Mutant p53 subverts p63 control over KLF4 expression in keratinocytes.

Genetic experiments established that p63 is crucial for the development and maintenance of pluri-stratified epithelia and KLF4 for the barrier function of the skin. KLF4 is one of the factors that reprogram differentiated cells to iPS. We investigated the relationship between p63 and KLF4 using RNA interference, overexpression, chromatin immunoprecipitation and transient transfections with reporter constructs. We find that p63 directly represses KLF4 in normal keratinocytes (KCs) by binding to upstream promoter sites. Unlike p63, KLF4 levels are high in the upper layers of human skin and increase upon differentiation of KCs in vitro. In HaCaT KCs, which harbor two mutant alleles of p53, inactivation of p63 and of mutant p53 leads to KLF4 repression. p63 and p53 mutants are bound to sites in the KLF4 core promoter. Importantly, expression of the H179Y and R282Q p53 mutants in primary KCs is sufficient to activate endogenous KLF4. Finally, immunohistochemical analysis of tissue arrays confirms increased coexpression of KLF4 and mutant p53 in squamous cell carcinomas. Our data indicate that suppression of KLF4 is part of the growth-promoting strategy of p63 in the lower layers of normal epidermis, and that tumor-predisposing p53 mutations hijack p63 to a different location on the promoter, turning it into an activator of this reprogramming factor.

The p63 target HBP1 is required for skin differentiation and stratification.

Genetic experiments established that p63 is crucial for the development and maintenance of pluristratified epithelia. In the RNA interference (RNAi) screening for targets of p63 in keratinocytes, we identified the transcription factor, High Mobility Group (HMG) box protein 1 (HBP1). HBP1 is an HMG-containing repressor transiently induced during differentiation of several cell lineages. We investigated the relationship between the two factors: using RNAi, overexpression, chromatin immunoprecipitations and transient transfections with reporter constructs, we established that HBP1 is directly repressed by p63. This was further confirmed in vivo by evaluating expression in p63 knockout mice and in transgenics expressing p63 in basal keratinocytes. Consistent with these findings, expression of HBP1 increases upon differentiation of primary keratinocytes and HaCaT cells in culture, and it is higher in the upper layers of human skin. Inactivation of HBP1 by RNAi prevents differentiation of keratinocytes and stratification of organotypic skin cultures. Finally, we analyzed the keratinocyte transcriptomes after HBP1 RNAi; in addition to repression of growth-promoting genes, unexpected activation of differentiation genes was uncovered, coexisting with repression of other genes involved in epithelial cornification. Our data indicate that suppression of HBP1 is part of the growth-promoting strategy of p63 in the lower layers of epidermis and that HBP1 temporally coordinates expression of genes involved in stratification, leading to the formation of the skin barrier.

p63 regulates the caspase-8-FLIP apoptotic pathway in epidermis.

The transcription factor p63, member of the p53 family, is crucial for epithelial development. An RNAi screening identified the apoptotic gene Procaspase-8 as a target activated by p63. The caspase-8 inhibitor FLIP is also under p63 control. We analysed and detailed the direct transactivation through the use of RNAi, reporter assays, ChIPs, western blots, confocal studies in HaCat, as well as in primary human keratinocytes. The direct DeltaNp63 regulation of these targets was confirmed in vivo using transgenic DeltaNp63 mice under the K5 promoter, as compared with p63 knockout mice, and in vitro in normal human primary keratinocytes following UV irradiation. Lowering the steady state of p63 protein levels changes the relative ratio of FLIP isoforms, causing the activation of the expressed, inactive Procaspase-8, into the active isoform thus triggering the proapoptotic cascade. Therefore, p63 fine-tunes the Procaspase-8-FLIP pro- and antiapoptotic pathway in keratinocytes.

Breast cancer metastases are molecularly distinct from their primary tumors.

Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether 'phenotypic' disease progression is really associated with 'molecular' progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.

A HOX complex, a repressor element and a 50 bp sequence confer regional specificity to a DPP-responsive enhancer.

