LIM Domain-binding Protein Ldb1 Research Articles

High level of fetal-globin reactivation by designed transcriptional activator-like effector

AbstractThe fetal-to-adult hemoglobin switch has been a focus of a long-standing effort to potentially treat sickle cell disease and β thalassemia by induction of fetal hemoglobin. In a continuation of this effort, we designed specific transcriptional activator-like effectors (TALEs) to target both the Gγ and Aγ-globin promoters. We fused the TALEs to a LIM domain binding protein (Ldb1) dimerization domain, followed by a T2A green fluorescent protein (GFP) cassette, which were assembled into a lentiviral vector. To prevent deletions caused by the repeats of TALEs during the lentivirus packing process, we changed the TALE encoding DNA by codon optimization. Intriguingly, 5 of 14 TALEs showed forced reactivation of fetal-globin expression in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, with a significant increase in the γ-globin mRNA level by more than 70-fold. We also observed a more than 50% reduction of β-globin mRNA. High-performance liquid chromatography analysis revealed more than 30% fetal globin in TALE-induced cells compared with the control of 2%. Among several promoters studied, the β-globin gene promoter with the locus control region (LCR) enhancer showed the highest TALE expression during CD34 erythroid differentiation. At day 19 of differentiation, 2 TALEs increased fetal-globin expression more than 40-fold in the mRNA level and up to 70% of the total globin protein. These TALEs have potential for clinical translation.

Necessity and Sufficiency of Ldb1 in the Generation, Differentiation and Maintenance of Non-photoreceptor Cell Types During Retinal Development.

During mammalian retinal development, the multipotent progenitors differentiate into all classes of retinal cells under the delicate control of transcriptional factors. The deficiency of a transcription cofactor, the LIM-domain binding protein Ldb1, has been shown to cause proliferation and developmental defects in multiple tissues including cardiovascular, hematopoietic, and nervous systems; however, it remains unclear whether and how it regulates retinal development. By expression profiling, RNA in situ hybridization and immunostaining, here we show that Ldb1 is expressed in the progenitors during early retinal development, but later its expression gradually shifts to non-photoreceptor cell types including bipolar, amacrine, horizontal, ganglion, and Müller glial cells. Retina-specific ablation of Ldb1 in mice resulted in microphthalmia, optic nerve hypoplasia, retinal thinning and detachment, and profound vision impairment as determined by electroretinography. In the mutant retina, there was precocious differentiation of amacrine and horizontal cells, indicating a requirement of Ldb1 in maintaining the retinal progenitor pool. Additionally, all non-photoreceptor cell types were greatly reduced which appeared to be caused by a generation defect and/or retinal degeneration via excessive cell apoptosis. Furthermore, we showed that misexpressed Ldb1 was sufficient to promote the generation of bipolar, amacrine, horizontal, ganglion, and Müller glial cells at the expense of photoreceptors. Together, these results demonstrate that Ldb1 is not only necessary but also sufficient for the development and/or maintenance of non-photoreceptor cell types, and implicate that the pleiotropic functions of Ldb1 during retinal development are context-dependent and determined by its interaction with diverse LIM-HD (LIM-homeodomain) and LMO (LIM domain-only) binding protein partners.

Role of the LIM domain binding protein LDB1 and the LIM only factor LMO4 in SHH subgroup medulloblastoma

Role of the LIM domain binding protein LDB1 and the LIM only factor LMO4 in SHH subgroup medulloblastoma

Incorporation of a Histone Demethylase, JARID1B, in a Novel ETS-E Box DNA-Binding Complex in Murine Erythroid Progenitors.

