Several HOX genes are aberrantly expressed in a wide range of cancers interfering with their development and resistance to treatment. This seems to be often caused by alterations in the methylation profiles of their promoters. The role of HOX gene products in cancer is highly 'tissue specific', relying ultimately on their ability to regulate oncogenes or tumor-suppressor genes, directly as transcriptional regulators or indirectly interfering with the levels of epigenetic regulators. Nowadays, different strategies have been tested the use of HOX genes as therapeutic targets for cancer diagnosis and treatment. Here, we trace the history of the research concerning the involvement of HOX genes in cancer, their connection with epigenetic regulation and their potential use as therapeutic targets.

Retinoic acid (RA), the major bioactive metabolite of retinol or vitamin A, induces a spectrum of pleiotropic effects in cell growth and differentiation that are relevant for embryonic development and adult physiology. The RA activity is mediated primarily by members of the retinoic acid receptor (RAR) subfamily, namely RARα, RARβ and RARγ, which belong to the nuclear receptor (NR) superfamily of transcription factors. RARs form heterodimers with members of the retinoid X receptor (RXR) subfamily and act as ligand-regulated transcription factors through binding specific RA response elements (RAREs) located in target genes promoters. RARs also have non-genomic effects and activate kinase signaling pathways, which fine-tune the transcription of the RA target genes. The disruption of RA signaling pathways is thought to underlie the etiology of a number of hematological and non-hematological malignancies, including leukemias, skin cancer, head/neck cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, liver cancer, glioblastoma and neuroblastoma. Of note, RA and its derivatives (retinoids) are employed as potential chemotherapeutic or chemopreventive agents because of their differentiation, anti-proliferative, pro-apoptotic, and anti-oxidant effects. In humans, retinoids reverse premalignant epithelial lesions, induce the differentiation of myeloid normal and leukemic cells, and prevent lung, liver, and breast cancer. Here, we provide an overview of the biochemical and molecular mechanisms that regulate the RA and retinoid signaling pathways. Moreover, mechanisms through which deregulation of RA signaling pathways ultimately impact on cancer are examined. Finally, the therapeutic effects of retinoids are reported.

Pre-activation of the retinoid signaling pathway by all-trans retinoic acid facilitates neuronal differentiation of mesenchymal stem cells. Using protein/DNA based screening assays, we identified activator protein 2α as an important downstream target of all-trans retinoic acid. Although all-trans retinoic acid treatment significantly increased activator protein 2α transcriptional activity, it did not affect its expression. Inhibition of activator protein 2α with dominant-negative mutants reduced ATRA-induced differentiation of mesenchymal stem cells into neurons and reversed its associated functional recovery of memory impairment in the cell-based treatment of a hypoxic-ischemic brain damage rat model. Dominant-negative mutants of activator protein 2α inhibited the expression of neuronal markers which were induced by retinoic acid receptor β activation. All-trans retinoic acid treatment increased phosphorylation of activator protein 2α and resulted in its nuclear translocation. This was blocked by siRNA-mediated knockdown of retinoic acid receptor β. Furthermore, we found that retinoic acid receptor β directly interacted with activator protein 2α. In summary, the regulation of all-trans retinoic acid on activator protein 2α transcriptional activity was mediated by activation of retinoic acid receptor β and subsequent phosphorylation and nuclear translocation of activator protein 2α. Our results strongly suggest that activator protein 2α transcriptional activity is essential for all-trans retinoic acid-induced neuronal differentiation of mesenchymal stem cells.

To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes.

The nuclear retinoic acid receptors interact with specific retinoic acid (RA) response elements (RAREs) located in the promoters of target genes to orchestrate transcriptional networks involved in cell growth and differentiation. Here we describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs. More than 15,000 DR5 RAREs were identified and analyzed for their localization and conservation in vertebrates. We selected 138 elements located ±10 kb from transcription start sites and gene ends and conserved across more than 6 species. We also validated the functionality of these RAREs by analyzing their ability to bind retinoic acid receptors (ChIP sequencing experiments) as well as the RA regulation of the corresponding genes (RNA sequencing and quantitative real time PCR experiments). Such a strategy provided a global set of high confidence RAREs expanding the known experimentally validated RAREs repertoire associated to a series of new genes involved in cell signaling, development, and tumor suppression. Finally, the present work provides a valuable knowledge base for the analysis of a wider range of RA-target genes in different species.

