Zn finger homeodomain 1


C. elegans homolog of Drosophila ZFH-1

Neurons acquire distinct cell identities and implement differential gene programs to generate their appropriate neuronal attributes. On the basis of position, axonal structure and synaptic connectivity, the 302 neurons of the nematode Caenorhabditis elegans are divided into 118 classes. The development and differentiation of many neurons require the gene zag-1, which encodes a deltaEF1/ZFH-1 Zn-finger-homeodomain protein. zag-1 mutations cause misexpression of neuron-specific genes, block formation of stereotypic axon branches, perturb neuronal migrations, and induce various axon-guidance, fasciculation and branching errors. A zag-1-GFP translational reporter is expressed transiently in most or all neurons during embryogenesis and in select neurons during the first larval stage. Analysis of the zag-1 promoter reveals that zag-1 is expressed in neurons and specific muscles, and that ZAG-1 directly represses its own expression. zag-1 activity also downregulates expression of genes involved in either the synthesis or reuptake of serotonin, dopamine and GABA. It is proposed that ZAG-1 acts as a transcriptional repressor to regulate multiple, discrete, neuron-specific aspects of terminal differentiation, including cell migration, axonal development and gene expression (Clark, 2003).

The Zn finger/homeodomain factor, zag-1 is required for particular aspects of axonal pathfinding. In zag-1 mutants, motorneuron commissures either branch prematurely or fail to branch at the correct point. Ventral cord interneurons show defects in the guidance towards the ventral cord and also in the ventral cord. Several neurons misexpress differentiation markers, including glutamate receptor subunits and chemosensory receptors. zag-1 is expressed transiently in embryonic and postembryonic neurons during differentiation as well as in some mesodermal tissues. Null mutants of zag-1 are unable to swallow food and die as L1 larvae with a starved appearance, indicating that zag-1 has an additional role in pharynx development. The vertebrate homolog, {delta}EF1, is highly conserved and acts as transcriptional repressor in various tissues. C. elegans zag-1 also acts as a transcriptional repressor controlling important aspects of terminal differentiation of neurons (Wacker, 2003).

Sensory neuron fates are distinguished by a transcriptional switch that regulates dendrite branch stabilization

Sensory neurons adopt distinct morphologies and functional modalities to mediate responses to specific stimuli. Transcription factors and their downstream effectors orchestrate this outcome but are incompletely defined. This study shows that different classes of mechanosensory neurons in C. elegans are distinguished by the combined action of the transcription factors LIM-type homeodomain protein MEC-3, bHLH PAS domain protein AHR-1, and Zn finger/homeodomain factor ZAG-1. Low levels of MEC-3 specify the elaborate branching pattern of PVD nociceptors, whereas high MEC-3 is correlated with the simple morphology of AVM and PVM touch neurons. AHR-1 specifies AVM touch neuron fate by elevating MEC-3 while simultaneously blocking expression of nociceptive genes such as the MEC-3 target, the claudin-like membrane protein HPO-30, that promotes the complex dendritic branching pattern of PVD. ZAG-1 exercises a parallel role to prevent PVM from adopting the PVD fate. The conserved dendritic branching function of the Drosophila AHR-1 homolog, Spineless, argues for similar pathways in mammals (Smith, 2013).

Sensory neurons display a wide range of morphological motifs and functional modalities that serve to transduce diverse types of external stimuli into specific physiological responses. Transcription factors define both the identity and number of each type of sensory neuron and thus are critical determinants of organismal behavior. The downstream pathways that distinguish the architectural and functional properties of different sensory neuron classes are largely unknown, however. This study shows that the conserved transcription factors MEC-3, AHR-1 and ZAG-1, function together to define distinct sensory neuron fates in C. elegans and identify downstream targets that are necessary for these roles (Smith, 2013).

The MEC-3 LIM homeodomain protein is expressed in both touch receptor neurons (TRNs) and in PVD but is responsible for distinctly different sets of characteristics displayed by these separate classes of mechanosensory neurons. In PVD neurons, MEC-3 promotes the creation of a highly branched dendritic arbor and nociceptive responses to harsh stimuli, whereas in the TRNs, MEC-3 is necessary for light touch sensitivity and for the adoption of a simple, unbranched morphology. Genetic ablation of mec-3 or its upstream regulator, the POU domain protein UNC-86, disrupts the function and morphological differentiation of both of these types of mechanosensory neurons. How are these different MEC-3-dependent traits produced? The results suggest that low levels of MEC-3 are sufficient to specify the PVD fate, whereas elevated MEC-3 drives TRN differentiation. The existence of this threshold effect is also supported by the finding that overexpression of MEC-3 induces TRN-specific gene expression in the PVD-like FLP neuron. This simple model is not sufficient, however, to explain why PVD nociceptor genes, which are turned on by low levels of MEC-3, are actually repressed in the TRNs as MEC-3 expression is elevated. The current findings now provide a mechanism for this effect. In the light touch AVM neuron, AHR-1 elevates MEC-3 expression while simultaneously blocking downstream MEC-3 targets that drive PVD branching and nociceptor function. It is suggested that ZAG-1 may exercise a similar role in PVM. This mechanism is robust because each of these TRNs is effectively transformed into a functional PVD-like neuron when either ahr-1 or zag-1 is genetically eliminated. Thus, this work has revealed the logic of alternative genetic regulatory pathways in which a single type of transcription factor (e.g., MEC-3) can specify the differentiation of two distinct classes of mechanosensory neurons. A related mechanism accounts in part for the dose-dependent effects of the homeodomain transcription factor Cut on the branching complexity of larval sensory neurons in Drosophila. The transcription factor Knot/Collier is selectively deployed in Type IV da neurons to antagonize expression of Cut targets that produce the dendritic spikes that are characteristic of Type III da neurons. In this case, however, Knot does not regulate Cut expression but functions in a parallel pathway. The finding that the Zinc-finger transcription factor ZAG-1 is required to prevent the PVM touch neuron from adopting a PVD nociceptor fate mirrors the recent observation that genetic ablation of the mammalian ZAG-1 homolog Zfhx1b (Sip1, Zeb2) results in cortical interneurons adopting the fate of striatal GABAerigic cells (McKinsey, 2013). The current results are suggestive of a potentially complex regulatory mechanism in which AHR-1 and ZAG-1 inhibit expression of nociceptor genes (e.g., hpo-30) whereas MEC-3 activates transcription of these targets. Additional upstream regulators of mec-3, UNC- 86, and ALR-1, are also likely involved in this pathway (Smith, 2013).

