Beadex
Hematopoietic stem cells are derived from ventral mesoderm during vertebrate development. Gene targeting experiments in the mouse have demonstrated key roles for the basic helix-loop-helix transcription factor SCL (related protein, Drosophila Helix loop helix protein 3B) and the GATA-binding protein GATA-1 in hematopoiesis. When overexpressed in Xenopus animal cap explants, SCL and GATA-1 are each capable of specifying mesoderm to become blood. Forced expression of either factor in whole embryos, however, does not lead to ectopic blood formation. This apparent paradox between animal cap assays and whole embryo phenotype has led to the hypothesis that additional factors are involved in specifying hematopoietic mesoderm. SCL and GATA-1 interact in a transcriptional complex with the LIM domain protein LMO-2. The Xenopus homolog of LMO-2 has been cloned and it has been shown to be
expressed in a pattern similar to SCL during development. LMO-2 can specify hematopoietic mesoderm in animal cap assays. SCL and LMO-2 act
synergistically to expand the blood island when overexpressed in whole embryos. Furthermore, co-expression of GATA-1 with SCL and LMO-2 leads
to embryos that are ventralized and have blood throughout the dorsal-ventral axis. The synergistic effect of SCL, LMO-2 and GATA-1, taken together with the findings that these factors can form a complex in vitro, suggests that this complex specifies mesoderm to become blood during embryogenesis (Mead, 2001).
Lmo1, Lmo2, and Lmo3 show individually unique but partially
overlapping patterns of expression in several regions of the adult mouse forebrain, including hippocampus, caudate putamen,
medial habenula, thalamus, amygdala, olfactory bulb, hypothalamus, and cerebral cortex. In the hippocampal formation, Lmo1,
Lmo2, and Lmo3 show different combinatorial patterns of expression levels in CA pyramidal and dentate granule neurons, and
Lmo1 is present in topographically restricted subpopulations of astrocytes. Kainic acid-induced limbic seizures differentially
regulate Lmo1, Lmo2, and Lmo3 mRNA levels in hippocampal pyramidal and granule neurons, such that Lmo1 mRNA
increases, whereas Lmo2 and Lmo3 mRNAs decrease significantly, with maximal changes at 6 hr after seizure onset and a return to
baseline by 24 hr. These findings show that Lmo1, Lmo2, and Lmo3 continue to be expressed in the adult mammalian CNS in a
cell type-specific manner, are differentially regulated by neuronal activity, and may thus be involved in cell phenotype-specific
regulatory functions (Hinks, 1997).
LMO4 is a novel member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors. LMO1, LMO2, and LMO4 have distinct expression patterns in adult tissue, and nuclear retention of LMO proteins is enhanced by the nuclear LIM interactor (NLI). In situ hybridization to early mouse embryos of 8-14.5 days reveals a complex pattern of LMO4 expression spatially overlapping with NLI and LHX genes. LMO4 expression in somites is repressed in mice mutant for the segment polarity gene Mesp2 (related to neurogenic HLH transcription factors) and expanded in Splotch (see Drosophila Paired) mutants. During jaw and limb outgrowth, LMO4 and LMO2 expression defines mesenchyme that is uncommitted to regional fates. Although both LMO2 and LMO4 are activated in thymic blast cells, only LMO4 is expressed in mature T cells. Mesenchymal and thymic blast cell expression patterns of LMO4 and LMO2 are consistent with the suggestion that LMO genes inhibit differentiation (Kenny, 1998).
Many vertebrate homologs of Drosophila genes important for wing patterning have been found to play a role in limb development. To determine
whether the LMO genes might also be involved, in situ hybridization was performed using the mouse LMO-2 cDNA on E10.5 mouse
embryos. LMO-2 RNA is present in the developing limb bud at E10.5. Expression is seen in a band centered on the D-V boundary of the developing limb bud. In situ hybridization to sections of the
limb bud reveals that LMO-2 mRNA is present in a broad field of the mesenchyme underlying the apical ectodermal ridge, a structure known to be an important organizing structure of the limb bud. Expression is also seen at the somite boundaries (Zeng, 1998).
