LIM Domain-binding Protein Research Articles

Promoting Exchange through Degradation

Transcription factor activity is regulated by interactions with cofactors, such that a given transcription factor may have both activator and repressor activities, depending on the cofactors present. Ostendorff et al. show that the repressive cofactor for the LIM homeodomain transcription factor (LIMTF), RING finger LIM domain-binding protein (RLIM), exhibits E3 ligase activity in vitro, resulting in the conjugation of ubiquitin to itself and two other LIMTF cofactors, CLIM and LMO, both of which serve to activate transcription. Overexpression of RLIM promoted CLIM and LMO degradation, and inhibition of RLIM (through transfection of dominant-negative mutants or RNA interference-mediated gene repression) resulted in enhanced CLIM immunofluorescence, suggesting increased protein abundance. Expression of RLIM inhibited reporter gene expression through both a CLIM-dependent and a CLIM-independent mechanism. The CLIM-dependent mechanism required an intact RING finger domain, whereas the CLIM-independent mechanism, which most likely involved recruitment of the histone deacetylase Sin3A, did not require the RING finger domain and most likely was not dependent on the E3 activity of RLIM. The last experiments in the paper attempted to determine DNA binding of the various cofactors and LIMTF and how RLIM affected these interactions. DNA binding of LIMTF and CLIM were inhibited in the presence of RLIM and an E2 ubiquitin conjugation enzyme, suggesting that RLIM can promote the ubiquitination and dissociation of the LIMTF-CLIM complex. Thus, ubiquitination by RLIM may represent one mechanism for promoting the exchange of cofactors on LIMTF. H. P. Ostandorff, R. I. Peirano, M. A. Peters, A. Schlüter, M. Bossenz, M. Scheffner, I. Bach, Ubiquitination-dependent cofactor exchange on LIM homeodomain transcription factors. Nature 416 , 99-103 (2002). [Online Journal]

Ubiquitination-dependent cofactor exchange on LIM homeodomain transcription factors

The interactions of distinct cofactor complexes with transcription factors are decisive determinants for the regulation of gene expression. Depending on the bound cofactor, transcription factors can have either repressing or transactivating activities. To allow a switch between these different states, regulated cofactor exchange has been proposed; however, little is known about the molecular mechanisms that are involved in this process. LIM homeodomain (LIM-HD) transcription factors associate with RLIM (RING finger LIM domain-binding protein) and with CLIM (cofactor of LIM-HD proteins; also known as NLI, Ldb and Chip) cofactors. The co-repressor RLIM inhibits the function of LIM-HD transcription factors, whereas interaction with CLIM proteins is important for the exertion of the biological activity conferred by LIM-HD transcription-factors. Here we identify RLIM as a ubiquitin protein ligase that is able to target CLIM cofactors for degradation through the 26S proteasome pathway. Furthermore, we demonstrate a ubiquitination-dependent association of RLIM with LIM-HD proteins in the presence of CLIM cofactors. Our data provide a mechanistic basis for cofactor exchange on DNA-bound transcription factors, and probably represent a general mechanism of transcriptional regulation.

Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes.

The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein-protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO-ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional (1)H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO-ldb1 interactions.

Functional repression of Islet-2 by disruption of complex with Ldb impairs peripheral axonal outgrowth in embryonic zebrafish.

Islet-2 is a LIM/homeodomain-type transcription factor of the Islet-1 family expressed in embryonic zebrafish. Two Islet-2 molecules bind to the LIM domain binding protein (Ldb) dimers. Overexpression of the LIM domains of Islet-2 or the LIM-interacting domain of Ldb proteins prevented binding of Islet-2 to Ldb proteins in vitro and caused similar in vivo defects in positioning, peripheral axonal outgrowth, and neurotransmitter expression by the Islet-2-positive primary sensory and motor neurons as the defects induced by injection of Islet-2-specific antisense morpholino oligonucleotide. These and other experiments, i.e., mosaic analysis, coexpression of full-length Islet-2, and overexpression of the chimeric LIM domains derived from two different Islet-1 family members, demonstrated that Islet-2 regulates neuronal differentiation by forming a complex with Ldb dimers and possibly with some other Islet-2-specific cofactors.