Abstract 2335The TAL1/SCL gene, originally discovered from its involvement by a recurrent chromosomal translocation in T-cell acute lymphoblastic leukemia, is important for hematopoietic stem cell and progenitor function and is essential for hematopoietic and vascular development. A member of the basic helix-loop-helix family of transcription factors, TAL1 binds a DNA sequence motif, CANNTG, termed the E box. We and others recently identified a novel TAL1-containing DNA-binding complex that includes an ETS protein, ELF2 (also known as NERF) in erythroid cells, and recognizes a bipartite sequence element containing an adjacent ETS binding site and E box. Our work showed this complex also contains proteins common to other TAL1 DNA-binding complexes described, including a LIM domain protein, LMO2 in erythroid cells, the LIM domain binding protein Ldb1, and putative single-stranded DNA-binding proteins SSBP2 and SSBP3. As both ELF2 and histone demethylase JARID1A, and later the related JARID1B, were identified using the same methodology (yeast two-hybrid analysis) to interact with LMO2, and multiple peptides derived from Jarid1b were identified by mass spectrometry analysis of highly purified Tal1-containing complexes from murine erythroleukemia (MEL) cells, we investigated whether JARID1B was present in the TAL1- and ELF2-containing complex. First, co-immunoprecipitation analysis identified Jarid1b in Tal1- or Elf2-containing immune precipitates and Tal1 in Jarid1b-containing immune precipitates from MEL cell extracts. Further, chromatin immunoprecipitation (ChIP) and re-ChIP analysis showed that Elf2 and Jarid1b co-occupied a region in the fourth intron of the Ssbp3 gene in MEL cells, previously demonstrated to be a physiologic target of the Tal1/Elf2 complex. Finally, knockdown of Elf2 and Jarid1b in MEL cells produced the same phenotype, decreased cellular proliferation. These results suggest a role for the JARID type histone demethylase JARID1B, and by inference histone demethylation, in the function of the ETS-E box DNA-binding complex and in proliferation of late-stage murine erythroid progenitors. Disclosures:No relevant conflicts of interest to declare.

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human Tcell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

The ETS Protein ELF2 Contributes to a Novel TAL1/SCL-Containing DNA-Binding Complex That Positively Regulates Erythroid Gene Expression and Cell Differentiation,

Abstract 3389The TAL1/SCL gene, originally discovered from its involvement by a recurrent chromosomal translocation in T-cell acute lymphoblastic leukemia, is important for hematopoietic stem cell and erythroid, megakaryocyte, and macrophage progenitor function and is essential for hematopoietic and vascular development. Although Tal1 can bind DNA with one of its E protein DNA binding partners, its function in erythroid cells has been thought to be mediated by a larger complex recognizing an E box-GATA or GATA sequence motif. In independent lines of investigation, a novel DNA-binding motif for TAL1 comprised of an ETS site immediately adjacent to an E box was identified by site selection analysis in K562 cells, and multiple peptides derived from a single ETS protein, E74-like factor 2 (Elf2, also known as NERF), were identified by tandem mass spectrometry analysis of Tal1-containing protein complexes purified from murine erythroleukemia (MEL) cells. To determine whether Elf2, previously shown to interact with Tal1's interaction partner LMO2, recognized this sequence element, electrophoretic mobility shift analysis was carried out using MEL cell nuclear extracts. This revealed the existence of a novel multiprotein ETS-E box DNA-binding complex containing Tal1, Elf2, Lmo2, the LIM domain-binding protein Ldb1, and Ldb1-interacting single-stranded DNA binding proteins Ssbp2 and Ssbp3. Structure-function analysis of this DNA-binding activity, which increased with chemical induction of MEL cell differentiation, showed that both the E box and ETS site were required for ternary complex formation, that preference existed for specific ETS flanking sequences, and that precise spacing between the two transcription factor binding sites was critical for complex formation. Finally, a physiological target for this complex was identified in the Ssbp3 gene, with Tal1, Elf2, and Ldb1 found to specifically co-occupy the third intron of this gene by chromatin immunoprecipitation analysis but not a similar ETS-E box element in the 11th intron. In accord, Ssbp3 expression, ETS-E box DNA-binding activity, and hemoglobin concentration increased with enforced expression of Elf2 in MEL cells, while Ssbp3 protein abundance, ETS-E box DNA-binding activity, and cellular differentiation decreased with Elf2 knockdown. These studies identify a novel TAL1- and ELF2-containing DNA-binding complex in erythroid progenitors that positively regulates gene expression and differentiation. In addition, they reveal a previously unknown role for the ETS protein ELF2 in erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