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 (Drosophila homolog: Chip), 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 product of the scl (also called tal-1 or TCL5) gene is a basic domain, helix-loop-helix (bHLH) transcription factor required for
the development of hematopoietic cells. Additionally, scl gene disruption and dysregulation, by either chromosomal translocations
or a site-specific interstitial deletion whereby 5' regulatory elements of the sil gene become juxtaposed to the body of the scl gene,
is associated with T-cell acute lymphoblastic leukemia (ALL) and T-cell lymphoblastic lymphoma. An
inappropriately expressed scl protein, driven by sil regulatory elements, can cause aggressive T-cell malignancies in collaboration
with a misexpressed LMO1 protein, thus recapitulating the situation seen in a subset of human T-cell ALL. Inappropriately expressed scl can interfere with the development of other tissues derived from mesoderm. An scl construct lacking the scl transactivation domain collaborates with misexpressed LMO1, demonstrating that the scl
transactivation domain is dispensable for oncogenesis, and supporting the hypothesis that the scl gene product exerts its oncogenic
action through a dominant-negative mechanism (Aplan, 1997).
The LIM-finger protein Lmo2, which is activated in T cell leukemias by chromosomal translocations, is required for yolk sac
erythropoiesis. Because Lmo2 null mutant mice die at embryonic day 9-10, it prevents an assessment of a role in other stages of
hematopoiesis. The hematopoietic contribution of homozygous mutant Lmo2 -/- mouse embryonic stem cells has been studied
and Lmo2 -/- cells are found not to contribute to any hematopoietic lineage in adult chimeric mice, but reintroduction of an
Lmo2-expression vector rescues the ability of Lmo2 null embryonic stem cells to contribute to all lineages tested. This disruption of
hematopoiesis probably occurs because interaction of Lmo2 protein with factors such as Tal1/Scl is precluded. Thus, Lmo2 is
necessary for early stages of hematopoiesis, and the Lmo2 master gene encodes a protein that has a central and crucial role in the
hematopoietic development (Yamada, 1998).
The LIM-only protein LMO2 is expressed aberrantly in acute T-cell leukemias as a result of the chromosomal translocations
t(11;14) (p13;q11) or t(7;11) (q35;p13). In a transgenic model of tumorigenesis by Lmo2, T-cell acute leukemias arise after an
asymptomatic phase in which an accumulation of immature CD4(-) CD8(-) double negative thymocytes occurs. Possible molecular
mechanisms underlying these effects have been investigated in T cells from Lmo2 transgenic mice. Isolation of DNA-binding sites
by CASTing and band shift assays demonstrates the presence of an oligomeric complex involving Lmo2 that can bind to a
bipartite DNA motif comprising two E-box sequences approximately 10 bp apart, which is distinct from that found in erythroid
cells. This complex occurs in T-cell tumors and is restricted to the immature CD4(- )CD8(-) thymocyte subset in asymptomatic
transgenic mice. Thus, ectopic expression of Lmo2 by transgenesis, or by chromosomal translocations in humans, may result in
the aberrant protein interactions causing abnormal regulation of gene expression, resulting in a blockage of T-cell differentiation
and providing precursor cells for overt tumour formation (Grutz, 1998).
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).
Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one
family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain
factor is known to be expressed in epidermis. Therefore, the conserved LIM-interaction domain
of Clim coregulators was used to screen for LIM domain factors in adult and embryonic mouse skin expression
libraries and a factor was isolated that is highly homologous to the previously described LIM-only proteins
LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in an overlapping manner with Clim-2 in
epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and
genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral
nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because
LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins,
LMO-4 was used as a bait to screen for such DNA-binding proteins in epidermis. Identified was the mouse
homolog of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein
that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element.
The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse
DEAF-1, distinct from the helix-loop-helix and GATA domains previously shown to interact with LMOs,
thus defining an additional LIM-interacting domain (Sugihara, 1998).
