retained

EVOLUTIONARY HOMOLOGS

The C. elegans hermaphrodite nervous system is composed of 302 neurons that fall into at least 118 diverse classes. cfi-1 contributes to the development of neuronal diversity. cfi-1 promotes appropriate differentiation of the URA sensory neurons and inhibits URA from expressing the male-specific CEM neuronal fate. The UNC-86 POU homeodomain protein is present in CEM and URA neurons, and can promote expression of CEM-specific genes in both CEM and URA, but CFI-1 inhibits expression of these genes in the URA cells. cfi-1 also promotes appropriate differentiation and glutamate receptor expression in the AVD and PVC interneurons. cfi-1 encodes a conserved neuron- and muscle-restricted DNA-binding protein containing an A/T rich interaction domain (ARID). In the eARID, CFI-1 is 70%, 73%, and 72% identical to the Drosophila Dead ringer protein, and the mammalian Dril1 (Bright) and Dril2 proteins, respectively. These proteins share other regions of similarity with CFI-1 as well, suggesting that they are orthologs. ARID proteins regulate early patterning and muscle fate in Drosophila, but they have not previously been implicated in the control of neuronal subtype identity (Shaham, 2002).

The four C. elegans CEM neurons are male-specific head neurons with morphological features suggesting a chemosensory function, perhaps in regulating male-specific behavior or attraction to hermaphrodites. The development of these cells shares some features with the development of the hermaphrodite-specific neurons (HSNs). For example, the CEMs migrate: they are born in the anterior of the animal and receive cues instructing them to migrate to their final, more posterior positions. CEMs also undergo programmed cell death in a sex-specific manner. However, in contrast to the HSN neurons, CEMs survive in males and die in hermaphrodites. In addition, during the transition from the L4 stage to the adult, the CEMs acquire a prominent nucleolus and begin to express the ion-channel proteins PKD-2 and LOV-1 (Shaham, 2002 and references therein).

CFI-1 regulates cell identity and promotes diversity in the C. elegans nervous system. In cfi-1 mutants, two differation responses are disrupted: the differentiation of URA and IL2 sensory neurons (both IL2s and CEMs are sensory neurons with dendritic processes that are physically exposed to the environment) and the differentiation of AVD and PVC interneurons (AVD, AVA, AVB, and PVC interneurons mediate responses initiated by the touch response cells). In the URA and IL2 neurons, cfi-1 mutations result in the inappropriate acquisition of aspects of the CEM neuronal fate. Because the dorsal URA neurons are sister cells of the dorsal CEM neurons, it is possible that cfi-1 responds to an asymmetric cell lineage signal that is differentially distributed between the dorsal URA and the dorsal CEM neurons. Cells unrelated by lineage to the CEMs (the IL2 neurons and the ventral URAs) can also acquire CEM fate in cfi-1 mutants, suggesting that cfi-1 may also respond to other kinds of cues that regulate cell fate (Shaham, 2002).

CFI-1 probably controls neuronal fate by regulating a specific set of target genes. Such a function is consistent with the sequence-specific DNA binding of CFI-1, and with the observation that the mouse Bright (Dril1) protein, a protein highly related to CFI-1, regulates expression from the immunoglobulin heavy chain enhancer. Interestingly, cfi-1 may function redundantly with other genes to regulate neuronal subtype identity, because considerable phenotypic variability is seen even in the strong cfi-1(ky651) mutant (Shaham, 2002).

Transcriptional regulators play important roles in creating diversity in the nervous system. The most well-understood cell fate regulators are the families of homeodomain proteins. The POU homeodomain proteins, including C. elegans UNC-86 (Drosophila homolog, ACJ6) affect development of many neurons; the Pax homeodomain proteins subdivide the vertebrate neural tube into distinct domains; and the LIM-homeodomain proteins generate a variety of motor neurons in the vertebrate spinal cord and contribute to the diversity of sensory neurons and interneurons in C. elegans. Homeodomain proteins typically function in a context that is defined by other DNA-binding proteins. In the URA and IL2 neurons, CFI-1 represents an important factor that modifies the activity of UNC-86 to match a particular cell type (Shaham, 2002).

