Orange Domain Research Articles

Identification and characterization of human HESL, rat Hesl and rainbow trout hesl genes in silico.

Activation of Notch signaling pathway leads to nuclear translocation of Notch intracellular domain (NIC), and transcriptional activation of target genes through the interaction between CSL proteins (RBPSUH or RBPSUHL) and NIC. HES1, HES5, HEY1 and HEY2 are Notch target genes. Mammalian HES/HEY family proteins as well as Drosophila Hairy and Enhancer of split are implicated in the cell fate determination. We have previously identified and characterized human HES2, HES3 and HES5 genes. Here, we identified and characterized human HES-like (HESL), rat Fesl, and rainbow trout fesl genes by using bioinformatics. Human HESL gene, located within AC093824.3 genome sequence, was mapped to human chromosome 4q35.1 between ACSL1 and SLC25A4 genes. Rat Fesl gene, located within AC111303.4 genome sequence, was mapped to rat chromosome 16q11. EST BX875070.2 was the representative rainbow trout fesl cDNA. HESL-ACSL1 locus was conserved among human, rat, mouse, and zebrafish genomes. Human HESL (241 aa) showed 92.9% total-amino-acid identity with rat Hesl (240 aa), 92.1% total-amino-acid identity with mouse Hesl (240 aa), and 68.0% total-amino-acid identity with rainbow trout hesl (235 aa). HESL orthologs consist of basic Helix-loop-helix (bHLH) domain and ORANGE domain. C-terminal region of HESL orthologs were divergent from that of HES, HEY, and DEC homologs. Phylogenetic analyses revealed that bHLH transcription factors with ORANGE domain were classified into the following three groups: (group I) HES1, HES2, HES4 and HES6 orthologs; (group II) HESL, HES5, HES7, HEY1, HEY2 and HEYL orthologs; (group III) HES3, DEC1/BHLHB2 and DEC2/BHLHB3 orthologs.

Sequences downstream of the bHLH domain of the Xenopus hairy-related transcription factor-1 act as an extended dimerization domain that contributes to the selection of the partners

XHRT1 is a member of the HRT/Hey protein subfamily that are known as Notch effectors. XHRT1 is expressed in the developing floor plate and encodes a basic helix-loop-helix (bHLH) transcription repressor. Here, we show that XHRT1 misexpression in the neural plate inhibits differentiation of neural precursor cells and thus may be important for floor plate cells to prevent them from adopting a neuronal fate. Deletion analysis indicated that inhibition of differentiation by XHRT1 requires the DNA-binding bHLH motif and either the Orange domain or the C-terminal region. XHRT1 could efficiently homodimerize and heterodimerize with hairy proteins. Among those hairy genes, Xhairy2b shows extensive overlap of expression with XHRT1 in floor plate precursors and may be a biologically relevant XHRT1 partner. Dimerization is mediated through both the bHLH and downstream sequences, the Orange domain being particularly important for the efficiency of the interaction. Using chimeric constructs between XHRT1 and the ESR9 bHLH-O protein that does not interact with Xhairy1 and Xhairy2b, we found that both the bHLH domain and downstream sequences of XHRT1 were required for heterodimerization with Xhairy2b, while only the XHRT1 sequences downstream of the Orange domain are required for the interaction with Xhairy1. Together, these results suggest that XHRT1 plays a role in floor plate cell development and highlight the importance of the Orange and downstream sequences in dimerization and in the selection of the bHLH partners.

Transcriptional repression by the bHLH-Orange factor XHRT1 does not involve the C-terminal YRPW motif

Hairy-related transcription factors (HRTs) constitute a recently identified subfamily of basic-helix-loop-helix transcription factors containing an Orange domain (bHLH-O factors). As compared to the related HES proteins, HRTs exhibit distinct DNA-binding activities in vitro and the molecular mechanisms underlying their transcriptional activity remain poorly understood. We have identified here the sequence “ggCACGTGcc” as predominant binding site for Xenopus HRT1 (XHRT1). In transiently transfected 3T3 cells, XHRT1 represses the expression of a luciferase reporter gene under the control of multimerized XHRT1 binding sites. Deletion analysis indicated that repression by XHRT1 requires the presence of the DNA-binding bHLH motif and the Orange domain. However, the presence of the sequence motif YRPWGTEIGAF located at the very C-terminus of XHRT1 is dispensable. Accordingly, the groucho co-repressor, which is known to mediate transcriptional repression by HES factors through binding their C-terminal WRPW sequence, does not recognize the related YRPW motif present in the C-terminal part of XHRT1 significantly in vitro. As the C-terminus of HRTs is well conserved, our observation indicates that this part of HRTs, unlike the corresponding part of HES proteins, does not recruit the groucho co-repressor efficiently.