It is now widely accepted that hemopoietic cells born intraembryonically are the best candidates for the seeding of definitive hemopoietic organs. To further
understand the mechanisms involved in the generation of definitive hemopoietic stem cells, the expression of the hemopoietic-related transcription factors
Lmo2 and GATA-3 during the early steps of mouse development (7-12 dpc) was analysed, with a particular emphasis on intraembryonic hemogenic sites. Both Lmo2 and GATA-3 are present in the intraembryonic regions known to give rise to hemopoietic precursors in vitro and in vivo, suggesting that they act together
at key points of hemopoietic development.
Lmo2 mRNA is observed in all the
sites endowed with a hemopoietic potential, where its expression
is tightly regulated spatiotemporally. The rapid modifications of
Lmo2 expression patterns suggest that it allocates specific
combinations of transcription factors during key points of
development. The overlapping expressions of Lmo2 and GATA-3
suggests combined functions during specific steps of
definitive hemopoietic development, namely: (1) endodermal
induction leading to the emergence of hemopoietic precursors
in the mesoderm; (2) determination of these cells from the
mesoderm, and (3) their production in the aortic region from 9
to 12 dpc as well as their release into the blood stream. Lmo2 and GATA-3 are expressed in the caudal mesoderm during the phase that determines intraembryonic precursors.
A highly transient concomitant expression is observed in the caudal intraembryonic definitive endoderm, suggesting that these factors are involved
in the specification of intraembryonic hemopoietic precursors. Lmo2 and GATA-3 are expressed within the hemopoietic clusters located in the aortic floor during
fetal liver colonization. Furthermore, a strong GATA-3 signal allowed the uncovering of previously unreported mesodermal aggregates beneath the aorta. Combined in
situ and immunocytological analysis strongly suggests that ventral mesodermal GATA-3 patches are involved in the process of intraembryonic stem cell generation (Manaia, 2000).
In the stretch-reflex system, proprioceptive sensory neurons make selective synaptic connections with different
subsets of motoneurons, according to the peripheral muscles they supply. To examine the molecular mechanisms that may influence the selection of these synaptic targets, single-cell cDNA libraries were constructed from sensory neurons that innervate antagonist muscles. Differential screening of these libraries identified a transcription regulatory co-factor of the LIM homeodomain proteins, the LIM domain only 4 protein Lmo4, expressed in most adductor but few sartorius sensory neurons. Differential patterns of Lmo4 expression were also seen in sensory neurons supplying three other muscles. A subset of motoneurons also expresses Lmo4 but the pattern of expression is not specific for motor pools. Differential expression of Lmo4 occurs early, as neurons develop their characteristic LIM homeodomain protein expression patterns. Moreover, ablation of limb buds does not block Lmo4 expression, suggesting that an intrinsic program controls the early differential expression of Lmo4. LIM homeodomain proteins are known to regulate several aspects of sensory and motor neuronal development. The results suggest that Lmo4 may participate in this differentiation by regulating the transcriptional activity of LIM homeodomain proteins (Chen, 2002).
Lmo4 can regulate the transcriptional activities of LIM homeodomain factors in several ways. Lmo transcriptional regulatory factors lack a DNA
binding domain but contain two protein-protein interaction LIM domains. Lmo proteins can compete for NLI with LIM homeodomain transcription
factors, and thereby regulate the formation of LIM homeodomain/NLI complexes and their transcriptional activity.
Drosophila Lmo can bind to Chip with higher affinity than the LIM homeodomain of Apterous and thereby regulate Apterous activity levels in vivo. Whether there is a differential affinity to NLI between Lmo4 and other LIM homeodomain proteins is not yet known (Chen, 2002).
Lmo4 may also compete for other co-factors besides NLI that are specific for individual LIM homeodomain proteins and could thus regulate the
expression of downstream target genes. For example, data based on the expression of chimeric LIM domains derived from different Islet family members (i.e. Isl1, Isl2 and ISL3) in zebrafish, has led to the conclusion that Isl2 probably forms a transcriptional complex with an Isl2-specific co-factor, in
addition to NLI. Interaction with an Isl2-specific co-factor could contribute to the role of Isl2 in the differentiation of primary motoneurons, neuronal
positioning, peripheral axonal outgrowth and neuronal transmitter expression in zebrafish (Chen, 2002).