A central theme during development and homeostasis is the generation of cell type-specific responses to the action of a limited number of extant signaling cascades triggered by extracellular ligands. The molecular mechanisms by which information from such signals are integrated in responding cells in a cell-type specific manner remain poorly understood. We have undertaken a detailed characterization of an enhancer that is regulated by DPP signaling and by the homeotic protein Labial and its partners, Extradenticle and Homothorax. The expression driven by this enhancer (lab550) and numerous deletions and point mutants thereof was studied in wild-type and mutant Drosophila embryos as well as in cultured cells. We find that the lab550 enhancer is composed of two elements, a Homeotic Response Element (HOMRE) and a DPP Response Element (DPPRE) that synergize. None of these two elements can reproduce the expression of lab550, either with regard to expression level or with regard to spatial restriction. The isolated DPPRE of lab550 responds extremely weakly to DPP. Interestingly, we found that the inducibility of this DPPRE is weak because it is tuned down by the action of a repressor element. This repressor element and an additional 50 bp element appear to be crucial for the cooperation of the HOMRE and the DPPRE, and might tightly link the DPP response to the homeotic input. The cooperation between the different elements of the enhancer leads to the segmentally restricted activity of lab550 in the endoderm and provides a mechanism to create specific responses to DPP signaling with the help of a HOX protein complex.

Nuclear interpretation of Dpp signaling in Drosophila.

Signaling by Decapentaplegic (Dpp), a member of the TGFbeta superfamily of signaling molecules similar to vertebrate BMP2 and BMP4, has been implicated in many developmental processes in Drosophila melanogaster. Notably, Dpp acts as a long-range morphogen during imaginal disc growth and patterning. Genetic approaches led to the identification of a number of gene products that constitute the core signaling pathway. In addition to the ligand-activated heteromeric receptor complex and the signal-transducing intracellular Smad proteins, Dpp signaling requires two nuclear proteins, Schnurri (Shn) and Brinker (Brk), to prime cells for Dpp responsiveness. A complex interplay between the nuclear factors involved in Dpp signaling appears to control the transcriptional readout of the Dpp morphogen gradient. It remains to be seen whether similar molecular mechanisms operate in the nucleus in vertebrate systems.

Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker.

The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct threshold concentrations during Drosophila development. An important, albeit indirect, mode by which Dpp controls the spatial extent of its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. Here we report the molecular dissection of the cis-regulatory sequences of optomotor-blind (omb), a Dpp target gene in the wing. We identify a minimal 284 bp Dpp response element and demonstrate that it is subject to Brinker (Brk) repression. Using this omb wing enhancer, we show that Brk is a sequence-specific DNA binding protein. Mutations in the high-affinity Brk binding site abolish responsiveness of this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. Our results therefore identify Brk as a novel transcription factor antagonizing Dpp signalling by directly binding target genes and repressing their expression.

Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context.

Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

Transcriptional activation by Oct-3: evidence for a specific role of the POU-specific domain in mediating functional interaction with Oct-1.

Oct-3, a member of the POU family of transcription factors, is expressed in pluripotent cells of early mammalian embryos and in undifferentiated embryonal carcinoma cell lines. Using a variety of Oct-3 mutants, we have identified two different domains of Oct-3 which activate transcription in transfected mammalian cells. One of these domains, located in the C-terminal part of the protein, plays a major role in transcriptional activation when Oct-3 is bound to its cognate site, the octamer motif. An Oct-3 mutant containing a single amino acid substitution in the POU homeodomain is unable to bind the octamer target in vitro, yet is still able to activate transcription in an octamer-dependent manner. We provide evidence that transactivation by this mutant involves protein-protein interactions with the ubiquitous octamer binding factor Oct-1. This interaction requires the POU-specific domain of Oct-3 and allows recruitment of Oct-3 to the target promoter even in the absence of Oct-3 DNA binding.

Regulation of the human HOXD4 gene by retinoids.

