Interactive Fly, Drosophila

Vertebrate Engrailed: Protein interactions

Specific residues located within the Pbx homeodomain (See Drosophila Extradenticle) are essential for cooperative DNA binding with Hox and Engrailed gene products. Within the N-terminal region of the Pbx homeodomain, a residue has been identified that is required for cooperative DNA binding with three Hox Antennapedia class gene products (Hoxb-7, Hoxb-8 and Hoxc-6) but not for cooperativity with Engrailed-2 (En-2). There are similarities between heterodimeric interactions involving the yeast mating type homeodomain proteins MATa1 and MATalpha2, and those that allow the formation of Pbx/Hox and Pbx/En-2 heterodimers. Specifically, residues located in the a1 homeodomain that form a hydrophobic pocket allowing the alpha2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/En-2 cooperativity (Peltenburg, 1997).

Three residues located at another site, in the turn between helix 1 and helix 2 are characteristic of many atypical homeodomain proteins. These residues, present in Pbx type homeodomains, are required for cooperative DNA binding involving both Hox and En-2. Replacement of the three residues located in the turn between helix 1 and helix 2 of the Pbx homeodomain with those of the atypical homeo-domain proteins controlling cell fate in the basidiomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2. The data suggest that the molecular mechanism of homeodomain protein interactions that control cell fate in Saccharomyces cerevisiae and in the basidiomycetes may well be conserved in part in multicellular organisms. While a number of structural determinates, such as the hydrophobic pocket, are required for cooperativity involving both Hox and Engrailed, others, such as the three amino acid insert, are clearly more specific (Peltenburg, 1997).

Homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains. Part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that Engrailed proteins (in addition to their well-known presence in the nucleus) are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion. Other homeoproteins, Emx-1 and Hoxa-7 have been found in the axon and nerve terminals of post-mitotic neurons. Emx-1 is found in the axon of the mouse olfactory receptors. Hoxa7 is present in the ascending and descending spinal tracts and can be found in the brain where no Hoxa-7 transcripts are present (Joliot, 1997).

Transcriptional regulation of vertebrate Engrailed

Polycomb-group (PcG) proteins form large multimeric protein complexes that are involved in maintaining the transcriptionally repressive state of genes. RING1 interacts with vertebrate Polycomb (Pc) homologs and is associated with or is part of a human PcG complex. However, very little is known about the role of RING1 as a component of the PcG complex. A detailed characterization of RING1 protein-protein interactions has been undertaken. By using directed two-hybrid and in vitro protein-protein analyses, it has been demonstrated that RING1, in addition to interacting with the human Pc homolog HPC2, can also interact with itself and with the vertebrate PcG protein BMI1. Distinct domains in the RING1 protein are involved in the self-association and in the interaction with BMI1. Further, the BMI1 protein can also interact with itself. To better understand the role of RING1 in regulating gene expression, the protein was overexpressed in mammalian cells and differences in gene expression levels were analyzed. This analysis shows that overexpression of RING1 strongly represses En-2, a mammalian homolog of the well-characterized Drosophila PcG target gene engrailed. Furthermore, RING1 overexpression results in enhanced expression of the proto-oncogenes c-jun and c-fos. The changes in expression levels of these proto-oncogenes are accompanied by cellular transformation, as judged by anchorage-independent growth and the induction of tumors in athymic mice. These data demonstrate that RING1 interacts with multiple human PcG proteins, indicating an important role for RING1 in the PcG complex. Further, deregulation of RING1 expression leads to oncogenic transformation by deregulation of the expression levels of certain oncogenes (Satijn, 1999).

The Xenopus homolog of Drosophila Enhancer of Zeste has been identified using a differential display strategy designed to identify genes involved in early anterior neural differentiation. XEZ codes for a protein of 748 amino acids that is very highly conserved in evolution and is 96% identical to both human and mouse EZ(H)2. In common with most other Xenopus Pc-G genes and unlike mammalian Pc-G genes, XEZ is anteriorly restricted. Zygotic expression of XEZ commences during gastrulation, much earlier than other anteriorly localized Pc-G genes; expression is restricted to the anterior neural plate and is confined later to the forebrain, eyes and branchial arches. XEZ is induced in animal caps overexpressing noggin; up-regulation of XEZ therefore represents a response to inhibition of BMP signaling in ectodermal cells. The midbrain/hindbrain junction marker En-2, and hindbrain marker Krox-20, are target genes of XEZ and that XEZ functions to repress these anteroposterior marker genes. Conversely, XEZ does not repress the forebrain marker Otx-2. XEZ overexpression results in a greatly thickened floor of the forebrain. These results implicate an important role for XEZ in the patterning of the nervous system (Barnett, 2001).