Although transcription factors are well-established determinants of sensory neuron fate, the downstream pathways that they regulate are largely unknown. As a solution to this problem for MEC-3, a cell-specific profiling strategy was used to detect mec-3-regulated transcripts in the PVD neuron. A combination of RNAi and mutant analysis was used to identify the subset of targets that affect PVD branching morphogenesis. Additional experiments with one of these hits, the claudin-like protein HPO-30, revealed a key role in the generation of PVD branches. It is noted that HPO-30 is expressed in the FLP neuron, where it also mediates the higher order branching morphology shared by FLP and PVD. Time-lapse imaging has revealed that PVD lateral or 2 branches may adopt either of two different modes of outgrowth along the inside surface of the epidermis: (1) fasciculation with existing motor neuron commissures or (2) independent extension as noncommissural or 'pioneer' dendrites. The results show that the principle role of HPO-30 is to stabilize pioneer 2 branches and, thus, that additional unknown factors may drive fasciculation with motor neuron commissures. Because claudins serve as key constituents of junctions between adjacent cells, it seems likely that HPO-30 functions in this case to link growing 2 dendrites with the nematode epidermis. It is noted that an additional membrane component, the LRR protein DMA-1, displays a mutant PVD branching phenotype strongly resembling that of Hpo-30 and therefore could also function in this pathway. The intimate association of topical sensory arbors with the skin and the broad conservation of junctional proteins across species point to the likelihood that homologs of HPO-30/Claudin and similar components could be widely utilized to pattern sensory neuron morphogenesis (Smith, 2013).

ahr-1 encodes a member of the bHLH-PAS family of transcription factors and is the nematode homolog of the aryl hydrocarbon receptor (AHR) protein. In mammals, AHR is activated by the xenobiotic compound dioxin to trigger a wide range of pathological effects. Invertebrate AHR proteins are not activated by dioxin, which suggests that this toxin-binding function represents an evolutionary adaptation unique to vertebrates. An ancestral role for AHR is suggested by AHR mutants in C. elegans and Drosophila that display distinct developmental defects in which a given cell type or tissue adopts an alternative fate. For example, stochastic expression of the Drosophila AHR homolog, Spineless, promotes the adoption of one specific photoreceptor sensory neuron identity at the expense of another (Smith, 2013).

The current results parallel those findings with the demonstration that AHR-1 function is required in C. elegans to distinguish between alternative types of mechanosensory neurons; in ahr-1 mutants, the unbranched light touch neuron, AVM, is transformed into a functional homolog of the highly branched PVD nociceptor. This role for ahr-1 in C. elegans is particularly notable because the AHR-1 homolog, Spineless, also regulates branching complexity in Drosophila. In spineless (Ss) mutants, Class I and II sensory neurons, which normally display simple branching patterns, adopt more complex dendritic arbors. This phenotype resembles the current finding in C. elegans that the simple morphology of the AVM neuron is transformed into the highly branched architecture of the PVD nociceptor in ahr-1 mutants. Ss mutants in Drosophila also show the opposite phenotype of more complex class III and class IV da neurons assuming simpler branching patterns, which could therefore reflect an additional role for spineless in this context of promoting the creation of dendritic branches. On the basis of these results, it is suggested that the striking conservation of the shared role of AHR homologs in regulating sensory neuron fate and branching complexity in nematodes and insects argues that this function is evolutionarily ancient and, thus, that the downstream effectors that have been identified in C. elegans may also pattern the dendritic architecture of vertebrate sensory neurons (Smith, 2013).

Fish and Frog homologs of Drosophila ZFH-1

Kheper is a novel member of the ZFH (zinc-finger and homeodomain protein)/deltaEF1 family in zebrafish. kheper transcripts are first detected in the epiblast of the dorsal blastoderm margin at the early gastrula stage and kheper is expressed in nearly all the neuroectoderm at later stages. kheper expression was expanded in noggin RNA-injected embryos and also in swirl mutant embryos and was reduced in bmp4 RNA-injected embryos and chordino mutant embryos, suggesting that kheper acts downstream of the neural inducers Noggin and Chordino. Overexpression of Kheper elicits ectopic expansion of the neuroectoderm-specific genes fkd3, hoxa-1, and eng3, and the ectopic expression of hoxa-1 is not inhibited by BMP4 overexpression. Kheper interacts with the transcriptional corepressors CtBP1 and CtBP2. Overexpression of either a Kheper mutant lacking the homeodomain or of a VP16-Kheper fusion protein disturbs the development of the neuroectoderm and head structures. These data underscore the role of Kheper in the development of the neuroectoderm and indicate that Kheper acts as a transcriptional repressor (Muraoka, 2000).

Xenopus SIP1 (XSIP1), Smad interacting protein, has been characterized from activin-treated animal caps by differential screening. The XSIP1 is very similar to mouse SIP1 in the protein coding region, including the zinc finger domain and homeodomain. The expression pattern was analyzed by RT-PCR and whole mount in situ hybridization. XSIP1 expression was initially restricted to the dorsal marginal zone in the late gastrula and was subsequently expressed at the lateral edge of neural plate and, in the tailbud stage, in the forebrain, neural tube, and eye. Overexpression of XSIP1 at the animal caps results in activation of anterior neural markers without mesodermal markers. Ectopic expression of XSIP1 induces enlargement of neural cells and disordered eye formation. In addition to abnormal head phenotypes, many embryos were short-tailed. These findings suggest that XSIP1 is a transcriptional repressor, which may be involved in the activin-dependent signal pathway (Eisaki, 2000).

A Xenopus homolog of the zinc finger/homeodomain-containing transcriptional repressor Smad-interacting protein-1 (SIP1) has been isolated from the mouse. XSIP1 is activated at the early gastrula stage and transcription occurs throughout embryogenesis. At the beginning of gastrulation, XSIP1 is strongly expressed in prospective neurectoderm. At the neurula stage, XSIP1 is highly expressed within the neural plate but weakly in the dorsal midline. At later stages of development transcripts are detected primarily within the neural tube and neural crest. In the adult, XSIP1 expression is detected at variable levels in several organs (van Grunsven, 2000).

Snail2 and Zeb2 repress P-Cadherin to define embryonic territories in the chick embryo

Snail and Zeb (see Drosophila Snail and Zinc finger homeodomain 1) transcription factors induce epithelial to mesenchymal transition (EMT) in embryonic and adult tissues by direct repression of E-Cadherin (see Drosophila Shotgun) transcription. The repression of E-Cadherin transcription by the EMT inducers Snail1 and Zeb2 plays a fundamental role in defining embryonic territories in the mouse, as E-Cadherin needs to be downregulated in the primitive streak and in the epiblast concomitant with the formation of mesendodermal precursors and the neural plate, respectively. This study shows that in the chick embryo, E-Cadherin is weakly expressed in the epiblast at pre-primitive streak stages where it is substituted by P-Cadherin. Snail2 and Zeb2 were shown to repress P-Cadherin transcription in the primitive streak and the neural plate, respectively. This indicates that E- and P-Cadherin expression patterns evolved differently between chick and mouse. As such, the Snail1/E-Cadherin axis described in the early mouse embryo corresponds to Snail2/P-Cadherin in the chick, but both Snail factors and Zeb2 fulfill a similar role in chick and mouse in directly repressing ectodermal Cadherins to promote the delamination of mesendodermal precursors at gastrulation and the proper specification of the neural ectoderm during neural induction (Acloque, 2017).

