Chip
LIM homeodomain (LIM-HD) and nuclear LIM-only proteins play important roles in a variety of developmental processes
in animals. In some cases their activities are modulated by a nuclear LIM binding protein family called Ldb/NLI/Clim, exemplified by Drosophila Chip. The Ldb/NLI/Clim ortholog ldb-1 of the nematode C. elegans has been characterized. Two alternatively spliced
variants exist, which differ in their amino-termini. The ldb-1 ortholog of Caenorhabditis briggsae has the same structure
as that of C. elegans and is highly conserved throughout the open reading frame, while conservation to fly and vertebrate
proteins is restricted to specific domains: the dimerization domain, the nuclear localization sequence, and the LIM
interaction domain. C. elegans ldb-1 is expressed in neurogenic tissues in embryos, in all neurons in larval and adult stages,
and in vulval cells, gonadal sheath cells, and some body muscle cells. C. elegans LDB-1 is able to specifically bind LIM
domains in yeast two-hybrid assays. RNA inactivation studies suggest that C. elegans ldb-1 is not required for the
differentiation of neurons that express the respective LIM-HD genes or for LIM-HD gene autoregulation. In contrast, ldb-1
is necessary for several neuronal functions mediated by LIM-HD proteins, including the transcriptional activation of mec-2,
the mechanosensory neuron-specific stomatin (Cassata, 2000).
LIM homeobox family members regulate a variety of cell fate choices during animal development. In C. elegans, mutations in the LIM homeobox gene lim-11 (most closely related to Drosophila Lim1) have been shown to alter the cell division pattern of a subset of the 2º lineage vulval cells. Multiple functions of lin-11 during vulval development have been demonstrated. The fate of vulval cells was examined in lin-11 mutant animals using five cellular markers: lin-11 is necessary for the patterning of both 1º and 2º lineage cells. In the absence of lin-11 function, vulval cells fail to acquire correct identity and inappropriately fuse with each other. The expression pattern of lin-11 reveals dynamic changes during development. Using a temporally controlled overexpression system, lin-11 is shown to be initially required in vulval cells for establishing the correct invagination pattern. This process involves asymmetric expression of lin-11 in the 2º lineage cells. Using a conditional RNAi approach, it has been shown that lin-11 regulates vulval morphogenesis. LDB-1, a NLI/Ldb1/CLIM2 family member, interacts physically with LIN-11, and is necessary for vulval morphogenesis. Together, these findings demonstrate that temporal regulation of lin-11 is crucial for the wild-type vulval patterning (Gupta, 2003).
LIM homeodomain proteins are developmental regulators whose functions depend on synergism with
LIM domain binding proteins (Ldb proteins); they are homologs of Drosophila Chip. Three members of the ldb gene family (Ldb1, Ldb2 and Ldb3)
from the zebrafish, Danio rerio, 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 tissues, especially the anterior central nervous system (CNS) and vasculature, but not all tissues; Ldb3
mRNA is expressed primarily in the anterior CNS. The expression of individual lbd and LIM proteins correlates in various regions and stages of embryogenesis. For example, lbd1 and ldh2 are expressed in the shield as are lim1 and lim6. Both lbd2 and lbd3 expression in the telencephalon overlap with that of lim5 and lim6, and three genes, lbd2, lim3 and lim5, are coexpressed in the epiphysis (Toyama 1998).
The crucial involvement of CLIM/NLI/Ldb cofactors for the exertion of the biological activity of LIM homeodomain transcription factors (LIM-HD) has been demonstrated. CLIM cofactors are widely expressed during zebrafish development with high protein levels in specific neuronal cell types where LIM-HD proteins of the Isl class are synthesized. The overexpression of a dominant-negative CLIM molecule (DN-CLIM) that contains the LIM interaction domain (LID) during early developmental stages of zebrafish embryos results in an impairment of eye and midbrain-hindbrain boundary (MHB) development and disturbances in the formation of the anterior midline. On a cellular level it has been shown that the outgrowth of peripheral but not central axons from Rohon Beard (RB) and trigeminal sensory neurons is inhibited by DN-CLIM overexpression. A further critical role of CLIM cofactors has been demonstrated for axonal outgrowth of motor neurons. Additionally, DN-CLIM overexpression causes an increase of Isl-protein expression levels in specific neuronal cell types, likely due to a protection of the DN-CLIM/LIM-HD complex from proteasomal degradation. These results demonstrate multiple roles of the CLIM cofactor family for the development of entire organs, axonal outgrowth of specific neurons and protein expression levels (Becker, 2002).
A novel protein and a new LIM-domain-binding factor, Ldb1, has been isolated on the basis of its ability
to interact with the LIM-HD protein Lhx1 (Lim1). High-affinity binding by Ldb1
requires paired LIM domains and is restricted to the related subgroup of LIM domains
found in LIM-HD and LMO proteins (see Drosophila Muscle LIM protein at 60A). The highly conserved Xenopus Ldb protein
XLdb1, interacts with Xlim-1, the Xenopus orthologue of Lhx1. When injected into
Xenopus embryos, XLdb1 (or Ldb1) can synergize with Xlim-1 in the formation of
partial secondary axes and in activation of the genes encoding goosecoid,
chordin, NCAM and XCG7, demonstrating a functional as well as a physical
interaction between the two proteins (Agulnick, 1996).