Identification and characterization of human HES2, HES3, and HES5 genes in silico

MMTV induces the mouse mammary tumor through the dysregulation of Notch, Wnt, or Fgf signaling pathway. Activation of Notch signaling pathway leads to transcriptional activation of Hes family genes through the interaction between Notch intracellular domain and RBPSUH (CSL). Hes family proteins are mammalian homologs of Drosophila Hairy and Enhancer of split. Hes family of transcriptional repressors with basic Helix-loop-helix (bHLH) and Orange domains are implicated in the cell fate determination of stem cells (or precursor cells) by suppressing the expression of tissue-specific transcriptional activators. Human HES1, HES4, HES6, and HES7 genes have been reported by other groups. Here, we identified and characterized human HES2, HES3 and HES5 genes by using bioinformatics. FLJ33803 (AK091122.1) was the representative human HES2 cDNA. HES2 gene, encoding a 173-aa protein, was located within human genome sequence AL031848.11. HES3 gene, encoding a 186-aa protein, was identified within human genome sequence AL031847.17. HES5 gene, encoding a 166-aa protein, was identified within human genome sequence AL139246.20. HES2 and HES3 genes were mapped to human chromosome 1p36.31, while HES5 gene to 1p36.32. HES2 mRNA was expressed in placenta, pancreatic cancer, colon cancer with RER, cervical cancer, and in head and neck tumors. HES5 mRNA was expressed in fetal heart, and brain tumors. Human HES family proteins were found consisting of bHLH, Orange, Proline-rich domains, and WRPW motif. Phylogenetic analyses revealed that HES family proteins were distantly related except a paralog pair of HES1 and HES4. HES family genes are pharmacogenomic targets in the field of regenerative medicine and oncology.

Her3, a zebrafish member of the hairy-E(spl) family, is repressed by Notch signalling.

her3 encodes a zebrafish bHLH protein of the Hairy-E(Spl) family. During embryogenesis, the gene is transcribed exclusively in the developing central nervous system, according to a fairly simple pattern that includes territories in the mesencephalon/rhombencephalon and the spinal cord. In all territories, the her3 transcription domain encompasses regions in which neurogenin 1 (neurog1) is not transcribed, suggesting regulatory interactions between the two genes. Indeed, injection of her3 mRNA leads to repression of neurog1 and to a reduction in the number of primary neurones, whereas her3 morpholino oligonucleotides cause ectopic expression of neurog1 in the rhombencephalon. Fusions of Her3 to the transactivation domain of VP16 and to the repression domain of Engrailed show that Her3 is indeed a transcriptional repressor. Dissection of the Her3 protein reveals two possible mechanisms for transcriptional repression: one mediated by the bHLH domain and the C-terminal WRPW tetrapeptide; and the other involving the N-terminal domain and the orange domain. Gel retardation assays suggest that the repression of neurog1 transcription occurs by binding of Her3 to specific DNA sequences in the neurog1 promoter. We have examined interrelationships of her3 with members of the Notch signalling pathway by the Gal4-UAS technique and mRNA injections. The results indicate that Her3 represses neurog1 and, probably as a consequence of the neurog1 repression, deltaA, deltaD and her4. Moreover, Her3 represses its own transcription as well. Surprisingly, and in sharp contrast to other members of the E(spl) gene family, transcription of her3 is repressed rather than activated by Notch signalling.

Helt, a Novel Basic-Helix-Loop-Helix Transcriptional Repressor Expressed in the Developing Central Nervous System

Neuronal differentiation is regulated by many basic-helix-loop-helix (bHLH) family transcriptional activators and repressors, and the balance of activity between these factors is important for the differentiation process. Here, we report the identification of a novel transcriptional repressor, designated Helt. Helt encoded a Hey-related bHLH protein containing the bHLH and Orange domains. Helt could homodimerize, and heterodimerize with Hes5 or Hey2. Both the bHLH and Orange domains were involved in the homodimerization. In contrast, only the bHLH domain was required for the heterodimerization with Hey2, whereas only the Orange domain mediated the interaction between Helt and Hes5. Thus, Helt has two dimerization domains, and these domains independently select a partner. Identification of preferred recognition sequences by CASTing experiments revealed that Helt bound to the E box, which was distinct from the Hes1 optimal sequence around the E box core. Not only the core sequence but also sequences flanking the E box were essential for the recognition by Helt and Hes1. Furthermore, Helt repressed transcription from an artificial promoter through binding to the optimal E box elements, as well as transcription from its own promoter. Using in situ hybridization and immunohistochemistry, Helt expression in embryos was investigated. Helt was mainly expressed in undifferentiated neural progenitors in some of the developing brain regions, including the mesencephalon and diencephalon, at the neurogenesis stage. These results suggest that Helt acts as a transcriptional repressor to regulate neuronal differentiation and/or identity.

Identification of BOIP, a novel cDNA highly expressed during spermatogenesis that encodes a protein interacting with the orange domain of the hairy‐related transcription factor HRT1/Hey1 in Xenopus and mouse

Hairy-related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix-loop-helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer-of-split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two-hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity.