Combinatorial interactions of Lmo4 with other transcription factors might provide additional mechanisms for the regulation of transcription during
neuronal development. In enkaphalin-producing neurons, Lmo4 interacts with the transcription factor DEAF1 (deformed epidermal autoregulatory
factor 1). DEAF1 has been implicated in opioid production by regulating enkaphalin transcription through a retinoic
acid-responsive element. Interestingly, in the fetal and adult mouse brain, Lmo4 expression is region specific: high levels of expression are present in
the limbic system and in regions involved in autonomic, motor and neuroendocrine regulation. Recently, studies in breast
cancer cell lines have demonstrated that Lmo4 expression is upregulated and forms a multiprotein complex with CtIP and BRCA1. A role for BRCA1 in neurons has not been explored (Chen, 2002).
Different LIM homeodomain proteins are known to activate different downstream target genes. The pattern of
neuronal generation in the ventral neural tube is achieved primarily by the spatially restricted expression of transcriptional repressors. By modulating the transcriptional activity of LIM homeodomain proteins, Lmo4 is likely to be involved in the specification of motor neuronal
identity. Its restricted expression in subsets of muscle sensory neurons suggests that it contributes to the specification of sensory neurons as well (Chen, 2002).
Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activate smooth muscle gene targets. CRP2 is found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocks proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converts pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibits the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation (Chang, 2003).
The proneural protein neurogenin 2 (NGN2) is a key transcription factor in regulating both neurogenesis and neuronal radial migration in the embryonic cerebral cortex. However, the co-factors that support the action of NGN2 in the cortex remain unclear. This study shows that the LIM-only protein LMO4 functions as a novel co-factor of NGN2 in the developing cortex. LMO4 and its binding partner nuclear LIM interactor (NLI/LDB1/CLIM2) interact with NGN2 simultaneously, forming a multi-protein transcription complex. This complex is recruited to the E-box containing enhancers of NGN2-target genes, which regulate various aspects of cortical development, and activates NGN2-mediated transcription. Correspondingly, analysis of Lmo4-null embryos shows that the loss of LMO4 leads to impairments of neuronal differentiation in the cortex. In addition, expression of LMO4 facilitates NGN2-mediated radial migration of cortical neurons in the embryonic cortex. These results indicate that LMO4 promotes the acquisition of cortical neuronal identities by forming a complex with NGN2 and subsequently activating NGN2-dependent gene expression (Asprer, 2011).
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, the cofactor CtIP was identified 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. The breast tumor suppressor BRCA1 was identified as an LMO4-associated protein. The C-terminal BRCT domains of BRCA1, previously shown to bind CtIP, also mediate interaction with LMO4. Tumor-associated mutations within the BRCT repeats that abolish interaction between BRCA1 and CtIP have 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 participates 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 (Sum, 2002).
The LIM domain protein Lmo2 and the basic helix-loop-helix transcription factor Scl/Tal1 (distantly related to Drosophila Twist) are expressed in early haematopoietic and endothelial progenitors and interact with each other in haematopoietic cells. While loss-of-function studies have shown that Lmo2 and Scl/Tal1 are essential for haematopoiesis and angiogenic remodelling of the vasculature, gain-of-function studies have suggested an earlier role for Scl/Tal1 in the specification of haemangioblasts, putative bipotential precursors of blood and endothelium. In zebrafish embryos, Scl/Tal1 can induce these progenitors from early mesoderm mainly at the expense of the somitic paraxial mesoderm. This restriction to the somitic paraxial mesoderm correlates well with the ability of Scl/Tal1 to induce ectopic expression of its interaction partner Lmo2. Co-injection of lmo2 mRNA with scl/tal1 dramatically extends its effect to head, heart, pronephros and pronephric duct mesoderm inducing early blood and endothelial genes all along the anteroposterior axis. Erythroid development, however, is expanded only into pronephric mesoderm, remaining excluded from head, heart and somitic paraxial mesoderm territories. This restriction correlates well with activation of gata1 transcription and co-injection of gata1 mRNA along with scl/tal1 and lmo2 induces erythropoiesis more broadly without ventralizing or posteriorizing the embryo. While no ectopic myeloid development from the Scl/Tal1-Lmo2-induced haemangioblasts was observed, a dramatic increase in the number of endothelial cells was found. These results suggest that, in the absence of inducers of erythroid or myeloid haematopoiesis, Scl/Tal1-Lmo2-induced haemangioblasts differentiate into endothelial cells (Gering, 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).
