grainy head


Identification and expression of Grainyhead family proteins

GRH has weak homology to the HLH motifs of myoD and myogen (Bray, 1989).

Human transcription factor LSF, which binds simian virus 40 late promoter has sequence homology to GRH (Shirra, 1994).

Human LBP-1, which binds to human immunodeficiency virus type 1 initiation site has sequence homology to GRH (Yoon, 1994).

CP2 is a cellular transcription factor that interacts with the alpha-globin promoter as well as with additional cellular and viral promoter elements. Genes for the murine and human mRNAs contain 16 and 15 exons, respectively. Both genes span approximately 30 kilobases of chromosomal DNA; among coding exons, all exon/intron boundaries are conserved. The human gene for CP2 resides on chromosome 12, while the murine gene maps to the distal end of chromosome 15, near Gdc-1, Wnt-1, and Rarg, a region syntenic with human chromosome 12. The murine and human promoters initiate mRNAs at multiple start sites in a conserved region that span more than 450 nucleotides. A study of the pattern of CP2 gene expression shows that the factor is expressed in all adult and fetal murine tissues examined from at least day 9.5 of development (Swendeman, 1994).

CP2-related proteins comprise a family of DNA-binding transcription factors that are generally activators of transcription and expressed ubiquitously. A differential display polymerase chain reaction fragment, Psc2, is reported that was expressed in a regulated fashion in mouse pluripotent cells in vitro and in vivo. The Psc2 cDNA contains an open reading frame homologous to CP2 family proteins. Regions implicated in DNA binding and oligomeric complex formation, but not transcription activation, were conserved. Psc2 expression in vivo during embryogenesis and in the adult mouse demonstrated tight spatial and temporal regulation, with the highest levels of expression in the epithelial lining of distal convoluted tubules in embryonic and adult kidneys. Functional analysis has demonstrated that PSC2 represses transcription 2.5-15-fold when bound to a heterologous promoter in ES, 293T, and COS-1 cells. The N-terminal 52 amino acids of PSC2 were shown to be necessary and sufficient for this activity and did not share obvious homology with reported repressor motifs. These results represent the first report of a CP2 family member that is expressed in a developmentally regulated fashion in vivo and that acts as a direct repressor of transcription. Accordingly, the protein has been named CP2-Related Transcriptional Repressor-1 (Rodda, 2001).

The Drosophila transcription factor Grainyhead regulates several key developmental processes. Three mammalian genes, CP2, LBP-1a and LBP-9 have been previously identified as homologues of grainyhead. This study reports the cloning of two new mammalian genes [Mammalian grainyhead (MGR) and Brother-of-MGR (BOM)] and one new Drosophila gene (dCP2) that rewrite the phylogeny of this family. MGR and BOM are more closely related to grh, whereas CP2, LBP-1a and LBP-9 are descendants of the dCP2 gene. MGR shares the greatest sequence homology with grh, is expressed in tissue-restricted patterns more comparable to grh and binds to and transactivates the promoter of the human Engrailed-1 gene, the mammalian homologue of the key grainyhead target gene, engrailed. This sequence and functional conservation indicates that the new mammalian members of this family play important developmental roles (Wilanowski, 2002).

To determine the extent of the functional homology between Grainyhead and MGR, whether the mammalian protein could bind to the well-characterised binding sites for the Drosophila factor in the dopa decarboxylase and PCNA gene regulatory regions was examined. Oligonucleotide probes encompassing these sites were incubated with nuclear extract from the human placental cell line JEG-3, which expresses both isoforms of MGR at mRNA and protein levels, and an electrophoretic mobility shift assay (EMSA) was performed. A specific protein/DNA complex was observed with the PCNA probe in the presence of preimmune sera (lanes 1 and 3). This complex supershifts with the addition of anti-p70 MGR specific antisera raised against peptides in the amino terminal region of the protein and ablates with the addition of anti-MGR antisera raised against peptides common to p49 and p70 MGR in the dimerisation domain of the protein. Similar results were obtained with the Dopa decarboxylase promoter probe (Wilanowski, 2002).

Many Drosophila genes regulated by Grainyhead have known mammalian homologues. In terms of functional homology, Engrailed-1 (En-1) is one of the best characterised. Mice expressing Drosophila engrailed in place of En-1 have a near complete rescue of the lethal En-1 mutant brain defect and most skeletal abnormalities. The En-1 promoter was examined for the grainyhead consensus DNA binding sequence derived from a comparison of the Drosophila Ultrabithorax, Dopa decarboxylase and fushi tarazu promoters. A highly conserved region was identified in the proximal En-1 promoter. Moreover, this sequence was also largely conserved in the DNAseI footprint attributed to grainyhead in the Drosophila engrailed promoter. The ability of this region of the human En-1 promoter to compete off MGR binding to the Ddc probe was examined in an EMSA with nuclear extract from JEG-3 cells. The specific MGR/DNA complex observed with the Ddc probe was supershifted with the addition of MGR antisera 67 and ablated with the addition of a 50-fold excess of unlabelled Ddc probe as competitor. Addition of a 10- or 20-fold excess of unlabelled En-1 probe also markedly reduced the binding of MGR to the Ddc probe. To determine the functional significance of this binding, this region of the En-1 promoter was linked to a minimal globin gene promoter/luciferase reporter gene construct and transfected into the MGR null cell line COS, in the presence of a p70 MGR mammalian expression vector or the empty vector. Transfection of the minimal promoter/reporter or the TK promoter linked to a Renilla luciferase gene with either vector served as the controls. Expression of p70 MGR dramatically enhanced the transcriptional activity of the En-1 promoter (Wilanowski, 2002).

In addition to the existence of specific isoforms, the grainyhead-like factors also achieve functional diversity through the formation of homo- and hetero-meric complexes. Therefore whether protein complexes could be formed between MGR, BOM and the CP2-like proteins was examined. Initial studies used the yeast two-hybrid assay system to assess these protein/protein interactions. The dimerisation domains of the four grainyhead-like factors were cloned into a yeast expression vector in frame with the GAL4 DNA binding domain and a second yeast vector in frame with the GAL4 activation domain. The constructs were co-transfected into yeast in various combinations and interactions assessed by activation of a histidine reporter gene and growth on selective media plates. MGR is capable of homodimerisation, as well as heterodimerisation with BOM. Very small colonies of questionable significance were detected with CP2 and LBP-1a. No colonies were observed with an unrelated transcription factor (SCL). Studies with BOM also demonstrated its homodimeric capabilities and confirmed the interactions between BOM and MGR. No significant interactions were observed with CP2, LBP-1a or SCL. Significant activation of the histidine reporter in these experiments was accompanied by concomitant activation of the second reporter, LacZ, with all colonies staining blue with X-gal (Wilanowski, 2002).