The homeobox genes Xlim-1 and goosecoid are coexpressed in the Spemann organizer and later in the prechordal plate
that acts as head organizer. Since gsc is a possible target gene for Xlim-1, the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages was studied 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 show that the Xlim-1 homeodomain and the 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 does not bind Xlim-1 or PV.1 but does 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. Using animal cap assays, it has been shown that the endogenous gsc gene is synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene is activated by Xlim-1/3m, and this activation is suppressed
by the posterior factor Xbra. Based on these data, a model is proposed for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation (Mochizuki, 2000).
Thus Gsc protein is capable of inhibiting the activity of its
own promoter in assays using reporters activated by Xlim-1,
Ldb1, and Otx2. Otx2 and Gsc belong to the same homeodomain
group in that both have a lysine residue at position
50 of the homeodomain and share binding specificity for
TAATCT and TAATCC. Since these two proteins recognize similar
target sequences, there may be competition between Otx2
and Gsc for binding to the C site of distal element, with Otx2 having
activating and Gsc inhibiting effects. Inhibition of the
mouse and human gsc promoter by Gsc requires the proximal element, suggesting that Gsc inhibition, just
like Xlim-1 activation, involves multiple sites in the complex
gsc promoter. Repression of the gsc promoter by Gsc and PV.1 proteins
is similarly effective under the experimental conditions
employed, but the biological roles of the two proteins are
different. In the case of Gsc autoinhibition, the rationale
may be to provide a feedback loop to limit gsc expression. In
contrast, PV.1, closely related to Xvent-1, is expressed
ventrally as a consequence of BMP signaling in a region of
the embryo where gsc is not expressed. It appears that PV.1 is a repressor
protein whose function is to maintain the character of
ventral mesoderm by inhibiting gsc expression in the non-organizer
regions of the marginal zone. Similarly, Xbra may
inhibit Gsc function in the notochord where gsc expression
diminishes during gastrulation. The ability of Xbra to
repress otx2 expression and of Gsc to repress Xbra
expression may play a role in restricting
gsc expression to the prechordal plate and Xbra expression
to the notochord at mid- to late gastrulation. However,
because Xbra is a transcriptional activator,
it is assumed that otx2 repression is indirect (Mochizuki, 2000).
These transcription factor interactions have been
incorporated into a model of dorsoventral and anteroposterior patterning in the gastrula embryo. In the prechordal plate, Xlim-1 and Ldb1, in addition to
contributing to chordin induction, maintain the expression
of otx2 and of gsc; the autoinhibitory action of the latter is
counteracted by the activating function of Otx2, while Xbra
expression is suppressed by Gsc. In the notochord, the high
initial level of Xbra prevents otx2 gene activation by Xlim-1
plus Ldb1, and in the absence of Otx2, the gsc gene turns
itself off by autorepression. Note that in the early gastrula,
gsc is active in the entire organizer, but its expression fades
in posterior axial mesoderm as gastrulation proceeds. In
ventral mesoderm, the strong repression of gsc and otx2 by
PV.1/Xvent-1 and Xbra maintains the ventral character of
this tissue. Clearly, this scheme is incomplete in that
additional factors are undoubtedly involved, yet it provides
a cogent model for the interactions of the factors considered
in this paper during axial patterning in the gastrula (Mochizuki, 2000).
LIM domain-containing transcription factors, including the LIM-only rhombotins and
LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages.
The zinc-binding LIM domains mediate protein-protein interactions; interactions between nuclear
LIM proteins and transcription factors with restricted expression patterns have been demonstrated. A novel protein, nuclear LIM interactor (NLI) has been isolated that specifically associates with a single
LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell
types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron
differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The
broad range of interactions between NLI and LIM-containing transcription factors suggests the
utilization of a common mechanism to impart unique cell fate instructions (Jurata, 1996).
Two highly homologous proteins specifically interact with the LIM
domains of P-Lim/Lhx3 and several other LIM homeodomain factors. Transcripts encoding these
factors can be detected as early as mouse E8.5, with maximal expression observed in regions of the
embryo in which the LIM homeodomain factors P-Lim/Lhx3, Isl-1, and LH-2 are selectively
expressed. These proteins can potentiate transactivation by P-Lim/Lhx-3 and are required for a
synergistic activation of the glycoprotein hormone alpha-subunit promoter by P-Lim/Lhx3 and a
pituitary Otx class homeodomain transcription factor (P-OTX/Ptx1), with which they also specifically associate. The two new genes are referred to as CLIM-1 and CLIM-2 (cofactor of LIM homeodomain proteins). The CLIM proteins are required for a transcriptional synergy between P-Lim/Lhx3 and P-OTX/Ptx1. The fact that CLIM-encoded mRNAs show a widely overlapping expression pattern with Otx1 and Otx2 in the developing mouse brain suggests that the CLIM protein family may play critical roles in the functional relationships of LIM homeoproteins and additional Otx factors (Bach, 1997).