LMO4 belongs to a family of transcriptional regulators that comprises two zinc-binding LIM domains. LIM-only (LMO) proteins appear to function as docking sites for other factors, leading to the assembly of multiprotein complexes. The transcription factor Deaf-1/NUDR (see Drosophila
Deaf-1) has been identified as one partner protein of LMO4. The Lmo4 and Deaf-1 genes were inactivated in mice to define their biological function in vivo. All Lmo4 mutants died shortly after birth and showed defects within the presphenoid bone, with 50% of mice also exhibiting exencephaly. Homeotic transformations were observed in Lmo4-null embryos and newborn mice, but with incomplete penetrance. These included skeletal defects in cervical vertebrae and the rib cage. Furthermore, fusions of cranial nerves IX and X and defects in cranial nerve V were apparent in some Lmo4(-/-) and Lmo4(+/-) mice. Remarkably, Deaf-1 mutants displayed phenotypic abnormalities similar to those observed in Lmo4 mutants. These included exencephaly, transformation of cervical segments, and rib cage abnormalities. In contrast to Lmo4 nullizygous mice, nonexencephalic Deaf-1 mutants remained healthy. No defects in the sphenoid bone or cranial nerves were apparent. Thus, Lmo4 and Deaf-1 mutant mice exhibit overlapping as well as distinct phenotypes. These data indicate an important role for these two transcriptional regulators in pathways affecting neural tube closure and skeletal patterning, most likely reflecting their presence in a functional complex in vivo (Hahm, 2004).
The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. Gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, was carried out to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, the effect of compound Lmo1/Lmo3 null mutations was assessed. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. These results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways (Tse, 2004).
Nuclear LIM domain-only proteins (LMOs), which consist of two closely spaced 50 amino acid Zn2+-finger protein interaction modules mediate interactions between several classes of transcription factors important for development. LMO2 is necessary for development of the entire hematopoietic system and overexpression of LMO1 or LMO2 results in human acute T cell leukemia. LMO4 is the most widely expressed LMO but its normal function is unknown. During development, LMO4 is expressed in dividing neuroepithelial cells within the ventricular zone along the entire rostrocaudal axis of the nervous system. In telencephalic and spinal cord regions of the CNS, LMO4 is highly expressed in ventral but is low in dorsal proliferating neuroepithelial cells. To understand the role of LMO4 during mouse development, a homozygous null mutation was generated in the gene. It was found that LMO4 is required for proper closure of the anterior neural tube. In the absence of LMO4, elevation, bending, and proliferation of the ventral neural epithelium and consequent fusion of the prospective dorsal ends of the neural tube do not occur. LMO4 mutant mice die embryonically and exhibit exencephaly, which is associated with abnormal patterns of cell proliferation and with high levels of apoptotic cell death within the neuroepithelium. LMO4 is thus essential for normal patterns of proliferation and for survival of neural epithelial cells in the rostral neural tube. LMO4 is also expressed in Schwann cell progenitors after these contact neurites, a process mediated in part by neuregulin (Lee, 2005).