This sequence, expression and functional analyses suggested that MGR represented the closest mammalian homologue of grh and BOM was a highly related member of this arm of the family. However, they also suggested that CP2/LBP-1a and LBP-9 were in a functionally distinct arm of the family. This raised the possibility that an additional Drosophila gene may exist, the homologue of CP2/LBP-1a/LBP-9. To address this, the EST databasewas screened using human CP2 as a query and a Drosophila entry was identified that differed significantly from grainyhead. Primers derived from this EST were used to amplify a probe from a Drosophila embryo cDNA library, which was subsequently employed to clone a full-length cDNA from a Drosophila head library. The cDNA encodes a novel Drosophila gene that has been called Drosophila CP2 (dCP2). Amino acid sequence comparison between this gene and the mammalian members of the grainyhead-like family revealed that dCP2 aligns more closely to CP2 and LBP-1a than MGR or BOM (Wilanowski, 2002).

To examine the grainyhead and dCP2-like genes in an evolutionary context, a phylogenetic analysis was performed. The previously identified homologues were included in this family, CP2, LBP-1a and LBP-9, the newly identified genes MGR, BOM and dCP2, as well as another mammalian grainyhead-like gene Sister-of-MGR (SOM). Two distinct branches of this tree exist. The first contains grainyhead and its closest mammalian homologue MGR and the highly related BOM and SOM. The second contains dCP2 and the mammalian CP2, LBP-1a and LBP-9 genes. This evolutionary divergence supports the hypothesis that these factors play distinct roles during mammalian development (Wilanowski, 2002).

The Drosophila gene grainyhead is the founding member of a large family of genes encoding developmental transcription factors that are highly conserved from fly to human. The family consists of two main branches, with grainyhead as the ancestral gene for one branch and the recently cloned Drosophila CP2 (Gemini) as the ancestral gene for the other. This family is now extended with the identification of another novel mammalian member, Sister-of-Mammalian Grainyhead (SOM), which is phylogenetically aligned with grainyhead. SOM is closely related to the other mammalian homologs of grainyhead, including Mammalian Grainyhead (MGR) and Brother-of-MGR, sharing a high degree of sequence identity with these factors in the functional DNA-binding, protein dimerization and activation domains. Protein interaction studies demonstrate that SOM can heterodimerize with MGR and Brother-of-MGR, but not with the more distant members of the family. Like grainyhead, the SOM gene too produces several distinct isoforms with differing functional properties through alternative splicing. The tissue distributions of these isoforms differ and all display highly restricted expression patterns. These findings indicate that SOM, like its family members, may play important roles in mammalian development (Ting, 2003a).

Interaction with DNA of Grainyhead family proteins

The mammalian transcription factor LSF (CP2/LBP-1c) binds cellular promoters modulated by cell growth signals. LSF-DNA-binding activity is strikingly regulated by induction of cell growth in human peripheral T lymphocytes. Within 15 min of mitogenic stimulation of these cells, the level of LSF-DNA-binding activity increases by a factor of five. The level of LSF protein in the nucleus remains constant throughout this interval. However, a rapid decrease in the electrophoretic mobility of LSF, attributable to phosphorylation, correlates with the increase in DNA-binding activity. pp44 (ERK1) phosphorylates LSF in vitro on the same residue that is phosphorylated in vivo, specifically at amino acid position 291, as indicated by mutant analysis. As direct verification of the causal relationship between phosphorylation and DNA-binding activity, treatment in vitro of LSF with phosphatase both increases the electrophoretic mobility of the protein and decreases LSF-DNA-binding activity. This modulation of LSF-DNA-binding activity as T cells progress from a resting to a replicating state reveals that LSF activity is regulated during cell growth and suggests that LSF regulates growth-responsive promoters (Volker, 1997).

The mammalian transcription factor LSF (also known as CP2 and LBP-1c) binds as a homo-oligomer to directly repeated elements in viral and cellular promoters. LSF and the Drosophila transcription factor NTF-1 (also known as Elf-1 and Grainyhead) share a similar DNA binding region, which is unlike any other established DNA binding motif. Dimeric NTF-1 can bind an LSF half-site, whereas LSF cannot. To characterize further the DNA binding and oligomerization characteristics of LSF, truncation mutants were used to demonstrate that between 234 and 320 amino acids of LSF are required for high affinity DNA binding. Mixing of a truncation mutant with full-length LSF in a DNA binding assay establishes that the form of LSF that binds DNA is larger than a dimer. Unexpectedly, one C-terminal deletion derivative, partially defective in oligomerization properties, can occupy odd numbers of adjacent, tandem LSF half-sites, unlike full-length LSF. The numbers of DNA-protein complexes formed on multiple half-sites with this mutant indicates that LSF binds DNA as a tetramer, although cross-linking experiments confirm a previous report concluding that LSF is primarily dimeric in solution. The DNA binding and oligomerization properties of LSF support models depicting novel mechanisms to prevent continual, adjacent binding by a protein that recognizes directly repeated DNA sequences (Shirra, 1998).

Protein interactions of Grainyhead family proteins

The neural protein Fe65 possesses three putative protein-protein interaction domains: one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1 and PID2); the most C-terminal of these domains (PID2) interacts in vivo with the Alzheimer's beta-amyloid precursor protein, whereas the WW domain binds to Mena, the mammalian homolog of Drosophila Enabled protein. By the interaction trap procedure, a cDNA clone encoding a possible ligand of the N-terminal PID/PTB domain of Fe65 (PID1) was isolated. Sequence analysis of this clone reveals that this ligand corresponds to the previously identified transcription factor CP2/LSF/LBP1. Co-immunoprecipitation experiments demonstrate that the interaction between Fe65 and CP2/LSF/LBP1 also takes place in vivo between the native molecules. The localization of both proteins was studied using fractionated cellular extracts. These experiments demonstrate that the various isoforms of CP2/LSF/LBP1 are differently distributed among subcellular fractions. At least one isoform, derived from alternative splicing (LSF-ID), is present outside the nucleus; Fe65 was found in both fractions. Furthermore, transfection experiments with an HA-tagged CP2/LSF/LBP1 cDNA demonstrate that Fe65 interacts also with the nuclear form of CP2/LSF/LBP1. Considering that the analysis of Fe65 distribution in fractionated cell extracts demonstrates that this protein is present both in nuclear and non-nuclear fractions, the expression of Fe65 deletion mutants was examined in the two fractions. This analysis results in the observation that a small region N-terminal to the WW domain is phosphorylated and is necessary for the presence of Fe65 in the nuclear fraction (Zambrano, 1998).