The modification of chromatin structure is an important regulatory mechanism for developmental gene expression. Differential expression of the mammalian ISWI genes, SNF2H and SNF2L, has suggested that they possess distinct developmental roles. This study describes the purification and characterization of the first human SNF2L-containing complex. The subunit composition suggests that it represents the human ortholog of the Drosophila nucleosome-remodeling factor (NURF) complex. Human NURF (hNURF) is enriched in brain, and it regulates human Engrailed, a homeodomain protein that regulates neuronal development in the mid-hindbrain. Furthermore, hNURF potentiates neurite outgrowth in cell culture. Taken together, these data suggess a role for an ISWI complex in neuronal growth (Barak, 2003).

The Drosophila ISWI protein exists in three multiprotein complexes, namely, ACF, CHRAC and NURF. Mammalian complexes corresponding to ACF and CHRAC have been purified and contain the SNF2H protein. Additional unique mammalian ISWI complexes have also been purified, including RSF, WICH, NoRC and SNF2H-cohesin, and these all comprise the SNF2H protein. Despite the growing list of mammalian ISWI complexes, a NURF equivalent or complexes containing the related protein SNF2L has been notably absent. The hNURF complex identified in this study contains BPTF and RbAP46/48. Surprisingly, hNURF does not contain the inorganic pyrophosphatase protein NURF38. Nonetheless, the biochemical activity of hNURF is similar; it displays predominantly nucleosome-stimulated ATPase activity, as well as potent chromatin-remodeling activity on oligonucleosomal arrays (Barak, 2003).

The brain-enriched expression profile of SNF2L prompted an examination of a role for hNURF in neuronal physiology. SNF2L chromatin-remodeling activity can induce neurite outgrowth in a tissue culture-based assay, and this is specific to SNF2L-containing ISWI complexes since SNF2H expression does not result in a similar induction. The conversion of a neuroblast to a differentiated neuron will require the modification of chromatin structure at numerous genes, for both activation and repression, and it is not likely to be restricted to the NURF complex. Nonetheless, these studies suggest that hNURF has a role in this process and, thus, identification of target genes will help elucidate the molecular pathways. In this regard, hNURF can regulate the mammalian engrailed genes, through a direct interaction at the promoters of these two homeotic loci. The murine engrailed genes are critical regulators of mid-hindbrain development; ablation leads to animals that are missing most of the colliculi and cerebellum. Although engrailed was identified previously as a NURF target gene through the characterization of flies harboring mutant ISWI or NURF301 genes, a neural defect was not appreciated due to the early lethality of these animals. As such, this may represent a novel function for the NURF complex (Barak, 2003).

The effect of chromatin-remodeling complexes on development is a well-established phenomenon. Linkages between chromatin remodeling and developmental disorders include ATRX and mental retardation, SMARCAL1 and Schimke immuno-osseous dysplasia, CSB and Cockayne syndrome, and SNF2H and Williams syndrome. It is hypothesized that the hNURF complex may represent another connection of a chromatin-remodeling protein to disorders of development, and it can be stated with confidence that the hNURF complex regulates other developmentally important genes. In this regard, the analysis of flies ablated for the NURF complex also suggests a role for the hNURF complex in hematopoietic development and the regulation of chromosome structure. However, such studies in mammals must await further dissection using in vivo model systems (Barak, 2003).

The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, whether Xenopus Zic1 acted through the Wnt pathway was examined. Using Wnt signaling inhibitors, it was demonstrated that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated in this study. Together, these data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates (Merzdorf, 2006).

