Identification of the Key LMO2-binding Determinants on Ldb1

Ryan, Daniel P.; Sunde, Margaret; Kwan, Ann H.; Marianayagam, Neelan J.; Nancarrow, Amy L; Hoven, Rachel N. vanden; Thompson, Lyndal S.; Baca, Manuel; Mackay, Joel P.; Visvader, Jane E.; Matthews, Jacqueline M.

doi:10.1016/j.jmb.2006.02.074

Cited by 31 publications

(50 citation statements)

References 41 publications

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“…To test whether Clim and LMO4 can form a complex on the BMP7 promoter, we performed double ChIP assays in the MCF7-LMO4-TetOff cells, first precipitating the tagged LMO4 with a Myc antibody and then the endogenous Clim2 protein with a Clim2 antibody. Under conditions of high LMO4 expression, Clim antibody could precipitate the LMO4 complex on the BMP7 promoter (Figure 4c, lane 6), indicating that both LMO4 and Clim2 bind to the BMP7 promoter, most likely in a complex given the high-affinity interaction between these proteins (Deane et al, 2004;Ryan et al, 2006). These results are specific because the Clim2 antibody was not able to precipitate IgG-precipitated BMP7 promoter (Figure 4c, lane 5).…”

Section: Identification Of Lmo4-responsive Genesmentioning

confidence: 97%

The LIM-only factor LMO4 regulates expression of the BMP7 gene through an HDAC2-dependent mechanism, and controls cell proliferation and apoptosis of mammary epithelial cells

Wang

Lin

et al. 2007

Oncogene

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The nuclear LIM-only protein 4 (LMO4) is upregulated in breast cancer, especially estrogen receptor-negative tumors, and its overexpression in mice leads to hyperplasia and tumor formation. Here, we show that deletion of LMO4 in the mammary glands of mice leads to impaired lobuloalveolar development due to decreased epithelial cell proliferation. With the goal of discovering potential LMO4-target genes, we also developed a conditional expression system in MCF-7 cells for both LMO4 and a dominant negative (DN) form of its co-regulator, cofactor of LIM domains (Clim/Ldb/Nli). We then used DNA microarrays to identify genes responsive to LMO4 and DN-Clim upregulation. One of the genes common to both data sets was bone morphogenic protein 7 (BMP7), whose expression is also significantly correlated with LMO4 transcript levels in a large dataset of human breast cancers, suggesting that BMP7 is a bona fide target gene of LMO4 in breast cancer. Inhibition of BMP7 partially blocks the effects of LMO4 on apoptosis, indicating that BMP7 mediates at least some functions of LMO4. Gene transfer studies show that LMO4 regulates the BMP7 promoter, and chromatin immunoprecipitation studies show that LMO4 and its cofactor Clim2 are recruited to the BMP7 promoter. Furthermore, we demonstrate that HDAC2 recruitment to the BMP7 promoter is inhibited by upregulation of LMO4 and that HDAC2 knockdown upregulates the promoter. These studies suggest a novel mechanism of action for LMO4: LMO4, Clim2 and HDAC2 are part of a transcriptional complex, and increased LMO4 levels can disrupt the complex, leading to decreased HDAC2 recruitment and increased promoter activity.

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Section: Identification Of Lmo4-responsive Genesmentioning

confidence: 97%

The LIM-only factor LMO4 regulates expression of the BMP7 gene through an HDAC2-dependent mechanism, and controls cell proliferation and apoptosis of mammary epithelial cells

Wang

Lin

et al. 2007

Oncogene

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“…21 That is, the binding affinity increases by $10-fold when the LIM2 domain is present. The availability of structures of both the individual LIM-LID modules (Lmo2 LIM1 -Ldb1 LID and Lmo2 LIM2 -Ldb1 LID , both determined by NMR spectroscopy), and the double LIM-LID complex (Lmo2 LIM1þ2 -Ldb1 LID ) gives us an opportunity to consider how the tandem binding domains from both proteins synergistically facilitate interactions in this complex.…”

Section: Resultsmentioning

confidence: 99%

“…These ordered regions from the single domain NMR structures correspond well with the results of mutagenic studies that indicated a single binding hotspot in Ldb1 LID , centered on Ldb1 I322 , that drives binding to the LIM domains of Lmo2, Lmo4, Lhx3, and Isl1. 21,25,27 The only significant difference in the structures of the LIM2 domain constructs lies at start of this domain. Residues Lmo2 G92/L93 form a well defined short b-strand in the double LIM domain X-ray structures, but are disordered at the start of the single LIM domain NMR structure [Fig 2(A), blue box]; Lmo2 A20-M23 is also poorly ordered in the NMR structure, Lmo2 (Fig 1, cyan).…”

Section: Resultsmentioning

confidence: 99%

“…19 Although the LIM1 domain from Lmo2 appears to be the primary determinant of Ldb1-binding activity, both LIM domains are required for high affinity binding. 16,20,21 Recombinant forms of Lmo2 tend to be insoluble, but it is possible to overcome this problem by creating fusion proteins in which either or both of the LIM domains are tethered to Ldb1 LID via a flexible linker. 20,21 Using this approach, we have previously determined the solution structure of Lmo2 LIM1 in complex with Ldb1 LID (Lmo2 LIM1 -Ldb1 LID ) 22 ; the crystal structure of the tandem LIM domain construct (Lmo2 LIM1þ2 -Ldb1 LID ) has also been determined using a similar strategy.…”

mentioning

confidence: 99%

“…16,20,21 Recombinant forms of Lmo2 tend to be insoluble, but it is possible to overcome this problem by creating fusion proteins in which either or both of the LIM domains are tethered to Ldb1 LID via a flexible linker. 20,21 Using this approach, we have previously determined the solution structure of Lmo2 LIM1 in complex with Ldb1 LID (Lmo2 LIM1 -Ldb1 LID ) 22 ; the crystal structure of the tandem LIM domain construct (Lmo2 LIM1þ2 -Ldb1 LID ) has also been determined using a similar strategy. 23 Here we report the solution structure of Lmo2 LIM2 -Ldb1 LID and compare this structure and the solution structure of Lmo2 LIM1 -Ldb1 LID with the crystal structures of Lmo2 LIM1þ2 -Ldb1 LID to gain a better understanding of how modular binding between the tandem LIM domains of Lmo2 and tandem linear motifs in Ldb1 facilitate complex formation.…”

mentioning

confidence: 99%

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Solution structure of a tethered Lmo2_LIM2/Ldb1_LID complex

Dastmalchi¹,

Wilkinson-White²,

Kwan³

et al. 2012

Protein Science

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LIM-only protein 2, Lmo2, is a regulatory protein that is essential for hematopoietic development and inappropriate overexpression of Lmo2 in T-cells contributes to T-cell leukemia. It exerts its functions by mediating protein-protein interactions and nucleating multicomponent transcriptional complexes. Lmo2 interacts with LIM domain binding protein 1 (Ldb1) through the tandem LIM domains of Lmo2 and the LIM interaction domain (LID) of Ldb1. Here, we present the solution structure of the LIM2 domain of Lmo2 bound to Ldb1 LID . The ordered regions of Ldb1 in this complex correspond well with binding hotspots previously defined by mutagenic studies. Comparisons of this Lmo2 LIM2 -Ldb1 LID structure with previously determined structures of the Lmo2/Ldb1 LID complexes lead to the conclusion that modular binding of tandem LIM domains in Lmo2 to tandem linear motifs in Ldb1 is accompanied by several disorder-to-order transitions and/ or conformational changes in both proteins.

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