Teleost fishes have extra Hox gene clusters owing to shared or lineage-specific genome duplication events in rayfinned fish (actinopterygian) phylogeny. Hence, extrapolating between genome function of teleosts and human or even between different fish species is difficult. We have sequenced and analyzed Hox gene clusters of the Senegal bichir (Polypterus senegalus), an extant representative of the most basal actinopterygian lineage. Bichir possesses four Hox gene clusters (A, B, C, D); phylogenetic analysis supports their orthology to the four Hox gene clusters of the gnathostome ancestor. We have generated a comprehensive database of conserved Hox noncoding sequences that include cartilaginous, lobe-finned, and ray-finned fishes (bichir and teleosts). Our analysis identified putative and known Hox cis-regulatory sequences with differing depths of conservation in Gnathostoma. We found that although bichir possesses four Hox gene clusters, its pattern of conservation of noncoding sequences is mosaic between outgroups, such as human, coelacanth, and shark, with four Hox gene clusters and teleosts, such as zebrafish and pufferfish, with seven or eight Hox gene clusters. Notably, bichir Hox gene clusters have been invaded by DNA transposons and this trend is further exemplified in teleosts, suggesting an as yet unrecognized mechanism of genome evolution that may explain Hox cluster plasticity in actinopterygians. Taken together, our results suggest that actinopterygian Hox gene clusters experienced a reduction in selective constraints that surprisingly predates the teleost-specific genome duplication.

Cellular senescence is an irreversible growth arrest that is activated in normal cells upon shortening of telomere and other cellular stresses. Bypassing cellular senescence is a necessary step for cells to become immortal during oncogenic transformation. During the spontaneous immortalization of Li-Fraumeni Syndrome (LFS) fibroblasts, we found that CREG1 (Cellular Repressor of E1A-stimulated Genes 1) expression was decreased during immortalization and increased in senescence. Moreover, we found that repression of CREG1 expression occurs via an epigenetic mechanism, promoter DNA methylation. Ectopic expression of CREG1 in the immortal LFS cell lines decreases cell proliferation but does not directly induce senescence. We confirmed this in osteosarcoma and fibrosarcoma cancer cell lines, cancers commonly seen in Li-Fraumeni Syndrome. In addition, we found that p16 (INK4a) is also downregulated in immortal cells and that coexpression of CREG1 and p16 (INK4a) , an inhibitor of CDK4/6 and Rb phosphorylation, has a greater effect than either CREG1 and p16 (INK4a) alone to reduce cell growth, induce cell cycle arrest and cellular senescence in immortal LFS fibroblasts, osteosarcoma and fibrosarcoma cell lines. Moreover, cooperation of CREG1 and p16 (INK4a) inhibits the expression of cyclin A and cyclin B by inhibiting promoter activity thereby decreasing mRNA and protein levels; these proteins are required for S-phase entry and G2/M transition. In conclusion, this is the first evidence to demonstrate that CREG1 enhances p16 (INK4a) -induced senescence by transcriptional repression of cell cycle-regulated genes.

The MultiPipMaker World Wide Web server (http://www.bx.psu.edu) provides a tool for aligning multiple DNA sequences and visualizing regions of conservation among them. This unit describes its use and gives an explanation of the resulting output files and supporting tools. Features provided by the server include alignment of up to 20 very long genomic sequences, output choices of a true, nucleotide-level multiple alignment and/or stacked, pairwise percent identity plots, and support for user-specified annotations of genomic features and arbitrary regions, with clickable links to additional information. Input sequences other than the reference can be fragmented, unordered, and unoriented.