Mammalian homologs of Drosophila ZFH-1

A nuclear factor delta EF1, which binds to the essential element of the delta 1-crystallin enhancer core, was molecularly cloned from the chicken. The protein organization of delta EF1 deduced from the cDNA sequence indicated that it has heterogeneous domains for DNA-binding, two widely separated zinc finger clusters and a homeodomain, analogous to Drosophila ZFH-1 protein. A cluster of four zinc-finger domains is present about one quarter of the way from the N-terminal. Another cluster of three zinc-finger domains is present at the C-terminal end. The C-terminal zinc fingers are responsible for binding to the delta 1-crystallin enhancer core sequence. Delta EF1 has proline-rich and acidic domains common to various transcriptional activators. During embryogenesis, delta EF1 expression is observed in the postgastrulation period in mesodermal tissues. Expression occurs initially in the notochord, followed by somites, nephrotomes and other components. Expression levels change dynamically in different tissues, possibly a reflection of the differentiation states of the constituent cells. In addition to mesoderm, delta EF1 is expressed in the nervous system and the lens, but other ectodermal tissues and endoderm remain very low in delta EF1 expression. Cotransfection experiments indicate that this factor acts as a repressor of delta 1-crystallin enhancer. The possession of heterogeneous DNA-binding domains and the observed dynamic change of expression in embryogenesis strongly suggest that delta EF1 acts in multiple ways depending on the cell type and the gene under its regulation (Funahashi, 1993).

A vertebrate homolog of Drosophila Zfh-1, called ZEB, is a negative regulator of muscle differentiation. ZEB binds to a subset of E boxes in muscle genes and functions by actively repressing transcription. Targets of this repression include members of the MEF-2 family, which synergize with proteins of the myogenic basic helix-loop-helix family (bHLH) (myoD, myf-5, myogenin and MRF-4) to induce myogenic differentiation. As muscle differentiation proceeds, myogenic bHLH proteins accumulate to levels sufficient to displace ZEB from the E boxes, releasing the repression and allowing bHLH proteins to further activate transcription. This mechanism of active transcriptional repression distinguishes ZEB from other negative regulators of myogenesis (Id, Twist and I-mfa) that inhibit muscle differentiation by simply binding and inactivating myogenic factors. The relative affinity of ZEB versus myogenic bHLH proteins varies for E boxes in different genes such that ZEB would be displaced from different genes at distinct times as myogenic bHLH proteins accumulate during myogenesis, thus providing a mechanism to regulate temporal order of gene expression (Postigo, 1997a).

ZEB, a vertebrate homolog of Zfh-1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c-Myb (see Drosophila Myb oncogene-like) and Ets (see Drosophila Pointed). These factors act synergistically to activate transcription; this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c-Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c-Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c-Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. One target of this positive/negative regulation in vivo is the alpha4 integrin, which plays a key role in normal hematopoiesis and thefunction of mature leukocytes. Stem cell alpha4 integrin, negatively regulated by ZEB, is known to bind VCAM-1 and fibronectin located in the stroma of hematopoetic organs. alpha4 integrin binding to stromal ligands is necessary for lymphoid differentiation (Postigo, 1997b).

ZEB is a zinc finger-homeodomain protein that represses transcription by binding to a subset of E-box sequences. ZEB inhibits muscle differentiation in mammalian systems, and its Drosophila orthologue, zfh-1, inhibits somatic and cardiac muscle differentiation during Drosophila embryogenesis. ZEB also binds to the promoter of pivotal hematopoietic genes (including those encoding interleukin-2, CD4, GATA-3, and alpha(4)-integrin), and mice in which ZEB has been genetically targeted show thymic atrophy, severe defects in lymphocyte differentiation, and increased expression of the alpha(4)-integrin and CD4. ZEB contains separate repressor domains that function in T lymphocytes and muscle, respectively. The most C-terminal domain inhibits muscle differentiation in mammalian cells by specifically blocking the transcriptional activity of the myogenic factor MEF2C. The more N-terminal domain blocks activity of hematopoietic transcription factors such as c-myb, members of the ets family, and TFE-III. These results demonstrate that ZEB has evolved with two independent repressor domains that target distinct sets of transcription factors and function in different tissues (Postigo, 1999b).

The rat Zfhep gene encodes a member of the Zfh family of transcription factors having a homeodomain-like sequence and multiple zinc fingers. Expression of Zfhep in the rat forebrain during embryonic and postnatal development was examined. Zfhep mRNA is strongly expressed in the progenitor cells of the ventricular zone around the lateral ventricles on E14 and E16, but shows little expression in cells that have migrated to form the developing cortex. Dual labeling with PCNA demonstrates expression of Zfhep mRNA in proliferating cells. Expression of Zfhep in the ventricular zone decreases during late development as the population of progenitor cells decreases. This pattern is distinctly different from other members of the Zfh family. The expression of Zfhep protein was examined during retinoic acid-induced neurogenesis of P19 embryonal carcinoma cells. Zfhep is highly expressed in P19 neuroblasts, and expression decreases by the time of morphological neurogenesis. Hence, both P19 cells and embryonic brain demonstrate a loss of Zfhep expression during the transition from proliferating precursor to differentiated neural cells. A possible link between Zfhep and proliferation was examined by treating human glial cell lines with Zfhep antisense phosphorothioate oligodeoxynucleotides. Two Zfhep antisense oligonucleotides repress proliferation of either U-138 or U-343 glioblastoma cells more than control oligonucleotides. Based on the expression patterns of Zfhep in vivo and in the P19 cell model of neurogenesis, it is suggested that Zfhep may play a role in proliferation or differentiation of neural cells (Yen, 2001).

The smad binding protein 1 gene (SMADIP1, MIM 605802) has been recently identified as a disease causing gene in a polytopic embryonic defect (MIM 235730) including midline anomalies, facial dysmorphic features and enteric nervous system malformation (Hirschsprung disease). To confirm the pleiotropic role of SMADIP1 during embryogenesis and investigate its role in neural crest cell derivatives differentiation, RNA in situ hybridization was performed at early stages of human development. According to the spectrum of malformations observed in patients, expression of SMADIP1 is observed in neural crest derived cells (peripheric nervous system, enteric nervous system, facial neurectoderm and cranial nerve ganglia), central nervous system, genital tubercle, muscles and kidneys. Surprisingly, SMADIP1 expression is also found in limbs and developing eye. Although congenital heart defects are frequently observed in patients with either a SMADIP1 large scale deletion or truncating mutation, no SMADIP1 expression could be detected in the developing heart at the stages studied (Espinosa-Parrilla, 2002).

SIP1, a member of the deltaEF1 family of two-handed zinc finger transcriptional repressors, has been identified as a Smad-binding protein. Mutations in the human SIP1 gene (ZFHX1B) have been implicated in Hirschsprung disease. The structure and transcriptional pattern of the mouse SIP1 gene (Zfhx1b) has been documented and it is compared to homologues from other species. The overall structure of Zfhx1b is highly similar to that of the deltaEF1 gene (Zfhx1a), confirming their close evolutionary relationship. In contrast to Zfhx1a, the 5' untranslated region of the SIP1-encoding mouse gene is very complex and includes several alternative exons. The corresponding 5'-UTR splicing pattern seems to be conserved between species and suggests a role in its transcriptional and/or translational regulation. The gene also codes for an antisense transcript that is highly conserved between human and mouse (Nelles, 2003).

