Interactive Fly, Drosophila



Cloning and expression of Daughterless homologs

Two alternatively spliced products of the human E2A gene, E12 and E47, encode helix-loop-helix DNA-binding proteins. Comparing two human da homologs( E2A and E2-2) with Daughterless, one finds a region in the middle of each protein, the loop-helix motif, which bears almost as much homology to that of Daughterless as do the bHLH domains (42-60% and 81% respectively). This is in contrast to 13% homology for the rest of the proteins. It is this loop-helix region that is responsible for transcriptional activation (Quong, 1993). Like its fly homolog, E2A is expressed in most tissues, particularly those that are undergoing rapid cell proliferation (Roberts, 1993).

The E proteins of mammals, and the related Daughterless (DA) protein of Drosophila, are ubiquitously expressed helix-loop-helix (HLH) transcription factors that play a role in many developmental processes. A related C. elegans protein, CeE/DA, has a dynamic and restricted distribution during development. CeE/DA is present embryonically in neuronal precursors, some of which are marked by promoter activity of a newly described achaete-scute-like gene hlh-3. In contrast, CeE/DA is not present in CeMyoD-positive striated muscle cells (CeMyoD is a homolog of Drosophila Nautilus). Although the majority of cells lose CeE/DA positive staining during the latter half of embryogenesis, certain pharyngeal muscle and gland cell nuclei maintain staining. The remaining cells staining after embryogenesis are located in the neuronal cluster between the nerve ring and the posterior pharyngeal bulb. CeE/DA dimerizes with HLH-3 while efficient interaction of CeE/DA with CeMyoD is not seen. These studies suggest multiple roles for CeE/DA in C. elegans development and provide evidence that both common and alternative strategies have evolved for the use of related HLH proteins in controlling cell fates in different species (Krause, 1997).

Dauterless homolog in C elegans involved in lateral signaling

The anchor cell/ventral uterine precursor cell (AC/VU) decision in Caenorhabditis elegans is a canonical example of lin-12/Notch-mediated lateral specification. Two initially equivalent cells interact via the receptor LIN-12 and its ligand LAG-2, so that one becomes the AC and the other a VU. During this interaction, feedback loops amplify a small difference in lin-12 activity, limiting lin-12 transcription to the presumptive VU and lag-2 transcription to the presumptive AC. hlh-2 appears to be required for the VU fate and directly activates lag-2 transcription in the presumptive AC. HLH-2 appears to accumulate selectively in the presumptive AC prior to differential transcription of lin-12 or lag-2, and is therefore the earliest detectable difference between the two cells undergoing the AC/VU decision. The restricted accumulation of HLH-2 to the presumptive AC reflects post-transcriptional down-regulation of HLH-2 in the presumptive VU. These observations suggest that hlh-2 is regulated as part of the negative feedback that down-regulates lag-2 transcription in the presumptive VU. The AC/VU decision in an individual hermaphrodite is biased by the relative birth order of the two cells, so that the first-born cell is more likely to become the VU. Models are proposed to suggest how birth order, HLH-2 accumulation, and transcription of lag-2 may be linked during the AC/VU decision (Karp, 2003).

These results suggest that hlh-2 functions in a negative feedback loop that operates during the AC/VU decision. It is proposed that activation of LIN-12 leads to expression or activation of a factor, X, that serves to down-regulate the level of HLH-2 in the presumptive VU post-transcriptionally. An interesting possibility is that the gene encoding X is a direct transcriptional target of the LAG-1-LIN-12intra complex (Karp, 2003).

A feedback loop involving post-transcriptional regulation of HLH-2 in the AC/VU decision is logically parallel to the circuit that operates during SOP specification in Drosophila, but differs in the mechanism by which a critical regulatory step occurs. In particular, the postulated factor X, a negative regulator of HLH-2 accumulation, plays the same formal role as the E(spl)/Gro complex, a negative regulator of AS-C transcription. In this context, it is noted that unc-37 and lin-22, the C. elegans counterparts of gro and E(spl), do not appear to function in the AC/VU decision, consistent with a different mode of regulation for hlh-2 (Karp, 2003).

Although this study focuses on the regulation of HLH-2 in the negative feedback loop, it is noted that there may also be positive feedback that leads to up-regulation of HLH-2 in the presumptive AC. Positive autoregulation of AS-C transcription underlies the positive feedback loop that operates in the presumptive SOP. However, if there is positive feedback control of HLH-2, the data would suggest that it also involves a post-transcriptional mechanism, since the level of hlh-2 transcription appears to be roughly the same in both Z1.ppp and (Karp, 2003).

In cultured mammalian cells, HES genes [E(spl) homologs] have been shown to be targets of Notch signaling; HES genes also serve as negative regulators of the transcription of homologs of AS-C, suggesting that the same negative feedback circuit that has been defined in Drosophila is conserved. However, it has also been reported that Notch activation can lead to rapid degradation of the human Achaete-Scute homolog 1 protein as well as degradation of the HLH-2 homologs encoded by the mammalian E2A gene. Perhaps these observations reflect a role for post-translational regulation of the transcriptional activators of Delta expression during cell fate decisions in mammals (Karp, 2003).

The key to understanding how the AC/VU decision is initiated is the nature of the stochastic event. At the molecular level, it has been envisaged that it is a random difference in the level of ligand or receptor activity; this might reflect something as simple as a random difference in the number of ligand or receptor molecules at the cell surface. Because gene expression itself is inherently stochastic, leading to heterogeneity in individual cells within a clonal population, it is unlikely that Z1.ppp and (which are not sisters, but lineal homologs that have not shared a common cellular ancestor for many cell divisions) have the same concentration of components of the LIN-12/Notch pathway. Experiments in Drosophila suggest that as little as a 2:3 ratio in Notch gene dosage can be efficiently amplified by the feedback mechanism that operates during SOP specification. Thus, it would seem in principle that amplification of random differences in transcription of LIN-12/Notch pathway components by feedback mechanisms could account for the accuracy of the AC/VU decision in wild-type hermaphrodites (Karp, 2003).

However, an additional influence on the AC/VU decision, birth order, has been identified. Z1.ppp and are born at the end of the first phase of the Z1 and Z4 lineages, in the late L1 or early L2 stage. There is a stochastic difference in the time of their birth, ranging from 2 min to 2 h, and that this difference strikingly correlates with cell fate; the first-born usually becomes a VU, and the second-born usually becomes an AC. The correlation is not absolute, since in one individual (of 13), the first-born cell became the AC, suggesting that birth order is biasing, rather than determining, the outcome of the AC/VU decision. Since birth order appears to be random, but highly correlated with the outcome of the AC/VU decision, it appears that birth order is the stochastic event that sets the feedback loops into play (Karp, 2003).

Since HLH-2 is the first detectable difference between the presumptive AC and the presumptive VU, it is tempting to try to forge a link between birth order and hlh-2 regulation. If the first-born cell has an advantage in activating LIN-12, then that may set into play the feedback loops that amplify the birth-order advantage, such as driving the accumulation of a negative regulator of HLH-2 (Factor X). Less HLH-2 would mean a disadvantage in the maintenance phase of lag-2 expression, thereby helping to bias the first-born cell against becoming a signaling cell, and therefore, toward becoming a receiving cell (Karp, 2003).