Functional conservation between members of an ancient duplicated transcription factor family, LSF/Grainyhead

The LSF/Grainyhead transcription factor family is involved in many important biological processes, including cell cycle, cell growth and development. In order to investigate the evolutionary conservation of these biological roles, two new family members in Caenorhabditis elegans and Xenopus laevis have been characterized. The C.elegans member, Ce-GRH-1, groups with the Grainyhead subfamily, while the X.laevis member, Xl-LSF, groups with the LSF subfamily. Ce-GRH-1 binds DNA in a sequence-specific manner identical to that of Drosophila melanogaster Grainyhead. The preference for an adenine immediately upstream and three thymidines immediately downstream of the central C(A/C/T)(T/G)G is consistent with the lack of binding in DNAs where these positions exhibit transversions. In addition, Ce-GRH-1 binds to sequences upstream of the C.elegans gene encoding aromatic L-amino-acid decarboxylase and genes involved in post-embryonic development, mab-5 and dbl-1. All three C.elegans genes are homologs of D.melanogaster Grainyhead-regulated genes. RNA-mediated interference of Ce-grh-1 results in embryonic lethality in worms, accompanied by soft, defective cuticles. These phenotypes are strikingly similar to those observed previously in D.melanogaster grainyhead mutants, suggesting conservation of the developmental role of these family members over the course of evolution. The phylogenetic analysis of the expanded LSF/GRH family (including other previously unrecognized proteins/ESTs) suggests that the structural and functional dichotomy of this family dates back more than 700 million years, i.e., to the time when the first multicellular organisms are thought to have arisen (Venkatesan, 2003).

Computational analysis of the expanded LSF/GRH family suggest that the phylogenetic division into two subfamilies reflects significant differences in structure and function between them. Functionally, Grainyhead, the best studied protein in its subfamily, is mainly a regulator of developmental control genes in D.melanogaster, such as Ultrabithorax, dopa decarboxylase, tailless and decapentaplegic. Fruit flies carrying grainyhead mutations display an embryonic lethal phenotype. Similarly, H.sapiens MGR (presumably the H.sapiens ortholog of grainyhead) binds to and can activate the promoter of engrailed, a gene involved in development. Ce-GRH-1, the putative C.elegans ortholog, binds the promoters of the gene encoding aromatic L-amino acid decarboxylase and genes involved in post-embryonic development, mab-5 and dbl-1. These genes are all homologs of D.melanogaster Grainyhead-regulated genes. It remains to be seen if Ce-GRH-1 regulates the transcription of these genes in vivo. Ce-grh-1 phenotypic knockout worms generated by RNAi analysis are late embryonic lethal and have cuticles apparently defective in rigidity. The observed cuticle rupturing in these RNAi worms suggests that their cuticles are too weak to maintain structural integrity. These phenotypes are strikingly similar to the phenotypes of grainyhead mutant fruit flies, which also die at the end of embryogenesis, have granular head skeletal structures and misshapen, weak cuticles that manifest distended bulges and rupture easily. In addition, Ce-GRH-1 binds to the promoter of the gene encoding aromatic L-amino acid decarboxylase, the D.melanogaster homolog of which is involved in cuticle hardening and is regulated by Grainyhead. These findings suggest that Grainyhead and Ce-GRH-1 regulate genes in the embryonic epidermis involved in cuticular morphogenesis pathways and that these developmental pathways have been conserved during the course of evolution (Venkatesan, 2003). Homo sapiens and M.musculus LSF/CP2, in contrast, bind to promoters of genes such as DNA polymerase ß, thymidylate synthase, c-fos, ornithine decarboxylase, alpha-globin and IL-4, involved in a wide variety of processes, including the cell cycle, growth and differentiation (Venkatesan, 2003).

Although the protein sequence profiles representing the two family divisions show conservation in the DNA binding-associated region as mapped by deletion analysis, they show no commonality in the oligomerization-associated region. Further, there is no protein-protein interaction observed between proteins belonging to different subfamilies. Taken together, these observations suggest fundamental differences in the quaternary DNA binding protein structure between the two subfamilies (Venkatesan, 2003).

Analysis of Ce-grh-1 is consistent with this family dichotomy, based both on phylogenetic and experimental data. The binding site preference for Ce-GRH-1 is identical to that of Grainyhead, although the data set is statistically small. In addition to competition analyses using mutated DNA binding sites, comparison of sequences of DNAs that bound Ce-GRH-1 versus those that did not (at least under the binding conditions used) indicated contiguous base positions important for interaction with Ce-GRH-1; these agree with the base preferences estimated in the DNA binding site count matrix (Venkatesan, 2003).

Among the known full-length proteins in the family, the H.sapiens, M.musculus, D.melanogaster and A.gambiae genomes have evolved members in both the GRH and LSF subfamilies. Although there is only a single identified member each in X.laevis and G.gallus (both in the LSF subfamily), the dbEST database contains additional X.laevis EST sequences with greater than 85% identity over their entire length to LBP-9 in the LSF subfamily and MGR in the GRH subfamily and a similar G.gallus EST corresponding to MGR. There is also EST evidence for the existence of genes similar to LSF and MGR in D.rerio (zebrafish), reflecting a similar gene duplication event. The B.amphitrite BCS-3 protein groups with the GRH subfamily in the tree, and its cDNA is selectively expressed in the larval stage. Given its phylogenetic placement, it is anticipated that there is at least one additional member belonging to the LSF subfamily in this genome (Venkatesan, 2003).