Repression of XMyoD expression and myogenesis by Xhairy-1 in Xenopus early embryo

Activated Notch-Delta signalling was shown to inhibit myogenesis, but whether and how it regulates myogenic gene expression is not clear. We analyzed the implication of Xenopus hairy-1 (Xhairy-1), a member of the hairy and enhancer-of-split (E(spl)) family that may function as nuclear effector of Notch signalling pathway, in regulating XMyoD gene expression at the initial step of myogenesis. Xhairy-1 transcripts are expressed soon after mid-blastula transition and exhibits overlapping expression with Notch pathway genes such as Delta-1 in the posterior somitic mesoderm. We show that overexpression of Xhairy-1 blocks the expression of XMyoD in early gastrula ectodermal cells treated with the mesoderm-inducing factor activin, and in the mesoderm tissues of early embryos. It inhibits myogenesis and produces trunk defects at later stages. Xhairy-1 also inhibits the expression of the pan-mesodermal marker Xbra, but expression of other early mesoderm markers such as goosecoid and chordin is not affected. These effects require the basic helix-loop-helix (bHLH) domain, as well as a synergy between the central Orange domain and the C-terminus WRPW-Groucho-interacting domain. Furthermore, overexpression in ectodermal cells of Xhairy-1/VP16, in which Xhairy-1 repressor domain is replaced by the activator domain of the viral protein VP16, induces the expression of XMyoD in the absence of protein synthesis. Interestingly, Xhairy-1/VP16 does not induce the expression of Xbra and XMyf5 in the same condition. During neurulation, the expression of XMyoD induced by Xhairy-1/VP16 declines and the expression of muscle actin gene was never detected. These results suggest that Notch signalling through hairy-related genes may specifically regulate XMyoD expression at the initial step of myogenesis in vertebrates.

Molecular Cloning and Characterization of DEC2, a New Member of Basic Helix-Loop-Helix Proteins

DEC1 is a basic helix-loop-helix (bHLH) protein related to Drosophila Hairy, Enhancer of split and HES, and involved in the control of proliferation and/or differentiation of chondrocytes, neurons, etc. We report here the identification and characterization of human, mouse and rat DEC2, a novel member of the DEC subfamily. DEC2 had high (97%) and moderate (52%) similarities in the bHLH region and the Orange domain with DEC1, respectively. However, DEC2, but not DEC1, had alanine and glycine-rich regions in the C-terminal half. Unlike Hairy, Enhancer of split and HES, DEC2 lacked the WRPW motif for interaction with the corepressor Groucho. The DEC2 gene was mapped to human chromosome 12p11.23-p12.1, mouse chromosome 6 G2-G3 and rat chromosome 4q43 distal–q4, where the conserved linkage homology has been identified among these species. Unlike DEC1, which was broadly expressed in many tissues, DEC2 showed a more restricted pattern of mRNA expression. The DEC subfamily proteins may play an important role in tissue development.

Characterization of the Human and Mouse HEY1, HEY2, and HEYL Genes: Cloning, Mapping, and Mutation Screening of a New bHLH Gene Family

Many basic helix-loop-helix (bHLH) transcription factors are known as key regulators of embryonic development or differentiation in various species. We have isolated and characterized three new hairy-related bHLH transcription factor genes from mouse and human (hairy and Enhancer-of-split related with YRPW motif; HEY1, HEY2, and HEYL). All three HEY genes have a similar genomic structure with five exons. Together with a highly related Drosophila homologue, they form a new bHLH gene subfamily that is different from both hairy and the known vertebrate Hes and Her genes. While the overall structure with the bHLH domain, Orange domain, and WRPW motif is similar, the last motif is changed to KPYRPWG in Hey1/2 and absent in HeyL. This and other sequence features suggest Hey proteins to have unique functional properties. The genes were mapped by fluorescence in situ hybridization and RH mapping to the following human chromosomes: (HEY1) 8q21, (HEY2) 6q21, and (HEYL) 1p34.3. Based on expression patterns and map location, HEY genes are candidates for several human or mouse disease loci. However, initial screening of DNA from affected individuals for two human disorders and four mouse mutants did not reveal any diagnostic alterations in the coding regions.

Identification and Expression of a Novel Family of bHLH cDNAs Related to Drosophila Hairy and Enhancer of Split

In this report we describe the initial characterization of murine, human, and Drosophila hesr-1 (for hairy and enhancer of split related-1) a novel evolutionary conserved family of hairy/enhancer of split homologs. Hesr-1 cDNAs display features typical of hairy and enhancer of split-type bHLH proteins including a N-terminal bHLH domain a conserved orange domain immediately C-terminal to the bHLH region. Despite their similarity to known hairy/enhancer of split homologs, hesr-1 cDNAs are divergent members of the hairy and enhancer of split bHLH family since the degree of sequence identity within the bHLH and their nearest homologs are relatively low. Moreover, the tetrapeptide motif, WRPW, which is found in all hairy and enhancer of split family members, is not present in hesr-1. Rather, a variant of this motif, YRPW, is found. Analysis of embryonic murine hesr-1 expression by in situ hybridization reveals strong expression in the somitic mesoderm, the central nervous system, the kidney, the heart, nasal epithelium, and limbs indicating a role for hesr-1 in the development of these tissues. Like the enhancer of split cDNAs in Drosophila, we show that hesr-1 expression depends critically on signaling through the notch pathway in murine embryos, suggesting that aspects of hesr-1 regulation and function might also be evolutionary conserved.

Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and hairy-mediated transcriptional repression.

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairy-interacting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. We identified a P-element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.