There are several different ways an advantage in activating LIN-12 in the first-born cell might be achieved. For example, a cell may begin to accumulate LIN-12 at birth, giving the first-born an advantage when the second-born cell appears and the two cells start to interact. In one variation on this theme, LIN-12, newly synthesized or even inherited from its parent, may be activated in the first-born cell by LAG-2 present on the parent of the second-born cell, or by a related ligand emanating from elsewhere (at a level that is not sufficient to promote the VU fate, but which can activate LIN-12 to a sufficient extent to account for the birth-order bias). Another way that the first-born cell might gain an advantage in activating LIN-12 - or that the second-born cell might be at a disadvantage - would be if other signal-transduction pathways act to influence LIN-12 activity. This kind of bias would be analogous to the establishment of polarity in the developing Drosophila ommatidium, in which positional cues cause a higher level of Frizzled activity in one cell of the R3/R4 pair, biasing that cell to lower Notch activity, and hence, to the R3 fate (Karp, 2003).

An alternative hypothesis is that the first-born cell has an advantage because it begins its progression through the cell cycle first. This hypothesis is inspired by evidence that passage through S phase into G2 is necessary for LIN-12 signal transduction to be manifest in the vulval precursor cells. If similar cell-cycle gating operates during the AC/VU decision, perhaps the first-born cell has a greater probability of reaching G2 first, and hence, adopting the VU fate associated with LIN-12 activation. Since the VU divides a few hours after it has been specified, whereas the AC is a terminally differentiated cell, a potential connection between LIN-12 activation and cell-cycle progression in the presumptive VU seems plausible (Karp, 2003).

A bHLH code for sexually dimorphic form and function of the C. elegans somatic gonad

How sexually dimorphic gonads are generated is a fundamental question at the interface of developmental and evolutionary biology. In C. elegans, sexual dimorphism in gonad form and function largely originates in different apportionment of roles to three regulatory cells of the somatic gonad primordium in young larvae. Their essential roles include leading gonad arm outgrowth, serving as the germline niche, connecting to epithelial openings, and organizing reproductive organ development. The development and function of the regulatory cells in both sexes requires the basic-helix-loop-helix (bHLH) transcription factor HLH-2, the sole ortholog of the E proteins mammalian E2A and Drosophila Daughterless, yet how they adopt different fates to execute their different roles has been unknown. This study shows that each regulatory cell expresses a distinct complement of bHLH-encoding genes-and therefore distinct HLH-2:bHLH dimers-and formulate a "bHLH code" hypothesis for regulatory cell identity. This hypothesis is supported by showing that the bHLH gene complement is both necessary and sufficient to confer particular regulatory cell fates. Strikingly, prospective regulatory cells can be directly reprogrammed into other regulatory cell types simply by loss or ectopic expression of bHLH genes, and male-to-female and female-to-male transformations indicate that the code is instructive for sexual dimorphism. The bHLH code appears to be embedded in a bow-tie regulatory architecture, wherein sexual, positional, temporal, and lineage inputs connect through bHLH genes to diverse outputs for terminal features and provides a plausible mechanism for the evolutionary plasticity of gonad form seen in nematodes (Sallee, 2017).

Structure of Daughterless homologs

Homodimeric complexes of members of the E protein family of basic helix-loop-helix (bHLH) transcription factors are important for tissue-specific activation of genes in B lymphocytes. A novel cis-acting transcriptional repression domain is present in the E protein family of bHLH transcription factors. This domain, the Rep domain, is present in each of the known vertebrate E proteins. Extensive mapping analysis demonstrates that this domain is an acidic region of 30 amino acids with a predicted loop structure. Fusion studies indicate that the Rep domain can repress both of the E protein transactivation domains (AD1 and AD2). Physiologically, the Rep domain plays a key role in maintaining E protein homodimers in an inactive state on myogenic enhancers. In addition, Rep domain mediated repression of AD1 is a necessary for the function of MyoD-E protein heterodimeric complexes. These studies demonstrate that the Rep domain is important for modulating the transcriptional activity of E proteins and provide key insights into both the selectivity and mechanism of action of E protein containing bHLH protein complexes (Markus, 2002).

Partners of Daughterless homologs

To some extent in transformed flies, the bHLH domain of a zebrafish-Daughterless homology can functionally replace the bHLH domain of Daughterless (Wulbeck, 1994).

Although the ubiquitous helix-loop-helix (HLH) protein E12 does not homodimerize efficiently, the myogenic factor MyoD forms an avid DNA-binding heterodimer with E12 through the conserved HLH dimerization domain. Members of the MyoD family can overcome the E12 dimerization inhibitory domain through a mechanism involving, in part, the negatively charged amino acid residues in helix 2 (Shirakata, 1995).

Twist is a transcription factor that is required for mesodermal cell fates in all animals studied to date. Mutations of this locus in humans have been identified as the cause of the craniofacial disorder Saethre-Chotzen syndrome. The C. elegans Twist homolog is required for the development of a subset of the mesoderm. A semidominant allele of the gene that codes for CeTwist, hlh-8, has defects that occur earlier in the mesodermal lineage than a previously studied null allele of the gene. The semidominant allele has a charge change (E29K) in the basic DNA-binding domain of CeTwist. Surprisingly, the mutant protein retains DNA-binding activity as both a homodimer and a heterodimer with its partner E/Daughterless (CeE/DA). However, the mutant protein blocks the activation of the promoter of a target gene. Therefore, the mutant CeTwist may cause cellular defects as a dominant negative protein by binding to target promoters as a homo- or hetero-dimer and then blocking transcription. Similar phenotypes as those caused by the E29K mutation were observed when amino acid substitutions in the DNA-binding domain that are associated with the human Saethre-Chotzen syndrome were engineered into the C. elegans protein. These data suggest that Saethre-Chotzen syndrome may be caused, in some cases, by dominant negative proteins, rather than by haploinsufficiency of the locus (Corsi, 2002).

The absolute conservation of the LDFS motif suggests that it may form a surface that mediates protein-protein interactions. The SAGA complex directly interacts with the amino-terminal activation domains of both E2A and Rtg3p in a manner that is dependent upon the integrity of the helix and the LDFS motif. Furthermore, both the molecular modeling and helical wheel analysis reveal that these conserved amino acids form groups on opposite faces of the proposed helix. It is suggested that these conserved residues directly interact with a subunit(s) present in the SAGA complex in a manner that requires contact from both sides of the helix. This raises the question: which component(s) of SAGA contact the LDFS motif? To address this issue, a series of pulldown assays was performed with GST-AD1 and subset of proteins found in the SAGA complex, including Ada1, Spt3, Spt7, Spt8 and Spt20, TAFII 17/20, TAFII 25, TAFII 60, TAFII 68, TAFII 90, and Tra1. None of these SAGA components were individually capable of interacting with the LDFS motif. The Ada complex, like SAGA, contains the Ada2, Ada3, and Gcn5 proteins. Since the AD1 domain does not bind to purified Ada complexes, it seems unlikely that the LDFS motif is interacting with the Ada2, Ada3, or Gcn5 components present within SAGA. Future studies will be directed at understanding the LDFS-SAGA interaction in greater detail. In particular, it will be interesting to determine whether opposing faces of the helix interact with one particular component of SAGA or with distinct polypeptides within the complex (Massari, 1999).