Finally, the C.elegans genome apparently has a single member, which clusters with the GRH subfamily. Similarly, there is evidence for GRH subfamily members in other nematodes such as Caenorhabditis briggsae (contig FPC2032 from assembly cb25.agp8, derived from BLAST analysis of C.briggsae genomic contigs) and S.stercoralis (BLAST analysis of dbEST). However, there is no evidence for a nematode protein in the LSF subfamily in terms of additional products in RT-PCR assays performed across C.elegans developmental stages, sequence similarities to nematode ESTs or sequence similarities to either the complete genome sequence of C.elegans or the available genome sequence of C.briggsae. Thus, the nematodes alone appear to have a representative from only one of these two subfamilies. The function of the other gene(s) may have been lost through the course of evolution in these genomes. There is precedent for gene loss in nematodes in the case of other gene families, including the HOX gene cluster. An alternative hypothesis is that the LSF subfamily functions may have been subsumed by the GRH member in nematodes (Venkatesan, 2003).

Phylogenetic analysis, along with the known functions and quaternary structures of the LSF/GRH family members, suggests that the family underwent a major gene duplication event more than 700 million years ago, when the first multicellular organisms are thought to have evolved. (Consistent with this estimated time line, the only EST from fungi with even weak sequence similarity to proteins in the LSF/GRH family is an EST from C.coronatus, a multicellular fungus.) This gene duplication event may have resulted in a distinct functional and structural division among its members (Venkatesan, 2003).

Targets of activity of Grainyhead family proteins

CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) that encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids, which is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor, or of a core region comprising amino acids 133 to 395, generates polypeptides with the DNA binding specificity of CP2. These results confirm the cloning of CP2 and delimit the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognize polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor (Lim, 1992).

CP2 is a transcription factor that interacts with the murine alpha-globin promoter. CP2 expressed in bacteria was significantly enriched and used in a series of DNase I footprinting and electrophoretic gel shift assays. The results suggest that CP2 binds a hyphenated recognition sequence motif spanning one DNA helix turn. The enriched bacterial protein activates transcription of alpha-globin promoter templates approximately 3- to 4-fold in vitro. The effect of elevating CP2 levels 2.5- to 5.5-fold in vivo was examined using both transient and stable transformation assays. When the intact murine alpha-globin promoter is introduced into these overexpressing cells, a 3- to 6-fold increase in promoter activity is observed, when compared to cells expressing normal levels of CP2. These results define the CP2 factor binding site in more detail and help characterize the activities of the factor in vivo (Lim, 1993).

The dimeric transcription factor CP2 binds a sequence element found near the transcription start site of the human immunodeficiency virus (HIV-1) long terminal repeat. Several groups have suggested that cellular factors binding this element might play a role in modulating HIV-1 promoter activity in vivo. For example, induction of latent HIV-1 gene expression in response to superinfection by either herpes simplex virus type 1 (HSV-1) or cytomegalovirus is thought to be mediated, in part, by factors binding the CP2 site. The relationship between CP2 and expression of the HIV-1 promoter has been examined. The effect HSV-1 infection of T cells has on the cellular levels of CP2 was examined. HSV-1 infection leads to a significant reduction in the level of CP2 DNA binding activity and protein within 20 h. The effect of overexpressing either the wild-type factor or a dominant negative variant of CP2 on HIV-1 promoter activity in vivo was also examined. CP2 has little effect or slightly represses HIV-1 promoter activity in vivo. In addition, these expression constructs have little effect on the induction of HIV-1 promoter activity elicited by HSV-1 infection (Zhong, 1994).

LBP-1 is a cellular protein that binds strongly to sequences around the human immunodeficiency virus type 1 (HIV-1) initiation site and weakly over the TATA box. LBP-1 represses HIV-1 transcription by inhibiting the binding of TFIID to the TATA box. Four similar but distinct cDNAs encoding LBP-1 (LBP-1a, -b, -c, and -d) have been isolated. These are products of two related genes, and each gene encodes two alternatively spliced products. Comparison of the amino acid sequence of LBP-1 with entries in the available protein data bases reveals the identity of LBP-1c to alpha-CP2, an alpha-globin transcription factor. These proteins are also homologous to Drosophila melanogaster Elf-1/NTF-1, an essential transcriptional activator that functions during Drosophila embryogenesis. Three of the recombinant LBP-1 isoforms show a DNA binding specificity that is identical to that of native LBP-1 and that bind DNA as a multimer. Antisera raised against recombinant LBP-1 recognize native LBP-1 from HeLa nuclear extract. Functional analyses in a cell-free transcription system demonstrate that recombinant LBP-1 specifically represses transcription from a wild-type HIV-1 template but not from an LBP-1 mutant template. LBP-1 can function as an activator both in vivo and in vitro, depending on the promoter context. Interestingly, one isoform of LBP-1 which is missing the region of the Elf-1/NTF-1 homology is unable to bind DNA itself and, presumably through heteromer formation, inhibits binding of the other forms of LBP-1, suggesting that it may function as a dominant negative regulator (Yoon, 1994).

The human stage selector protein (SSP) has been implicated in the developmental regulation of the globin genes. Binding of SSP to the stage selector element (SSE) in the proximal gamma-globin promoter is integral to the competitive silencing of a linked beta-promoter in embryonic/fetal stage erythroleukemia (K562) cells. SSP has been purified from a K562 cell nuclear extract. It has been demonstrated that the ubiquitously expressed transcription factor CP2 is pivotal to, but not sufficient for, SSP binding activity. Although addition of anti-CP2 antiserum disrupts the formation of the SSP-SSE complex in the electrophoretic mobility shift assay (EMSA), recombinant CP2 fails to bind to the SSE. Binding of CP2 to the SSE requires a heterodimeric partner present in K562 cells. The molecular weight of the partner protein is 40-45 kDa. An element analogous to the human SSE has previously been demonstrated in the chicken beta A-gene-promoter. The effects of this element are dependent on the binding of the chicken stage selector protein, NF-E4. Comparative studies between human CP2 and chicken NF-E4 demonstrate homology between the protein complexes. SSP binds to the chicken SSE; formation of this complex is ablated by the addition of anti-CP2 antiserum or a monoclonal antibody to NF-E4. Western analysis of partially purified NF-E4 using anti-CP2 antiserum or the NF-E4 monoclonal antibody both demonstrate a dominant band at 66 kDa. Similarly, the NF-E4 antibody recognizes the 66 kDa human CP2 protein in Western analysis of the SSP-SSE complex (Jane, 1995).

