expression that stayed refractory to OB differentiation. BMSC from MM patients compared to normals also had decreased levels of H3K9Ac at the Runx2 gene promoter. MM co-culture induced binding of Gfi1 to the Runx2 promoter in pre-OB with increased occupancy at 36 and 48 h, concomitantly with increased histone deacetylase HDAC1 (erases H3K9ac) and the PRC2 complex methyltransferase subunit EZH2 (adds H3K27me3). Further, ectopic Gfi1 also bound Runx2 and recruited these histone modifiers. We found that Gfi1 knockdown in pre-OB MC4 prevented MM-dependent HDAC1 and EZH2 recruitment, resulting in less Runx2 de-acetylation, lack of repressive H3K27 tri-methylation and rescue of differentiation-induced Runx2 mRNA expression. Additionally, the use of the selective inhibitors MC1293 (HDAC1i) and GSK126 (EZH2i) also prevented Runx2 mRNA suppression in MM treated pre-OB. The LIM-domain protein family member Ajuba was reported to serve as a Gfi1 corepressor on a subset of Gfi1 target genes in macrophages. Therefore, we investigated if Gfi1 requires Ajuba to repress Runx2 in pre-OB. After 36-48 h MM exposure of MC4 cells, enrichment of Ajuba occupancy was co-localized to the Gfi1 binding site in the Runx2 promoter concurrently with the recruitment of Gfi1. Biotin-oligo pulldown of Gfi1 brought down Ajuba as well. Further, co-transfection of Ajuba and Gfi1 revealed that Ajuba enhanced repression by suboptimal doses of Gfi1 of both a Runx2-luciferase reporter as well as the endogenous Runx2 gene in pre-OB. Studies using deletion constructs showed that the LIM region of Ajuba in conjunction with Gfi1 is necessary and sufficient for Runx2 repression, and the pre-LIM portion of Ajuba does not affect Runx2 luciferase expression. Transfected Ajuba exhibits cytoplasmic localization in MC4 cells unless co-expressed with full-length Gfi1, which brings it into the nucleus. Nuclear co-localization of Ajuba with Gfi1 was uncoupled in MC4 cells when Ajuba was co-transfected with Gfi1 containing only the DNA binding region (aa 239-423). Transfected 239-423 Gfi1 binds the endogenous Runx2 promoter, but fails to repress transcription, likely due to impaired recruitment of Ajuba and histone co-repressors. Importantly, knockdown of Ajuba caused decreased recruitment of Gfi1 to the Runx2 gene in pre-OB and prevented the Gfi1 repression of a Runx2 reporter. Collectively these data show that Ajuba functions as a required Gfi1 co-factor recruiting HDAC1 and EZH2 to establish long-termepigenetic suppression of Runx2 transcription in OB lineage cells in MM bone disease. Disclosures Roodman: Amgen: Consultancy; Eli Lilly: Research Funding.

Proteomic identification of the LIM domain protein FHL1 as the gene-product mutated in reducing body myopathy

2009 ◽  
Vol 40 (01) ◽  
Author(s):  
J Schessl ◽  
Y Zou ◽  
MJ McGrath ◽  
BS Cowling ◽  
B Maiti ◽  
...  
Keyword(s):  
Gene Product ◽  
Lim Domain ◽  
Lim Domain Protein ◽  
Proteomic Identification ◽  
Domain Protein

scholarly journals Four-and-a-half LIM domain protein 2 (FHL2) deficiency protects mice from diet-induced obesity and high FHL2 expression marks human obesity

Metabolism ◽  
2021 ◽  
pp. 154815
Author(s):  
Maria P. Clemente-Olivo ◽  
Jayron J. Habibe ◽  
Mariska Vos ◽  
Roelof Ottenhoff ◽  
Aldo Jongejan ◽  
...  
Keyword(s):  
Lim Domain ◽  
Human Obesity ◽  
Diet Induced Obesity ◽  
Lim Domain Protein ◽  
Domain Protein

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

scholarly journals The Catenin p120ctnInteracts with Kaiso, a Novel BTB/POZ Domain Zinc Finger Transcription Factor

1999 ◽  
Vol 19 (5) ◽  
pp. 3614-3623 ◽  
Author(s):  
Juliet M. Daniel ◽  
Albert B. Reynolds
Keyword(s):  
Transcription Factor ◽  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Mammalian Cells ◽  
Yeast Two Hybrid ◽  
Amino Terminal ◽  
Juxtamembrane Region ◽  
Carboxy Terminal ◽  
Two Hybrid ◽  
E Cadherin

ABSTRACT p120 ctn is an Armadillo repeat domain protein with structural similarity to the cell adhesion cofactors β-catenin and plakoglobin. All three proteins interact directly with the cytoplasmic domain of the transmembrane cell adhesion molecule E-cadherin; β-catenin and plakoglobin bind a carboxy-terminal region in a mutually exclusive manner, while p120 binds the juxtamembrane region. Unlike β-catenin and plakoglobin, p120 does not interact with α-catenin, the tumor suppressor adenomatous polyposis coli (APC), or the transcription factor Lef-1, suggesting that it has unique binding partners and plays a distinct role in the cadherin-catenin complex. Using p120 as bait, we conducted a yeast two-hybrid screen and identified a novel transcription factor which we named Kaiso. Kaiso’s deduced amino acid sequence revealed an amino-terminal BTB/POZ protein-protein interaction domain and three carboxy-terminal zinc fingers of the C2H2 DNA-binding type. Kaiso thus belongs to a rapidly growing family of POZ-ZF transcription factors that include the Drosophila developmental regulators Tramtrak and Bric à brac, and the human oncoproteins BCL-6 and PLZF, which are causally linked to non-Hodgkins’ lymphoma and acute promyelocytic leukemia, respectively. Monoclonal antibodies to Kaiso were generated and used to immunolocalize the protein and confirm the specificity of the p120-Kaiso interaction in mammalian cells. Kaiso specifically coprecipitated with a variety of p120-specific monoclonal antibodies but not with antibodies to α- or β-catenin, E-cadherin, or APC. Like other POZ-ZF proteins, Kaiso localized to the nucleus and was associated with specific nuclear dots. Yeast two-hybrid interaction assays mapped the binding domains to Arm repeats 1 to 7 of p120 and the carboxy-terminal 200 amino acids of Kaiso. In addition, Kaiso homodimerized via its POZ domain but it did not heterodimerize with BCL-6, which heterodimerizes with PLZF. The involvement of POZ-ZF proteins in development and cancer makes Kaiso an interesting candidate for a downstream effector of cadherin and/or p120 signaling.

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