The NSM cells of the nematode Caenorhabditis elegans differentiate into serotonergic neurons, while their sisters, the NSM sister cells, undergo programmed cell death during embryogenesis. The programmed death of the NSM sister cells is dependent on the cell-death activator EGL-1, a BH3-only protein required for programmed cell death in C. elegans, and can be prevented by a gain-of-function (gf) mutation in the cell-death specification gene ces-1, which encodes a Snail-like DNA-binding protein. The genes hlh-2 and hlh-3, which encode a Daughterless-like and an Achaete-scute-like bHLH protein, respectively, are required to kill the NSM sister cells. A heterodimer composed of HLH-2 and HLH-3, HLH-2/HLH-3, binds to Snail-binding sites/E-boxes in a cis-regulatory region of the egl-1 locus in vitro that is required for the death of the NSM sister cells in vivo. Hence, it is proposed that HLH-2/HLH-3 is a direct, cell-type specific activator of egl-1 transcription. Furthermore, the Snail-like CES-1 protein can block the death of the NSM sister cells by acting through the same Snail-binding sites/E-boxes in the egl-1 locus. In ces-1(gf) animals, CES-1 might therefore prevent the death of the NSM sister cells by successfully competing with HLH-2/HLH-3 for binding to the egl-1 locus (Thellmann, 2003).

Achaete-Scute basic helix-loop-helix (bHLH) proteins promote neurogenesis during metazoan development. A C. elegans Achaete-Scute homolog, HLH-14, has been characterized in this study. A number of neuroblasts express HLH-14 in the C. elegans embryo, including the PVQ/HSN/PHB neuroblast, a cell that generates the PVQ interneuron, the HSN motoneuron and the PHB sensory neuron. hlh-14 mutants lack all three of these neurons. The fact that HLH-14 promotes all three classes of neuron indicates that C. elegans proneural bHLH factors may act less specifically than their fly and mammalian homologs. Furthermore, neural loss in hlh-14 mutants results from a defect in an asymmetric cell division: the PVQ/HSN/PHB neuroblast inappropriately assumes characteristics of its sister cell, the hyp7/T blast cell. It is argued that bHLH proteins, which control various aspects of metazoan development, can control cell fate choices in C. elegans by regulating asymmetric cell divisions. Finally, a reduction in the function of hlh-2, which encodes the C. elegans E/Daughterless bHLH homolog, results in similar neuron loss as hlh-14 mutants and enhances the effects of partially reducing hlh-14 function. It is proposed that HLH-14 and HLH-2 act together to specify neuroblast lineages and promote neuronal fate (Frank, 2003).

Aside from its conserved sequence, there are a number of similarities between hlh-14 and previously characterized A-S genes. The most obvious similarity is that hlh-14 mutants lack neurons. A-S family members in Drosophila specify external sense organs. In the absence of A-S genes, neuronal cell types needed for the function of these organs are lost. Vertebrate A-S family members generate a wide variety of neuronal precursors, including the progenitors of the cerebral cortex and progenitors in the ventral telencephalon. This study clearly establishes that hlh-14 function is required for normal PVQ, HSN, and PHB neuron development (Frank, 2003).

Yet a close look at the types of neurons specified by hlh-14 reveals an important difference between hlh-14 and other A-S genes. While Drosophila and mammalian A-S genes appear to specify neuroblast lineages dedicated to generating neuronal cells of a particular type or coordinated function, hlh-14 specifies a neuroblast lineage dedicated to generating three disparate types of neuron: an interneuron (PVQ), a motoneuron (HSN) and a sensory neuron (PHB). There is no known coordinated function that these three neurons perform. Why is hlh-14 less specific than fellow A-S family members in this regard? One possibility is that C. elegans must adapt the function of neural bHLH genes such as hlh-14 in order to generate its diverse collection of 302 neurons (of 118 distinct types) out of only 959 somatic cells. Such adaptation may allow hlh-14 to specify lineally related, yet functionally distinct, collections of neurons (Frank, 2003)

A second similarity between hlh-14 and other A-S-like genes is the genetic interaction between hlh-14 and hlh-2, the C. elegans E/DA homolog. In Drosophila, heterodimers between DA and A-S family members are essential for neurogenesis. In C. elegans, the A-S factor HLH-3 can bind E/DA HLH-2 in vitro, and the expression patterns of HLH-3 and HLH-2 overlap in a number of neuronal lineages. Taken together, four facts suggest that HLH-14 and HLH-2 act together in the PVQ/HSN/PHB lineage to regulate neuronal development. (1) A-S proteins, as well as other types of bHLH proteins, are known to interact physically with E/DA family members to form functional heterodimers in a number of organisms. (2) Both HLH-14 and HLH-2 are expressed in the PVQ/HSN/PHB lineage. (3) Loss of function of either gene results in the loss of neurons in this lineage. (4) A weak hlh-2 mutant can enhance the partial neuronal loss defects of hlh-14(RNAi) (Frank, 2003).

Another similarity that HLH-14 shares with certain A-S family members is that it possesses proneural characteristics. Not only is hlh-14 necessary for neuron development, but also it is expressed early in neurogenesis. hlh-14::gfp expression is seen in the PVQ/HSN/PHB neuroblast, the first cell in this lineage solely dedicated to generating neurons (Frank, 2003).

The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1

The combinatorial interaction among transcription factors is believed to determine hematopoietic cell fate. Stem cell leukemia (SCL, also known as TAL1 [T-cell acute lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix (bHLH) factor that plays a central function in hematopoietic development; however, its target genes and molecular mode of action remain to be elucidated. This study shows that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, the role of SCL is defined as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, it was demonstrated by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. It is therefore concluded that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, these observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells (Lécuyer, 2002).

SCL assembles a multifactorial complex that determines glycophorin A expression

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).

TAL-1/SCL and its partners E47 and LMO2 up-regulate VE-cadherin expression in endothelial cells

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).

E protein silencing by the leukemogenic AML1-ETO fusion protein

The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 15% of acute myeloid leukemia (AML) cases. This study shows that AML1-ETO, as well as ETO (Drosophila homolog: Nervy), inhibits transcriptional activation by E proteins (e.g. Drosophila Daughterless) through stable interactions that preclude recruitment of p300/CREB-binding protein (CBP) coactivators. These interactions are mediated by a conserved ETO TAF4 homology domain and a 17-amino acid p300/CBP and ETO target motif within AD1 activation domains of E proteins. In t(8;21) leukemic cells, very stable interactions between AML1-ETO and E proteins underlie a t(8;21) translocation-specific silencing of E protein function through an aberrant cofactor exchange mechanism. These studies identify E proteins as AML1-ETO targets whose dysregulation may be important for t(8;21) leukemogenesis, as well as an E protein silencing mechanism that is distinct from that associated with differentiation-inhibitory proteins (Zhang, 2004).

HEB and E2A function as SMAD/FOXH1 cofactors

Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. A new motif, termed SMAD complex-associated (SCA), was identified that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. Two basic helix-loop-helix (bHLH) proteins-HEB and E2A-bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation (Yoon, 2011).

ChIP-seq was used to generate genome-wide occupancy maps for the Nodal signaling factors SMAD2/3, SMAD3, SMAD4, and FOXH1 in both hESCs and derived endoderm. This study sought to identify novel SMAD complex cofactors by performing de novo motif discovery on the SMAD/FOXH1 genomic targets. Three nonrepetitive motifs were identified that were consistently enriched in all data sets (SMAD2/3, SMAD3, SMAD4, and FOXH1) and in both cell types, hESCs and endoderm. The first and second motifs contain the canonical SMAD- and FOXH1-binding sites, respectively, confirming their genome-wide cooperativity in regulating Nodal signaling and further validating the antibodies used for ChIP. The third motif, CCTGCTG, has not previously been shown to associate with any of the SMAD/FOXH1 complex proteins.This element is referred to as the SCA (SMAD complex-associated) motif (Yoon, 2011).