The mesenchymal cells that contribute to oral and facial hard tissues are derived from cranial neural crest cells, whereas limb mesenchyme cells are derived from axial mesoderm. The outgrowth of facial processes has been compared with limb bud outgrowth; the tooth bud enamel knot has been identified as a signaling center with similarities to both the limb ZPA and AER signaling centers. In addition, several homeobox-containing genes have been implicated in both branchial arch and limb development, such as members of the Msx, Dlx and Lim-homeobox families. The expression of the Lim-domain gene Lhx-6, and its closely associated family member Lhx-7 are largely restricted to anterior mesenchymal cells of the mandibular and maxillary arches. Clim-2 (NLI, Lbd1) is one of two related mouse proteins that interact with Lim-domain homeoproteins. In the
mouse, embryonic expression of Clim-2 is particularly pronounced in facial ectomesenchyme and limb bud
mesenchyme in association with Lim genes Lhx-6 and Lmx-1, respectively. In common with both
these Lim genes, Clim-2 expression is regulated by signals from overlying epithelium. In both the developing face
and the limb buds Fgf-8 is identified as the likely candidate signaling molecule that regulates Clim-2 expression. In the mandibular arch, as in the limb, Fgf-8 functions in combination with CD44, a cell surface
binding protein that has been considered to be a hyaluronan receptor. Blocking CD44 binding results in inhibition of Fgf8-induced expression of Clim-2 and
Lhx-6. CD44 has been shown to be required for presentation of Fgf-8 to its receptor, rather than as a hyaluronan receptor. Regulation of gene expression by Fgf8 in association with CD44 is thus conserved between limb and
mandibular arch development (Tucker, 1999).
LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged
Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have
restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for
cell type specification in a variety of developing tissues. A recently identified and widely expressed protein,
NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and
in vivo. A 38-amino-acid fragment of NLI is sufficient for the association of
NLI with nuclear LIM domains. NLI has been shown to form high affinity homodimers through
the amino-terminal 200 amino acids, but dimerization of NLI is not required for association with the
LIM homeodomain protein Lmxl. Chemical cross-linking analysis reveals higher-order complexes
containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not
interfere with LIM domain interactions. NLI forms complexes with Lmx1 on the rat
insulin I promoter and inhibits the LIM domain-dependent synergistic transcriptional activation by
Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I minienhancer. These studies
indicate that NLI contains at least two functionally independent domains and may serve as a negative
regulator of synergistic transcriptional responses that require direct interaction via LIM domains.
Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining
specific partner interactions (Jurata, 1997).
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. A widely expressed protein, Ldb1, has been identified 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 inhibits cellular
maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during
erythroid differentiation. Overexpression of the LMO2 gene also inhibits erythroid differentiation. These
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 (Visvader, 1997).
The LIM-only protein Lmo2, activated by chromosomal translocations in T-cell leukemias, is normally
expressed in hematopoiesis. It interacts with TAL1 and GATA-1 proteins, but the function of the
interaction is unexplained. In erythroid cells Lmo2 forms a novel DNA-binding
complex with GATA-1, TAL1, E2A, and the recently identified LIM-binding protein, Ldb1/NLI.
This oligomeric complex binds to a unique, bipartite DNA motif comprising an E-box (CAGGTG),
followed approximately 9 bp downstream by a GATA site. In vivo assembly of the DNA-binding
complex requires interaction of all five proteins and establishes a transcriptional transactivating
complex. These data demonstrate one function for the LIM-binding protein Ldb1 and establish a
function for the LIM-only protein Lmo2 as an obligatory component of an oligomeric, DNA-binding
complex, which may play a role in hematopoiesis (Wadman, 1997).
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). 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. Evidence is presented
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. It is concluded that Rlim is a novel corepressor
that recruits histone deacetylase-containing complexes to the LIM domain (Bach, 1999).
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 either Islet-2 or the LIM-interacting domain of Ldb proteins prevents binding of Islet-2 to Ldb proteins in vitro and causes similar in vivo defects in positioning, peripheral axonal outgrowth, and neurotransmitter expression by the Islet-2-positive primary sensory and motor neurons. Inhibition of Islet-2 translation with antisense morpholino oligonucleotide against Islet-2 mRNA reproduces the defects caused by overexpression of LIM domains of Isl-2. 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, demonstrate that Islet-2 regulates neuronal differentiation by forming a complex with Ldb dimers and possibly with some other Islet-2-specific cofactors (Segawa, 2001).