Ubiquitin-dependent and Ubiquitin-independent Control of Subunit Stoichiometry in the SWI/SNF Complex

The mammalian SWI/SNF chromatin remodeling complex is a key player in multiple chromatin transactions. Core subunits of this complex, including the ATPase, Brg-1, and various Brg-1-associated factors (BAFs), work in concert to maintain a functional remodeling complex. This intra-complex regulation is supervised by protein-protein interactions, as stoichiometric levels of BAF proteins are maintained by proteasomal degradation. We show that the mechanism of BAF155-mediated stabilization of BAF57 involves blocking its ubiquitination by preventing interaction with TRIP12, an E3 ubiquitin ligase. Consequently, as opposed to complexed BAF57, whose principal lysines are unavailable for ubiquitination, uncomplexed BAF57 can be freely ubiquitinated and degraded by the proteasome. Additionally, a BAF57 mutant, which contains no lysine residues, was found to retain its ability to be stabilized by interaction with BAF155, suggesting that in addition to the ubiquitin-dependent mechanism of BAF57 degradation, there exists a ubiquitin-independent mechanism that may involve the direct interaction of BAF57 with the proteasome. We propose that this regulatory mechanism exists to ensure functional fidelity of the complex and prevent the accumulation of uncomplexed proteins, which may disrupt the normal activity of the complex.

Single-Stranded DNA Binding Proteins (SSBPs) Interact with LDB1 Homodimers to Facilitate DNA Looping.

Abstract 1469Poster Board I-492The LIM domain binding protein LDB1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in hematopoiesis and other developmental programs. We have shown that LDB1 and its LIM-HD and LMO interaction partners are protected from ubiquitylation by a small family of SSBPs. Recently, we demonstrated that these SSBPs bind to LDB1 homodimers to promote formation of a ternary complex containing two molecules of LDB1 and SSBP. This was dependent on an intact LDB1 dimerization domain (DD) and produced a shift, directly or indirectly, in the equilibrium between LDB1 monomer and dimer. In the present study, we introduced a 24-glycine linker between two full-length LDB1 peptide-coding sequences and expressed this forced or tethered LDB1 dimer (TD-LDB1) in vitro and in vivo. First, both TD-LDB1, introduced into cells by transfection, and endogenous LDB1 were found to have the same turnover rate, indicating that protection from ubiquitylation was independent of dimerization status. Second, TD-LDB1 was fused to the DNA binding domain of GAL4 (GAL4DBD) and in a second construct to the activation domain of herpesvirus VP16 (VP16AD) and these constructs were expressed in cells with a GAL4 reporter plasmid. Co-expression of GAL4-LDB1 and VP16-LDB1 significantly increased reporter luciferase activity as a result of dimerization, while co-expression of GAL4-TD-LDB1 with VP16-LDB1 did not, ruling out formation of LDB1 trimers. Likewise, co-expression of GAL4-TD-LDB1 with VP16-TD-LDB1 did not significantly affect luciferase activity, indicating that LDB1 also cannot form protein tetramers. In contrast, TD-LDB1 was able to bind SSBP2 and SSBP3 in both chemical cross-linking and mammalian two-hybrid assays, consistent with the SSBP interacting with preformed LDB1 dimers. Finally, the complete 200-amino acid DD of LDB1, reported as necessary and sufficient for protein dimerization, was confirmed in cross-linking analysis to act as a dominant negative inhibitor of LDB1 dimerization. When the DD was introduced into Lhx2-, Ldb1-, and Ssbp3-expressing cells, application of a modified electrophoretic mobility assay that can detect linking of two DNA probes in solution revealed that the DD reduced formation of a ‘looped' complex containing two DNA probes and led to the appearance of a new complex containing Lhx2, Ssbp3, and Ldb1, apparently in a monomeric form. In summary, this work elucidates a novel function of SSBPs in enhancing LDB1 dimerization and, ultimately, long-range communication between cis regulatory regions in genes. In addition, it suggests that SSBPs bind dimeric LDB1 and induce an allosteric change in the adjacent SSBP interaction domain rather than vice versa. Finally, these results lead to the prediction that an SSBP- and LDB1-containing complex could promote looping between promoter-proximal and promoter-distal LIM-HD binding elements. Disclosures:No relevant conflicts of interest to declare.