This study presents strong genomic, biochemical, and functional evidence that E2A and HEB interact with SMAD2/3/4 and FOXH1 to regulate transcription of Nodal target genes. E2A and HEB associate with SMAD2/3 and FOXH1 at the SCA consensus site, which is functionally conserved between frogs and humans. The genomic identification of this site using the power of large sequence reads in multiple data sets provided inroads into testing the interaction of E2A, HEB, and the SMAD/FOXH1 complex. Using biochemical approaches, this study shows that these proteins interact in a DNA-independent manner, but then associate with similar target regions. Based on evidence presented in this study, it is hypothesized that a complex consisting of E2A, HEB, SMAD2/3, and FOXH1 forms within the nucleus in response to Nodal, but that maintenance of this complex is independent of continual Nodal signaling. Overall, it is suggested that E2A and HEB are key regulators of SMAD2/3-mediated transcriptional responses, and thus are fundamental Nodal cofactors that have not previously been implicated in this important developmental pathway (Yoon, 2011).

While genomic and biochemical association is suggestive of a key signaling role, the phenotypic effect of knocking down e2a in X. tropicalis embryos is highly reminiscent of phenotypes resulting from perturbation of other key Nodal signaling factors, such as overexpression of a dominant-negative Nodal receptor or of the Nodal antagonists Cerberus-short and Lefty. Furthermore, it was shown epistatically that e2a knockdown inhibits the ability of both Activin and Xnr1 to induce bottle cell formation, strongly suggesting a key downstream role in the pathway. In the mouse, the roles of HEB and E2A and their family member, E2-2, have been extensively characterized as essential factors in hematopoiesis. The phenotypes of single-gene knockout models for E2A and HEB demonstrated that E2A was the primary E-protein member driving B-cell development, but that both E2A and HEB were required for proper T-cell development. Interestingly, however, there is very strong evidence that these proteins are highly redundant due to their heterodimerizing abilities. Dominant-negative HEB, which can also disrupt E2A function through nonproductive heterodimer formation, causes a stronger phenotype than the heb-null mutation. In B-cell development, HEB, driven by the E2A promoter, can rescue E2A loss of function. These complex genetics and the associated lethality of some compound mutants have made investigation of the roles of these proteins in early embryonic development difficult, and a role for E2A or HEB in early embryogenesis or SMAD/FOXH1 signaling has never been identified. Conditional genetic approaches to ablate several family members during gastrulation will more accurately address the role of E2A and HEB during mammalian germ layer formation. It is noted with interest that loss of e2a function in X. tropcialis achieves an effect on gastrulation not seen in the mouse. It is hypothesized that the expansion of the Nodal pathway in frogs during evolution may have generated less redundancy between the E proteins; this is currently being tested by evaluating compound MOs. Overall, further investigation of the mechanisms used by E2A and HEB to modulate Nodal signal transduction will elucidate new insights into how this important pathway is diversified to induce cell lineages within distinct species (Yoon, 2011).

Signaling upstream of Daughterless homologs

E47 is inhibited by mammalian Deltex (see Drosophila Deltex) , a second Notch-interacting protein. Evidence is provided that Notch and Deltex may act on E47 by inhibiting signaling through Ras (see Drosophila Ras). The EGR-1 promoter (see Huckebein) is known to be stimulated by Ras through the action of mitogen-activated protein kinases (MAPKs) on a ternary complex involving ETS proteins (e.g., ELK1) and Serum response factor. The activity of a CAT reporter under the control of the EGR-1 promoter is inhibited by Deltex, both in the presence and in the absence of Ras stimulation by platelet-derived growth factor. To reduce the complexity of the effects, a series of GAL4 promoter fusions were used and their abilities to activate a minimal promoter containing GAL4 binding sites was assessed. GAL4-Jun includes a portion of the c-Jun protein whose activity is dependent on signaling from Ras to SAPK/JNK. A promoter fragment lacking the CBF1 interaction domain inhibits GAL4-Jun activity but has no effect on GAL4-CREB. Similarly, Deltex inhibits GAL4-Jun activity and has no effect on GAL4-CREB. Although it is likely that N2-IC and Deltex have somewhat different effects on cells, these results clearly show that both Notch and Deltex inhibit signaling by Ras, as measured by the ability to stimulate SAPK/JNK activity. It is proposed that this is the mechanism by which Notch and Deltex inhibit E47 (Ordentlich, 1998).

Daughterless homologs: C. elegans anchor cell gene expression

During C. elegans vulval development, the anchor cell (AC) in the somatic gonad expresses lin-3, coding for a novel member of the epidermal growth factor (EGF) family activating the EGF receptor signaling pathway in vulval precursor cells (VPCs) and thereby inducing and patterning VPCs. Previous studies with lin-3 mutants and transgene expression have revealed that the level of LIN-3 in the AC must be precisely regulated for proper vulval development. To understand how lin-3 expression is achieved in the AC, a 59 bp lin-3 enhancer sufficient to activate lin-3 transcription solely in the AC has been identifed. The enhancer contains two E-box elements, and one FTZ-F1 nuclear hormone receptor (NHR) binding site that is mutated in a vulvaless mutant, lin-3(e1417). Mutagenesis studies show that both E-boxes and the NHR binding site are necessary to express lin-3 in the AC. In vitro DNA-binding studies and in vivo functional assays indicate that distinct trans-acting factors, including the E-protein/Daughterless homolog HLH-2 and unidentified nuclear hormone receptor(s), are necessary for lin-3 transcription in the AC and thus are involved in vulval development (Hwang, 2004).

Thus the C. elegans E-protein homolog, HLH-2, binds to the enhancer element to activate the lin-3 transcription, and NHR-25 also binds to the enhancer. E-protein/Daughterless proteins generally recognize target DNA sequences as a heterodimer with other bHLH proteins. However, the model that a HLH-2 homodimer activates lin-3 transcription in the AC is preferred since purified HLH-2 proteins alone recognize the E-box, and hlh-2 is expressed in the AC but not in the VU cells. The nhr-25 gene is expressed in the AC, and its protein binds to the wild-type form of the NHR binding site. However, nhr-25 appears not to be the NHR that activates lin-3 transcription in the AC since RNAi against nhr-25 does not eliminate lin-3::gfp expression in the AC. About 270 nhr genes are predicted in C. elegans and most of them are not pseudogenes; this contrasts with 21 nhr genes in Drosophila and 50 in human. All of the C. elegans NHRs are orphan receptors for which ligands have not been identified, but evidence indicates the presence of unidentified ligands such as steroids, metabolic intermediates and external materials from the environment. Furthermore, although the amino acid sequences of the ligand-binding domains in C. elegans NHRs are evolutionarily less conserved than those of the DNA-binding domains, structural modeling indicates that many of the C. elegans ligand binding domain sequences are compatible with the X-ray crystal structures of the known ligand-binding domains, suggesting they may bind to ligands (Hwang, 2004).