ZFH-1 homologs bind to E-boxes

Transcription factor AREB6 has a unique structure composed of two zinc-finger clusters in N- and C-terminal regions, and one homeodomain in the middle between them. AREB6 has been known to regulate the expression of the Na, K-ATPase alpha 1 subunit, interleukin 2 and delta-crystallin genes. The optimal binding sites for the N-terminal zinc-finger cluster have been determined by the CASTing method (cyclic amplification and selection of targets) as GTCACCTGT or TGCACCTGT and for the C-terminal zinc-finger cluster as C/TACCTG/TT. The additional consensus sequence GTTTC/G, in conjunction with the CACCTGT sequence, is selected by the second CASTing for the entire coding region. The N-terminal zinc-finger cluster binds strongly to DNA when the DNA has GTTTC/G in conjunction with the CACCTGT sequence. The homeodomain has no specific DNA binding activity but is found to interact with the N-terminal zinc-finger cluster. Analyses of zinc-finger mutation proteins reveals that the contribution to DNA binding of each N-terminal zinc-finger motif is altered depending on the presence of the additional consensus. Transient transfection assays shows that AREB6 represses the human 70-kDa heat-shock gene promoter harboring the CACCTGT sequence, together with the additional consensus, and that AREB6 activates the promoter harboring the CACCTGT sequence without the additional consensus. These results suggest that AREB6 has multiple conformational states, leading to both positive and negative regulations of gene transcription (Ikeda, 1995).

SIP1, a Smad-interacting protein, and deltaEF1, a transcriptional repressor involved in skeletal and T-cell development, belong to the same family of DNA binding proteins. SIP1 and deltaEF1 contain two separated clusters of zinc fingers, one N-terminal and one C-terminal. These clusters show high sequence homology and are highly conserved between SIP1 and deltaEF1. Each zinc finger cluster binds independently to a 5'-CACCT sequence. However, high-affinity binding sites for full-length SIP1 and deltaEF1 in the promoter regions of candidate target genes like Xenopus Xbra2, and human alpha4-integrin and E-cadherin, are bipartite elements composed of one CACCT and one CACCTG sequence; the orientation and spacing of these sequences can vary. Using transgenic Xenopus embryos, it has been demonstrated that the integrity of these two sequences is necessary for correct spatial expression of a Xbra2 promoter-driven reporter gene. Both zinc finger clusters must be intact for the high-affinity binding of SIP1 to DNA and for its optimal repressor activity. These results show that SIP1 binds as monomer and contacts one target sequence with the first zinc finger cluster, and the other with the second cluster. This work redefines the optimal binding site and, consequently, candidate target genes for vertebrate members of the deltaEF1 family (Remacle, 1999).

Transcriptional downregulation of E-cadherin appears to be an important event in the progression of various epithelial tumors. SIP1 (ZEB-2) is a Smad-interacting, multi-zinc finger protein that shows specific DNA binding activity. Expression of wild-type but not of mutated SIP1 downregulates mammalian E-cadherin transcription via binding to both conserved E2 boxes of the minimal E-cadherin promoter. SIP1 and Snail bind to partly overlapping promoter sequences and show similar silencing effects. SIP1 can be induced by TGF-beta treatment and shows high expression in several E-cadherin-negative human carcinoma cell lines. Conditional expression of SIP1 in E-cadherin-positive MDCK cells abrogates E-cadherin-mediated intercellular adhesion and simultaneously induces invasion. SIP1 therefore appears to be a promoter of invasion in malignant epithelial tumors (Comijn, 2001).

Repressor function of ZFH-1 homologs

Counteraction between activators and repressors is crucial for the regulation of a number of cell-specific enhancers, where an activator and a repressor are mutually competitive in binding to the same site. DeltaEF1 is a repressor protein of delta1-crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator deltaEF3 from binding to the overlapped site. It has two zinc finger clusters N-fin and C-fin, close to N- and C-termini, respectively, and a homeodomain in the middle. deltaEF1 also binds to the E2-box sequence CACCTG, and represses E2-box-dependent enhancers. The mechanism of the repressor action of deltaEF1 has been investigated by examining various deletion mutants of deltaEF1 for their activity to repress a delta1-crystallin enhancer fragment HN that contains DC5 sequence and an additional activator site. Both zinc finger clusters are essential for DNA binding and repression, but the homeodomain is not. In addition, the NR domain close to the N-terminus is required for full repression. The NR domain shows active repression when fused to the Gal4 DNA binding domain. Active repression by deltaEF1, dependent on the NR domain, is also demonstrated in a situation where the binding sites of deltaEF1 and deltaEF3 are separated. N-fin and C-fin in their isolated forms bind the 5'-(T/C)ACCTG-3' and 5'-(t/C)ACCT-3' sequences, respectively, while the homeodomain shows no DNA binding activity. An analysis of DNA binding of the delta(Int)F form, having both N-fin and C-fin, has indicated that a single DNA binding domain is assembled from two zinc finger clusters. It is concluded that two mechanisms are involved in the repressor action of deltaEF1: (1) a binding site competition with an activator that depends on the integrity of both zinc finger clusters, and (2) an active repression to silence an enhancer that is attributed to the NR domain (Sekido, 1997).

deltaEF1, a representative of the zinc finger-homeodomain protein family, is a transcriptional repressor that binds E2-box (CACCTG) and related sequences and counteracts the activators through transrepression mechanisms. It has been shown that the N-proximal region of the protein is involved in the transrepression. deltaEF1 has a second mechanism of transrepression recruiting CtBP1 or CtBP2 as its corepressor. A two-hybrid screen of mouse cDNAs with various portions of deltaEF1 identified these proteins, which bind to deltaEF1 in a manner dependent on the PLDLSL sequence located in the short medial (MS) portion of deltaEF1. CtBP1 is the mouse orthologue of human CtBP, known as the C-terminal binding protein of adenovirus E1A, while CtBP2 is the second homologue. Fusion of mouse CtBP1 or CtBP2 to Gal4DBD (Gal4 DNA binding domain) made them Gal4 binding site-dependent transcriptional repressors in transfected 10T1/2 cells, indicating their involvement in a transcriptional repression mechanism. When the MS portion of deltaEF1 was fused to Gal4DBD and used to transfect cells, a strong transrepression activity was generated, but this activity was totally dependent on the PLDLSL sequence that served as the site for interaction with endogenous CtBP proteins, indicating that CtBP1 and -2 can act as corepressors. Exogenous CtBP1/2 significantly enhances transcriptional repression by deltaEF1, and this enhancement is lost if the PLDLSL sequence is altered, demonstrating that CtBP1 and CtBP2 act as corepressors of deltaEF1. In the mouse, CtBP1 is expressed from embryo to adult, but CtBP2 is mainly expressed during embryogenesis. In developing embryos, CtBP1 and CtBP2 are expressed broadly with different tissue preferences. Remarkably, their high expression occurs in subsets of deltaEF1-expressing tissues, e.g., cephalic and dorsal root ganglia, spinal cord, posterior-distal halves of the limb bud mesenchyme, and perichondrium of forming digits, supporting the conclusion that CtBP1 and CtBP2 play crucial roles in the repressor action of deltaEF1 in these tissues (Furusawa, 1999).