The CYRENE Project focuses on the study of cis-regulatory genomics and gene regulatory networks (GRN) and has three components: a cisGRN-Lexicon, a cisGRN-Browser, and the Virtual Sea Urchin software system. The project has been done in collaboration with Eric Davidson and is deeply inspired by his experimental work in genomic regulatory systems and gene regulatory networks. The current CYRENE cisGRN-Lexicon contains the regulatory architecture of 200 transcription factors encoding genes and 100 other regulatory genes in eight species: human, mouse, fruit fly, sea urchin, nematode, rat, chicken, and zebrafish, with higher priority on the first five species. The only regulatory genes included in the cisGRN-Lexicon (CYRENE genes) are those whose regulatory architecture is validated by what we call the Davidson Criterion: they contain functionally authenticated sites by site-specific mutagenesis, conducted in vivo, and followed by gene transfer and functional test. This is recognized as the most stringent experimental validation criterion to date for such a genomic regulatory architecture. The CYRENE cisGRN-Browser is a full genome browser tailored for cis-regulatory annotation and investigation. It began as a branch of the Celera Genome Browser (available as open source at http://sourceforge.net/projects/celeragb /) and has been transformed to a genome browser fully devoted to regulatory genomics. Its access paradigm for genomic data is zoom-to-the-DNA-base in real time. A more recent component of the CYRENE project is the Virtual Sea Urchin system (VSU), an interactive visualization tool that provides a four-dimensional (spatial and temporal) map of the gene regulatory networks of the sea urchin embryo.

The MultiPipMaker World Wide Web server (http://www.bx.psu.edu) provides a useful tool for aligning multiple sequences and visualizing regions of conservation between them. This unit describes the use of the MultiPipMaker server and gives an explanation of the resulting output files and supporting tools. Features provided by the server include alignment of up to 20 very long genomic sequences, output choices of a true, nucleotide-level multiple alignment or stacked, pairwise percent identity plots, and user-specified annotations for genomic features and elements of choice, with clickable links to additional information. Alignments can include unordered, unoriented secondary sequences.

Hox genes encode transcription factors that are involved in the regulation of normal development and are mutated in some diseases and malformations. Chicken HOX genes have been extensively studied in the chick limb and other developmental models. To date while the chicken HOXA cluster has been completely sequenced many other chicken HOX genes are known only from partial mRNAs or unfinished genome assemblies. Furthermore, although a finished sequence of the HOXA cluster is available, the sequence has not yet been annotated. We have therefore manually annotated the available HOX sequences and improved the sequences by sequencing PCR fragments that bridge existing gaps in the genome sequences. These sequences complement the published sequences, including the currently incomplete WashUC Gallus_gallus-2.1 build, to give an improved coverage of the cluster. We used phylogenetic footprinting to map the genomic location of 398 Ultra Conserved Regions in the HOX complex 248 of which do not overlap with any known annotated coding exon. These included the hox-related microRNAs miR-10 and miR-196. The chicken HOX clusters appear to be broadly comparable to their human counterparts. A few human orthologues were not recovered from the chicken, presumably because of incomplete sequence.

AP2alpha and p53 form nuclear complexes that establish a functional partnership, which regulates the expression of certain genes involved in cell growth and metastasis. The growth effects of AP2alpha are mediated through p21WAF1/CIP1 and the ability for AP2alpha to coactivate p21 requires p53. Herein, we have localized the AP2-binding region of p53 to amino acids 305-375. Analysis of 26 distinct p53 alleles established a correlation between AP2alpha binding and transcriptional coactivation. The L350P point mutation was the only nonbinding allele that retained normal transcriptional activity by reporter assay. Although both wild-type and L350P alleles facilitated binding of AP2alpha to the p21 promoter, the L350P allele was significantly reduced in its ability to induce the endogenous p21 gene, demonstrating a striking difference in activity comparing reporter assays with activation of endogenous p53 target genes. Interestingly, expression of AP2 in the absence of radiation repressed p53-mediated induction of p21 and this effect was explained by a reduction in p53 stability induced by AP2alpha overexpression. We conclude that AP2alpha has competing effects on p53 activity through coactivation and decreased stability. These findings may provide a mechanism to account for the discrepancies reported for the association between AP2 and p21 expression in tumor tissue.