Daughterless homologs and myogenesis

The murine bHLH protein Twist has been shown to inhibit muscle differentiation in mammalian cells. This inhibition is cell autonomous and does not alter cell proliferation. By overexpression of E12, the inhibitory mechanisms of Twist and the dominant negative HLH factor Id (Drosophila homolog Extra macrochaetae) can be distinguished. A difference is seen both for the native muscle-specific enhancers of myogenin and myosin light chain 1/3 and for an enhancer consisting of only four E-boxes. Mutagenesis experiments reveal that both the basic region and an evolutionarily conserved carboxy-terminal domain are required for the Twist-specific type of inhibition. Loss of either of these regions renders Twist less efficient and more similar to Id. Twist can bind to the muscle creatine-kinase E-box and inhibit DNA binding of E12 heterodimers with myogenic bHLH transcription factors like MyoD. However, a fourfold excess of Twist compared to MyoD is required for both effects. These results suggest that Twist inhibits muscle-specific gene activation by formation of actively inhibitory complexes rather than by sequestering E-proteins (Hebrok, 1997).

The muscle LIM protein (MLP) (Drosophila homolog: Muscle LIM protein at 60A) is a muscle-specific LIM-only factor that exhibits a dual subcellular localization, being present in both the nucleus and in the cytoplasm. Overexpression of MLP in C2C12 myoblasts enhances skeletal myogenesis, whereas inhibition of MLP activity blocks terminal differentiation. Thus, MLP functions as a positive developmental regulator, although the mechanism through which MLP promotes terminal differentiation events remains unknown. While examining the distinct roles associated with the nuclear and cytoplasmic forms of MLP, it was found that nuclear MLP functions through a physical interaction with the muscle basic helix-loop-helix (bHLH) transcription factors MyoD, MRF4, and myogenin. This interaction is highly specific since MLP does not associate with nonmuscle bHLH proteins E12 or E47 or with the myocyte enhancer factor-2 (MEF2) protein, which acts cooperatively with the myogenic bHLH proteins to promote myogenesis. The first LIM motif in MLP and the highly conserved bHLH region of MyoD are responsible for mediating the association between these muscle-specific factors. MLP also interacts with MyoD-E47 heterodimers, leading to an increase in the DNA-binding activity associated with this active bHLH complex. Although MLP lacks a functional transcription activation domain, it is proposed that it serves as a cofactor for the myogenic bHLH proteins by increasing their interaction with specific DNA regulatory elements. Thus, the functional complex of MLP-MyoD-E protein reveals a novel mechanism for both initiating and maintaining the myogenic program and suggests a global strategy for how LIM-only proteins may control a variety of developmental pathways (Kong, 1997).

Basic helix-loop-helix (bHLH) proteins perform a wide variety of biological functions. Most bHLH proteins recognize the consensus DNA sequence CAN NTG (the E-box consensus sequence is in bold) via the DNA-binding basic region (BR) but acquire further functional specificity by preferring distinct internal and flanking bases. In addition, induction of myogenesis by MyoD-related bHLH proteins depends on myogenic basic region and BR-HLH junction residues, both of which are unessential for binding to a muscle-specific site, implying that their BRs may be involved in other critical interactions. An investigation has been carried out to see whether the myogenic residues influence DNA sequence recognition and how MyoD, Twist, and their E2A partner proteins (Daughterless in Drosophila) prefer distinct CAN NTG sites. In MyoD, the myogenic BR residues establish specificity for particular CAN NTG sites indirectly, by influencing the conformation through which the BR helix binds DNA. An analysis of DNA binding by BR and junction mutants suggests that an appropriate BR-DNA conformation is necessary but not sufficient for myogenesis, supporting the model that additional interactions with this region are important. The sequence specificities of E2A and Twist proteins require the corresponding BR residues. In addition, mechanisms that position the BR allow E2A to prefer distinct half-sites as a heterodimer with MyoD or Twist, indicating that the E2A BR can be directed toward different targets by dimerization with different partners. These findings indicate that E2A and its partner bHLH proteins bind to CAN NTG sites by adopting particular preferred BR-DNA conformations, from which they derive differences in sequence recognition that can be important for functional specificity (Kophengnavong, 2000).

In part, the specificity with which bHLH proteins function derives from preferential recognition of different classes of CAN NTG sites by different bHLH protein subgroups. The HLH segment consists of a parallel, left-handed, four-helix bundle. The BR is unstructured in solution but when bound to DNA, it extends N terminally from the HLH segment as a helix that crosses the major groove. Crystallographic analyses have revealed some differences in how these proteins bind DNA. For example, in Myc family and related bHLH proteins, an arginine residue at BR position 13 specifies recognition of CACGTG sites by contacting bases in the center. However, it still is not understood how bHLH proteins that have a different amino acid at BR position 13 bind preferentially to distinct CAN NTG sites or how bHLH proteins establish differences in flanking sequence selectivity that can be of biological importance (Kophengnavong, 2000 and references therein).

Many bHLH proteins that lack R13, including MyoD and other E2A partners, can bind to similar DNA sequences in vitro but they act on different tissue-specific genes. Cooperative or inhibitory relationships with other transcriptional regulators might contribute to this specificity, but it is not likely to derive entirely from other lineage-specific factors, because MyoD can induce myogenesis in many different cell types. Initiation of myogenesis by MyoD and other myogenic bHLH proteins depends on three residues that are located within the BR and the BR-HLH junction (A5, T6, and K15). These residues, which are referred to in this study as myogenic are not essential for binding a muscle-specific site in vitro or in vivo, suggesting that they are involved in other critical interactions. These interactions have been proposed to involve distinct cofactors and the unmasking of an activation domain in MyoD or the myogenic cofactor MEF2. In the MyoD-DNA structure, K15 is oriented away from the DNA, but A5 and T6 face the major groove and could not contact other proteins directly. However, the latter two residues could influence protein-protein interactions indirectly, by affecting how the BR helix is positioned on the DNA. Although substitutions at these positions might not substantially impair binding to particular CAN NTG sites, it is important to determine whether they might have more subtle influences on sequence specificity that could reflect conformational effects (Kophengnavong, 2000 and references therein).

The myogenic residues A5 and T6 establish the characteristic MyoD sequence preference, which includes a CAGCTG core. Individual substitutions at these BR positions simultaneously alter preferences for multiple bases that MyoD does not contact directly, indicating that these preferences are determined indirectly, by how the BR helix is positioned on the DNA. This mechanism is distinct from the standard model for sequence specificity, in which preferred bases are contacted directly. The corresponding BR residues are also required for the sequence preferences of E2A proteins, which can recognize either of two distinct half-sites depending on their dimerization partner. E2A homodimers and E2A-MyoD heterodimers bind to asymmetric sites that include a CACCTG core. In contrast, as a heterodimer with the bHLH protein Twist, E2A binds preferentially to half of the symmetric sequence CATATG. The preference of E2A for the former asymmetric sites depends not only on the BR sequence but also on BR positioning that involves the junction region. An analysis of DNA binding by MyoD and E2A junction and BR mutants indicates that a MyoD-like sequence specificity is associated with, but not sufficient for, myogenesis. This supports the model that the BR-junction region is also involved in other critical interactions. The results suggest that E2A and its partner bHLH proteins bind DNA by adopting a limited number of preferred BR conformations, each of which is associated with a characteristic DNA sequence preference. They also indicate that binding of cofactors to the MyoD BR might be influenced by how it is positioned on the DNA and are consistent with the idea that relatively subtle differences in binding sequence recognition can modulate bHLH protein activity in vivo (Kophengnavong, 2000).