The cellular factor, LBP-1, can repress HIV-1 transcription by preventing the binding of TFIID to the promoter. The effect of recombinant LBP-1 on HIV-1 transcription in vitro was examined by using a "pulse-chase" assay. LBP-1 has no effect on initiation from a preformed preinitiation complex and elongation to position +13 ("pulse"). However, addition of LBP-1 after RNA polymerase is stalled at +13 strongly inhibits further elongation ("chase") by reducing RNA polymerase processivity. Severe mutations of the high affinity LBP-1 binding sites between -4 and +21 do not relieve the LBP-1-dependent block. However, LBP-1 can bind independently to upstream low affinity sites (-80 to -4), suggesting that these sites mediate the effect of LBP-1 on elongation. These results demonstrate a novel function of LBP-1, restricting HIV-1 transcription at the level of elongation. In addition, the HIV coded protein Tat is found to suppress the antiprocessivity effect of LBP-1 on HIV-1 transcription in nuclear extracts. These findings strongly suggest that LBP-1 may provide a natural mechanism for restricting the elongation of HIV-1 transcripts and that this may be a target for the action of Tat in enhancing transcription (Parada, 1995).

A subpopulation of stably infected CD4+ cells capable of producing virus upon stimulation has been identified in human immunodeficiency virus (HIV)-positive individuals. Few host factors that directly limit HIV-1 transcription and could support this state of nonproductive HIV-1 infection have been described. YY1, a widely distributed human transcription factor, is known to inhibit HIV-1 long terminal repeat (LTR) transcription and virus production. LSF (also known as LBP-1, UBP, and CP-2) has been shown to repress LTR transcription in vitro, but transient expression of LSF has no effect on LTR activity in vivo. Both YY1 and LSF participate in the formation of a complex that recognizes the initiation region of the HIV-1 LTR. These factors cooperate in the repression of LTR expression and viral replication. This cooperative function may account for the divergent effects of LSF previously observed in vitro and in vivo. Thus, the cooperation of two general cellular transcription factors may allow for the selective downregulation of HIV transcription. Through this mechanism of gene regulation, YY1 and LSF could contribute to the establishment and maintenance of a population of cells stably but nonproductively infected with HIV-1 (Romerio, 1997).

Lens-specific transcriptional activation of the chicken alphaA-crystallin gene is controlled by the distal and proximal enhancers, alphaCE1 and alphaCE2, respectively. Analysis using specific monoclonal antibodies against purified alphaCE1-binding factor alphaCEF1 reveals that alphaCEF1 is composed of two distinct subunits. One of the subunits of alphaCEF1 is encoded by chicken ubiquitous transcription factor CP2 (cCP2), which is homologous to mouse CP2, and human CP2/LBP-1/LSF-1. Electrophoretic mobility shift assays and cross-linking experiments have shown that alphaCEF1 and bacterially expressed cCP2 form a tetramer. Overexpression of cCP2 activates transcription through alphaCE1, but a mutant cCP2 lacking the DNA-binding domain reduces the transcription to basal levels. Although cCP2 binds to the CP2 template from the mouse alpha-globin promoter, it fails to promote transcription through this template. Element substitution experiments between alphaCE1 and the CP2 template reveals that the lens-specific enhancer activity of alphaCE1 is due to the 6 bp sequence [-139/-134; lens-specific element (LSE)] adjacent to the 3' end of the cCP2 binding site within alphaCE1. It is concluded that the tetrameric transcription factor cCP2 is essential for lens-specific transcription of the chicken alphaA-crystallin gene, despite being ubiquitously expressed. A model is suggested where cCP2 cooperates with a putative lens-specific factor that binds to LSE (Murata, 1998).

Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2 is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells (Keane-Myers, 2000).

The cholesterol side-chain cleavage enzyme, cytochrome P450scc, initiates the biosynthesis of all steroid hormones. Adrenal and gonadal strategies for P450scc gene transcription are essentially identical and depend on the orphan nuclear receptor steroidogenic factor-1, but the placental strategy for transcription of P450scc employs cis-acting elements different from those used in the adrenal strategy and is independent of steroidogenic factor-1. Because placental expression of P450scc is required for human pregnancy, factors were sought that bind to the -155/-131 region of the human P450scc promoter, which participates in its placental but not adrenal or gonadal transcription. A yeast one-hybrid screen of 2.4 x 10 6 cDNA clones from human placental JEG-3 cells yielded two unique clones; one is the previously described transcription factor LBP-1b, which is induced by HIV, type I infection of lymphocytes, and the other is a new factor, termed LBP-9, that shares 83% amino acid sequence identity with LBP-1b. When expressed in transfected yeast, both factors bound specifically to the -155/-131 DNA; antisera to LBP proteins supershifted the LBP-9.DNA complex and inhibited formation of the LBP-1b.DNA complex. Reverse transcriptase-polymerase chain reaction detected LBP-1b in human placental JEG-3, adrenal NCI-H295A, liver HepG2, cervical HeLa, and monkey kidney COS-1 cells, but LBP-9 was detected only in JEG-3 cells. When the -155/-131 fragment was linked to a minimal promoter, co-expression of LBP-1b increased transcription 21-fold in a dose-dependent fashion, but addition of LBP-9 suppressed the stimulatory effect of LBP-1b. The roles of LBP transcription factors in normal human physiology have been unclear. Their modulation of placental but not adrenal P450scc transcription underscores the distinctiveness of placental strategies for steroidogenic enzyme gene transcription (Huang, 2000).

The reduced level of CP2 suppresses the mouse alpha- and beta-globin gene expression and hemoglobin synthesis during terminal differentiation of mouse erythroleukemia (MEL) cells in vitro. Human alpha-, epsilon-, and gamma- globin genes are also suppressed by the reduced expression of CP2 in K562 cells. To address how much CP2 contributes in the regulation of globin gene expression, transcriptional activities of the wild type alpha-globin promoter and its various factor-binding sites mutants were measured in erythroid and nonerythroid cells. Interestingly, CP2 site dependent transcriptional activation occurs in an erythroid-cell specific manner, even though CP2 is ubiquitously expressed. In addition, CP2 site mutation within the alpha-promoter severely suppresses promoter activity in differentiated, but not in undifferentiated MEL cells, suggesting that the CP2 binding site is needed for the enhanced transcription of globin genes during erythroid differentiation. When the human beta-globin locus control region is linked to the alpha-promoter, suppression is more severe in the CP2 site mutant in differentiated MEL cells. Overall data indicate that CP2 is a major factor in the regulation of globin expression in human and mouse erythroid cells, and CP2 binding to the globin gene promoter is essential for the enhanced transcription of globin genes in erythroid differentiation (Chae, 2003).