unc-86 is essential for the development of the URA and IL2 neurons, where it induces expression of cfi-1. cfi-1, in turn, activates normal URA and IL2 gene expression and prevents inappropriate expression of CEM-specific genes. If cfi-1 is absent from URA and IL2 neurons, unc-86 promotes expression of the CEM-specific marker pkd-2::GFP instead, as it would normally do in CEM neurons. These results indicate that during normal development a cfi-1-dependent activity prevents unc-86-dependent induction of CEM-specific genes in URA and IL2 neurons. Such a cfi-1-dependent activity could function by qualitatively changing UNC-86 from an activator of gene expression to a repressor. Alternatively, a cfi-1-dependent activity could prevent the association of UNC-86 or an UNC-86 target protein with regulatory regions of CEM-specific genes. In either model, the absence of CFI-1 function in CEM neurons would allow UNC-86 to activate pkd-2 in these cells (Shaham, 2002).

CFI-1 and the Drosophila Dead ringer protein are very similar and are likely orthologs. dead ringer is required for anterior-posterior patterning and muscle development in the Drosophila embryo. dead ringer RNA is maternally contributed to embryos and is expressed in a restricted pattern during embryogenesis. Initially, dead ringer is expressed in three major bands in the embryo, with the anterior and posterior bands being most prominent. Following gastrulation, dead ringer expression becomes restricted to mesoderm (including muscles), pharyngeal muscles, a small group of cells in the brain, a regular array of cells in the ventral nervous system, and a small number of other tissues including the hindgut. This expression pattern is reminiscent of CFI-1 expression in C. elegans, where expression is detected primarily in head and tail cells, in pharyngeal and head muscles, in a small group of neurons near the nerve ring (the brain of the animal), and in ventral cord neurons. In contrast to Dead ringer, no early ubiquitous expression of CFI-1 is detected in C. elegans, nor is CFI-1 expressed in the gut or germ line, but cfi-1::GFP is expressed in posterior gut nuclei (Shaham, 2002).

Their similar sequences, DNA-binding properties, and expression patterns suggest that CFI-1 and Dead ringer proteins function in similar ways to regulate cell fate. It is speculated that Dead ringer contributes to neuronal diversity in Drosophila. Although the expression patterns of the mammalian CFI-1-related proteins Bright and Dril2 have not been described in the developing nervous system, it is proposed that these, or other related proteins, may function during development of the mammalian nervous system as well (Shaham, 2002).

B lymphocyte-restricted transcription of immunoglobulin heavy-chain (IgH) genes is specified by elements within the variable region (VH) promoter and the intronic enhancer (E mu). The gene has been cloned that encodes a protein that binds a VH promoter proximal site necessary for induced mu-heavy-chain transcription. This B-cell specific protein, termed Bright (B cell regulator of IgH transcription), is found in both soluble and matrix insoluble nuclear fractions. Bright binds the minor groove of a restricted ATC sequence that is sufficient for nuclear matrix association. This sequence motif is present in previously described matrix-associating regions (MARs) proximal to the promoter and flanking E mu. Bright can activate E mu-driven transcription by binding these sites, but only when they occur in their natural context and in cell lines permissive for E mu activity. To bind DNA, Bright requires a novel tetramerization domain and a previously undescribed domain that shares identity with several proteins, including SWI1, a component of the SWI/SNF complex (Herrscher, 1995)

The Drosophila gene dead ringer (dri) was isolated as a novel gene encoding a sequence-specific DNA-binding protein. Dri is a founding member of a growing protein family whose members share a conserved DNA binding domain termed the A/T-rich interaction domain. dri is developmentally regulated, being expressed in a restricted set of cells including some neural cells and differentiating cells of the gut and salivary gland ducts. The mouse homolog of dri, bright, has been shown to be expressed in mature B-cells in the immune system; its product trans-activates expression through an IgH enhancer in transient transfection assays. A human dri/bright homolog, termed DRIL1, has been cloned. The exon-intron structure of DRIL1 is reported and physical linkage within 80 kb to the D19S886 marker on 19p13.3 has been demonstrated. Since this marker is intimately linked to the Peutz-Jeghers syndrome in several large pedigrees, human dri (DRIL1) is a candidate gene for this disorder (Kortschak, 1998).