Hox genes are developmentally regulated in mammalian embryogenesis, according to temporally and spatially restricted patterns which are affected by retinoids, vitamin A derivatives which have a function as, or at least mimic the action of, axis-specifying morphogens. In the human embryonal carcinoma cell line NT2/D1, HOX gene clusters are activated by at least two retinoids, all-trans- and 9-cis-retinoic acid (RA), in a 3' to 5' sequential cascade which reproduces the activation pattern observed in early embryogenesis. We have studied the regulation of the early activated HOXD4 gene, which is expressed in human embryogenesis in multiple transcripts generated by the developmentally controlled use of alternative transcription start sites and polyadenylation signals. Transfection of a 2.9 kb HOXD4 upstream genomic region linked to a reporter gene in NT2/D1 cells, allowed the identification of two different promoters and a distal enhancer element necessary for RA-dependent gene activation. This element confers to a heterologous promoter the ability to be induced by RA in NT2/D1 cells, and transactivated by alpha, beta and gamma retinoic acid receptors (RARs), but not retinoid X receptor (RXR), in COS-7 cells. DNase I footprinting analysis allowed the identification of four sequences which bind nuclear factors from both RA-induced NT2/D1 cells and embryonic tissues with similar patterns. The use of specific antibodies allowed the identification of at least RAR beta in some of the DNA-protein complexes, although the four sequences bind single RARs transfected in COS cells much less efficiently, or not at all, when compared to a canonical RAR responsive element. Induction of the HOXD4 promoter-enhancer in the presence of a selective RAR alpha antagonist indicated that the RAR alpha-dependent RAR beta activation is nevertheless a necessary step in HOX gene activation. Our results indicate that the ligand-dependent activity of RARs upon specific, cis-acting regulatory elements may have a key role in the induction of early activated HOX genes in response to retinoids. However, RARs represent only a fraction of the transcription factors interacting with the RA-responsive HOXD4 enhancer, which appears to be a complex element requiring specific combinations of nuclear factors for its proper function.

A novel N-terminal motif for palmitoylation of G-protein alpha subunits.

We have examined the post-translational processing of G alpha subunits expressed endogenously in rat PC12 and NG108-15 rat/mouse hybrid cells, and after transfection of cDNA expression constructs into COS cells. Thioester-linked palmitoylation of alpha o, alpha s, alpha q/alpha 11 and alpha 12 has been detected by metabolic labelling with [3H]palmitate and immunoprecipitation. Palmitoylation of alpha o occurs post-translationally in cells treated with protein-synthesis inhibitors, suggesting possible dynamic acylation. Palmitoylation of the C-terminal CAAX motif has been excluded. Site-directed mutagenesis of alpha o has been used to implicate the site of modification as a cysteine residue next to the N-terminal myristoylated glycine, in a novel protein-lipid modification motif Met-Gly-Cys. The non-palmitoylated alpha o mutant is still myristoylated but shows reduced membrane binding, suggesting that reversible palmitoylation may regulate G alpha localization and function.

Oct-3 and mammalian development: correction of discussion.

The title of the 5 June report on page 1445 by R. C. deL. Milton et al. should have been "Total chemical synthesis of a D-enzyme: The enantiomers of HIV-1 protease show reciprocal chiral substrate specificity." Figure 3 in the same report (p. 1447) was inadvertently printed upside down. The labels "L-HIV protease" and "D-HIV protease" were therefore under the wrong illustrations. The correct figure is printed below. [See figure in the PDF file]

Chromosomal location of the octamer transcription factors, Otf-1, Otf-2, and Otf-3, defines multiple Otf-3-related sequences dispersed in the mouse genome.

Chromosomal locations have been assigned for the octamer transcription factor, Otf, gene family (previously named the octamer-binding protein, Oct, gene family) using an interspecific backcross of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice and the BXH recombinant inbred strains. Molecular probes for Otf-1 and Otf-2 recognized single loci on mouse chromosomes 1 and 7, respectively, whereas probes for Otf-3 recognized a minimum of eight independently segregating loci (designated Otf-3a through Otf-3h). Members of the Otf-3 family mapped to mouse chromosomes 1, 2, 3, 6, 14, 17, and the X chromosome, indicating that the Otf family has become widely dispersed during evolution. Several Otf loci mapped near developmental mutations, raising the possibility that these mutations result from defects in Otf family members.

A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo.

The murine oct-3 gene encodes a transcription factor containing a POU-specific domain and a homeodomain. In marked contrast to other homeodomain-encoding genes, oct-3 is expressed in the totipotent and pluripotent stem cells of the pregastrulation embryo and is down-regulated during differentiation to endoderm and mesoderm, suggesting that it has a role in early development. The oct-3 gene is also expressed in primordial germ cells and in the female germ line.

Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: pharmacological characterization.