Zfh-1 is a zinc finger/homeodomain transcriptional repressor in Drosophila that regulates differentiation of muscle and gonadal cells and is also expressed in the central nervous system (CNS). Binding sites for Zfh-1 overlap with those for Snail, and like Snail, it recruits the corepressor CtBP-1. The protein ZEB-1 appears to be a vertebrate homologue of zfh-1 and is expressed in several tissues including muscle, CNS, and T lymphocytes, and during skeletal differentiation. Mutation of the ZEB-1 gene leads to a severe T cell phenotype and skeletal defects but, interestingly, no defects are evident in other ZEB-1-expressing tissues. These results suggested that another ZEB-1-related factor may compensate for the loss of ZEB-1 in other tissues. Such a ZEB-1-related protein has now been characterized and termed ZEB-2. The overall organization of ZEB-2 is similar to ZEB-1 and Zfh-1 and it has similar biochemical properties: it binds E boxes and interacts with CtBP-1 to repress transcription. However, there are also differences between ZEB-1 and ZEB-2, both in activity and tissue distribution. Whereas ZEB-1 and ZEB-2 overlap in skeletal muscle and CNS (providing an explanation for why mutation of ZEB-1 alone has little effect in these tissues), they show a different pattern of expression in lymphoid cells. ZEB-1, but not ZEB-2, is expressed in T cells from the thymus; ZEB-2 appears to be expressed on splenic B cells. Additionally, ZEB-2 inhibits a wider spectrum of transcription factors than ZEB-1 (Postigo, 2000).

Some repressors contain more than one repressor domain. This feature allows versatility in their mechanism of repression and, therefore, in their targets of repression. Thus, Drosophila Hairy is able to interact with CtBP-1 as well as with the corepressor Groucho. ZEB-1 contains multiple RDs and interaction with CtBP-1 does not account for all of its repressor activity. The overall sequence similarity between ZEB-1 and ZEB-2 in the RD is quite limited outside the CID region (~30%). However, the same is also true for ZEB-1 and Zfh-1 despite the fact that their RDs appear similar in activity (around 12% sequence similarity in their RD). Therefore, whether ZEB-2 shares the same pattern of repressor activity as ZEB-1 and Zfh-1 was investigated. For this purpose, RD-ZEB-2 was fused to the DNA binding domain of the bacterial protein LexA and its ability to repress a number of transcriptional activators was checked. ZEB-2, as was previously found for ZEB-1, represses the activity of c-myb, the ets protein PU.1, NF-kappaB p65, MEF2C, CTF, and VP16. However, factors that are refractory to ZEB-1, such as the B cell transcription factor ITF-1 and muscle transcription factor myoD, are also repressed by ZEB-2. This finding suggests that ZEB-1 and ZEB-2 may use distinct mechanisms of repression and ZEB-1 may only target a subset of genes targeted by ZEB-2. These results also indicate more functional similarity between Zfh-1 and ZEB-1 than to ZEB-2, suggesting that although sequence-wise both ZEB-1 and ZEB-2 are equally similar to Zfh-1, somehow during evolution the pattern of transcriptional repressor specificity segregates more to ZEB-1 than to ZEB-2 and that ZEB-2 is the more divergent family member (Postigo, 2000).

ZEB-1 repressor activity is segregated to different regions of the protein. While most factors, including hematopoietic factors, are repressed by region 1 (between the N-terminal zinc fingers and the homeodomain), region 3 (close to the C-terminal zinc fingers) represses exclusively the myogenic factor MEF2C and inhibits muscle differentiation. Whether a similar functional organization is present in ZEB-2 was investigated. Constructs encoding the corresponding regions of ZEB-1 and ZEB-2 were constructed, fused to LexA, and their ability to repress different transcriptional activators was checked. Some factors, such as c-myb, TFE-3, CTF, or NF-kappaB p65 are repressed by the same region of ZEB-2 and ZEB-1 (region 1). However, other factors are repressed by different regions of the proteins. For example, the ets factor PU.1 and the myogenic factor MEF2C are repressed by regions 1 and 3 of ZEB-1, respectively; however, repression of these factors by ZEB-2 seems to require the entire RD (Postigo, 2000).

ZEB-1/Zfh-1 regulates myogenic differentiation in both mammalian cells and Drosophila. In mammals, muscle differentiation is regulated by two families of positive factors: (1) MRF proteins that induce muscle differentiation by binding E box sequences in the promoter regions of muscle genes and activating their transcription and (2) the MEF2 proteins, which synergize with MRF proteins to regulate muscle differentiation and activate transcription either by binding to specific DNA sequences or through interaction with the MRF proteins. Another difference between ZEB-1 and ZEB-2 is that ZEB-2 is able to repress myoD whereas ZEB-1 cannot. Repression of the MRF member myoD by ZEB-2 is the result of the activity of region 1. Although ZEB-1 can block myogenic differentiation, this appears to be due to its ability to repress the activity of MEF2C. ZEB-2 also blocks MEF2C transcriptional activity (Postigo, 2000).

Another specific target of ZEB-2 repression is the B cell factor ITF-1. ITF-1, along with TFE-3, has a key role in the regulation of the heavy chain Ig enhancer. Both ITF-1 and TFE-3 are repressed by region 1 of ZEB-2. This may have significance because ZEB-1 is expressed in T cells, whereas ZEB-2 appears to be restricted to B cells (Postigo, 2000).

Protein interactions of ZFH-1 homologs

ZEB is an active transcriptional repressor that regulates lymphocyte and muscle differentiation in vertebrates. Its homolog in Drosophila (Zfh-1) is also essential for differentiation of somatic and cardiac muscle. ZEB and Zfh-1 are shown to interact with the corepressor CtBP to repress transcription. ZEB and Zfh-1, both contain the sequence PLDLS in the same region of the repressor domain, and this sequence is shown to bind CtBP-1 and -2. In vertebrate species, ZEB contains two additional CtBP-like binding sites (variations of the PLDLS sequence) that also bind CtBP proteins and are required for full repressor activity. The three sites have an additive effect, and mutation of all three sites is necessary to abolish both binding to CtBP and repressor activity. Finally, the interaction of CtBP with ZEB at the promoter is shown to be necessary for repressor activity (Postigo, 1999a).