Members of the AP-2 transcription factor family have critical roles in many aspects of embryonic development. The zebrafish tfap2a mutant lockjaw (low) displays defects in skeletal and pigment cell derivatives of the neural crest. Here we show essential roles for tfap2a in subsets of embryonic cartilages and pigment cells. Defects in cartilage of the hyoid arch in low correlate with a loss of Hox group 2 gene expression and are suggestive of a transformation to a mandibular fate. In contrast, loss of joints in the mandibular arch and defects in certain types of pigment cells suggest a requirement for tfap2a independent of Hox regulation. Early melanophores do not develop in low mutants, and we propose that this results in part from a loss of kit function, leading to defects in migration, as well as kit-independent defects in melanophore specification. Iridophores are also reduced in low, in contrast to xanthophores, revealing a role for tfap2a in the development of pigment subpopulations. We propose a model of tfap2a function in the neural crest in which there are independent functions for tfap2a in specification of subpopulations of pigment cells and segmental patterning of the pharyngeal skeleton through the regulation of Hox genes. Developmental Dynamics 229:87-98, 2004.

Comparisons of DNA sequences among evolutionarily distantly related genomes permit identification of conserved functional regions in noncoding DNA. Hox genes are highly conserved in vertebrates, occur in clusters, and are uninterrupted by other genes. We aligned (PipMaker) the nucleotide sequences of the HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human, and mouse, which are separated by approximately 500 million years of evolution. In support of our approach, several identified putative regulatory elements known to regulate the expression of Hox genes were recovered. The majority of the newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac database). The regulatory intergenic regions located between the genes that are expressed most anteriorly in the embryo are longer and apparently more evolutionarily conserved than those at the other end of Hox clusters. Different presumed regulatory sequences are retained in either the Aalpha or Abeta duplicated Hox clusters in the fish lineages. This suggests that the conserved elements are involved in different gene regulatory networks and supports the duplication-deletion-complementation model of functional divergence of duplicated genes.

A lipophilic analog of vasoactive intestinal peptide (VIP), stearyl-Nle(17)-neurotensin(6-11)VIP(7-28) (SNH), that inhibited lung cancer growth, has been previously described. The mechanism of SNH inhibition of cancer growth is still being elucidated. The present study examined the effects of SNH on homeobox genes in the colon cancer cell line HT 29 that expresses VIP receptors. Homeobox genes contain a characteristic DNA sequence, coding for a stretch of 61 amino acid homeodomain that binds specific DNA motifs. While the HOX gene family contains a single homeodomain, the POU gene family contains an additional DNA binding homeodomain. HT 29 cells were incubated with SNH; RNA was extracted and subjected to reverse-transcription-polymerase chain reaction (RT-PCR) with primers that matched the conserved area of the various HOX or POU genes. The PCR products that were altered by SNH treatment were sequenced. Three candidate SNH-responsive genes, the HOX A4, the HOX B5 and the PUO V transcription factor I (Oct-3) were identified. Semi-quantitative RT-PCR with specific primers confirmed the increase in HOX A4 and the decrease in Oct-3 expression levels following SNH treatment. Thus, the HOX A4 and the Oct-3 homeobox genes may partially mediate SNH activity on cancer cells.