Rhabdomyosarcomas are characterized by expression of myogenic specification genes, such as MyoD and/or Myf5, and some muscle structural genes in a population of cells that continues to replicate. Because MyoD is sufficient to induce terminal differentiation in a variety of cell types, attempts were made to determine the molecular mechanisms that prevent MyoD activity in human embryonal rhabdomyosarcoma cells. This study shows that a combination of inhibitory Musculin:E-protein complexes and a novel splice form of E2A compete with MyoD for the generation of active full-length E-protein:MyoD heterodimers. A forced heterodimer between MyoD and the full-length E12 robustly restores differentiation in rhabdomyosarcoma cells and broadly suppresses multiple inhibitory pathways. These studies indicate that rhabdomyosarcomas represent an arrested progress through a normal transitional state that is regulated by the relative abundance of heterodimers between MyoD and the full-length E2A proteins. The demonstration that multiple inhibitory mechanisms can be suppressed and myogenic differentiation can be induced in the RD rhabdomyosarcomas by increasing the abundance of MyoD:E-protein heterodimers suggests a central integrating function that can be targeted to force differentiation in muscle cancer cells (Yang, 2009).

Daughterless homologs and neurogenesis

bHLH transcription factors function in neuronal development in organizms as diverse as worms and vertebrates. In the C. elegans male tail, a neuronal sublineage clonally gives rise to the three cell types (two neurons and a structural cell) of each sensory ray. The bHLH genes lin-32 and hlh-2 are necessary for the specification of multiple cell fates within this sublineage, and for the proper elaboration of differentiated cell characteristics. Mutations in lin-32, a member of the atonal family, can cause failures at each of these steps, resulting in the formation of rays that lack fully-differentiated neurons, neurons that lack cognate rays, and ray cells defective in the number and morphology of their processes. Mutations in hlh-2, the gene encoding the C. elegans E/daughterless ortholog, enhance the ray defects caused by lin-32 mutations. In vitro, LIN-32 can heterodimerize with HLH-2 and bind to an E-box-containing probe. Mutations in these genes interfere with this activity in a manner consistent with the degree of ray defects observed in vivo. It is proposed that LIN-32 and HLH-2 function as a heterodimer to activate different sets of targets, at multiple steps in the ray sublineage. During ray development, lin-32 performs roles of proneural, neuronal precursor, and differentiation genes of other systems (Portman, 2000).

Loss-of-function and ectopic expression analyses have shown that lin-32 function is both necessary and sufficient for hypodermal seam cells to enter the ray sublineage, leading to the idea that lin-32 functions as the proneural gene for the rays. Weak loss-of-function alleles of lin-32 and hlh-2 in combination with specific markers for ray cell fates show that these genes are also required for later steps. In these mutants, partial ray sublineages are observed and also clonal groups in which some, but not all, cells have fully differentiated. These results demonstrate that the development of individual ray cell types can be uncoupled from each other by loss of lin-32 and hlh-2 function, indicating that these genes have separable functions required at different points in ray development. Thus lin-32, in addition to having a proneural-like competence function in the ray neuroblast, specifies later aspects of ray cell determination and differentiation as well (Portman, 2000).

Based upon the genetic evidence presented in this study, it cannot be determined how direct the functions of lin-32 and hlh-2 are on the steps for which they are required. Indeed, it is possible that the LIN-32:HLH-2 heterodimer is acting only at an early step, perhaps in ray precursor cells, to activate a variety of targets, each required for a different subsequent step of ray development. These intermediates might then be segregated as determinants into different branches of the sublineage, allowing them to function in the proper cells at the proper time. Since lin-32 and hlh-2;lin-32 mutations can disrupt these steps separately, it is clear that the multiple functions of these genes are independent of each other to at least some degree, and that failure to activate one target or set of targets can occur without serious effects on another. The alternative hypothesis, that LIN-32:HLH-2 complexes might be activating different targets at different times, seems more likely, and is supported by the observation that the expression of lin-32 reporter genes and HLH-2 protein continues until the final division of the ray sublineage (Portman, 2000).

ID4 levels dictate the stem cell state in mouse spermatogonia

Spermatogenesis is a classic model of cycling cell lineages that depend on a balance between stem cell self-renewal for continuity and formation of progenitors as the initial step in production of differentiated cells. However, the mechanisms guiding the continuum of spermatogonial stem cell (SSC) to progenitor spermatogonial transition and precise identifiers of subtypes in the process are undefined. This study used an Id4-eGfp reporter mouse to discover that EGFP intensity is predictive of the subsets with the ID4-EGFPBright population being mostly, if not purely, SSCs while the ID4-EGFPDim population is in transition to the progenitor state. These subsets are also distinguishable by transcriptome signatures. Moreover, using a conditional overexpression mouse model, this study found that transition from the stem cell to immediate progenitor state requires down-regulation of Id4 (see Drosophila Daughterless) coincident with a major change in the transcriptome. Collectively, these results demonstrate that the level of ID4 is predictive of stem cell or progenitor capacity in spermatogonia and dictates the interface of transition between the different functional states (Helsel, 2017).

Tcf4 regulates synaptic plasticity, DNA methylation, and memory function

Human haploinsufficiency of the bHLH transcription factor Tcf4 (see Drosophila Daughterless) leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS), which is associated with severe language impairment and development delay. This study demonstrates that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4+/- mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, small-molecule HDAC inhibitors were found to normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified "memory-associated" genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, it was observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4+/- mice (Kennedy, 2016).

Daughterless homologs and pancreas function

Chronic exposure of pancreatic beta-cells to supraphysiologic glucose concentrations results in decreased insulin gene transcription. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta, Drosophila homolog: Slow border cells) is identified as a repressor of insulin gene transcription in conditions of supraphysiological glucose levels. C/EBPbeta is expressed in primary rat islets. After exposure to high glucose concentrations, the beta-cell lines HIT-T15 and INS-1 express increased levels of C/EBPbeta. The rat insulin I gene promoter contains a consensus binding motif for C/EBPbeta (CEB box) that binds C/EBPbeta. In non-beta-cells C/EBPbeta stimulates the activity of the rat insulin I gene promoter through the CEB box. Paradoxically, in beta-cells C/EBPbeta inhibits transcription, directed by the promoter of the rat insulin I gene by direct protein-protein interaction with a heptad leucine repeat sequence within activation domain 2 of the basic helix-loop-helix transcription factor E47. This interaction leads to the inhibition of both dimerization and DNA binding of E47 to the E-elements of the insulin promoter, thereby reducing functionally the transactivation potential of E47 on insulin gene transcription. It is suggested that the induction of C/EBPbeta in pancreatic beta-cells by chronically elevated glucose levels may contribute to the impaired insulin secretion in severe type II diabetes mellitus (Lu, 1997).