Repression of Islet-2 function either by overexpressing the LIM domains of Isl-2 or injecting antisense against Islet-2 mRNA impairs outgrowth of the peripheral axons from the primary sensory neurons, while keeping their central axons intact. In vitro study using the explants of the chick dorsal root ganglion (DRG) has demonstrated that the DRG neurons extend their axons into the peripheral tissue only in the presence of the nerve growth factor (NGF), while they extend the axons into the CNS tissue irrespective of the presence of NGF. This suggests that NGF may selectively promote the peripheral outgrowth of the DRG neurons. Recently, double mutant mice for the genes encoding the proapoptotic BCL-2 homolog BAX and NGF/TrkA were generated. All DRG neurons (which would normally die in the absence of the NGF/TrkA signaling) survive if BAX is also eliminated. In BAX-/-;NGF-/- or BAX-/-;TrkA-/- mice, only the peripheral axons of the DRG neurons are lost, while their central axons remain intact. Therefore, the NGF/TrkA signaling regulates outgrowth of the peripheral axons from the DRG neurons. In view of this, it would be intriguing to examine whether Islet-2 is involved in the NGF/TrkA signaling (Segawa, 2001).
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.
RLIM has been identified as a ubiquitin protein ligase that is able to
target CLIM cofactors for degradation through the 26S proteasome
pathway. Furthermore, this study demonstrates a ubiquitination-dependent
association of RLIM with LIM-HD proteins in the
presence of CLIM cofactors. These data provide a mechanistic basis
for cofactor exchange on DNA-bound transcription factors, and probably represent a general mechanism of transcriptional regulation (Ostendorff, 2002).
The LIM domain-binding protein 1 (Ldb1) is found in multi-protein complexes containing various combinations of LIM-homeodomain, LIM-only, bHLH, GATA and Otx transcription factors. These proteins exert key functions during embryogenesis. Targeted deletion of the Ldb1 gene in mice results in a pleiotropic phenotype. There is no heart anlage and head structures are truncated anterior to the hindbrain. In about 40% of the mutants, posterior axis duplication is observed. There are also severe defects in mesoderm-derived extraembryonic structures, including the allantois, blood islands of the yolk sack, primordial germ cells and the amnion. Abnormal organizer gene expression during gastrulation may account for the observed axis defects in Ldb1 mutant embryos. The expression of several Wnt inhibitors is curtailed in the mutant, suggesting that Wnt pathways may be involved in axial patterning regulated by Ldb1 (Mukhopadhyay, 2003).
The Xenopus LIM homeodomain (LIM-HD) protein, Xlim-1, is expressed in the Spemann organizer and cooperates with its positive regulator, Ldb1, to activate organizer gene expression. While this activation is presumably mediated through Xlim-1/Ldb1 tetramer formation, the mechanisms regulating proper Xlim-1/Ldb1 stoichiometry remain largely unknown. The Xenopus ortholog (XRnf12) of the RING finger protein Rnf12/RLIM has been isolated and its functional interactions with Xlim-1 and Ldb1 have been explored. Although XRnf12 functions as an E3 ubiquitin ligase for Ldb1 and causes proteasome-dependent degradation of Ldb1, co-expression of a high level of Xlim-1 suppresses Ldb1 degradation by XRnf12. This suppression requires both the LIM domains of Xlim-1 and the LIM interaction domain of Ldb1, suggesting that Ldb1, when bound to Xlim-1, escapes degradation by XRnf12. A high level of Ldb1 suppresses the organizer activity of Xlim-1/Ldb1, suggesting that excess Ldb1 molecules disturb Xlim-1/Ldb1 stoichiometry. Consistent with this, Ldb1 overexpression in the dorsal marginal zone suppresses expression of several organizer genes including postulated Xlim-1 targets, and importantly, this suppression is rescued by co-expression of XRnf12. These data suggest that XRnf12 confers proper Ldb1 protein levels and Xlim-1/Ldb1 stoichiometry for their functions in the organizer. Together with the similarity in the expression pattern of Ldb1 and XRnf12 throughout early embryogenesis, Rnf12/RLIM is proposed as a specific regulator of Ldb1 to ensure its proper interactions with LIM-HD proteins and possibly other Ldb1-interacting proteins in the organizer as well as in other tissues (Hiratani, 2003).
LMO2 and LMO4 are members of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. LMO2 is essential for hemopoiesis and angiogenesis, and inappropriate overexpression of this protein leads to T-cell leukemias. LMO4 is developmentally regulated in the mammary gland and has been implicated in breast oncogenesis. Both proteins comprise two tandemly repeated LIM domains. LMO2 and LMO4 interact with the ubiquitous nuclear adaptor protein ldb1/NLI/CLIM2, which associates with the LIM domains of LMO and LIM homeodomain proteins via its LIM interaction domain (ldb1-LID). This study reports the solution structures of two LMO:ldb1 complexes (PDB: 1M3V and 1J2O) and shows that ldb1-LID binds to the N-terminal LIM domain (LIM1) of LMO2 and LMO4 in an extended conformation, contributing a third strand to a beta-hairpin in LIM1 domains. These findings constitute the first molecular definition of LIM-mediated protein-protein interactions and suggest a mechanism by which ldb1 can bind a variety of LIM domains that share low sequence homology (Deane, 2003).
Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (Ldb1). A high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, has been determined in complex with Ldb1-LID, providing the first example of a tandem LIM:Ldb1-LID complex and the first structure of a type-B LIM domain. The complex possesses a highly modular structure with Ldb1-LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone-backbone hydrogen bonds. A mutagenic screen of Ldb1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM-HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains (Deane, 2004).
The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. This study shows using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1. However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1, whereas the second LIM domain increases binding by an order of magnitude. Mutagenesis was used in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding 'hot spots' within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia (Ryan, 2006).
SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. This study identified the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and shows that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, these observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression (Lahlil, 2004).
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) have been shown to interact with Ldb1 and are also important in developmental programs. 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 β-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 (Xu, 2007).
The transcriptional activity of LIM-homeodomain (LIM-HD) proteins is
regulated by their interactions with various factors that bind to the LIM
domain. Reduced expression of single-stranded DNA-binding protein
1 (Ssdp1), which encodes a co-factor of LIM domain interacting
protein 1 (Ldb1), in the mouse mutant headshrinker (hsk)
disrupts anterior head development by partially mimicking Lim1
mutants. Although the anterior visceral endoderm and the anterior definitive
endoderm, which together comprise the head organizer, are able to form
normally in Ssdp1hsk/hsk mutants, development of the
prechordal plate was compromised. Head development is partially initiated in
Ssdp1hsk/hsk mutants, but neuroectoderm tissue anterior to
the midbrain-hindbrain boundary is lost, without a concomitant increase in
apoptosis. Cell proliferation is globally reduced in
Ssdp1hsk/hsk mutants, and approximately half also exhibit
smaller body size, similar to the phenotype observed in Lim1 and
Ldb1 mutants. Ssdp1 contains an activation domain
and is able to enhance transcriptional activation through a Lim1-Ldb1 complex
in transfected cells, and Ssdp1 interacts genetically with
Lim1 and Ldb1 in both head development and body growth.
These results suggest that Ssdp1 regulates the development of late head
organizer tissues and body growth by functioning as an essential activator
component of a Lim1 complex through interaction with Ldb1 (Nishioka, 2005).
Ssdp1 mutants exhibit a global reduction in cell proliferation
after E8.5 and an increase in apoptosis in somites at E9.0. These changes may
be at the root of the abnormalities such as growth retardation and kinked
neural tube that were observed in Ssdp1 mutants. Although the
mechanism by which Ssdp1 regulates cell proliferation is unknown at
present, growth retardation of
Ssdp1+/hsk;Lim1+/- and
Ssdp1+/hsk;Ldb1+/- compound mutants suggests
involvement of a Ssdp1-Lim1-Ldb1 complex in this process. A shortened body
axis was also observed in embryos lacking either Ldb1 or
Lim1, supporting this hypothesis.
However, if the Lim1 complex plays a major role in the regulation of cell
proliferation and cell death, it must be through an indirect mechanism, since
Lim1 is not expressed in all of the affected cells. It is conceivable
that defective gastrulation movements or the
inability of cells with reduced Lim1 complex activity to induce lateral plate
mesoderm genes secondarily affects the proliferation and survival of
surrounding cells. Furthermore, it is possible that Ssdp1 may also function
independently of Lim1, in which case the Ldb1-Ssdp1 complex may regulate cell
proliferation in a cell-autonomous manner by controlling the activities of
transcription factors involved in cell cycle regulation and cell survival.
Alternatively, Ssdp1 might play a direct role in the DNA replication process
as a single stranded DNA-binding protein (Nishioka, 2005).
Analysis of hsk mutants shows that disruption of the
Ssdp1 gene and the resulting reduction in Ssdp1 expression
causes defects in the prechordal plate development and anterior truncations,
with some mutants also exhibiting smaller body size. In vitro data have
demonstrated that Ssdp1 acts as a coactivator that enhances transcriptional
activation by the Lim1-Ldb1 complex. Moreover, genetic interactions between
Ssdp1 and Lim1 or Ldb1 suggest that the phenotypes
observed in Ssdp1 mutants very probably reflect reduced activity of a
Lim1 complex. Together, these data demonstrate that Ssdp1 acts as an essential
activator component of a Ssdp1-Lim1-Ldb1 complex in the development of the
prechordal plate and body growth (Nishioka, 2005).
Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes. This study has identified a number of novel Ldb1 interacting proteins in erythroleukaemic cells, in particular the repressor protein Eto-2 (and its family member Mtgr1), the cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated knockdowns in zebrafish show these factors to be essential for definitive haematopoiesis. In accordance with the zebrafish results these factors are coexpressed in prehaematopoietic cells of the early mouse embryo, although the complex was originally identified in late erythroid cells. Based on the change in subcellullar localisation of Eto-2 it is postulated that it plays a central role in the transition from the migration and expansion phase of the prehaematopoietic cells to the establishment of definitive haematopoietic stem cells (Meier, 2006).