Single-Stranded DNA-Binding Proteins (SSBPs) Promote the Oligomerization of LIM-Domain Binding Protein Ldb1

The multifunctional LIM domain-binding protein Ldb1 is important in multiple developmental programs, including hematopoiesis. An evolutionarily conserved family of proteins with single-stranded DNA-binding activity, the SSBPs, has been shown to act as Ldb1 partners and augment its biological actions. We recently established that Ssbp2 and Ssbp3 were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing the LIM-only protein Lmo2 and transcription factors Tal1, E2A, and Gata1 and showed these SSBPs stimulated E box-GATA DNA-binding activity and inhibited Ldb1 ubiquitination and subsequent proteasomal degradation (Genes & Dev. 21:942–955, 2007). As its SSBP interaction domain (Ldb1/Chip conserved domain or LCCD) is adjacent to Ldb1's N-terminal dimerization domain (DD), we sought to determine whether SSBP binding affected Ldb1 dimerization. To investigate, the Ldb1 coding region was fused to the DNA-binding domain of the yeast transcription factor GAL4 (GAL4DBD) and in a second construct to the activation domain of herpesvirus VP16 (VP16AD). These fusion proteins were then expressed in mammalian cells with a luciferase reporter linked to a promoter with iterated GAL4 binding sites. Luciferase activity became detectable with coexpression of the VP16AD-Ldb1 and GAL4DBD-Ldb1 fusions, presumably from Ldb1 dimerization, which increased markedly with simultaneous expression of SSBP2. In contrast, SSBP2 (ΔLUFS) and Ldb1 (ΔLCCD) mutants incapable of interacting with Ldb1 and SSBPs, respectively, were inactive, suggesting that SSBP2 augmentation of Ldb1 dimerization involved direct protein-protein interactions. To exclude an effect of SSBP2 on turnover of Ldb1 fusion proteins, radiolabeled full-length Ldb1 and SSBP3 were prepared by in vitro transcription/translation, mixed, and subjected to chemical crosslinking. Addition of the crosslinker bis(sulfosuccinimidyl)-suberate (BS3) to Ldb1, but not SSBP3, led to the appearance of a radiolabeled protein with mobility in denaturing polyacrylamide gels approximately twice that of Ldb1, consistent with an Ldb1 homodimer. When SSBP3 and Ldb1 were mixed together and crosslinked, a dose-related increase was noted in a more retarded species predicted to contain two molecules each of Ldb1 and SSBP3, together with a decrease in monomeric Ldb1. Finally, two well-characterized dimerization-defective Ldb1 mutants, Ldb1(200–375) and Ldb1(50–375), failed to support the formation of the higher molecular weight species or to homodimerize. Thus, the SSBPs promoted assembly of ternary complexes incorporating both SSBP and Ldb1 in a manner dependent on Ldb1 dimerization. The failure to observe Ldb1-SSBP heterodimers in cross-linking experiments suggests, further, that the SSBPs interacted with preformed Ldb1 dimers. In summary, either through an allosteric effect on Ldb1's DD or by altering the equilibrium between monomeric and dimeric species, the SSBPs promote Ldb1 oligomerization. Together with inhibition of Ldb1 ubiquitination and turnover, this would serve to augment Ldb1 function.

The LIM-domain binding protein Ldb1 is required for proper endocardial cushion formation during heart development in Mus musculus

The LIM-domain binding protein Ldb1 is required for proper endocardial cushion formation during heart development in Mus musculus

Single-Stranded DNA-Binding Proteins (SSBPs) Regulate the Abundance of the LIM-Homeodomain Protein LHX2 and Augment Its Transcriptional Activity.