Expression of vertebrate Hox genes is regulated by retinoids such as retinoic acid (RA) in cell culture and in early embryonic development. Retinoic acid response elements (RAREs) have been identified in Hox gene regulatory regions, suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. Previously, two RAREs located 3' of the murine Hoxb1 gene, a DR(2) RARE and a DR(5) RARE, have been shown to regulate Hoxb1 mRNA expression in the neural epithelium and the foregut region, respectively; the foregut develops into the esophagus, liver, pancreas, lungs, and stomach. We have now examined the functional roles of these two types of 3' RAREs in regulating Hoxb1 expression at different stages of gestation, from embryonic day 7.5 to 13.5, in transgenic mice carrying specific RARE mutations. We demonstrate that the DR(5) RARE is required for the regulation of Hoxb-1 transgene region-specific expression in the gut and extraembryonic tissues, as well as for the RA-induced anteriorization of Hoxb-1 transgene expression in the gut. In contrast, expression of the Hoxb1 transgene in the neural epithelium requires only the DR(2) RARE. By in situ hybridization, we have identified a new site of Hoxb1 expression in the developing forelimbs at approximately day 12.5, and we show that, in transgenic embryos, expression in the forelimb buds requires that either the DR(2) or the DR(5) RARE is functional. Attainment of a high level of Hoxb1 transgene expression in other regions, such as in rhombomere 4 (r4) and in the somites, requires that both the DR(2) and DR(5) RAREs are functional. In addition, our transgenic data indicate that the Hoxb1 gene is expressed in other tissues such as the hernia gut, genital eminence, and lung. Our analysis shows that endogenous retinoids act through individual DR(2) and DR(5) RAREs to regulate Hoxb1 expression in different regions of the embryo and that functional redundancy between these DR(2) and DR(5) RAREs does not exist with respect to neural epithelium and the gut Hoxb1 expression.

The upregulation of left ventricular atrial natriuretic peptide (ANP) serves as a molecular marker of cardiac hypertrophy. The precise mechanisms underlying this gene induction are unclear, since the presently cloned 3.6 kilo base (kb) rat ANP promoter failed to substantially induce coupled reporter genes in chronically hypertrophied hearts. The aim of this study was to clone and to functionally analyse the upstream ANP promoter.

Upstream of the known ANP promoter, a 1.5 kb segment was cloned by the promoter walker method and found to harbour a putative CCAAT-binding site as well as multiple putative transcription factor binding sites. This newly cloned segment was ligated with a reporter gene, in vivo transfected into rat myocardium, and analysed under basal conditions or after stimulation with both acute (isovolumetric contractions in the Langendorff apparatus) and chronic wall stress (aortic banding).

Reporter gene constructs carrying the newly cloned segment conferred only little promoter activity. In hearts exposed to acute wall stress, the previously cloned 3.6 kb ANP promoter as well as a constitutive promoter (pGL3 promoter vector) were active but markedly suppressed after extension with the newly cloned upstream promoter (-88.1 and -85.5%; P < 0.05 respectively). Site directed mutagenesis of two AP-2 transcription factor binding sites (base pairs -3946 to -3954 or -4192 to -4200) eliminated this silencing effect. In hearts with chronic pressure overload hypertrophy as well as in normal, unstimulated hearts the activity of the 3.6 kb ANP promoter was weak and also abolished after ligation with the 1.5 kb upstream segment. Moreover, both putative AP-2 binding sites within the upstream rat ANP promoter bound specifically to nuclear proteins of unstimulated, acute and chronic pressure overloaded hearts as demonstrated by electrophoresis mobility shift assays.

Novel silencer elements were cloned, localized to two AP-2 binding sites in the upstream ANP promoter, and functionally characterized. Given that the putative upregulation of left ventricular ANP by the extensively studied 3.6 kb proximal promoter region is substantially diminished by the newly cloned segment, the functional significance of regulatory elements within the proximal promoter region should be re-evaluated. The molecular mechanism causing ANP mRNA induction in left ventricular hypertrophy remains obscure.