Activation of transforming growth factor beta receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. A novel Smad-interacting protein, SIP1, is described that was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast and in vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation. SIP1 is a new member of the deltaEF1/Zfh-1 family of two-handed zinc finger/homeodomain proteins. Like deltaEF1, SIP1 binds to 5'-CACCT sequences in different promoters, including the Xenopus brachyury promoter. Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2 promoter in vitro, prevents expression of the endogenous Xbra gene in early Xenopus embryos. Therefore, SIP1, like deltaEF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways. The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor beta members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo (Verschueren, 1999).

Binding of TGFß/BMP factors to their receptors leads to translocation of Smad proteins to the nucleus where they activate transcription of target genes. The two-handed zinc finger proteins encoded by Zfhx1a and Zfhx1b, ZEB-1/deltaEF1 and ZEB-2/SIP1, respectively, regulate gene expression and differentiation programs in a number of tissues. ZEB proteins are also crucial regulators of TGFß/BMP signaling with opposing effects on this pathway. Both ZEB proteins bind to Smads, but while ZEB-1/deltaEF1 synergizes with Smad proteins to activate transcription, promote osteoblastic differentiation and induce cell growth arrest, the highly related ZEB-2/SIP1 protein has the opposite effect. Finally, the ability of TGFß to mediate transcription of TGFß-dependent genes and induce growth arrest depends on the presence of endogenous ZEB-1/deltaEF1 protein (Postigo, 2003a).

ZEB proteins are members of a large family of zinc finger proteins known as Zinc finger homeodomain, that was first identified in Drosophila. The genes encoding ZEB-1/deltaEF1 and ZEB-2/SIP1 proteins (Zfhx1 and Zfhx1b, respectively) appear to have evolved from a single Drosophila gene named zfh-1. zfh-1 is crucial for mesodermal (gonadal, skeletal and cardiac muscle) and neural differentiation in flies. The human ortholog of Drosophila zfh-2 seems to be ATBF-1, which has two isoforms: ATBF-1A and ATBF-1B. As in the case of ZEB proteins, a recent report demonstrated that ATBF-1A and ATBF-1B have opposing effects on the regulation of muscle differentiation. These results raise the interesting possibility that in vertebrates, the Drosophila zfh family of zinc finger proteins may have evolved into proteins with opposing activities to balance signaling pathways during tissue differentiation and embryonic development. ZEB-1/deltaEF1 and ZEB-2/SIP1 are structurally quite similar and both repress transcription of a number of genes involved in differentiation and development. However, the results presented here indicate that ZEB proteins function antagonistically in the regulation of TGFß/BMP signaling (Postigo, 2003a).

Balancing signals derived from the TGFbeta family are crucial for regulating cell proliferation and differentiation, and in establishing the embryonic axis during development. TGFbeta/BMP signaling leads to the activation and nuclear translocation of Smad proteins, which activate transcription of specific target genes by recruiting P/CAF and p300. The two members of the ZEB family of zinc finger factors (ZEB-1/deltaEF1 and ZEB-2/SIP1) regulate TGFbeta/BMP signaling in opposite ways: ZEB-1/deltaEF1 synergizes with Smad-mediated transcriptional activation, while ZEB-2/SIP1 represses it. These antagonistic effects by the ZEB proteins arise from the differential recruitment of transcriptional coactivators (p300 and P/CAF) and corepressors (CtBP) to the Smads. Thus, while ZEB-1/deltaEF1 binds to p300 and promotes the formation of a p300-Smad transcriptional complex, ZEB-2/SIP1 acts as a repressor by recruiting CtBP. This model of regulation by ZEB proteins also functions in vivo, where they have opposing effects on the regulation of TGFbeta family-dependent genes during Xenopus development (Postigo, 2003b).

deltaEF1 and SIP1 (or Zfhx1a and Zfhx1b, respectively) are the only known members of the vertebrate Zfh1 family of homeodomain/zinc finger-containing proteins. Similar to other transcription factors, both Smad-interacting protein-1 (SIP1) and deltaEF1 are capable of repressing E-cadherin transcription through binding to the E2 boxes located in its promoter. In the case of deltaEF1, this repression has been proposed to occur via interaction with the corepressor C-terminal binding protein (CtBP). In this study, it is shown by coimmunoprecipitation that SIP1 and CtBP interact in vivo and that an isolated CtBP-binding SIP1 fragment depends on CtBP for transcriptional repression. However, and most importantly, full-length SIP1 and deltaEF1 proteins do not depend on their interaction with CtBP to repress transcription from the E-cadherin promoter. Furthermore, in E-cadherin-positive kidney epithelial cells, the conditional synthesis of mutant SIP1 that cannot bind to CtBP, abrogates endogenous E-cadherin expression in a similar way as wild-type SIP1. These results indicate that full-length SIP1 can repress E-cadherin in a CtBP-independent manner (van Grunsven, 2003).

Phosphorylation of ZFH-1 homologs

Zinc finger homeodomain enhancer-binding protein (Zfhep/Zfhx1a) is a transcription factor essential for immune system development, skeletal patterning, and life. Regulation of the interleukin-2 gene in T cells has been suggested to depend on post-translational processing of Zfhep, however, no modifications of Zfhep are known. This study demonstrates that Zfhep is present in both hyperphosphorylated and hypophosphorylated forms. Western blot analysis demonstrates two forms of Zfhep with different mobilities. Differences in phosphorylation are sufficient to explain the difference in mobilities. Zfhep is primarily phosphorylated on Ser and Thr residues since PP2A dephosphorylates the slower mobility band. Importantly, post-translational processing is cell-specific. Doublets of Zfhep were detected in five cell lines, whereas 6 cell lines contain only, or predominantly, non-phosphorylated Zfhep, and Saos-2 cells contain predominantly the phosphorylated form. These data provide the first demonstration that Zfhep is post-translationally modified (Constantino, 2002).

Effects of mutations in ZFH-1 homologs

Delta EF1 is a DNA binding protein containing a homeodomain and two zinc finger clusters, and is regarded as a vertebrate homologue of zfh-1 in Drosophila. In the developing embryo, delta EF1 is expressed in the notochord, somites, limb, neural crest derivatives and a few restricted sites in the brain and spinal cord. To elucidate the regulatory function of delta EF1 in mouse embryogenesis, delta EF1 null mutant ( delta EF1[null(lacZ)] ) mice were generated. The delta EF1[null(lacZ)] homozygotes develope to term, but never survived postnatally. In addition to severe T cell deficiency of the thymus, the delta EF1[null(lacZ)] homozygotes exhibit skeletal defects of various lineages:

  1. Craniofacial abnormalities of neural crest origin: cleft palate, hyperplasia of Meckel's cartilage, dysplasia of nasal septum and shortened mandible.
  2. Limb defects: shortening and broadening of long bones, fusion of carpal/tarsal bone and fusion of joints.
  3. Fusion of ribs.
  4. Sternum defects: split and asymmetric ossification pattern of the sternebrae associated with irregular sternocostal junctions.
  5. Hypoplasia of intervertebral discs.
These results indicate that delta EF1 has an essential role in regulating development of these skeletal structures. Since the skeletal defects are not observed in delta EF1(delta C727) mice lacking the C-terminal portion of the protein, but only the thymus defect, delta EF1 bears distinct regulatory activities that are dependent on different domains of the molecule (Takagi, 1998).