The B cell regulator of Ig heavy chain transcription (Bright) is a DNA-binding protein that was originally discovered in a mature Ag-specific B cell line after stimulation with IL-5 and Ag. It binds to the intronic heavy chain enhancer and 5' of the V1 S107 family V(H) promoter. Several studies suggested that Bright may increase transcription of the heavy chain locus, and expression in cell lines was limited to those representing mature B cells. Normal hemopoietic tissues have been analyzed for the expression of Bright during B lymphocyte differentiation. Bright is expressed in a subset of mature spleen cells, but also in a subset of normal B lymphocytic progenitors in both adult bone marrow (BM) and in fetal liver as early as day 12 of gestation. Bright is also expressed in the small percentage of CD4(low) cells in the thymus that are newly arrived from the BM and are not yet committed to the T lymphocyte lineage, but is not observed at later stages of T cell differentiation in either the spleen or thymus. Bright mRNA is not detected in the immature B lymphocytes that initially populate the spleen after migration from the BM. In addition, new splice variants of Bright have been observed in fetal tissues. Thus, Bright expression is highly regulated in normal murine lymphocytes and occurs both early and late during B cell differentiation. These findings may have important implications for the function of Bright in regulating Ig transcription (Webb, 1998).

The gene BDP has been cloned; it encods a protein with homology to the retinoblastoma-binding proteins Rbp1 and Rbp2. It also has homology to DNA-binding proteins such as Bright, a B-cell-specific trans-activator, and the Drosophila melanogaster dead ringer gene product. Like MyoD, Bdp binds to the COOH-terminal region of pRb through its conserved region and to hypophosphorylated pRb. It also binds to the MAR of the immunoglobulin heavy-chain locus. Thus Bdp may contribute to the transcriptional regulation of genes involved in differentiation and tissue-specific expression (Numata, 1999).

Nuclear matrix attachment regions (MARs) flanking the immunoglobulin heavy chain intronic enhancer (Emu) are the targets of the negative regulator NF-muNR, which is found in non-B and early pre-B cells. Expression library screening with NF-muNR binding sites yielded a cDNA clone encoding an alternatively spliced form of the Cux/CDP homeodomain protein. Cux/CDP fulfills criteria required for NF-muNR identity. It is expressed in non-B and early pre-B cells but not mature B cells. It binds to NF-muNR binding sites within Emu with appropriate differential affinities. Antiserum specific for Cux/CDP recognizes a polypeptide of the predicted size in affinity-purified NF-muNR preparations and binds NF-muNR complexed with DNA. Cotransfection with Cux/CDP represses the activity of Emu via the MAR sequences in both B and non-B cells. Cux/CDP antagonizes the effects of the Bright transcription activator at both the DNA binding and functional levels. It has been proposed that Cux/CDP regulates cell-type-restricted, differentiation stage-specific Emu enhancer activity by interfering with the function of nuclear matrix-bound transcription activators (Wang, 1999).

The jumonji gene was originally identified by the gene trap strategy and encodes a protein that is partially homologous to the AT-rich interaction domain (ARID) of the DNA binding proteins such as Dead Ringer in Drosophila, Bright in mouse and SWI1 in yeast. Homozygous jumonji (jmj^-/jmj^-) mice exhibit hepatic hypoplasia and defective hematopoiesis in the liver and die at around embryonic day 15.5 (E15.5), suggesting that jmj is essential for liver development. In order to gain insight into the mechanism of liver development, the expression and function of jmj was examined in fetal hepatocytes. The number of hepatocytes in jmj^-/jmj^- mice is markedly reduced in comparison with control mice and the expression of jmj in hepatocytes increases along with development. Since jmj^-/jmj^- embryos die by E15.5, an in vitro culture system was employed in which fetal hepatocytes differentiate in response to oncostatin M. The proliferation potential of jmj^-/jmj^- hepatocytes was comparable to that of wild type cells in vitro, however maturation of hepatocytes as evidenced by the expression of liver enzymes such as tyrosine amino transferase is severely impaired by the jmj gene inactivation. These results suggest that jmj plays a pivotal role in the development of mid-fetal hepatocytes to the neonatal stage (Anzai, 2003).