This work reports the molecular cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme (mME) gene. The proximal promoter region has features of housekeeping genes like high G + C-content and absence of TATA or CCAAT boxes. Deletion analysis of the 5' region of the mME showed that maximal transcriptional activity is located within the -205/+86 region. Footprinting analysis showed two protected regions, one comprising potential overlapped AP-1, CREB, and AP-4 sites and a second one encompassing AP-2 and several Sp1 ci-acting elements. Mutation of putative AP-1/AP-4/CREB sites reduced basal promoter activity to less than 50%. Supershift assays demonstrated the specific binding of Sp1 and AP-2 proteins. Moreover, experiments in Drosophila SL2 cells lacking endogenous Sp1 demonstrated that the Sp1 site(s) is essential to maintain a normal basal rate of transcription of this gene. A low-level expression of AP-2 enhanced the activity of a mME promoter construct in HepG2 cells and this effect was prevented by disruption of the putative AP-2 element. In contrast, higher levels of expression of AP-2 induced a DNA-independent inhibitory response. A biphasic regulation of endogenous mME gene is also shown in HepG2 cells transfected with an AP-2 expression plasmid, suggesting that availability of AP-2 protein may control this gene under physiological conditions. A recombinant lambda genomic clone containing a mME pseudogene was also isolated. The high degree of sequence conservation seems to indicate a recent emergency of this human pseudogene.

AP-2 transcription factors are sequence-specific DNA-binding proteins expressed in neural crest and other tissues during mammalian development. Three mammalian genes, AP-2alpha, AP-2beta, and AP-2gamma, have been reported previously. A partial predicted AP-2 gene was identified in tandem with AP-2beta on human chromosome 6p12-p21.1. The orthologous mouse gene, which we named Ap-2delta, was identified from a fetal mouse head cDNA library. Northern analysis revealed two transcripts in embryonic and newborn mouse brain, with markedly higher steady-state levels in the former. The predicted Ap-2delta protein comprised 452 amino acids and was highly similar to other AP-2 proteins across the DNA-binding and dimerization domains. Ap-2delta formed homodimers and heterodimers in vitro, bound an optimized AP-2 consensus DNA sequence, and transactivated gene expression in eukaryotic cells. Ap-2delta dimers bound poorly to an AP-2 binding sequence from the human metallothionein IIa promoter in vitro, revealing a sequence specificity not previously observed among other AP-2 proteins. The PY motif and critical residues in the transactivation domain, which are highly conserved in the AP-2 family and believed necessary for transactivation, were divergent in Ap-2delta. The unique protein sequence and functional features of Ap-2delta suggest mechanisms, besides tissue-specific AP-2 gene expression, for specific control of target gene activation.

The identification and interpretation of the regulatory signals within the human genome remain among the greatest goals and most difficult challenges in genome analysis. The ability to predict the temporal and spatial control of transcription is likely to require a combination of methods to address the contribution of sequence-specific signals, protein-protein interactions and chromatin structure. We present here a new procedure to identify clusters of transcription factor binding sites characteristic of sequence modules experimentally verified to direct transcription selectively to liver cells. This algorithm is sufficiently specific to identify known regulatory sequences in genes selectively expressed in liver, promising acceleration of experimental promoter analysis. In combination with phylogenetic footprinting, this improvement in the specificity of predictions is sufficient to motivate a scan of the human genome. Potential regulatory modules were identified in orthologous human and rodent genomic sequences containing both known and uncharacterized genes.

Normal embryonic development and survival in utero is dependent on an adequate supply of vitamin A. Embryos from vitamin A-deficient (VAD) pregnant rats fed an inadequate amount of all-trans retinoic acid (atRA; 12 microg per g of diet or approximately 230 microg per rat per day) exhibit severe developmental abnormalities of the anterior cardinal vein and hindbrain by embryonic day (E) 12.5 and die shortly thereafter.

In the present study, we sought to determine whether supplementation of VAD-RA supported (12 microg per g of diet) pregnant rats with retinol (ROL) at the late-gastrula (presomite or rat E9.5) or early somite stages (E10.5), or provision of higher levels of atRA throughout this period could prevent abnormalities in the developing cardiovascular and nervous systems.