Hirschsprung disease (HSCR) is sometimes associated with a set of characteristics including mental retardation, microcephaly, and distinct facial features, but the gene mutated in this condition has not yet been identified. Mutations in SIP1, encoding Smad interacting protein-1, has been shown to cause disease in a series of cases. SIP1 is located in the deleted segment at 2q22 from a patient with a de novo t(2;13)(q22;q22) translocation. SIP1 seems to have crucial roles in normal embryonic neural and neural crest development (Wakamatsu, 2001).

Hirschsprung disease (HD) has been described in association with microcephaly, mental retardation and characteristic facial features, delineating a syndrome possibly caused by mutations localized at chromosome 2q22--q23. A de novo translocation breakpoint at 2q22 was analyzed in one patient presenting with this syndrome, and a gene, SIP1, was identified that is disrupted by this chromosomal rearrangement. SIP1 encodes Smad interacting protein 1, a new member of the delta EF1/Zfh-1 family of two-handed zinc finger/homeodomain transcription factors. The genomic structure and expression of the human SIP1 gene was determined. Further analysis of four independent patients showed that SIP1 is altered by heterozygous frameshift mutations causing early truncation of the protein. SIP1, among other functions, seems to play crucial roles in normal embryonic development of neural structures and neural crest. Its deficiency, in altering function of the TGF beta/BMP/Smad-mediated signalling cascade, is consistent with some of the dysmorphic features observed in this syndrome, in particular the enteric nervous system defect that underlies HD (Cacheux, 2001).

Mutations in ZFHX1B, the gene that encodes Smad-interacting protein-1 (SIP1), are implicated in the etiology of a dominant form of Hirschsprung disease-mental retardation syndrome in humans. To clarify the molecular mechanisms underlying the clinical features of SIP1 deficiency, mice were generated that bear a mutation comparable to those found in several human patients. Zfhx1b-knockout mice do not develop postotic vagal neural crest cells, the precursors of the enteric nervous system that is affected in patients with Hirschsprung disease, and they display a delamination arrest of cranial neural crest cells, which form the skeletomuscular elements of the vertebrate head. This suggests that Sip1 is essential for the development of vagal neural crest precursors and the migratory behavior of cranial neural crest in the mouse. Furthermore, Sip1 is involved in the specification of neuroepithelium (Van de Putte, 2003).

Dlx1&2-dependent expression of Zfhx1b (Sip1, Zeb2) regulates the fate switch between cortical and striatal interneurons

Mammalian pallial (cortical and hippocampal) and striatal interneurons are both generated in the embryonic subpallium, including the medial ganglionic eminence (MGE). This study demonstrates that the Zfhx1b (Sip1, Zeb2) zinc finger homeobox gene is required in the MGE, directly downstream of Dlx1&2, to generate cortical interneurons that express Cxcr7, MafB, and cMaf. In its absence, Nkx2-1 expression is not repressed, and cells that ordinarily would become cortical interneurons appear to transform toward a subtype of GABAergic striatal interneurons. These results show that Zfhx1b is required to generate cortical interneurons, and suggest a mechanism for the epilepsy observed in humans with Zfhx1b mutations (Mowat-Wilson syndrome) (McKinsey, 2013).

Targets of Zfh-1 homologs

Xenopus Brachyury (Xbra) plays a key role in mesoderm formation during early development. One factor thought to be involved in the regulation of Xbra is XSIP1 (Drosophila homolog: Zn finger homeodomain 1), a zinc finger/homeodomain-like DNA-binding protein that belongs to the deltaEF1 family of transcriptional repressors. Xbra and XSIP1 are co-expressed at the onset of gastrulation, but that expression subsequently refines such that Xbra is expressed in prospective mesoderm and XSIP1 in anterior neurectoderm. This refinement of the expression patterns of the two genes is due in part to the ability of XSIP1 to repress expression of Xbra. This repression is highly specific, in the sense that XSIP1 does not repress the expression of other regionally expressed genes in the early embryo, and that other members of the family to which XSIP1 belongs, such as deltaEF1 and its Xenopus homolog ZEB, cannot regulate Xbra expression. The function of XSIP1 was studied further by making an interfering construct comprising the open reading frame of XSIP1 fused to the VP16 transactivation domain. Experiments using this chimeric protein suggest that XSIP1 is required for normal gastrulation movements to occur and for the development of the anterior neural plate (Papin, 2002).

Sip1, a Smad-binding zinc-finger homeodomain transcription factor, a homolog of Zfh-1, has essential functions in embryonic development, but neither its role in individual tissues nor the significance of its interaction with Smad proteins has been fully characterized. In the lens lineage, Sip1 expression is activated after lens placode induction, and as the lens develops, the expression is localized in the lens epithelium and bow region where immature lens fibers reside. The lens-lineage-specific inactivation of the Sip1 gene was performed using mice homozygous for floxed Sip1 that carry a lens-specific Cre recombinase gene. This caused the development of a small hollow lens connected to the surface ectoderm, identifying two Sip1-dependent steps in lens development. The persistence of the lens stalk resembles a defect in Foxe3 mutant mice, and Sip1-defective lenses lose Foxe3 expression, placing Foxe3 downstream of Sip1. In the Sip1-defective lens, ß-crystallin-expressing immature lens fiber cells were produced, but gamma-crystallin-expressing mature fiber cells were absent, indicating the requirement for Sip1 activity in lens fiber maturation. A 6.2 kb Foxe3 promoter region controlled lacZ transgene expression in the developing lens, where major and minor lens elements were identified upstream of -1.26 kb. Using transfection assays, the Foxe3 promoter was activated by Sip1 and this activation is further augmented by Smad8 in the manner dependent on the Smad-binding domain of Sip1. This Sip1-dependent activation and its augmentation by Smad8 occur using the proximal 1.26 kb promoter, and are separate from lens-specific regulation. This is the first demonstration of the significance of Smad interaction in modulating Sip1 activity (Yoshimoto, 2005).

Smad-interacting protein-1 (Sip1) [Zinc finger homeobox (Zfhx1b)] is a transcription factor implicated in the genesis of Mowat-Wilson syndrome in humans. Sip1 expression in the dorsal telencephalon of mouse embryos has been documented from E12.5. The gene was inactivated specifically in cortical precursors. This resulted in the lack of the entire hippocampal formation. Sip1 mutant mice exhibited death of differentiating cells and decreased proliferation in the region of the prospective hippocampus and dentate gyrus. The expression of the Wnt antagonist Sfrp1 was ectopically activated, whereas the activity of the noncanonical Wnt effector, JNK, was down-regulated in the embryonic hippocampus of mutant mice. In cortical cells, Sip1 protein was detected on the promoter of Sfrp1 gene and both genes showed a mutually exclusive pattern of expression suggesting that Sfrp1 expression is negatively regulated by Sip1. Sip1 is therefore essential to the development of the hippocampus and dentate gyrus, and is able to modulate Wnt signaling in these regions (Miquelajauregui, 2007).