cENS-1/cERNI genes have been shown to be expressed very early during chicken embryonic development and as well as in pluripotent chicken embryonic stem (CES) cells. A promoter region has been identified that is specifically active in CES cells compared to differentiated cells. In order to understand the molecular mechanisms that regulate the cENS-1/cERNI promoter, the cis-acting elements of this promoter were analyzed in CES and differentiated cells. A short sequence, named the B region, 5'-CAAG TCCAGG CAAG-3', has been identified that exhibits a strong enhancer activity in CES and differentiated cells. Mutation of the B region in the whole cENS-1 promoter strongly decreases the promoter activity in CES cells, suggesting that this region is essential for activating the promoter. The B region is similar to the previously described response element for the transcription factor CP2 and it is shown by supershift experiments that a protein complex containing CP2 is bound to this B response element. All these results identify a nuclear factor belonging to the CP2 transcription factor family that is crucial for the activation of the cENS-1/cERNI promoter. The pattern of expression of cCP2 in early chicken embryo before gastrulation is very similar to that of cENS-1/cERNI: this strongly suggests that cCP2 also plays an essential role in gene expression early in embryonic development (Acloque, 2004).

Effects of mutation of Grainyhead family proteins

The NTF-like family of transcription factors have been implicated in developmental regulation in organisms as diverse as Drosophila and man. The two mammalian members of this family, CP2 (LBP-1c/LSF) and LBP-1a (NF2d9), are highly related proteins sharing an overall amino acid identity of 72%. CP2, the best characterized of these factors, is a ubiquitously expressed 66-kDa protein that binds the regulatory regions of many diverse genes. Consequently, a role for CP2 has been proposed in globin gene expression, T-cell responses to mitogenic stimulation, and several other cellular processes. To elucidate the in vivo role of CP2, mice nullizygous for the CP2 allele have been generated. These animals were born in a normal Mendelian distribution and displayed no defects in growth, behavior, fertility, or development. Specifically, no perturbation of hematopoietic differentiation, globin gene expression, or immunological responses to T- and B-cell mitogenic stimulation was observed. RNA and protein analysis confirmed that the nullizygous mice expressed no full-length or truncated version of CP2. Electrophoretic mobility shift assays with nuclear extracts from multiple tissues demonstrated loss of CP2 DNA binding activity in the -/- lines. However, a slower migrating complex that was ablated with antiserum to NF2d9, the murine homolog of LBP-1a, was observed with these extracts. Furthermore, recombinant LBP-1a can bind to known CP2 consensus sites and form protein complexes with previously defined heteromeric partners of CP2. These results suggest that LBP-1a/NF2d9 may compensate for loss of CP2 expression in vivo and that further analysis of the role of the NTF family of proteins requires the targeting of the NF2d9 gene (Ramamurthy, 2001).

The neural tube defects (NTDs) spina bifida and anencephaly are widely prevalent severe birth defects. The mouse mutant curly tail (ct/ct) has served as a model of NTDs for 50 years, even though the responsible genetic defect remained unrecognized. By gene targeting, mapping and genetic complementation studies it has been shown that a mouse homolog of the Drosophila grainyhead (grh) gene, grainyhead-like-3 (Grhl3), is a compelling candidate for the gene underlying the curly tail phenotype. The NTDs in Grhl3-null mice are more severe than those in the curly tail strain, since the Grhl3 alleles in ct/ct mice are hypomorphic. Spina bifida in ct/ct mice is folate resistant, but its incidence can be markedly reduced by maternal inositol supplementation periconceptually. The NTDs in Grhl3-/- embryos are also folate resistant, but unlike those in ct/ct mice, they are resistant to inositol. These findings suggest that residual Grhl3 expression in ct/ct mice may be required for inositol rescue of folate-resistant NTDs (Ting, 2003b)

LBP-1a and CP2 are ubiquitously expressed members of the grainyhead transcription factor family, sharing significant sequence homology, a common DNA binding motif, and modulating a range of key regulatory and structural genes. CP2-null mice are viable with no obvious abnormality. LBP-1a provides redundant function in this context. Mice lacking LBP-1a expression develop intrauterine growth retardation at embryonic day 10.5, culminating in death 1 day later. No focal intraembryonic cause for this CP2-independent defect is evident. In contrast, a significant reduction in the thickness of the labyrinthine layer of the placenta is observed in LBP-1a-/- animals. However, expression of trophoblast differentiation markers is unperturbed in this context, and complementation studies utilizing tetraploid wild-type cells failed to rescue or ameliorate the LBP-1a-/- phenotype, excluding a primary trophoblast defect. An explanation for these observations is provided by the prominent angiogenic defect observed in the mutant placentas. LBP-1a-/- allantoic blood vessels fail to penetrate deeply and branch into the complex embryonic vasculature characteristic of the normal placenta. Interestingly, a similar defect in angiogenesis is observed in the yolk sac vasculature: primary endothelial cell-lined capillary tubes, although present, fail to connect into a characteristic intricate vascular network. Collectively, these results demonstrate that LBP-1a plays a critical role in the regulation of extraembryonic angiogenesis (Parekh, 2004).

Primary neurulation in mammals has been defined by distinct anatomical closure sites, at the hindbrain/cervical spine (closure 1), forebrain/midbrain boundary (closure 2), and rostral end of the forebrain (closure 3). Zones of neurulation have also been characterized by morphologic differences in neural fold elevation, with non-neural ectoderm-induced formation of paired dorso-lateral hinge points (DLHP) essential for neural tube closure in the cranial and lower spinal cord regions, and notochord-induced bending at the median hinge point (MHP) sufficient for closure in the upper spinal region. This study identified a unifying molecular basis for these observations based on the function of the non-neural ectoderm-specific Grainy head-like genes in mice. Using a gene-targeting approach it was shown that deletion of Grhl2 results in failed closure 3, with mutants exhibiting a split-face malformation and exencephaly, associated with failure of neuro-epithelial folding at the DLHP. Loss of Grhl3 alone defines a distinct lower spinal closure defect, also with defective DLHP formation. The two genes contribute equally to closure 2, where only Grhl gene dosage is limiting. Combined deletion of Grhl2 and Grhl3 induces severe rostral and caudal neural tube defects, but DLHP-independent closure 1 proceeds normally in the upper spinal region. These findings provide a molecular basis for non-neural ectoderm mediated formation of the DLHP that is critical for complete neuraxis closure (Rifat, 2010).