LIM-only proteins (LMO), which consist of LMO1, LMO2, LMO3, and LMO4, are involved in cell fate determination and differentiation during embryonic development. Accumulating evidence suggests that LMO1 and LMO2 act as oncogenic proteins in T-cell acute lymphoblastic leukemia, whereas LMO4 has recently been implicated in the genesis of breast cancer. However, little is known about the role of LMO3 in either tumorigenesis or development. This study identified LMO3 and HEN2, which encodes a neuronal basic helix-loop-helix protein, as genes whose expression levels were higher in unfavorable neuroblastomas compared with those of favorable tumors. Immunoprecipitation and immunostaining experiments showed that LMO3 was associated with HEN2 in mammalian cell nucleus. Human neuroblastoma SH-SY5Y cells stably overexpressing LMO3 showed a marked increase in cell growth, a promotion of colony formation in soft agar medium, and a rapid tumor growth in nude mice compared with the control transfectants. More importantly, the increased expression of LMO3 and HEN2 was significantly associated with a poor prognosis in 87 primary neuroblastomas. These results suggest that the deregulated expression of neuronal-specific LMO3 and HEN2 contributes to the genesis and progression of human neuroblastoma in a lineage-specific manner (Aoyama, 2005).
Defective permeability barrier is an important feature of many skin diseases and causes mortality in premature infants. To investigate the control of barrier formation, this study characterized the epidermally expressed Grainyhead-like epithelial transactivator (Get-1)/Grhl3, a conserved mammalian homologue of Grainyhead, which plays important roles in cuticle development in Drosophila. Get-1 interacts with the LIM-only protein LMO4, which is co-expressed in the developing mammalian epidermis. The epidermis of Get-1(-/-) mice showed a severe barrier function defect associated with impaired differentiation of the epidermis, including defects of the stratum corneum, extracellular lipid composition and cell adhesion in the granular layer. The Get-1 mutation affects multiple genes linked to terminal differentiation and barrier function, including most genes of the epidermal differentiation complex. Get-1 therefore directly or indirectly regulates a broad array of epidermal differentiation genes encoding structural proteins, lipid metabolizing enzymes and cell adhesion molecules. Although deletion of the LMO4 gene had no overt consequences for epidermal development, the epidermal terminal differentiation defect in mice deleted for both Get-1 and LMO4 is much more severe than in Get-1(-/-) mice with striking impairment of stratum corneum formation. These findings indicate that the Get-1 and LMO4 genes interact functionally to regulate epidermal terminal differentiation (Yu, 2006).
The basic helix-loop-helix TAL-1/SCL essential for hematopoietic development is also required during vascular development for embryonic angiogenesis. TAL-1 acts positively on postnatal angiogenesis by stimulating endothelial morphogenesis. This study investigated the functional consequences of TAL-1 silencing in human primary endothelial cells. It was found that TAL-1 knockdown caused the inhibition of in vitro tubulomorphogenesis, which was associated with a dramatic reduction in vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Consistently, silencing of TAL-1 as well as of its cofactors E47 and LMO2 down-regulated VE-cadherin at both the mRNA and the protein level. Endogenous VE-cadherin transcription could be activated in nonendothelial HEK-293 cells by the sole concomitant ectopic expression of TAL-1, E47, and LMO2. Transient transfections in human primary endothelial cells derived from umbilical vein (HUVECs) demonstrated that VE-cadherin promoter activity was dependent on the integrity of a specialized E-box associated with a GATA motif and was maximal with the coexpression of the different components of the TAL-1 complex. Finally, chromatin immunoprecipitation assays showed that TAL-1 and its cofactors occupied the VE-cadherin promoter in HUVECs. Together, these data identify VE-cadherin as a bona fide target gene of the TAL-1 complex in the endothelial lineage, providing a first clue to TAL-1 function in angiogenesis (Deleuze, 2007).
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).