Drosophia ZFH-2 and its homologs

A 40-bp upstream regulatory region of the DOPA decarboxylase gene (Ddc) has been found to be important for cell-specific expression in the Drosophila central nervous system (CNS). This region contains two redundant elements that when simultaneously mutated result in lowered DDC expression in serotonin neurons. A factor has been cloned that binds to the site. This factor is the product of the zfh-2 gene, a complex homeodomain/zinc finger protein previously identified by binding to an opsin regulatory element. The in vivo profile of ZFH-2 in the larval CNS shows intriguing overlap with DDC in specific serotonin and dopamine neurons. ZFH-2 is related to a human transcription factor ATBF1. The multiple homeodomain and zinc finger motifs in these two proteins show a similar linear arrangement, implying coordinate action among the motifs. In addition, the homology defines a new homeodomain subtype (Lundell, 1992).

A mouse ATBF1 cDNA has been isolated which is 12-kb long and capable of encoding a 406-kDa protein containing four homeodomains and 23 zinc-finger motifs. Mouse ATBF1 is 94% homologous to the human ATBF1-A transcription factor. Northern blot and RNase protection analysis show that levels of ATBF1 transcripts are low in adult mouse tissues, but high in developing brain, consistent with a role for ATBF1 in neuronal differentiation (Ido, 1996).

ATBF1 is a transcription factor containing four homeodomains and 17 zinc fingers. Since the Drosophila homolog ZFH-2 is implicated in neurogenesis, ATBF1 expression was examined in developing mouse brain and in P19 mouse embryonal carcinoma cells during differentiation. Pre- and postnatal mouse brains express high levels of ATBF1 mRNA, but the adult brain contain only a small amount of ATBF1 transcripts. In P19 cells, ATBF1 transcripts are undetectable before differentiation; however, 1 day after induction of neuronal differentiation with retinoic acid, ATBF1 mRNA is expressed at a high level. This increased level reaches a maximum on the 4th day and then declines. No comparable level of ATBF1 mRNA is expressed when P19 cells are treated with dimethyl sulfoxide to induce muscle cells. These temporal patterns of ATBF1 expression in vivo and in vitro suggest that ATBF1 may play a role in neuronal differentiation (Ido, 1994).

The human ATBF1 cDNA, now termed ATBF1-B, encodes a 306-kDa protein containing 4 homeodomains and 18 zinc fingers including one pseudo zinc finger motif. A second ATBF1 cDNA, 12 kilobase pairs long, termed ATBF1-A has been isolated. The deduced ATBF1-A protein is 404 kDa in size and differs from ATBF1-B by a 920-amino acid extention at the N terminus. Analysis of 5'-genomic sequences shows that the 5'-noncoding sequences specific to ATBF1-A and ATBF1-B transcripts are contained in distinct exons that could splice to a downstream exon common to the ATBF1-A and ATBF1-B mRNAs. The expression of ATBF1-A transcripts increases to high levels when P19 and NT2/D1 cells are treated with retinoic acid to induce neuronal differentiation. Preferential expression of ATBF1-A transcripts is also observed in developing mouse brain. Transient transfection assays show that the 5.5-kilobase pair sequence upstream of the ATBF1-A-specific exon (exon 2) supports expression of the linked chloramphenicol acetyltransferase gene in neuronal cells derived from P19 cells but not in undifferentiated P19 or in F9 cells, which do not differentiate into neurons. These results show that ATBF1-A and ATBF1-B transcripts are generated by alternative promoter usage combined with alternative splicing, and that the ATBF1-A-specific promoter is activated during neuronal differentiation (Miura, 1995).

ATBF1 is a 306-kDa protein containing four homeodomains, 17 zinc finger motifs, and several segments potentially involved in transcriptional regulation. At least one of the homeodomains of ATBF1 binds to an AT-rich element in the human alpha-fetoprotein (AFP) enhancer (enhancer AT motif). In the present work, the transcriptional regulatory activity of ATBF1 is analyzed with respect to the enhancer AT motif and similar AT-rich elements in the human AFP promoter and the human albumin promoter and enhancer. ATBF1 binds efficiently to the AFP enhancer AT motif; however, it binds weakly or not at all to other AT-rich elements in the AFP and albumin regulatory regions studied. Alterations of the enhancer AT motif by site-specific mutagenesis results in the loss of binding of ATBF1. ATBF1 suppresses the activity of AFP enhancer and promoter regions containing AT-rich elements. This suppression is reduced when the mutated AT motifs with low affinity to ATBF1 are linked to the CAT gene. The ATBF1 suppression of AFP promoter and enhancer activities appears to be due, at least in part, to competition between ATBF1 and HNF1 for the same binding site. In contrast to the AFP promoter and enhancer, the albumin promoter and enhancer are not affected by ATBF1, although they contain homologous AT-rich elements. These results show that ATBF1 is able to distinguish AFP and albumin AT-rich elements, leading to selective suppression of the AFP promoter and enhancer activities (Yasuda, 1994).

Zfh-4 is a new member of a recently recognized zinc finger-homeodomain (zfh) family of putative transcription factors. Zfh-4 homeodomains are related to those of ATBF1. Only a fragment of zfh-4 has been cloned, and this fragment shares a 55% amino acid identity with a region of ATBF1 that extends from homeodomain 2 to homeodomain 3. zfh-4 expression is prominent in developing muscle and brain. In both tissues, zfh-4 RNA levels are highest embryonically, then decrease gradually to barely detectable levels in adults. In myogenic cell lines, far more zfh-4 is expressed in proliferating myoblasts than in myotubes, suggesting a cellular basis for the developmental regulation observed in vivo. In contrast, zfh-4 RNA in brain is more abundant in postmitotic cells of the marginal zone than in proliferating cells of the ventricular zone. Within the brain, zfh-4 RNA is regionally localized: expression is highest in midbrain, readily detectable in hindbrain, and very low in cerebral cortex. Its patterns of expression, and its homology to known DNA binding proteins, support the idea that Zfh-4 may be a regulator of gene expression in developing brain and muscle (Kostich, 1995).

Other zinc-finger homeodomain proteins

A cDNA encoding the complete reading frame of a novel homeodomain-containing protein, named zhx-1, has been cloned from a mouse bone marrow stromal cell line. The ORF encodes a protein of 873 amino acids, in which two C2-H2 zinc-fingers and five homeodomains have been identified. These features classify zhx-1 as a member of the ZF class of homeodomain transcription factors. The five zhx-1 homeodomains are more closely related to each other than to either the ATBF1 class homeodomain or to the Antp class of homeodomain, indicating only a distant relationship. zhx-1 mRNA is widely expressed in adult mouse, although it is preferentially expressed in brain. At least two homologous mRNAs are present in humans suggesting that zhx-1 is the first member of a novel subclass of homeodomain factors (Barthelemy, 1996).

Zn finger homeodomain 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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