L-Glutamate neurotoxicity at the N-methyl-D-aspartate (NMDA) receptor was characterized in cultured cerebellar granule cells. When deprived of glucose for 40 min, these cells were killed by 20-60 microM L-glutamate. However, the neurons were resistant to glutamate at concentrations as high as 5 mM when glucose and Mg2+ were present throughout. Both competitive and non-competitive NMDA receptor antagonists completely blocked neurotoxicity due to glutamate and other NMDA receptor agonists. CPP [+/-)-3-(2-carboxypiperazin-4-yl)-prophyl-1-phosphonic acid) was the most effective competitive antagonist with full protection at 100 microM while MK-801 [+/-)-10,11-dihydro-5-methyl-5H-dibenzo[a,d]-cyclohepten-5,10-imin e) was the most effective non-competitive antagonist with full protection at 20 nM. Other antagonists with higher selectivity for other subtypes of glutamate receptors were ineffective. We conclude that glutamate toxicity in energy-deprived cerebellar granule cells is mediated by NMDA receptors. Results are discussed in terms of an hypothesis offering an explanation for the transition of glutamate from neurotransmitter to neurotoxin which emphasizes the responsiveness of the receptor to agonists rather than focusing on the presence of high concentrations of agonist.

Energy-related neurotoxicity at the NMDA receptor: a possible role in Alzheimer's disease and related disorders.

Using rat cerebellar granule cells in primary culture as our model system, we have shown that excitatory amino acids (EAAs) become neurotoxic via the NMDA (N-methyl-D-aspartate) receptor when neuronal energy levels are compromised. Omission of glucose, exclusion of oxygen, or inclusion of inhibitors of oxidative phosphorylation or of Na+/K+-ATPases enables NMDA receptor agonists to express their neurotoxic potential. Both competitive and noncompetitive NMDA receptor antagonists are potent blockers of EAA neurotoxicity, with MK-801 fully effective at 20 nM. We interpret these results as indicating that glucose metabolism, ATP production, and functioning ion pumps are necessary to generate a resting potential sufficient to maintain the voltage-dependent Mg++ block of the NMDA receptor channel; relief of the block enables EAAs to act persistently at the NMDA receptor causing an excessive ion influx which leads to neuronal death by a mechanism not yet understood. These findings are discussed in the context of the potential role for NMDA receptor-mediated neurotoxicity in Alzheimer's disease and related disorders.

Characterization of muscarinic receptors in guinea pig ileum longitudinal smooth muscle.

Heterogeneity in the muscarinic receptor population of guinea pig ileum longitudinal smooth muscle was found in competition binding experiments against N-methyl[3H]scopolamine using either a cardioselective (AF-DX 116) or a smooth muscle-selective (hexahydrosiladifenidol) antimuscarinic compound. AF-DX 116 recognized 65% of the total receptors with high affinity and 35% with low affinity. Hexahydrosiladifenidol distinguished 24% of the total receptors with high affinity and 76% with low affinity. The two affinity binding constants displayed in smooth muscle by the compounds were similar to those of heart and glands, suggesting that the muscarinic receptor population in the smooth muscle is formed of about 30% glandular type and 70% cardiac type of the M2 receptors. In dissociation experiments, the rate of breakdown of the N-methyl[3H]scopolamine receptor complex in the smooth muscle was rapid and similar to the dissociation of N-methyl[3H]scopolamine from muscarinic receptors in cardiac membranes, supporting the evidence for the presence of a large fraction of the cardiac receptor type in smooth muscle. To further characterize the population of the smooth muscle receptors recognized as glandular type, we performed protection experiments with hexahydrosiladifenidol, which binds to glandular M3 receptors with high affinity. Smooth muscle membranes were initially incubated with this compound and then phenoxybenzamine was added to irreversibly alkylate the remaining unprotected receptors. Data from competition and dissociation binding experiments showed that, under these conditions, this protected fraction of the total receptor population in ileum smooth muscle had all the characteristics of the glandular type, i.e., slow N-methyl[3H]scopolamine dissociation and affinity constants for a series of selective and nonselective muscarinic antagonists in the same order of magnitude as those found in the glandular tissue. These findings, together with the known observation that hexahydrosiladifenidol is more potent in inhibiting the functional activation of muscarinic receptors in smooth muscle relative to heart, lead to the hypothesis that smooth muscle contractility is mediated by a muscarinic receptor subtype similar to that found in glandular tissue.