Xlim-1 and LIM Domain Binding Protein 1 Cooperate with Various Transcription Factors in the Regulation of the goosecoid Promoter

The homeobox genes Xlim-1 and goosecoid (gsc) are coexpressed in the Spemann organizer and later in the prechordal plate that acts as head organizer. Based on our previous finding that gsc is a possible target gene for Xlim-1, we studied the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages, by using gsc–luciferase reporter constructs injected into animal explants. A 492-bp upstream region of the gsc promoter responds to Xlim-1/3m, an activated form of Xlim-1, and to a combination of wild-type Xlim-1 and Ldb1, a LIM domain binding protein, supporting the view that gsc is a direct target of Xlim-1. Footprint and electrophoretic mobility shift assays with GST–homeodomain fusion proteins and embryo extracts overexpressing FLAG-tagged full-length proteins showed that the Xlim-1 homeodomain or Xlim-1/Ldb1 complex recognize several TAATXY core elements in the 492-bp upstream region, where XY is TA, TG, CA, or GG. Some of these elements are also bound by the ventral factor PV.1, whereas a TAATCT element did not bind Xlim-1 or PV.1 but did bind the anterior factors Otx2 and Gsc. These proteins modulate the activity of the gsc reporter in animal caps: Otx2 activates the reporter synergistically with Xlim-1 plus Ldb1, whereas Gsc and PV.1 strongly repress reporter activity. We show further, using animal cap assays, that the endogenous gsc gene was synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene was activated by Xlim-1/3m, and this activation was suppressed by the posterior factor Xbra. Based on these data, we propose a model for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation.

RLIM inhibits functional activity of LIM homeodomain transcription factors via recruitment of the histone deacetylase complex.

LIM domains are required for both inhibitory effects on LIM homeodomain transcription factors and synergistic transcriptional activation events. The inhibitory actions of the LIM domain can often be overcome by the LIM co-regulator known as CLIM2, LDB1 and NLI (referred to hereafter as CLIM2; refs 2-4). The association of the CLIM cofactors with LIM domains does not, however, improve the DNA-binding ability of LIM homeodomain proteins, suggesting the action of a LIM-associated inhibitor factor. Here we present evidence that LIM domains are capable of binding a novel RING-H2 zinc-finger protein, Rlim (for RING finger LIM domain-binding protein), which acts as a negative co-regulator via the recruitment of the Sin3A/histone deacetylase corepressor complex. A corepressor function of RLIM is also suggested by in vivo studies of chick wing development. Overexpression of the gene Rnf12, encoding Rlim, results in phenotypes similar to those observed after inhibition of the LIM homeodomain factor LHX2, which is required for the formation of distal structures along the proximodistal axis, or by overexpression of dominant-negative CLIM1. We conclude that Rlim is a novel corepressor that recruits histone deacetylase-containing complexes to the LIM domain.

A brain region-specific gene product Lhx6.1 interacts with Ldb1 through tandem LIM-domains.

LIM-homeodomain (LHX) transcription factors play critical roles in cell fate determination during development, in particular, in CNS. The transcriptional activity of several LHX proteins is postulated to be regulated by interaction with an LIM-domain binding protein, Ldb1. We have now identified a novel LHX molecule, termed Lhx6.1, that is closely related to a recently reported Lhx6 molecule. The Lhx6.1 transcript is found in several restricted regions in the developing CNS, mostly within the embryonic forebrain. We further show that Lhx6.1 interacts with Ldb1 through tandem LIM-domains, implying transcriptional regulation of Lhx6.1 by Ldb1.

Isolation and cloning of a novel cDNALDB1 encoding human LIM domain binding protein

Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain binding protein Ldbl. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A 2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldbl,Xenopus Xldbl andDrosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been namedLDB1 ( LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts ofLDBI in heart, brain and lung were considerably higher than those in other tissues.

Characterization of Lhx9, a novel LIM/homeobox gene expressed by the pioneer neurons in the mouse cerebral cortex

In order to explain the phenotype observed in Lhx2 mutant embryos, we previously proposed that an Lhx2 related gene might exist. We now have cloned a new LIM/homeobox gene called Lhx9. Lhx9 is closely related to Lhx2 and is expressed in the developing central nervous system (CNS). Lhx9 and Lhx2 have expression patterns that overlap in some areas but are distinct in others. Thus, in some developmental domains these two highly related proteins may be functionally redundant. Lhx9 is expressed in the pioneer neurons of the cerebral cortex, while Lhx2 is expressed throughout the cortical layers. Postnatally, Lhx9 is expressed in the inner nuclei of the cerebellum, while Lhx2 is in the granular layer. In the developing limbs, both genes are highly expressed in a similar pattern. Based on the expression pattern and the developmental regulation of Lhx9, we propose that Lhx9 may be involved in the specification or function of the pioneer neurons of the cerebral cortex. We show that both Lhx9 and Lhx2 bind the LIM domain binding protein Ldb1/Nli1/Clim2.