Histone methylation is a posttranslational modification regulating chromatin structure and gene regulation. BHC110/LSD1 has been described as a histone demethylase that reverses dimethyl histone H3 lysine 4 (H3K4). This study shows that JARID1d, a JmjC-domain-containing protein, specifically demethylates trimethyl H3K4. Detailed mapping analysis revealed that besides the JmjC domain, the BRIGHT and zinc-finger-like C5HC2 domains are required for maximum catalytic activity. Importantly, isolation of native JARID1d complexes from human cells revealed the association of the demethylase with a polycomb-like protein Ring6a/MBLR. Ring6a/MBLR not only directly interacts with JARID1d but also regulates its enzymatic activity. JARID1d and Ring6a occupy human Engrailed 2 gene and regulate its expression and H3K4 methylation levels. Depletion of JARID1d enhances recruitment of the chromatin remodeling complex, NURF, and the basal transcription machinery near the transcriptional start site, revealing a role for JARID1d in regulation of transcriptional initiation through H3K4 demethylation (Lee, 2007).

Zebrafish Engrailed

Generation of cell diversity in the vertebrate central nervous system starts during gastrulation stages in the ectodermal germ layer and involves specialized cell groups, such as the organizer located at the midbrain-hindbrain boundary (MHB). Mutations in the zebrafish no isthmus (noi) gene alter development of the MHB, and affect the pax2.1 gene (formerly pax(zf-b), see Drosophila Sparkling). Analysis of the structure of pax2.1 reveals at least 12 normal splice variants. The noi alleles can be arranged, by molecular and phenotypic criteria, into a series of five alleles of differing strength, ranging from a null allele to weak alleles. In keeping with a role in development of the MHB organizer, gene expression is already affected in the MHB primordium of the gastrula neural ectoderm in noi mutants. engrailed gene eng3 activation depends completely on noi function, and eng2 activation is strongly dependent on noi function. In contrast, onset of wnt1, fgf8 and her5 expression occurs normally in the null mutants, but is eliminated later on. These observations suggest that three signaling pathways, involving pax2.1, wnt1 and fgf8, are activated independently in the early anterior-posterior patterning of this area. In addition, analysis of the allelic series unexpectedly suggests that noi activity is also required during dorsal-ventral patterning of the MHB in somitogenesis stages, and possibly in a later eng expression phase. It is proposed that noi/pax2.1 participates in sequential signaling processes as a key integrator of midbrain-hindbrain boundary development (Lun, 1998).

The ventral branches of the segmental peripheral nerves in the zebrafish embryo are pioneered by the caudal primary (CaP) motor axons, which extend midsegmentally at the interface of the somite and the notochord. The signals that define the CaP pathway are not well understood. To gain insight into the nature of the guidance cues, the environment of the CaP motor axons have been examined by using electron microscopy and histochemistry. Specifically, the distributions of the transcription factor engrailed, of a chondrotin sulfate epitope, and of the recognition molecules zebrafish semaphorin z1b and zebrafish tenascin C have been mapped. Ultrastructural examination of dye-labeled CaP motor axons reveals a close association with the medial surface of the somite but not with the notochord. The CaP axons are always accompanied by cells that appeared to migrate at the interface of somite and notochord. These cells are confined to the posterior half of the somite. Some of the cells may be neural crest derived; many others are probably of sclerotomal origin. The putative migratory cells express a chondroitin sulfate epitope that is a marker of sclerotome in the chick. The pathway of the CaP axon and the distribution of the putative neural crest and sclerotome cells correlate with a subdivision of the myotome into anterior and posterior components, which are evident at the histological level and by the expression of the markers engrailed, semaphorin z1b, chondroitin sulfate, and tenascin C. It is suggested that both the pathway choice of the CaP axon and the route of migratory cells reflect this anterior-posterior bipartition of the myotome (Bernhardt, 1998).