LMO2, a member of the LIM-only protein family, is essential for the regulation of hematopoietic stem cells and formation of erythroid cells. It is found in a transcriptional complex comprising LMO2, TAL1, E47, GATA-1, and LDB1 which regulates erythroid genes. While TAL1 has been shown to induce erythroid differentiation, LMO2 appears to suppress fetal erythropoiesis. In addition to LMO2, the closely related LMO4 gene is expressed in hematopoietic cells, but has unknown functions. This study demonstrates that LMO2 and LMO4 are expressed at the same level in erythroid colonies from mouse bone marrow, implying a function in erythroid differentiation. However, while LMO2 induced erythroid differentiation, LMO4 had no such effect. Interestingly, both LMO2 and TAL1 were able to partially suppress myeloid differentiation, implying that they activate erythroid differentiation in uncommitted bone marrow progenitors. Both LMO2 and LMO4 interacted strongly to LDB1, which was required for their localization to the nucleus (Hansson, 2007).
Spinal motor neurons (MNs) and V2 interneurons (V2-INs) are specified by two related LIM-complexes, MN-hexamer and V2-tetramer, respectively. This study shows how multiple parallel and complementary feedback loops are integrated to assign these two cell fates accurately. While MN-hexamer response elements (REs) are specific to MN-hexamer, V2-tetramer-REs can bind both LIM-complexes. In embryonic MNs, however, two factors cooperatively suppress the aberrant activation of V2-tetramer-REs. First, LMO4 blocks V2-tetramer assembly. Second, MN-hexamer induces a repressor, Hb9, which binds V2-tetramer-REs and suppresses their activation. V2-INs use a similar approach; V2-tetramer induces a repressor, Chx10, which binds MN-hexamer-REs and blocks their activation. Thus, this study uncovers a regulatory network to segregate related cell fates, which involves reciprocal feedforward gene regulatory loops (Lee, 2008).
A SELEX study revealed that the Lhx3-binding sites deviate between HxRE and TeRE in sequence. As HxRE is recognized by MN-hexamer but not by V2-tetramer, the conformation of Lhx3 in MN-hexamer and V2-tetramer is likely different. This may involve an allosteric structural change in the DNA binding domain of Lhx3 in MN-hexamer, induced by the Isl1:Lhx3 interaction. As MN-hexamer is assembled only in MNs, HxRE-containing genes would be stimulated specifically in MNs but not in V2-INs. In contrast, TeRE is activated by both V2-tetramer and MN-hexamer in vitro, suggesting that TeRE-containing V2 genes could be inappropriately induced in MNs. However, TeRE is a V2-specific response element in the developing embryos due to a collaborative action of Hb9 and LMO4 to silence TeRE in MNs. Thus, these studies demonstrate that DNA-REs for specific transcription complexes are sufficient to confer gene expressions to proper cell types in developing embryos (Lee, 2008).
Although Lhx4 and their cofactor NLI (Ldb, CLIM, Chip) dimerization is dispensable for the DNA-binding activity of V2-tetramer and MN-hexamer, it is essential for their robust transactivation. Thus, reiterated TeRE1/2s and HxRE1/2s are necessary for functional TeREs and HxREs. Indeed, the MN-specific enhancer of Hb9 has two functional HxRE1/2s spaced ~150 nt apart. Similarly, three evolutionarily conserved TeRE1/2 sequences were found in the Chx10-TeRE region. Three possible advantages can be proposed for the NLI dimerization in V2-tetramer and MN-hexamer. First, the requirement for multiple repeats of TeRE1/2 and HxRE1/2 may impose higher stringency for functional target gene selection. Second, NLI dimerization bridges two NLI-interacting transcription factors bound to their DNA-binding sites separated by a relatively long spacer region in Drosophila (Heitzler, 2003; Morcillo, 1997). This raises the possibility that V2-tetramer and MN-hexamer may integrate the transcriptional activity of multiple TeRE1/2s or HxRE1/2s located within a single target gene or across multiple target genes. For instance, both Chx10 and Lin-52 have additional TeRE1/2s within their gene. Thus, it will be interesting to test whether the Chx10-TeRE region is required to regulate both Chx10 and Lin-52 by V2-tetramer and whether the multiple TeRE1/2s throughout these two genes enable V2-tetramer to temporally coordinate expression of these genes during development. Third, NLI dimerization may potentiate the transcriptional activity of LIM-complexes by stabilizing the LIM-complexes and/or facilitating recruitment of transcriptional coactivators and chromatin remodeling complexes. Indeed, single-stranded DNA-binding proteins have been found to interact with NLI and augment the transactivation of NLI-containing complexes (Lee, 2008).