The distal portion of the rat insulin I gene 5'-flanking DNA contains two sequence elements, the Far and FLAT elements, that can function in combination, but not separately, as a beta-cell-specific transcriptional enhancer. Several cDNAs encoding proteins that bind to the FLAT element have been isolated. Two of these cDNAs, cdx-3 and lmx-1, represent homeo box containing mRNAs with restricted patterns of expression. The protein encoded by lmx-1 also contains two amino-terminal cysteine/histidine-rich 'LIM' domains. Both cdx-3 and lmx-1 can activate transcription of a Far/FLAT-linked gene when expressed in a normally non-insulin-producing fibroblast cell line. Furthermore, in fibroblasts expressing transfected beta-cell lmx-1, the addition of the Far-binding, basic helix-loop-helix protein shPan-1 (the hamster equivalent of human E47) causes a dramatic synergistic activation. ShPan-1 causes no activation in fibroblasts expressing transfected cdx-3 or the related LIM-homeodomain protein isl-1. Deletion of one or both of the LIM domains from the 5' end of the lmx-1 cDNA removes this synergistic interaction with shPan-1 without any loss of basal transcriptional activation. It is concluded that beta-cell lmx-1 functions by binding to the FLAT element and interacting through the LIM-containing amino terminus with shPan-1 bound at the Far element. These proteins form the minimal components for a functional mini-enhancer complex (German, 2002).

Daughterless homologs - lymphocyte and erythrocyte development

The basic-helix-loop-helix (bHLH) proteins encoded by the E2A gene are broadly expressed transcription regulators that function through binding to the E-box enhancer sequences. The DNA binding activities of E2A proteins are directly inhibited upon dimerization with the Id1 gene product. It has been shown that disruption of the E2A gene leads to a complete block in B-lymphocyte development and a high frequency of neonatal death. Nearly half of the surviving E2A-null mice develop acute T-cell lymphoma between 3 to 10 months of age. Disruption of the Id1 gene improves the chance of postnatal survival of E2A-null mice, indicating that Id1 is a canonical negative regulator of E2A and that the unbalanced ratio of E2A to Id1 may contribute to the postnatal death of the E2A-null mice. However, the E2A/Id1 double-knockout mice still develop T-cell tumors once they reach the age of 3 months. This result suggests that E2A may be essential for maintaining the homeostasis of T lymphocytes during their constant renewal in adult life (Yan, 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 (Drosophila homolog: Chip). 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-homeodomain proteins (See Drosophila Apterous and Islet) direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-homeodomain protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) protein E47/Pan-1 (Drosophila homolog: Daughterless) to activate the insulin promoter in transfected fibroblasts. Two proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.1 and Lmx1.2. Yeast genetic systems were used to delineate the functional domains of the Lmx1 proteins and to characterize the physical interactions between Lmx1 proteins and E47/Pan-1 that produce synergistic transcriptional activation. The LIM domains of the Lmx1 proteins, and particularly the second LIM domain, mediate both specific physical interactions and transcriptional synergy with E47/Pan-1. The LIM domains of the LIM-homeodomain protein Isl-1, which cannot mediate transcriptional synergy with E47/Pan-1, do not interact with E47/Pan-1. In vitro studies demonstrate that the Lmx1.1 LIM2 domain interacts specifically with the bHLH domain of E47/Pan-1. These studies provide the basis for a model of the assembly of LIM-homeodomain-containing complexes on DNA elements that direct cell-type-restricted transcription in differentiated tissues (Johnson, 1997).

E47 is a widely expressed transcription factor that activates B-cell-specific immunoglobulin gene transcription and is required for early B-cell development. In an effort to identify processes that regulate E47, and potentially B-cell development, it was found that activated Notch1 (see Drosophila Notch) and Notch2 effectively inhibit E47 activity. Only the intact E47 protein is inhibited by Notch. Fusion proteins containing isolated DNA binding and activation domains are unaffected. Although overexpression of the coactivator p300 partially reverses E47 inhibition, results of several assays indicate that p300/CBP is not a general target of Notch. Notch inhibition of E47 does not correlate with its ability to activate CBF1/RBP-Jkappa, the mammalian homolog of Suppressor of Hairless, a protein that associates physically with Notch and defines the only known Notch signaling pathway in Drosophila (Ordentlich, 1998).

The class I helix-loop-helix (HLH) proteins, which include E2A, HEB, and E2-2, have been shown to be required for lineage-specific gene expression during T and B lymphocyte development. Additionally, the E2A proteins function to regulate V(D)J recombination, possibly by allowing access of variable region segments to the recombination machinery. The mechanisms by which E2A regulates transcription and recombination, however, are largely unknown. A novel motif, LDFS, present in the vertebrate class I HLH proteins as well as in a yeast HLH protein has been identified that is essential for transactivation. Genetic and biochemical evidence is provided that the highly conserved LDFS motif stimulates transcription by direct recruitment of the SAGA histone acetyltransferase complex (Massari, 1999).

Normal B-cell development requires the E2A gene and its encoded transcription factors E12 and E47. Current models predict that E2A promotes cell differentiation and inhibits G1 cell cycle progression. The latter raises the conundrum of how B cells proliferate while expressing high levels of E2A protein. To study the relationship between E2A and cell proliferation, a tissue culture-based model was established in which the activity of E2A can be modulated in an inducible manner using E47R, an E47-estrogen fusion construct, and E47ERT, a dominant negative E47-estrogen fusion construct. The two constructs were subcloned into retroviral vectors and expressed in the human pre-B-cell line 697, the human myeloid progenitor cell line K562, and the murine fibroblastic cell line NIH 3T3. In both B cells and non-B cells, suppression of E2A activity by E47ERT inhibits G1 progression and is associated with decreased expression of multiple cyclins including the G1-phase cyclin D2 and cyclin D3. Consistent with these findings, E2A null mice express decreased levels of cyclin D2 and cyclin D3 transcripts. In complementary experiments, ectopic expression of E47R promotes G1 progression and is associated with increased levels of multiple cyclins, including cyclin D2 and cyclin D3. The induction of some cyclin transcripts occurs even in the absence of protein synthesis. It is concluded that, in some cells, E2A can promote cell cycle progression, contrary to the present view that E2A inhibits G1 progression (Zhao, 2001).

There are numerous published results that are inconsistent with the present view that E2A suppresses cell proliferation. The E2A homolog in Drosophila daughterless is required for normal cell proliferation. Absence of daughterless causes defects in proliferation and abnormal loss of cyclin B expression in cells of the imaginal disc. Expression of E2A is highest in the proliferating B cells of the germinal centers of lymph nodes. Ectopic expression of E2A in the kidney embryonic cell line 293T causes an increase in the S-phase fraction. Ectopic expression of E47 does not decrease the S-phase fraction of the T-cell lymphoma cell line 1.F9 that was derived from an E2A null mouse. These findings are more consistent with the hypothesis that E2A can actually promote cell proliferation (Zhao, 2001).

The mechanism by which E2A promotes cell proliferation probably involves induction of multiple cell cycle regulatory genes. Among these, E2A regulation of cyclin D2 and cyclin D3 may explain the effects of E2A on G1 progression and on serum dependence. The complex composed of cyclin D-cdk4 or -cdk6 phosphorylates the retinoblastoma tumor suppressor protein (Rb), which activates E2F to induce genes important for S phase such as cyclin A, cdc2, cyclin E, thymidylate synthase, and DNA polymerase alpha. Suppression of cyclin D inhibits G1 progression, while ectopic expression of cyclin D promotes G1 progression and decreased dependence on serum. These effects are virtually identical to the effects seen with modulation of E2A activity and suggest that E2A promotes G1 progression by inducing cyclin D3. Induction of cyclin D3 by E2A is consistent with the observations that proliferating B cells express very high levels of E2A and cyclin D3 proteins. E2A also appears to regulate the expression of cyclin A, cyclin B, and cdc2, which are important regulatory proteins of S-phase entry and mitosis (Zhao, 2001).