Xenopus Engrailed

Gain-of-function assays in Xenopus have demonstrated that Xwnt-3a (see Drosophila Wingless) can pattern neural tissue by reducing the expression of anterior neural genes, and elevating the expression of posterior neural genes. To date, no loss-of-function studies have been conducted in Xenopus to show a requirement of endogenous Wnt signaling for patterning of the neural ectoderm along the anteroposterior axis. Expression of a dominant negative Wnt in Xenopus embryos causes a reduction in the expression of posterior neural genes, and an elevation in the expression of anterior neural genes, thereby confirming the involvement of endogenous Wnt signaling in patterning the neural axis. The ability of Xwnt-3a to decrease expression of anterior neural genes in noggin-treated explants (noggin is a neural inducer) is dependent on a functional FGF signaling pathway, while the elevation of expression of posterior neural genes does not require FGF signaling. In Xenopus, eGFG, FGF3 and XFGF-9 are expressed in the posterior dorsal mesoderm during gastrulation, consistent with potential roles in neural patterning. The previously reported ability of FGF to elevate the expression of posterior neural genes in noggin-treated explants is found to be dependent on endogenous Wnt signaling. It is concluded that neural induction occurs initially in a Wnt-independent manner, but that generation of complete anteroposterior neural pattern requires the cooperative actions of Wnt and FGF pathways. Noggin induces the anterior markers Xanf-1 and Otx-2 in animal cap explants but in the presence of Xwnt-3a, expression of both markers is reduced. At the same time there is an elevation in expression of the posterior neural markers En-2 and Krox-2, although not the spinal cord marker Hox B9. In the presence of FGF, noggin (in contrast) does not reduce the expression of Xanf-1 or Otx-2, while there is a concurrent induction of posterior genes, including Hox B9. Thus Wnts and FGF can both pattern neural tissues but these factors exhibit differences in their neural patterning activities. Xwnt-3a cannot suppress anterior neural genes in the absence of FGF signaling, indicating that the two pathways work together in neural patterning (McGrew, 1997).

In Drosophila, the Polycomb-group constitutes a set of structurally diverse proteins that act together to silence target genes. Many mammalian Polycomb-group proteins have also been identified and show functional similarities with their invertebrate counterparts. To begin to analyze the function of Polycomb-group proteins in Xenopus development, a Xenopus homolog of Drosophila Polycomblike, XPcl1, has been cloned. XPcl1 mRNA is present both maternally and zygotically, with prominent zygotic expression in the anterior central nervous system. Misexpression of Pcl1 by RNA injection into embryos produces defects in the anterior central nervous system. The forebrain and midbrain contain excess neural tissue at the expense of the ventricle and include greatly thickened floor and roof plates. The eye fields are present but Rx2A, an eye-specific marker, is completely repressed. Overexpression of Pcl1 in Xenopus embryos alters two hindbrain markers, repressing En-2 and shifting it and Krox-20 in a posterior direction. Similar neural phenotypes and effects on the En-2 expression pattern were produced by overexpression of three other structurally unrelated Polycomb-group proteins: M33 (homolog of Drosophila Polycomb), XBmi-1 (homolog of Drosophila Polycomb), and mPh2 (homolog of Drosophila Polyhomeotic). These observations indicate an important role for the Polycomb-group in regulating gene expression in the developing anterior central nervous system (Yoshitake, 1999).

The molecular mechanisms that govern early patterning of anterior neuroectoderm (ANE) for the prospective brain region in vertebrates are largely unknown. Screening a cDNA library of Xenopus ANE led to the isolation of a Hairy and Enhancer of split- (HES)-related transcriptional repressor gene, Xenopus HES-related 1 (XHR1). XHR1 is specifically expressed in the midbrain-hindbrain boundary (MHB) region at the tailbud stage. The localized expression of XHR1 is detected as early as the early gastrula stage in the presumptive MHB region, an area just anterior to the involuting dorsal mesoderm, demarcated by the expression of the gene Xbra. Expression of XHR1 is detected much earlier than that of other known MHB genes (XPax-2 and En-2) and also before the formation of the expression boundary between Xotx2 and Xgbx-2, suggesting that the early patterning of the presumptive MHB is independent of Xotx2 and Xgbx-2. Instead, the location of XHR1 expression appears to be determined in relation to the Xbra expression domain, since reduced or ectopic expression of Xbra alters the XHR1 expression domain according to the location of Xbra expression. In functional assays using mRNA injection, overexpression of dominant-negative forms of XHR1 in the MHB region led to marked reduction of XPax-2 and En-2 expression, and this phenotype was rescued by coexpression of wild-type XHR1. Furthermore, ectopically expressed wild-type XHR1 near the MHB region enhances En-2 expression only in the MHB region but not in the region outside the MHB. These data suggest that XHR1 is required, but not sufficient by itself, to initiate MHB marker gene expression. Based on these data, it is proposed that XHR1 demarcates the prospective MHB region in the neuroectoderm in Xenopus early gastrulae (Shinga, 2001).