DNA-REs affect the protein-protein interaction properties of their cognate transcription factors. Sox2 and Pou factor family members Oct1 or Oct4 dimerize onto different DNA-REs in distinct conformational arrangements, offering one molecular explanation for the wide spectrum of developmental functions for Sox/Pou factors. Thus, it will be interesting to interrogate the role of TeRE and HxRE on the spatial alignment of DNA-protein complex of Lhx3/TeRE1/2 and of Isl1:Lhx3/HxRE1/2 (Lee, 2008).
As p2 and pMN cells are exposed to relatively similar concentration of Shh, deregulation of the transcriptional events downstream of Shh often results in V2-MN fate conversion or hybrid phenotypes. The initial segregation of V2 and MN pathways appears to involve transcriptional crossrepression of progenitor factors Irx3 and Olig2 in neuroepithelial cells. However, additional mechanisms are likely to be needed, as both differentiating V2 and MN cells express Lhx3/4, which are necessary for their cell fates. The results reveal an efficient feedforward gene regulatory circuitry in which a cell-type specific LIM-complex triggers expression of a transcriptional repressor, which in turn binds and represses the DNA-RE of another related LIM-complex, thereby blocking the unwanted choice of alternative fates. This likely contributes to establishing a precise cell identity once a specific LIM-complex is assembled and activates the downstream target genes in neural precursors (Lee, 2008).
First, MN-hexamer upregulates Hb9, which in turn refines MN-gene expression by silencing V2-genes. Hb9 selectively binds TeREs, which prevents undesirable recruitment of MN-hexamer (and any V2-tetramer) to TeREs and represses their inappropriate activation in embryonic MNs. Indeed, V2 genes are upregulated in MNs lacking Hb9. Hb9, a transcriptional repressor, may suppress TeRE-containing V2 genes by recruiting corepressors to their TeREs. Mnr2, an Hb9 paralog, is also known to recruit Ctbp-like corepressor. Thus, TeRE-containing V2 genes are likely to be activated by V2-tetramer in V2-INs, while they are simultaneously silenced by Hb9 in MNs (Lee, 2008).
Second, V2-tetramer directly binds Chx10-TeRE and upregulates Chx10 in chick spinal cord, suggesting that Chx10 is a direct target gene of V2-tetramer. Although Chx10 regulates retinal development, neither its function in the developing spinal cord nor its in vivo target genes are known. Interestingly, Chx10 binds Hb9-HxRE through its homeodomain and represses both the basal and MN-hexamer-induced levels of transcriptional activity mediated by HxRE, consistent with a previous report that Chx10 primarily functions as a transcriptional repressor. In V2 cells, Chx10 could be necessary to completely shut off any leaky expression of HxRE-containing MN genes or to actively block erroneous activation of MN genes by other transcription factors shared between V2-INs and MNs via recruiting corepressors to HxREs. Overall, repression of HxRE by Chx10 is likely to contribute to further refining V2 identity by suppressing unwanted MN-gene expression in V2 cells. Analysis of V2 specification in Chx10 mutant embryos should help examine this possibility genetically (Lee, 2008).
Overall, these studies reveal a sequential regulatory cascade of gene expression that operates to ensure the high fidelity in gene regulation required to specify two closely related, but distinct neural subtypes during vertebrate CNS development. This cascade resembles feedforward loops described in other organisms such as E. Coli, yeast, C. elegans and Drosophila. In particular, this study highlights the key role for DNA-REs in feedfoward gene regulatory loop and combinatorial transcription code, which have been underappreciated previously. Both HxRE and TeRE function as binary switches in the developing spinal cord; i.e., TeRE is off-switch in MNs and on-switch in V2-INs, while HxRE is on-switch in MNs and off-switch in V2-INs. Importantly, these strategies should reinforce the distinct gene expression outcomes in MNs and V2-INs, as expression of HxRE- and TeRE-containing genes would be precisely coregulated to opposite directions depending on the cell context. Thus, cell-type-specific DNA-RE alone is capable of decoding all the cell-fate-specifying genetic programs installed in each cell type, sensing both transcriptional activation and repression machineries. This model also predicts that a set of genes with TeRE or HxRE would be synonymously regulated during cell fate specification. Thus, the defined consensus TeRE and HxRE sequences could be useful in bioinformatics approaches to find a group of genes, which are specifically expressed in V2-INs and MNs and direct V2 and MN differentiations and maturations. Together, these findings provide a prototypic gene regulatory network for cell-type specification in development, which involves feedforward gene regulatory loops (Lee, 2008).
Hb9-MNe of Hb9 gene consists of functional HxRE and E-box elements that recruit proneural basic helix-loop-helix (bHLH) factors. MN-hexamer transcriptionally synergizes with proneural bHLH factors Ngn2 and NeuroM to fully activate Hb9 gene and subsequently specify MNs in the developing spinal cord and P19 cells. This synergistic interaction of MN-hexamer and Ngn2/NeuroM requires DNA bindings of these transcription factors in proximity. Thus, full activation of Chx10-TeRE in the neural tube may also need other transcription factors bound elsewhere in Chx10. It will be interesting to test whether V2-tetramer indeed cooperates with other transcription factors involved in V2 specification, such as Mash1, GATA2, FoxN4, or SCL, to promote V2-IN fate (Lee, 2008).