The ARID domain protein dril1 is necessary for TGF(beta) signaling in Xenopus embryos

ARID domain proteins are members of a highly conserved family involved in chromatin remodeling and cell-fate determination. Dril1 is the founding member of the ARID family and is involved in developmental processes in both Drosophila and Caenorhabditis elegans. This study describes he first embryological characterization of this gene in chordates. Dril1 mRNA expression is spatiotemporally regulated and is detected in the involuting mesoderm during gastrulation. Inhibition of dril1 by either a morpholino or an engrailed repressor-dril1 DNA binding domain fusion construct inhibits gastrulation and perturbs induction of the zygotic mesodermal marker Xbra and the organizer markers chordin, noggin, and Xlim1. Xenopus tropicalis dril1 morphants also exhibit impaired gastrulation and axial deficiencies, which can be rescued by coinjection of Xenopus laevis dril1 mRNA. Loss of dril1 inhibits the response of animal caps to activin and secondary axis induction by smad2. Dril1 depletion in animal caps prevents both the smad2-mediated induction of dorsal mesodermal and endodermal markers and the induction of ventral mesoderm by smad1. Mesoderm induction by eFGF is uninhibited in dril1 morphant caps, reflecting pathway specificity for dril1. These experiments identify dril1 as a novel regulator of TGFβ signaling and a vital component of mesodermal patterning and embryonic morphogenesis (Callery, 2005).

A DNA binding consensus sequence for murine dril1 has been identified, and the MatInspector program (Genomatix) was used to investigate whether several TGFβ-responsive Xenopus promoters contain this putative dril1 binding site. The activin-inducible gene, Xbra, which is down-regulated in dril morphants, has three putative dril1 binding sites in its promoter and is thus a promising candidate for direct regulation by dril1. The induction of Xlim1 by smad2 is also dril1 dependent, and the intronic region of Xlim1 that mediates activin responsiveness also contains two possible dril1 binding sites. A third activin-inducible gene, HNF1α, contains six sequences matching the dril1 binding consensus, four of which have no overlapping sequence, so it will be interesting to investigate whether dril1 is involved in transcriptional regulation of this gene. Interestingly, putative dril1 binding sites were not identified in several promoters that contain either activin- or BMP-responsive elements (AREs or BREs), including the goosecoid, mix2, and bambi promoters. dril1 is required for induction of goosecoid by smad2, and it was found that both the dril1 morpholino and EnR–dril impair the activation of mix2 by smad2. How might dril1 regulate the expression of these genes if no binding sites are identified in their promoters? If dril1 acts as a regulator of chromatin architecture, it may bind regulatory elements further upstream than the promoter sequences analyzed in this study. Alternatively, the dril1-dependent genes whose promoters lack a dril1 binding site may be indirect targets whose transcription is activated by an intermediary protein. A third possibility is that dril1 can bind to sequences other than the canonical consensus identified by the MatInspector program; however, it is also possible that the consensus sequences identified in Xbra, Xlim1, and HNF1α may not function as dril1 binding sites in vivo. Therefore, it is important to note that silico analysis, while a useful preliminary step, cannot substitute for an empirical investigation of promoter binding (Callery, 2005).

Considering that the regulation of gastrulation by brachyury is conserved among vertebrates, it is likely that dril1 plays a role in gastrulation throughout this group. The involvement of dril1 in gastrulation may be conserved throughout deuterostomes because dril1 has recently been shown to be necessary for gastrulation movements in the echinoderm S. purpuratus. However, there is little similarity between the regulatory networks modulated by dril in the two deuterostome groups: dril depletion has no effect on brachyury, lim1, or bmp4 expression in the echinoderm or on the battery of endomesodermal patterning genes assayed. Even within the echinoderms, the presence of brachyury in the presumptive mesoderm is quite variant -- its roles in endoderm patterning and invagination appear more ancient. A contributory factor in the failure of gastrulation in dril-depleted sea urchin embryos may be the inhibition of goosecoid, which is required for gastrulation and greatly reduced by dril depletion. In vertebrates, smad2 is involved in activation of the goosecoid promoter. The role of smad signaling in echinoderm development is unknown so it is not possible to determine whether dril mediates its effects on this deuterostome group through inhibition of these transcription factors. However, because components of the TGFβ regulatory pathway are known to function in flies and worms, it will be interesting to determine whether dril also modulates this pathway in protostomes (Callery, 2005).

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