A small family of proteins with putative single-stranded DNA-binding activity has been shown to augment the biological actions of LIM-homeodomain (LIM-HD) transcription factors through the mediation of the LIM domain-binding protein LDB1. We recently established that two of these SSBPs, Ssbp2 and Ssbp3, were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing transcription factors Tal1, E2A, and Gata-1 and LIM-only protein Lmo2 and showed that Ssbp2 stimulated E box-GATA DNA-binding activity by inhibiting Ldb1 ubiquitination and Ldb1 and Lmo2 turnover (Genes & Dev. 21:942–955, 2007). Since LIM-HD proteins are substrates of different E3 ubiquitin ligases than LIM-only proteins and have the additional property of binding DNA, we sought to determine the effect of SSBPs on LIM-HD expression and function. Using the prototype LIM-HD protein Lhx2 and one of its best-characterized target genes, Cga, for analysis, we found that an Ssbp3-, Ldb1-, and Lhx2-containing complex associated with an Lhx2 binding element in the Cga promoter in vitro and in mouse pituitary cells (alphaT3-1 cell line) in vivo. We then showed that enforced expression of Ssbp2 and Ssbp3 in alphaT3-1 cells increased Lhx2 and Ldb1 protein abundance, Lhx2 DNA-binding activity, and Cga expression and augmented Lhx2 transcriptional activity in an Ldb1-dependent fashion. While Lhx2-Ldb1-Ssbp3 DNA-binding activity increased in Ssbp3- relative to vector-transfected cells, the affinity of this complex for DNA was unaltered. Similar to the effect of Ssbp2 on Lmo2 in murine erythroleukemia (MEL) cells, overexpressed Ssbp3 reduced Lhx2 protein turnover in cycloheximide-treated alphaT3-1 cells without affecting Lhx2 RNA levels. In contrast, knockdown of endogenous Ssbp3, but not Ssbp2 which is expressed at much lower levels in these cells, reduced Lhx2 and Ldb1 abundance, Lhx2 DNA-binding activity, Lhx2, Ldb1, and Ssbp3 loading onto the Cga promoter, Cga promoter activity, and endogenous Cga gene expression. Significantly, neither overexpression nor knockdown of Ssbp2 in MEL cells, which express both the LIM-only protein Lmo2 and LIM-HD protein Lhx2, affected Lhx2 protein abundance, and Lhx2 DNA-binding activity was undetectable in nuclear extracts from these cells despite the presence of immunoreactive Lhx2. These studies indicate that SSBP augmentation of LIM-HD function results from Ldb1-mediated inhibition of LIM-HD protein turnover and increased assembly of a LIM-HD/LDB1/SSBP DNA-binding complex. The much greater affinity for LDB1 of LIM-only compared to LIM-HD proteins is likely a major determinant of the SSBP effect on LIM-HD protein abundance. Finally, these findings are consistent with cell type-specific contributions of different SSBPs, even for similar LDB1-dependent actions.

Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins.

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.

Regulation of Ldb1 Stability and Transcriptional Complex Assembly by Single-Stranded DNA-Binding Protein SSDP2.