Multiple excitatory and inhibitory interneurons form the motor circuit with motor neurons in the ventral spinal cord. Notch signaling initiates the diversification of immature V2-interneurons into excitatory V2a-interneurons and inhibitory V2b-interneurons. This study provides a transcriptional regulatory mechanism underlying their balanced production. LIM-only protein LMO4 controls this binary cell fate choice by regulating the activity of V2a- and V2b-specific LIM complexes inversely. In the spinal cord, LMO4 induces GABAergic V2b-interneurons in collaboration with bHLH factor SCL and inhibits Lhx3 from generating glutamatergic V2a-interneuons. In LMO4;SCL compound mutant embryos, V2a-interneurons increase markedly at the expense of V2b-interneurons. LMO4 nucleates the assembly of a novel LIM-complex containing SCL, Gata2, and LIM domain-binding protein NLI. This complex activates specific enhancers in V2b-genes consisting of binding sites for SCL and Gata2, thereby promoting V2b-interneuron fate. Thus, LMO4 plays essential roles in directing a balanced generation of inhibitory and excitatory neurons in the ventral spinal cord (Joshi, 2009).
LIM-HD codes are crucial in implementing cell-type-specific transcription by directing different types of LIM-complexes in a cell context-dependent manner. These studies expand the LIM codes to include bHLH and Gata proteins as these two factors form an atypical LIM-complex via a non-DNA binding LIM factor LMO4. Unlike typical LIM-complexes such as the V2-tetramer complex, which utilize LIM-HD proteins for recognition of specific DNA response elements (Lee, 2008), SCL and Gata2 serve as the major DNA-binding components in the V2b complex. A couple of unique advantages of assembling the V2b complex can be proposed in cell fate specification (Joshi, 2009).
First, these results suggest that the V2b complex allows integration of SCL and Gata2 functions by selecting a group of target genes that bear both SCL- and GATA-recognition sites. This should ensure the expression of V2b-target genes specifically in cells coexpressing SCL and Gata2. It was found that the enhancers of Gata2 and Gata3 genes display striking similarity in that they contain reiterated bipartite elements composed of E-box (CAnnTG) and/or atypical E-box (CAnnnTG) for SCL-binding and GATA sites for recruiting Gata proteins. E-boxes and GATA sites occur relatively often in the genome due to their short sequences and serve as binding motifs for multiple bHLH and Gata factors. Thus, simultaneous recognition of paired E-box-GATA composite elements by the V2b complex is expected to provide the required stringency in choosing the target genes coregulated by SCL and Gata2 (Joshi, 2009).
Second, this study found that formation of the V2b complex facilitates the transcriptional synergy among its components by enabling the recruitment of coactivators including SSDP1. Coexpression of SSDP1 allowed a potent transcriptional activation by the V2b complex on its physiological targets, Gata2/3-enhancers. Given that SCL and Gata2 are relatively weak transcriptional activators in Gal4-DBD fusion transcription assays, the transcriptional synergy between SCL and Gata2 resulting from forming a complex may be due, at least in part, to the recruitment of SSDP1. The facilitated recruitment of SSDP1 and possibly other coactivators may account for the necessity of the V2b complex formation for inducing the V2b-IN genes (Joshi, 2009).
Deciphering molecular events required for full transformation of normal cells into cancer cells remains a challenge. In T-cell acute lymphoblastic leukemia (T-ALL), the genes encoding the TAL1/SCL and LMO1/2 transcription factors are recurring targets of chromosomal translocations, whereas NOTCH1 is activated in >50% of samples. This study shows that the SCL and LMO1 oncogenes collaborate to expand primitive thymocyte progenitors and inhibit later stages of differentiation. Together with pre-T-cell antigen receptor (pre-TCR) signaling, these oncogenes provide a favorable context for the acquisition of activating Notch1 mutations and the emergence of self-renewing leukemia-initiating cells in T-ALL. All tumor cells harness identical and specific Notch1 mutations and Tcrβ clonal signature, indicative of clonal dominance and concurring with the observation that Notch1 gain of function confers a selective advantage to SCL-LMO1 transgenic thymocytes. Accordingly, a hyperactive Notch1 allele accelerates leukemia onset induced by SCL-LMO1 and bypasses the requirement for pre-TCR signaling. Finally, the time to leukemia induced by the three transgenes corresponds to the time required for clonal expansion from a single leukemic stem cell, suggesting that SCL, LMO1, and Notch1 gain of function, together with an active pre-TCR, might represent the minimum set of complementing events for the transformation of susceptible thymocytes (Tremblay, 2010).
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