Avian Engrailed and limb, brain and eye development

In quail neuroretinas, it has been observed that Engrailed (En-1) is expressed both in the ganglionic and the amacrine cell layers, similar to the expression patterns of Pax-6 (Drosophila homolog: Eyeless). Because a decrease of Pax-6 expression is observed in the neuroretina of hatched animals, the effect of the chicken En-1 and En-2 proteins on Pax-6 expression was examined. En-1 and to some extent En-2 are able to repress the basal and the p46Pax-6-activated transcription from the two Pax-6 promoters. Infection of retinal pigmented epithelium by a virus encoding the En-1 protein represses the endogenous Pax-6, and a similar effect is observed with a homeodomain-deleted En-1. In vitro interaction indicates that En proteins are able to interact with the p46Pax-6 through the paired domain. This interaction negatively regulates the DNA-binding properties of the p46Pax-6. These results suggest an interplay between En-1 and Pax-6 during the central nervous system development and indicate that En-1 may be a negative regulator of Pax-6 (Plaza, 1997).

In the early chick embryo, the dorsal ventral (DV) boundary organizes the apical ectodermal ridge (AER) structure in the limb bud field. Engrailed-1 (En-1), a homolog of the Drosophila segment polarity gene engrailed, expressed in the ventral limb ectoderm, participates in AER formation at the DV boundary of the limb bud. Restricted ectopic expression of En-1 in the dorsal side of the limb bud by transplantation of En-1-overexpressing ectoderm induces ectopic AER at the boundary of En-1-positive and -negative cells. The results suggest that En-1 is involved in AER formation at the DV boundary of the limb bud (Tanaka, 1998).

In vertebrates, the limb bud arises from a flat bilayered tissue consisting of ectoderm and underlying lateral plate mesoderm. Epithelial-mesenchymal interactions lead to primary outgrowth followed by induction of a specialized ectodermal structure, the apical ectodermal ridge (AER), at the distal tip of the bud. The apical ridge runs along the anteroposterior axis of the bud at the dorsoventral interface. Expression of certain genes is conserved between vertebrate limbs and Drosophila appendages although their distribution has changed. Ventral ectoderm and ventral apical ridge express Engrailed-1 (En-1). Dorsal ectoderm expresses Wnt-7a, and one of the fringe homologs, Radical-Fringe (R-Fng). Dorsal mesoderm expresses Lmx1 (called Lmx1b in mouse), an apterous homolog. Using lineage tracers, dorsal and ventral ectodermal compartments have been shown along the sides of the body in chick embryos. The compartments are formed both in presumptive limb-forming regions where they position the apical ridge and also in presumptive interlimb (flank). The ventral compartment coincides with the Engrailed-1 (En-1) domain of expression. This coincidence suggests that En-1 might maintain the ventral compartment and be necessary for apical ridge formation. To test this hypothesis, En-1 was ectopically expressed via retroviral transfer and then limb development and cell lineage restriction in the ectoderm were examined. En-1 misexpression can completely prevent formation of both normal limbs and ectopic limbs induced in the flank by application of FGF-2. In both cases, there are no morphological signs of apical ectodermal ridge formation and expression of ridge-associated genes is undetectable. In striking contrast, the lineage restriction between dorsal and ventral ectoderm is not altered. Therefore, En-1 is involved in the regulation of ridge formation but not compartment maintenance. The genes that are expressed in a dorsoventrally localized pattern seem to fulfill three different, but possibly not exclusive, functions: (1) encoding compartment identity as selector genes; (2) participating in signaling pathways leading to ridge formation; and (3) specifying dorsoventral pattern in the underlying mesoderm. According to this analysis, it is predicted that changes in expression of dorsoventrally restricted genes (e.g., Wnt-7a) in En-1 null mice do not reflect a change in dorsoventral lineage restriction. The gene(s) that initiate and/or maintain dorsoventral ectodermal cell lineage restriction remain to be identified (Altabef, 2000).