LMO4 disrupts the assembly of V2-tetramer in newborn MNs by displacing Lhx3 from NLI and suppresses V2-IN development in chick embryos. Among LMOs, LMO4 is most highly expressed in differentiating MNs in chick and mouse embryos and it binds NLI with a 2-fold higher affinity than LMO2. Thus, LMO4 is a good candidate to regulate the formation of LIM-complexes in MNs. Under the condition of 1:1 interactions, the affinities of Lhx3 and Isl1 for NLI binding are comparable, suggesting that LMO4 inhibits similarly the formation of V2-tetramer and MN-hexamer through competition for NLI binding. However, the data indicate that LMO4 functions as a selective competitor to disrupt V2-tetramer over MN-hexamer assembly. Interestingly, the binding of NLI and Isl1 without Lhx3 is also sensitive to LMO4, suggesting that the resistance of NLI:Isl1-Lhx3 binding to LMO4 is not simply due to the differences in the binding affinities between NLI:Lhx3 interaction and NLI:Isl1 interaction. Rather, the differences in the complex architecture of MN-hexamer and V2-tetramer may contribute to the distinct sensitivity of the two complexes to LMO4. Relative to V2-tetramer, MN-hexamer is a higher-order multiprotein complex. Thus, it could be more stable through multiple protein-protein interactions and less sensitive to a competitor such as LMO4. In MNs, LMO4 should increase the population of MN-hexamer, as MN-hexamer is formed at the expense of V2-tetramer. Consistently, deletion of LMO4 results in progressive increase of V2-INs. Although V2-MN hybrid cells are consistently found in LMO4 mutants, the phenotype is relatively subtle in LMO4 single mutant and greatly enhanced in LMO4:Hb9 compound mutants. Thus, LMO4 may provide a fine-tuning mechanism to control the stoichiometry of LIM-complexes in the developing spinal cord by increasing MN-hexamer concentration in MNs (Lee, 2008).
The results demonstrate that Hb9 and LMO4 cooperate to silence V2 genes. Why is the cooperative action of Hb9 and LMO4 necessary to inhibit V2 genes in MNs? LMO4 seems to function as a modulator, rather than an active MN fate selector, to promote MN-hexamer formation over other possible LIM-complexes in MNs. Likewise, although Hb9 is dominant over MN-hexamer for TeRE binding and thus blocks the access of MN-hexamer to TeRE-containing V2 genes, Hb9 may have intrinsically modest affinity to TeRE (weaker than V2-tetramer). Thus, Hb9 alone could be inefficient in blocking binding of V2-tetramer to TeRE, and may not completely shut down V2-gene expression in MNs, unless LMO4 helps Hb9 to bind TeRE more readily by destabilizing V2-tetramer, Hb9's competitor to bind TeRE. The loss of LMO4 and Hb9 in LMO4:Hb9-DKO likely permits both V2-tetramer and MN-hexamer to bind TeREs and upregulate V2 genes. As a consequence, both TeRE-containing V2 genes and HxRE-containing MN genes are activated in MNs, thereby resulting in MN-V2 hybrid cells. Thus, the functional cooperation between Hb9 and LMO4 is expected to be a critical component in the overall strategy to suppress expression of V2 genes in MNs. Together, these findings underscore the importance of actively suppressing alternative fate choice to generate correct neuronal subtype (Lee, 2008).
The data demonstrate that transcriptionally active endogenous MN-hexamer is assembled in MMCm-MNs. Interestingly, LMO4 is maintained mainly in MMCm-MNs among motor columns. Thus, the HxRE may mediate not only MN specification but also postmitotic MN diversification to MMCm cells and LMO4 may also antagonize the unwanted formation of V2-tetramer in MMCm cells. V2-INs also undergo further diversification to excitatory Chx10+ V2a-INs and inhibitory GATA2/3+ V2b-INs. Analogous to MN development, Lhx3 is maintained in V2a-INs, but extinguished in V2b-INs. Thus, TeRE activity could be maintained only in V2a subtype in which V2-tetramer is assembled. In comparison, V2b-INs express GATA2/3, SCL, and LMO4, which could assemble a transactivating complex similar to a hematopoietic complex containing NLI, GATA1, SCL, and LMO2. This raises the possibility that LMO4 might control the V2 subtype segregation by acting as an activator in V2b and a repressor in V2a (Lee, 2008).
In summary, these studies have established that subtle differences in DNA-REs can direct segregation of lineage-specific transcription pathways in the developing nervous system by concertedly mobilizing the action of transcriptional activators and repressors. This regulatory network likely represents a prototypic genetic mechanism for segregating related but distinct cell fates during the nervous system development. Importantly, this knowledge should provide a rational strategy to direct stem/progenitor cells into MNs in vitro (Lee, 2008).
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