How E2A regulates the cell cycle regulatory genes remains to be elucidated. Possible mechanisms include (1) direct activation mediated by binding of E2A to the promoters of the cell cycle regulatory genes, (2) indirect activation of the cell cycle regulatory genes that results from activation of cell proliferation pathways by E2A, and (3) normal induction of cell cycle regulatory genes secondary to changes in the cell cycle profile. While the last possibility is consistent with the effects of E47ERT, it cannot explain the effects of E47R. Tamoxifen treatment of 697/E47R and K562/E47R cells growing in 10% serum induces many cell cycle regulatory genes without changing the cell cycle profile. Of the two remaining mechanisms, a direct transcriptional activation by E2A is favored because multiple cell cycle regulatory genes are induced even in the absence of protein synthesis. Consistent with this hypothesis, the murine cyclin D3 contains three E boxes in the 5'-flanking region. The positions of two of the E boxes relative to the transcription start site appear conserved in the human cyclin D3 5'-flanking region. However, an indirect effect mediated by changes in cyclin D3 message stability or by stimulation of a signal transduction pathway leading to a proliferation transcription program cannot be completely excluded. Future studies to characterize the mechanisms will increase understanding of E2A function during normal and neoplastic cell proliferation (Zhao, 2001).

Lymphocyte development and differentiation are regulated by the basic helix-loop-helix (bHLH) transcription factors encoded by the E2A and HEB genes. These bHLH proteins bind to E-box enhancers in the form of homodimers or heterodimers and, consequently, activate transcription of the target genes. E2A homodimers are the predominant bHLH proteins present in B-lineage cells and are shown genetically to play critical roles in B-cell development. E2A-HEB heterodimers, the major bHLH dimers found in thymocyte extracts, are thought to play a similar role in T-cell development. However, disruption of either the E2A or HEB gene leads to only partial blocks in T-cell development. The exact role of E2A-HEB heterodimers and possibly the E2A and HEB homodimers in T-cell development cannot be distinguished in simple disruption analysis due to a functional compensation from the residual bHLH homodimers. To further define the function of E2A-HEB heterodimers, a dominant negative allele of HEB, which produces a physiological amount of HEB proteins capable of forming nonfunctional heterodimers with E2A proteins, was generated and analyzed. Mice carrying this mutation show a stronger and earlier block in T-cell development than HEB complete knockout mice. The developmental block is specific to the alpha/beta T-cell lineage at a stage before the completion of V(D)J recombination at the TCRbeta gene locus. This defect is intrinsic to the T-cell lineage and cannot be rescued by expression of a functional T-cell receptor transgene. These results indicate that E2A-HEB heterodimers play obligatory roles both before and after TCRbeta gene rearrangement during the alpha/beta lineage T-cell development (Barndt, 2000).

Immunoglobulin (Ig) and T cell receptor (TCR) genes are assembled during lymphocyte maturation through site-specific V(D)J recombination events. E2A proteins act in concert with RAG1 and RAG2 to activate Ig VK1J but not Iglambda VlambdaIII-Jlambda1 rearrangement in an embryonic kidney cell line. In contrast, EBF, but not E2A, promotes VlambdaIII-Jlambda1 recombination. Either E2A or EBF activate IgH DH4J recombination but not V(D)J rearrangement. The Ig coding joints are diverse, contain nucleotide deletions, and lack N nucleotide additions. IgK VJ recombination requires the presence of the E2A transactivation domains. These observations indicate that in nonlymphoid cells a diverse Ig repertoire can be generated by the mere expression of the V(D)J recombinase and a transcriptional regulator (Romanow, 2000).

Pancreatic beta-cell type-specific transcription of the insulin gene is mediated, in part, by factors in the basic helix-loop-helix (bHLH) family that act on a site within the insulin enhancer, termed the E1-box. Expression from this element is regulated by a heteromeric protein complex containing ubiquitous (i.e. the E2A- and HEB-encoded proteins) and islet-enriched members of the bHLH family. Recent studies indicate that the E2A- and HEB-encoded proteins contain a transactivation domain, termed AD2, that functions more efficiently in transfected beta-cell lines. In the present report, this observation is extended by demonstrating that expression of full-length E2A proteins (E47, E12, and E2/5) activates insulin E element-directed transcription in a beta-cell line-selective manner. Stimulation requires functional interactions with other key insulin gene transcription factors, including its islet bHLH partner as well as those that act on the RIPE3b1 and RIPE3a2 elements of the insulin gene enhancer. The conserved AD2 domain in the E2A proteins is essential in this process. The effect of the E2A- and HEB-encoded proteins on insulin gene expression was also analyzed in mice lacking a functional E2A or HEB gene. There was no apparent difference in insulin production between wild type, heterozygote, and homozygous mutant E2A or HEB mice. These results suggest that neither the E2A- or HEB-encoded proteins are essential for insulin transcription and that one factor can substitute for the other to impart normal insulin E1 activator function in mutant animals (Sharma, 1997).

Plasmacytoid dendritic cells (PDCs) represent a unique immune cell type specialized in type I interferon (IFN) secretion in response to viral nucleic acids. The molecular control of PDC lineage specification has been poorly understood. This paper reports that basic helix-loop-helix transcription factor (E protein) E2-2/Tcf4 is preferentially expressed in murine and human PDCs. Constitutive or inducible deletion of murine E2-2 blocked the development of PDCs but not of other lineages and abolished IFN response to unmethylated DNA. Moreover, E2-2 haploinsufficiency in mice and in human Pitt-Hopkins syndrome patients was associated with aberrant expression profile and impaired IFN response of the PDC. E2-2 directly activated multiple PDC-enriched genes, including transcription factors involved in PDC development (SpiB, Irf8) and function (Irf7). These results identify E2-2 as a specific transcriptional regulator of the PDC lineage in mice and humans and reveal a key function of E proteins in the innate immune system (Cisse, 2008).

Daughterless homologs and leukemia

Oncogenic mutation of nuclear transcription factors often is associated with altered patterns of subcellular localization that may be of functional importance. The leukemogenic transcription factor gene E2A-PBX1 is created through fusion of the genes E2A and PBX1 (Drosophila homolog: Extradenticle) as a result of t(1;19) in acute lymphoblastic leukemia. Subcellular localization patterns of E2A-PBX1 protein were evaluated in transfected cells using immunofluorescence. Full-length E2A-PBX1 is exclusively nuclear and is concentrated in spherical domains termed chimeric-E2A oncoprotein domains (CODs). In contrast, nuclear fluorescence for wild-type E2A or PBX1 proteins is diffuse. Enhanced concentrations of RNA polymerase II within many CODs and the requirement for an E2A-encoded activation domain suggest transcriptional relevance. However, in situ co-detection of nascent transcripts labeled with bromouridine fails to confirm altered transcriptional activity in relation to CODs. CODs also fail to co-localize with foci of DNA replication as well as with other proteins known to occupy functional nuclear compartments, including the transcription factor PML, the spliceosome-associated protein SC-35 and the adenovirus replication factor DBP. Co-transfection of Hoxb7, a homeodomain protein capable of enhancing DNA binding by PBX1, impairs COD formation, suggesting that CODs contain E2A-PBX1 protein not associated with DNA. It is concluded that as a 'gain of function' phenomenon requiring protein elements from both E2A and PBX1, COD formation may be relevant to the biology of E2A-PBX1 in leukemogenesis (LeBrun, 1997).

daughterless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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