Grainyhead-like 2 regulates neural tube closure and adhesion molecule expression during neural fold fusion

Defects in closure of embryonic tissues such as the neural tube, body wall, face and eye lead to severe birth defects. Cell adhesion is hypothesized to contribute to closure of the neural tube and body wall; however, potential molecular regulators of this process have not been identified. This study identified an ENU-induced mutation in mice that reveals a molecular pathway of embryonic closure. Line2F homozygous mutant embryos fail to close the neural tube, body wall, face, and optic fissure, and they also display defects in lung and heart development. Using a new technology of genomic sequence capture and high-throughput sequencing of a 2.5Mb region of the mouse genome, a mutation was discovered in the grainyhead-like 2 gene (Grhl2). Microarray analysis revealed Grhl2 affects the expression of a battery of genes involved in cell adhesion and E-cadherin protein is drastically reduced in tissues that require Grhl2 function. The tissue closure defects in Grhl2 mutants are similar to that of AP-2α null mutants and AP-2α has been shown to bind to the promoter of E-cadherin. Therefore, a possible interaction between these genes was tested. However, it was found that Grhl2 and AP-2α do not regulate each other's expression, E-cadherin expression is normal in AP-2α mutants during neural tube closure, and Grhl2;AP-2α trans-heterozygous embryos are morphologically normal. Taken together, these studies point to a complex regulation of neural tube fusion and highlight the importance of comparisons between these two models to understand more fully the molecular pathways of embryonic tissue closure (Pyrgaki, 2011).

Grainy head proteins and epidermis

Morphogen-dependent epidermal-specific transacting factors have not been defined in vertebrates. A member of the grainyhead transcription factor family, Grainyhead-like 1 (XGrhl1) has been identified that is essential for ectodermal ontogeny in Xenopus laevis. Expression of this factor is restricted to epidermal cells. Moreover, XGrhl1 is regulated by the BMP4 signaling cascade. Disruption of XGrhl1 activity in vivo results in a severe defect in terminal epidermal differentiation, with inhibition of XK81A1 epidermal keratin gene expression, a key target of BMP4 signaling. Furthermore, transcription of the XK81A1 gene is modulated directly by binding of XGRHL1 to a promoter-localized binding motif that is essential for high-level expression. These results establish a novel developmental role for XGrhl1 as a crucial tissue-specific regulator of vertebrate epidermal differentiation (Tao, 2005).

These studies demonstrate that XGrhl1 is a downstream epidermal-specific target of the BMP signaling. This observation varies significantly from hat reported in Drosophila, in which grh expression modulates BMP4 activity. This evolutionary divergence raises three questions: (1) is XGrhl1 necessary for BMP4-dependent epidermal specification; (2) if not, what is the role of XGrhl1 in the pathway; (3) is XGrhl1 involved in other epidermal-specific signaling events (Tao, 2005)?

Ectopic expression of BMP4 or of immediate early response (IER) factors, such as Xmad1, result in epidermal re-specification in cellular progeny of blastomeres with a neural fate. Similarly, co-expression of IER factors and BMP antagonists/inhibitors induces epidermal specification in injected ectodermal cells, with coincident repression of neural gene expression. Given the temporal pattern of endogenous expression, a similar outcome is expected with enforced expression of XGrhl1. Differing sharply from the effects of IER factors, ectopic expression of XGrhl1 fails to induce epidermal specification. These observations suggest that XGrhl1 activity is dispensable for this process, a conclusion supported by the inability of injection of Delta227XGrhl1-encoding transcripts (227XGRHL1 lacks a N-terminal activation domain encoding the first 227 amino acids, dimerizes with wild-type XGRHL1 and has comparable binding affinity to XGRHLl for a consensus Grh-binding motif) or XGrhl1-specific MOs to affect germ layer specification (Tao, 2005).

These studies suggest an alternate model, XGrhl1 functioning downstream of the IER factors in the BMP-signaling cascade. In this context, AP2 and Dlx-like factors have been shown to be essential for appropriate epidermal differentiation. However, it remains unclear how these factors achieve tissue specificity given their wider pattern of gene expression. Induction of epidermal keratin gene XK81A1 expression is dependent on appropriate XGrhl1 function. Like Dlx3, expression of XGrhl1 does not induce expression of the epidermal structural gene XK81A1 in the absence of a functional BMP4 pathway, suggesting that morphogen-induced expression of other factors is necessary. One candidate may be AP2, given its ability to rescue the epidermal defect induced by dominant-negative truncated BMP4-specific receptor (tBR) expression in a similar manner to IER regulatory factors. Furthermore, like XGrhl1, AP2 fails to repress expression of pan-neural gene markers, a divergence from the effects of IER factor expression. These observations, together with studies of the XK81A1 promoter, indicate that XGrhl1 functions predominantly downstream of the IER factors in the BMP4 signaling cascade. Furthermore, studies of the XK81A1 promoter demonstrate that both AP2 and XGRHL1 are required for XK81A1 expression (see below). Thus, it is suggested that characterization of the expression of XGrhl1 and its mechanism of action represents a significant new insight into the regulation of BMP4-responsive epidermal-specific targets, this tissue-specific factor modulating structural gene expression in concert with the more widely expressed regulator AP2 during terminal differentiation (Tao, 2005).

The Drosophila cuticle is essential for maintaining the surface barrier defenses of the fly. Integral to cuticle resilience is the transcription factor grainy head, which regulates production of the enzyme required for covalent cross-linking of the cuticular structural components. Formation and maintenance of the epidermal barrier in mice are dependent on a mammalian homolog of grainy head, Grainy head-like 3. Mice lacking this factor display defective skin barrier function and deficient wound repair, accompanied by reduced expression of transglutaminase 1, the key enzyme involved in cross-linking the structural components of the superficial epidermis. These findings suggest that the functional mechanisms involving protein cross-linking that maintain the epidermal barrier and induce tissue repair are conserved across 700 million years of evolution (Ting, 2005).