A newly described defect in the sinuatrial venus valve along with enlarged anterior cardinal veins and nervous system abnormalities and the later death of embryos are prevented by supplementing pregnant animals with ROL on the morning of E9.5. If ROL supplementation is delayed by 1 day (E10.5), most embryos are abnormal and die by E18.5. Supplementation of VAD rats with atRA (250 microg per g of diet) between E8.5 and E10.5 also prevents the cardiovascular and nervous system abnormalities and a significant number of these embryos survive to parturition. Thus, high levels of atRA can obviate the need for ROL between E9.5 and E10.5.

These results support an essential role for retinoid signaling between the late gastrula and early somite stages in the rat embryo for normal morphogenesis of the primitive heart tube and the posterior hindbrain. Further, these results suggest that embryonic death occurring at midgestation in the VAD rat may be linked to the abnormal development of one or both of these embryonic structures.

Reconstructing the evolutionary history of Hox cluster origins will lead to insights into the developmental and evolutionary significance of Hox gene clusters in vertebrate phylogeny and to their role in the origins of various vertebrate body plans. We have isolated two Hox clusters from the horn shark, Heterodontus francisci. These have been sequenced and compared with one another and with other chordate Hox clusters. The results show that one of the horn shark clusters (HoxM) is orthologous to the mammalian HoxA cluster and shows a structural similarity to the amphioxus cluster, whereas the other shark cluster (HoxN) is orthologous to the mammalian HoxD cluster based on cluster organization and a comparison with noncoding and Hox gene-coding sequences. The persistence of an identifiable HoxA cluster over an 800-million-year divergence time demonstrates that the Hox gene clusters are highly integrated and structured genetic entities. The data presented herein identify many noncoding sequence motifs conserved over 800 million years that may function as genetic control motifs essential to the developmental process.

Transcription of the human tissue-type plasminogen activator (t-PA) gene is regulated by a multi-hormonal responsive enhancer at -7 kb. Transient co-transfections of Drosophila SL2 and human HT1080 fibrosarcoma cells with t-PA reporter constructs showed that Sp1 and Sp3 activate the t-PA promoter. Moreover Sp1 (but not Sp3) binding to the promoter is involved in induction by retinoic acid (RA), a response mediated through the enhancer. The role of Sp1 is specific, since mutation of the CRE element in the promoter did not affect response to RA. In contrast, the glucocorticoid induction mediated by the enhancer is independent of these Sp1 and CRE elements.

The Drosophila NK-1 homeobox gene belongs to the NK-1 class that includes a large number of vertebrate homeobox genes and is shown to be expressed in specific muscle founder cells and a subset of neuronal cells in the ventral nerve cord during embryogenesis. To determine the cis-acting regulatory elements controlling the cell-specific expression of NK-1, we measured transiently expressed chloramphencol acetyl transferase (CAT) reporter gene activities from transfected C2C12 myoblasts and NG108-15 neuroblastoma cells using various CAT constructs containing different 5' upstream regions of NK-1. From the initial analysis of 3.9 kb of the 5' upstream region, we have found that the regions from -1865 to -476 and from -476 to +100 contained strong negative and positive regulatory elements, respectively. Within the positive cis-acting region an 86-bp DNA fragment (from -435 to -350) was sufficient to activate the reporter gene in C2C12 cells, whereas additional regions (from -157 to -28 and from -510 to -425) were required for optimal activity in NG108-15 cells. Gel shift and DNaseI footprinting assays have defined a plausible binding site for C/EBP, 5'-TTTCGCAAG-3' (-424 to -416), and a novel binding site for unknown factors, 5'-AATTACTCACATCC-3' (-370 to -357). Further mutation analysis has revealed that the novel binding sequence for unknown factors is necessary and sufficient for transcriptional activity for reporter gene expression in C2C12 myoblast cells in an orientation-independent manner.