The LIM domain-binding protein Ldb1 is known to form higher order complexes with LIM-homeodomain and LIM-only (LMO) proteins to regulate diverse developmental programs, including hematopoiesis. The level of Ldb1 is critical for its cellular roles, and its turnover is regulated by the E3 ubiquitin ligase RLIM. Single-stranded DNA-binding protein (SSDP), an Ldb1-interacting partner, is an essential gene for embryonic development and has been shown to regulate axis formation in Xenopus and wing development in Drosophila through Ldb1; however, the mechanisms by which SSDPs regulate these and other developmental programs are still obscure. We previously reported that a DNA-binding complex containing the basic helix-loop-helix protein TAL1/SCL, its DNA-binding partner E47, zinc finger protein GATA-1, LIM domain protein LMO2, and Ldb1 stimulates Protein 4.2 (P4.2) transcription in erythroid progenitors through tandem E box-GATA elements in the gene's proximal promoter. We have now established that SSDP2 is associated with this complex (by supershift analysis) and occupies the promoter of this gene (by chromatin immunoprecipitation analysis) in murine erythroleukemia (MEL) cells. Further, enforced expression of SSDP2 in these cells stimulated P4.2 reporter activity and accumulation of P4.2 and beta-globin mRNAs, and cotransfection of SSDP2 with the five originally identified components of this complex further increased promoter activity in reporter analysis. Importantly, overexpression of SSDP2 in MEL cells significantly increased Ldb1 protein half-life and steady-state levels of Ldb1 and LMO2 protein. This effect on Ldb1 stability required the Ldb1-interacting domain of SSDP2, consisting of its first 94 amino acids (SSDP2(1–94)), and was also observed in Cos7L and CHO cells. We showed, in addition, that SSDP2 or SSDP2(1–94), but not an Ldb1 interaction-defective mutant, prevented RLIM-mediated degradation of both Ldb1 and LMO2 in transfected cells, that SSDP2 protection of LMO2 degradation required Ldb1, and that SSDP2 directly inhibited RLIM-mediated ubiquitination of Ldb1. Immunoprecipitation analysis revealed that overexpression of SSDP2 or SSDP2(1–94) significantly decreased interaction between Ldb1 and RLIM. Finally, SSDP2 protein expression in differentiating MEL cells paralleled this multi-protein DNA-binding activity and overexpression of SSDP2 in these cells dramatically increased E box-GATA DNA-binding activity, with maximal formation of the ternary complex requiring coexpression of SSDP2, Ldb1, and LMO2. Together, these studies reveal a positive role for SSDP2 in erythroid gene expression and identify a biochemical function for SSDP2 in regulating Ldb1 stability and transcriptional complex assembly. The mechanism mediating Ldb1 stabilization appears to involve competitive inhibition of RLIM interaction with Ldb1.

Proteomic Identification of TAL1/SCL-Interacting Proteins: ETO-2 and MTGR1 Interact with TAL1 in Erythroid Progenitors.

The TAL1/SCL gene, originally identified from its involvement by a recurrent chromosomal translocation in T-cel acute lymphoblastic leukemia, encodes a basic helix-loop-helix (bHLH) transcription factor essential for hematopoietic and vascular development. Although TAL1 is believed to regulate transcription of specific sets of target genes, the mechanisms underlying TAL1-directed gene expression are poorly understood. Previous studies have shown, in fact, that it can act as either an activator or repressor depending on the coregulator(s) with which it interacts. To comprehensively identify TAL1's interaction partners in erythroid cells, we stably expressed a tandem epitope-tagged mouse TAL1 protein in murine erythroleukemia (MEL) cells and determined the composition of affinity-purified TAL1-containing complexes by multidimensional mass spectrometry. From this analysis, we identified all known members of a TAL1-containing DNA binding complex previously characterized in erythroid cells, including TAL1, its E protein DNA-binding partners, the zinc finger transcription factor GATA-1, the LIM-only protein LMO2, and the LIM domain-binding protein Ldb1, as well as proteins described to interact with GATA-1 (FOG-1), LMO2 (ELF2A2), and Ldb1 (SSDP2 and SSDP3). In addition, we identified a number of other DNA binding proteins, chromatin modifying proteins, and transcriptional regulators, including the ETO family members ETO-2 and MTGR1. TAL1 interaction with ETO-2 and MTGR1 was verified by coimmunoprecipitation analysis in MEL cells expressing these proteins at endogenous levels, in MEL cells stably expressing an epitope-tagged TAL1 protein, and in COS cells transiently transfected with TAL1 and ETO-2 or MTGR1 expression vectors. Mapping analysis with GAL4 fusion proteins identified the bHLH domain as the region in TAL1 responsible for interaction with these ETO family proteins. Significantly, expression of MTGR1 enhanced ETO-2 interaction with TAL1-GAL4 protein. Finally, transient transfection analysis with a luciferase reporter construct linked to multiple GAL4 DNA binding sites showed greater than additive augmentation of TAL1-directed gene repression with coexpression of the two ETO-related proteins compared to that observed with ETO-2 or MTGR1 transfected individually. These results identify ETO-2 and MTGR1 as authentic TAL1 interacting proteins and suggest that a hetero-oligomeric complex of the two contributes to TAL1-directed repression in erythroid progenitors.

Identification of a TAL1 target gene reveals a positive role for the LIM domain-binding protein Ldb1 in erythroid gene expression and differentiation.