The abnormal barrier in the Grhl3-null mice appears comparable to the phenotype seen in Drosophila grh mutants, which exhibit fragile cuticles. Similarly, C. elegans embryos in which expression of a grainy head-like gene had been reduced by RNA interference fail to hatch and display a cuticle defect consistent with a loss of rigidity. Grh was initially identified as a factor involved in the transcriptional regulation of Ddc, which functions to pigment and harden the insect cuticle through the generation of quinones that cross-link the cuticular proteins. The identification of TGase1, the predominant enzyme involved in the generation of high molecular weight polymers of cross-linked CE proteins, as a putative target of Grhl3 indicates that the grh family is involved in the regulation of protein cross-linking in barrier formation across 700 million years of evolution. The grh mutant cuticular defects are unlikely to be caused solely by grh regulation of the Ddc gene, because null Ddc mutants do not express the above cuticular phenotype, and the hypopigmentation of cuticular structures in the grh mutants is not as severe as that in the null Ddc mutants. Similarly, the Grhl3-null epidermal defects suggest the presence of additional target genes other than TGase1. It is conceivable that some of these genes may be conserved from fly to human, a hypothesis strengthened by the demonstration of identical DNA binding consensus sequences for grh and Grhl3 (Ting, 2005).

The recent identification of wound response enhancers in the Drosophila Ddc and pale (ple) genes that mediate protective functions of the epidermal wound response and require binding sites for grh, and grh genetic function, provides an additional compelling link between grh family members, cross-linking enzymes, and the integrity of the surface epithelium (Mace, 2005). Ple encodes tyrosine hydroxylase, which is also involved in the generation of quinones for protein/chitin cross-linking. It is of interest that the regulation of cross-linking enzyme genes by grh-like factors has been conserved, even though the cross-linking genes themselves have diversified from fly to human. No change was found in Ddc gene expression in the Grhl3-null mice, consistent with the fact that this gene is not linked to protein cross-linking in the mammalian epidermis (Ting, 2005).

These studies identify an essential function for the grh family in maintenance of the integument barrier in diverse species. However, the grh gene is also critical for other aspects of epidermal and epithelial development in the fly, including cell polarity and tubular morphogenesis. The complexity of gene function in the fly may also be seen in the mouse Grhl factors (Ting, 2005).

Duct epithelial structure is an essential feature of many internal organs, including exocrine glands and the kidney. The ducts not only mediate fluid transfer but also help to maintain homeostasis. For instance, fluids and solutes are resorbed from or secreted into the primary fluid flowing through the lumen of the ducts in the exocrine glands and kidneys. The molecular mechanism underlying the functional maturation of these ducts remains largely unknown. This study shows that a grainyhead-related transcription factor, CP2-like 1 (CP2L1), is required for the maturation of the ducts of the salivary gland and kidney. In the mouse, Cp2l1 is specifically expressed in the developing ducts of a number of exocrine glands, including the salivary gland, as well as in those of the kidney. In Cp2l1-deficient mice, the expression of genes directly involved in functional maturation of the ducts was specifically reduced in both the salivary gland and kidney, indicating that Cp2l1 is required for the differentiation of duct cells. Furthermore, the composition of saliva and urine was abnormal in these mice. These results indicate that Cp2l1 expression is required for normal duct development in both the salivary gland and kidney (Yamaguchi, 2006; full text of article).

Defective permeability barrier is an important feature of many skin diseases and causes mortality in premature infants. To investigate the control of barrier formation, this study characterized the epidermally expressed Grainyhead-like epithelial transactivator (Get-1)/Grhl3, a conserved mammalian homologue of Grainyhead, which plays important roles in cuticle development in Drosophila. Get-1 interacts with the LIM-only protein LMO4, which is co-expressed in the developing mammalian epidermis. The epidermis of Get-1(-/-) mice showed a severe barrier function defect associated with impaired differentiation of the epidermis, including defects of the stratum corneum, extracellular lipid composition and cell adhesion in the granular layer. The Get-1 mutation affects multiple genes linked to terminal differentiation and barrier function, including most genes of the epidermal differentiation complex. Get-1 therefore directly or indirectly regulates a broad array of epidermal differentiation genes encoding structural proteins, lipid metabolizing enzymes and cell adhesion molecules. Although deletion of the LMO4 gene had no overt consequences for epidermal development, the epidermal terminal differentiation defect in mice deleted for both Get-1 and LMO4 is much more severe than in Get-1(-/-) mice with striking impairment of stratum corneum formation. These findings indicate that the Get-1 and LMO4 genes interact functionally to regulate epidermal terminal differentiation (Yu, 2006).

Epidermal wound repair is regulated by the planar cell polarity signaling pathway

The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. This study shows that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, the flamingo homolog Celsr1, off-track homolog PTK7, and Scrb1, and the Grainyhead transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3-/- mice, RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, was identified as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair (Caddy, 2010).

Meta-analysis of Grainyhead-like dependent transcriptional networks: A roadmap for identifying novel conserved genetic pathways

Mutations affecting Drosophila grainyhead (grh) and vertebrate Grainyhead-like (Grhl) transcription factors lead to a developmental and adult onset epithelial disease, such as aberrant skin barrier formation, facial/palatal clefting, impaired neural tube closure, age-related hearing loss, ectodermal dysplasia, and importantly, cancers of epithelial origin. Recently, mutations in the family member GRHL3 have been shown to lead to both syndromic and non-syndromic facial and palatal clefting in humans. Large-scale datasets have been generated to explore the grh/Grhl-dependent transcriptome, following ablation or mis-regulation of grh/Grhl-function. A meta-analysis was performed of all 41 currently published grh and Grhl RNA-SEQ, and microarray datasets, in order to identify and characterise the transcriptional networks controlled by grh/Grhl genes across disparate biological contexts. Moreover, this study has also cross-referenced the results with published ChIP and ChIP-SEQ datasets, in order to determine which of the critical effector genes are likely to be direct grh/Grhl targets, based on genomic occupancy by grh/Grhl genes. Lastly, to interrogate the predictive strength of this approach, the expression of the top 10 candidate grhl target genes in epithelial development were experimentally validated, in a zebrafish model lacking grhl3, and found that orthologues of seven of these (cldn23, ppl, prom2, ocln, slc6a19, aldh1a3, and sod3) were significantly down-regulated at 48 hours post-fertilisation. Therefore, this study provides a strong predictive resource for the identification of putative grh/grhl effector target genes (Mathiyalagan, 2019).

grainy head: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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