Regionalization of a simple neural tube is a fundamental event during the development of the central nervous system. To analyze in vivo the molecular mechanisms underlying the development of the mesencephalon, expressed Engrailed, which is expressed in developing mesencephalon, was ectopically expressed in the brain of chick embryos by in ovo electroporation. Misexpression of Engrailed causes a rostral shift of the di-mesencephalic boundary, and causes transformation of dorsal diencephalon into tectum, a derivative of dorsal mesencephalon. Ectopic Engrailed rapidly represses Pax-6, a marker for diencephalon, which precedes the induction of mesencephalon-related genes, such as Pax-2, Pax-5, Fgf8, Wnt-1 and EphrinA2. In contrast, a mutant Engrailed, En-2(F51 to E), bearing mutation in the EH1 domain, which has been shown to interact with a co-repressor, Groucho, does not show the phenotype induced by wild-type Engrailed. Furthermore, VP16- Engrailed chimeric protein, the dominant positive form of Engrailed, causes a caudal shift of di-mesencephalic boundary and ectopic Pax-6 expression in mesencephalon. These data suggest that (1) Engrailed defines the position of dorsal di-mesencephalic boundary by directly repressing diencephalic fate, and (2) Engrailed positively regulates the expression of mesencephalon-related genes by repressing the expression of their negative regulator(s) (Araki, 1999).

The chick optic tectum consists of 16 laminae. This study reports contribution of En2 to laminar formation in chick optic tecta. En2 is specifically expressed in laminae g-j of stratum griseum et fibrosum superficiale (SGFS). Misexpression of En2 resulted in disappearance of En2-expressing cells from the superficial layers (laminae a-f of SGFS), where endogenous En2 is not expressed. Misexpression of En2 before postmitotic cells had left the ventricular layer indicated that En2-misexpressing cells stopped at the laminae of endogenous En2 expression and that they did not migrate into the superficial layers. Induction of En2 misexpression using a tetracycline-inducible system after the postmitotic cells had reached superficial layers also resulted in disappearance of En2-expressing cells from the superficial layers. Time-lapse analysis showed that En2-misexpressing cells migrated back from the superficial layers towards the middle layers, where En2 is strongly expressed endogenously. These results suggest a potential role of En2 in regulating cell migration and positioning in the tectal laminar formation (Omi, 2014).

Mammalian Engrailed: Transcriptional targets

Fibroblast growth factor-8 (Fgf8) plays a critical role in vertebrate development and is expressed normally in temporally and spatially restricted regions of the vertebrate embryo. This study reports the identification of regions of Fgf8 important for its transcriptional regulation in murine ES cell-derived embryoid bodies. Stable transfection of ES cells, using a human growth hormone reporter gene, was employed to identify regions of the Fgf8 gene with promoter/enhancer activity. A 2-kilobase 5' region of Fgf8 contains promoter activity. A 0.8-kilobase fragment derived from the large intron of Fgf8 enhances 3-4 fold the human growth hormone expressed from the Fgf8 promoter, in an orientation dependent manner. The intronic fragment contains DNA-binding sites for the AP2, Pbx1, and Engrailed transcription factors. Gel shift and Western blot experiments document the presence of these transcription factors in nuclear extracts from ES cell embryoid bodies. In vitro mutagenesis of the Engrailed or Pbx1 site demonstrate that these sites modulate the activity of the intronic fragment. In addition, in vitro mutagenesis of both Engrailed and Pbx1 sites indicates that other unidentified sites are responsible for the transcriptional enhancement observed with the intronic fragment (Gemel, 1999).

The MAP1B (Mtap1b) promoter presents two evolutionary conserved overlapping homeoproteins and Hepatocyte nuclear factor 3ß (HNF3ß/Foxa2) cognate binding sites (defining putative homeoprotein/Fox sites, HF1 and HF2). Accordingly, the promoter domain containing HF1 and HF2 is recognized by cerebellum nuclear extracts containing Engrailed and Foxa2 and has regulatory functions in primary cultures of embryonic mesmetencephalic nerve cells. Transfection experiments further demonstrate that Engrailed and Foxa2 interact physiologically in a dose-dependent manner: Foxa2 antagonizes the Engrailed-driven regulation of the MAP1B promoter, and vice versa. This led to an investigation to see if Engrailed and Foxa2 interact directly. Direct interaction was confirmed by pull-down experiments, and the regions participating in this interaction were identified. In Foxa2 the interacting domain is the Forkhead box DNA-binding domain. In Engrailed, two independent interacting domains exist: the homeodomain and a region that includes the Pbx-binding domain. Finally, Foxa2 not only binds Engrailed but also Lim1, Gsc and Hoxa5 homeoproteins and in the four cases Foxa2 binds at least the homeodomain. Based on the involvement of conserved domains in both classes of proteins, it is proposed that the interaction between Forkhead box transcription factors and homeoproteins is a general phenomenon (Foucher, 2003).