The TAL1 (or SCL) gene, originally identified from its involvement by a recurrent chromosomal translocation, encodes a basic helix-loop-helix transcription factor essential for erythropoiesis. Although presumed to regulate transcription, its target genes are largely unknown. We show here that a nuclear complex containing TAL1, its DNA-binding partner E47, zinc finger transcription factor GATA-1, LIM domain protein LMO2, and LIM domain-binding protein Ldb1 transactivates the protein 4.2 (P4.2) gene through two E box GATA elements in its proximal promoter. Binding of this complex to DNA was dependent on the integrity of both E box and GATA sites and was demonstrated to occur on the P4.2 promoter in cells. Maximal transcription in transiently transfected cells required both E box GATA elements and expression of all five components of the complex. This complex was shown, in addition, to be capable of linking in solution double-stranded oligonucleotides corresponding to the two P4.2 E box GATA elements. This DNA-linking activity required Ldb1 and increased with dimethyl sulfoxide-induced differentiation of murine erythroleukemia (MEL) cells. In contrast, enforced expression in MEL cells of dimerization-defective mutant Ldb1, as well as wild-type Ldb1, significantly decreased E box GATA DNA-binding activities, P4.2 promoter activity, and accumulation of P4.2 and beta-globin mRNAs. These studies define a physiologic target for a TAL1- and GATA-1-containing ternary complex and reveal a positive role for Ldb1 in erythroid gene expression and differentiation.

The LIM Domain Protein LMO4 Interacts with the Cofactor CtIP and the Tumor Suppressor BRCA1 and Inhibits BRCA1 Activity

LMO4 belongs to the LIM-only (LMO) group of transcriptional regulators that appear to function as molecular adaptors for protein-protein interactions. Expression of the LMO4 gene is developmentally regulated in the mammary gland and is up-regulated in primary breast cancers. Using LMO4 in a yeast two-hybrid screen, we have identified the cofactor CtIP as an LMO4-binding protein. Interaction with CtIP appeared to be specific for the LMO subclass of LIM domain proteins and could be mediated by a single LIM motif of LMO4. We further identified the breast tumor suppressor BRCA1 as an LMO4-associated protein. The C-terminal BRCT domains of BRCA1, previously shown to bind CtIP, also mediated interaction with LMO4. Tumor-associated mutations within the BRCT repeats that abolish interaction between BRCA1 and CtIP had no effect on the association of BRCA1 with LMO4. A stable complex comprising LMO4, BRCA1, and CtIP was demonstrated in vivo. The LIM domain binding-protein Ldb1 also participated in this multiprotein complex. In functional assays, LMO4 was shown to repress BRCA1-mediated transcriptional activation in both yeast and mammalian cells. These findings reveal a novel complex between BRCA1, LMO4, and CtIP and indicate a role for LMO4 as a repressor of BRCA1 activity in breast tissue.

Interactions between LIM Domains and the LIM Domain-binding Protein Ldb1

LIM domains mediate protein-protein interactions and, within LIM-homeodomain proteins, act as negative regulators of the transcriptional activation function of the protein. The recently described protein Ldb1 (also known as NLI; LIM domain-binding protein) binds LIM domains in vitro and synergizes with the LIM-homeodomain protein Xlim-1 in frog embryo microinjection experiments. In this study we localized the transcriptional activation domain of Xlim-1 to its carboxyl-terminal region, and characterized the interactions of the amino-terminally located LIM domains with Ldb1. Ldb1 binds LIM domains through its carboxyl-terminal region, and can form homodimers via its amino-terminal region. Optimal binding to Ldb1 required tandem LIM domains, while single domains could bind at lower but clearly measurable efficiency. In animal explant experiments, synergism of Ldb1 with Xlim-1 in the activation of downstream genes required both the region containing the dimerization domain of Ldb1 and the region containing the LIM-binding domain. The role of Ldb1 may be to recruit other transcriptional activators depending on the promoter context and LIM-homeodomain partner involved.

The LIM-domain binding protein Ldb1 and its partner LMO2 act as negative regulators of erythroid differentiation.

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.