Genomic structure, alternative transcripts and chromosome location of the human LIM domain binding protein 1 gene LDB1

By protein interaction screening using a radioactive LMO2 protein probe we have isolated a LIM domain binding protein. The gene shows high homology to independently isolated genes from mouse, Xenopus and Drosophila called Ldb1/Nli/Clim-2, Xldb1 and Chip, respectively. The human and mouse genes differ by only two amino acids, suggesting that the gene that we have isolated is the human homologue. Here we describe the genomic organization, alternative transcript forms and the chromosome mapping of the human gene LDB1 (alias NLI). The gene is spread over at least 12 kb and has 11 exons. Preceding the described ATG initiation site in the mouse a highly conserved region between mouse, chicken and human was detected with a second possible in frame initiation site coding for further 36 amino acids. An alternative splice site adding six nucleotides corresponding to the addition of two amino acids at the end of exon 10 was found. The gene was mapped to chromosome 10q24→q25 by in situ hybridization, a region frequently deleted in many types of cancer. Fine mapping with a radiation hybrid panel localized the gene in the interval between the markers D10S603 and D10S540.

Characterization of the porcine Lhx3/LIM-3/P-Lim LIM homeodomain transcription factor

Lhx3/LIM-3/P-Lim is a LIM homeodomain transcription factor which is essential in mice for the development of anterior and intermediate lobes of the pituitary gland. We report the cloning and characterization of porcine Lhx3. The porcine Lhx3 protein exhibits strong similarity to murine Lhx3 within the amino terminal LIM domains and the homeodomain, however, it is diverged in regions outside these motifs. Expression vectors for porcine Lhx3 activated murine and porcine alpha-glycoprotein reporter genes in transfection assays, and recombinant porcine Lhx3 protein specifically bound to a target site within the porcine alpha-glycoprotein gene upstream sequence. In addition, porcine Lhx3 synergistically induced transcription from prolactin enhancer/promoter reporter genes in cooperation with the Pit-1 pituitary transcription factor. Porcine Lhx3 protein interacted with Pit-1 protein in solution and also with the LIM domain-binding protein NLI/Lbd1/CLIM. Together, these data indicate that many aspects of Lhx3 function in the mammalian pituitary are conserved and that Lhx3 may be involved in the activation of trophic hormone genes during early and late stages of pituitary organogenesis. Divergence in the Lhx3 amino acid sequence between mammalian species may suggest distinct activities for this protein in some species and may help identify important functional domains of this key developmental transcription factor.

Expression of LIM-domain binding protein (ldb) genes during zebrafish embryogenesis

LIM homeodomain proteins are developmental regulators whose functions depend on synergism with LIM domain binding proteins (Ldb proteins). We have isolated four members of the ldb gene family from the zebrafish, Danio rerio. Ldb1, Ldb2 and Ldb3 share 95%, 73% and 62% amino acid identity with mouse Ldb1, respectively. In overlay assays, Ldb proteins bind LIM homeodomain proteins and LMO1, but not zyxin or MLP. Whole mount in situ hybridization showed that zebrafish ldb1 is expressed ubiquitously from gastrulation onward. Ldb2 is ubiquitous at gastrulation, and later is found in many but not all tissues, especially the anterior central nervous system (CNS) and vasculature. Ldb3 mRNA was expressed primarily in the anterior CNS.

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.

Genomic Structure and Chromosomal Localization of the Mouse LIM Domain-Binding Protein 1 Gene,Ldb1

The LIM domain is a structural motif that is well conserved throughout evolution in a variety of factors known to play important roles in development and cell regulation.Ldbgenes encode LIM domain-binding (Ldb) factors. Here we report on the structural organization and chromosomal localization of the mouseLdb1gene. It contains at least 10 exons and spans approximately 4 kb of genomic DNA. The transcription initiation site is located 462 bp upstream of the translation initiation codon ATG as determined by 5′-RACE. Sequencing analysis of the 5′-flanking region shows TATA and CCAAT motifs as well as potential binding sites for GATA, CF-1, PEA3, HRE, APRRE, RARE, Myc, and c-Jun.Ldb1maps to the distal region of mouse chromosome 19 that is syntenic with human chromosome 10q.

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.