Mapping of the interacting domains identified the Forkhead box binding domain in Foxa2 as the only domain interacting with Engrailed, Hoxa5, Lim1, and Gsc and Otx2. Similarly, for all homeoproteins tested, the homeodomain alone binds Foxa2. However, and in contrast with Foxa2, four out of these five homeoproteins contained additional Foxa2-interacting regions: Engrailed, Hoxa5, Gsc (in all three cases in the N-terminal sequence) and Otx2 [in its C-terminal sequence]. A detailed analysis of the interacting domains has been done for Engrailed only and the mapping of the other homeoproteins has been limited to the homeodomain, and its flanking N- and C-terminal regions, at large. In the case of Engrailed, in addition to the homeodomain, a short sequence (amino acids 146-199) overlapping the Pbx-interacting domain also binds Foxa2. This latter domain and the homeodomain bind independently to Foxa2 and the possibility that they interact with different sub-regions of the Forkhead box domain was not investigated. Such an additional non-homeodomain Foxa2 interacting domain was also present in the N-terminal sequences of Hoxa5 and Gsc, but not in Lim1. With the exception of the hexapeptide sequence present in Engrailed and Hoxa5, no further similarities were found between the Foxa2-binding domains identified outside the homeodomain in Engrailed, Hoxa5, Gsc and Otx2. It is thus possible that, in addition to the homeodomain, different homeoproteins have evolved separate Foxa2-binding regions with regulatory functions (Foucher, 2003).

In this context it is interesting that the fragment 146-199 of Engrailed includes the EH2 (homologous to hexapeptide in Hox proteins) and EH3 domains of Engrailed, both of which are implicated in functional interactions with Exd/Pbx homeoproteins. The same observation also holds for Hoxa5, for which the N-terminal sequence containing the hexapeptide sequence binds Foxa2. Both Pbx and Foxa2 might bind Engrailed (or Hoxa5) to form a tripartite complex or, alternatively, that Foxa2 and Pbx binding are mutually exclusive. Also intriguing is the fact that Engrailed and Gsc, as well as different Forkhead box proteins -- including BF1 and Foxa2 -- interact with co-factors of the Groucho/TLE family. Since the Groucho/TLE-interacting domains of Engrailed and Foxa2 have been mapped to the EH1 and CRII domains, respectively (two domains not involved in the Foxa2-Engrailed interaction) it is possible that larger complexes involving Groucho/TLE proteins, Forkhead transcription factors and homeoproteins form in vivo (Foucher, 2003).

Repeated mutation of a developmental enhancer contributed to human thermoregulatory evolution

Humans sweat to cool their bodies and have by far the highest eccrine sweat gland density among primates. Humans' high eccrine gland density has long been recognized as a hallmark human evolutionary adaptation, but its genetic basis has been unknown. In humans, expression of the Engrailed 1 (EN1; see Drosophila Engrailed) transcription factor correlates with the onset of eccrine gland formation. In mice, regulation of ectodermal En1 expression is a major determinant of natural variation in eccrine gland density between strains, and increased En1 expression promotes the specification of more eccrine glands. This study shows that regulation of EN1 has evolved specifically on the human lineage to promote eccrine gland formation. Using comparative genomics and validation of ectodermal enhancer activity in mice, a human EN1 skin enhancer, hECE18 was identified. Multiple epistatically interacting derived substitutions in the human ECE18 enhancer were shown to increase its activity compared with nonhuman ape orthologs in cultured keratinocytes. Repression of hECE18 in human cultured keratinocytes specifically attenuated EN1 expression, indicating this element positively regulates EN1 in this context. In a humanized enhancer knock-in mouse, hECE18 increased developmental En1 expression in the skin to induce the formation of more eccrine glands. This study uncovers a genetic basis contributing to the evolution of one of the most singular human adaptations and implicates multiple interacting mutations in a single enhancer as a mechanism for human evolutionary change (Aldea, 2021).

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