The human insulin-like growth factor II (IGF-II) gene contains four promoters that are differentially active during cell growth and development. Promoter 3 (P3) is the most active promoter in fetal and non-hepatic adult tissues. In addition to its expression during development, P3 is also the major promoter in many tumour tissues and IGF-II-expressing cell lines. Here we show that AP-2 has a dual function in P3 regulation in vivo as well as in vitro. In cells expressing low levels of endogenous AP-2, AP-2 overexpression activates P3, whereas P3 promoter activity is inhibited in cells containing abundant AP-2. Four potential AP-2-binding sites were identified in footprinting studies with recombinant AP-2. One of these AP-2-binding sites is located within the previously identified element P3-4 that contains two adjacent binding sites for IGF-II promoter-binding proteins IPBP3 and IPBP4/5. By applying binding competition assays and mutational analysis it is shown that AP-2 interferes with IPBP3 binding and transactivation in vivo as well as in vitro. Furthermore, AP-2 can bind additional elements in the proximal P3 promoter that also contribute to AP-2-mediated transactivation as shown by transient transfection assays. From these results we conclude that AP-2 is an important regulator in vivo and in vitro of IGF-II P3 activity.

The purpose of this study was to make an explicit test of the idea that a retinoid could act as a morphogen, differentially activating genes and specifying anteroposterior (a-p) level in the developing vertebrate central nervous system (CNS). Our approach was to characterize the concentration-dependent effects of retinoic acid (RA) on the neural expression of a set of a-p patterning genes, both in vivo and in an in vitro system for neural patterning. Our results indicate that a retinoid is unlikely to specify a-p level along the entire CNS. Instead, our data support the idea that the developing hindbrain may be patterned by a retinoid gradient. Sequentially more posterior hindbrain patterning genes were induced effectively by sequentially higher RA concentration windows. The most posterior CNS level induced under our RA treatment conditions corresponded to the most posterior part of the hindbrain.

Skeletal formation is a fundamental element of body patterning and is strictly regulated both temporally and spatially by a variety of molecules. Among these, retinoic acid (RA) has been shown to be involved in normal skeletal development. However, its pleiotropic effects have caused difficulty in identifying its crucial target cells and molecular mechanisms for each effect. Development of cartilage primordia is an important process in defining the skeletal structures. To address the role of RA in skeletal formation, we have generated mice expressing a dominant-negative retinoic acid receptor (RAR) in chondrogenic cells by using the type II collagen alpha1 promoter, and we have analyzed their phenotypes. These mice exhibited small cartilage primordia during development and retarded skeletal formation in both embryonic and postnatal periods. They also showed selective degeneration in their cervical vertebrae combined with homeotic transformations, but not in their extremities. The cervical phenotypes are reminiscent of phenotypes involving homeobox genes. We found that the expression of Hoxa-4 was indeed reduced in the cartilage primordia of cervical vertebrae of embryonic day 12.5 embryos. These observations demonstrate that endogenous RA acts directly on chondrogenic cells to promote skeletal growth in both embryonic and growing periods, and it regulates the proper formation of cervical vertebrae. Furthermore, RA apparently specifies the identities of the cervical vertebrae through the regulation of homeobox genes in the chondrogenic cells. Great similarities of the phenotypes between our mice and reported RAR knockout mice revealed that chondrogenic cells are a principal RA target during complex cascades of skeletal development.

Shuttle vectors are useful tools for studying DNA replication and mutagenesis. SV40-based shuttle vectors are popular because of their ease of use and quick results. However, one complication with the use of SV40-based shuttle vectors is the interaction of cellular p53 protein with the T-antigen of SV40. Wild-type, but not mutant p53 has been shown to be involved in DNA replication and DNA repair. To address this concern, we have modified an SV40-based shuttle vector, pZ189, by exchanging the wt T-antigen for a mutant SV40 T-antigen, which is unable to bind with p53. This shuttle vector, pZ402, provides us with a tool to study DNA replication and genomic instability in cells with varying genetic backgrounds without interference from the interaction of T-antigen with p53.