Repression of TFII-I-dependent transcription by nuclear exclusion.

TFII-I is an unusual transcription factor possessing both basal and signal-induced transcriptional functions. Here we report the characterization of a TFII-I-related factor (MusTRD1/BEN) that regulates transcriptional functions of TFII-I by controlling its nuclear residency. MusTRD1/BEN has five or six direct repeats, each containing helix--loop--helix motifs, and, thus, belongs to the TFII-I family of transcription factors. TFII-I and MusTRD1/BEN, when expressed individually, show predominant nuclear localization. However, when the two proteins are coexpressed ectopically, MusTRD1/BEN locates almost exclusively to the nucleus, whereas TFII-I is largely excluded from the nucleus, resulting in a loss of TFII-I-dependent transcriptional activation of the c-fos promoter. Mutation of a consensus nuclear localization signal in MusTRD1/BEN results in a reversal of nuclear residency of the two proteins and a concomitant gain of c-fos promoter activity. These data suggest a means of transcriptional repression by competition at the level of nuclear occupancy.

An efficient cis-element discovery method using multiple sequence comparisons based on evolutionary relationships.

The discovery of cis-element control motifs in noncoding DNA poses a difficult problem in genome analysis. Functional analysis by means of reporter constructs expressed in transgenic organisms is the most reliable method, but is by itself time-consuming and expensive. Searching noncoding DNA for known control motifs by sequence analysis is problematic, since protein binding motifs are short, in the range of 8-10 bp, and occur frequently by chance. Heretofore, the most reliable sequence analysis method has been the comparison of homologous sequence domains in related but moderately evolutionarily divergent species such as, for example, mouse and human. In such pairwise combinations, control regions are conserved because they serve a vital function and can be identified by their similar sequences. Single pairwise comparisons, however, allow the discovery of conserved sequence strings only at low resolution and without specific identity. We have investigated the possibility of using multiple sequence comparisons to correct these shortcomings. We applied this method to the Hoxc8 early enhancer region that has been previously analyzed in depth by functional methods and through its application successfully identified known protein binding cis-element motifs. Candidate protein binding sites could also be identified. This method, based on evolutionarily related sequence comparisons, should be quite useful as a prescreening step prior to functional analysis with corresponding savings in time and resources.

Isolation and characterization of BEN, a member of the TFII-I family of DNA-binding proteins containing distinct helix-loop-helix domains.

The transcriptional regulation of the Hoxc8 gene is controlled during early mouse embryogenesis by an enhanceosome-like control region, termed the early enhancer (EE), located 3 kb upstream from the Hoxc8 translation start site. The EE is involved in establishing the posterior expression pattern of Hoxc8 at embryonic day (E) 8.5-9. 0. Genetic and biochemical data have shown that nuclear factors interact with this region in a sequence-specific manner. We have used a yeast one-hybrid screen in a search for transcription factors that bind to EE motifs and have isolated a novel murine DNA-binding protein, termed BEN (binding factor for early enhancer). The ORF of BEN encodes a protein of 1072 amino acids and contains six helix-loop-helix domains, a hydrophobic leucine zipper-like motif, and a serine-rich repeat. The murine BEN gene is structurally similar to the human gene TFII-I in that both genes encode unique 95-amino acid long helix-loop/span-helix domains. The BEN gene produces several major transcripts (3.6, 4.4, and 5.9 kb) present in most adult tissues and shows discrete spatial and temporal domains of expression in areas of epithelial-mesenchymal interaction during mouse embryogenesis from E9.5 to E12.5. Several BEN-encoded polypeptides of different sizes ranging from 165 to 40 kDa were identified by Western blot analysis using BEN-specific polyclonal Abs. We propose, on the bases of sequence homology, that BEN is the mouse ortholog of the recently described human gene, WBSCR11, known also as GTF2IRD1, GTF3, Cream1, and MusTRD1. This gene is deleted hemizygously in individuals with Williams Syndrome, an autosomal dominant genetic condition characterized by complex physical, cognitive, and behavioral traits resulting from a perturbed developmental process.

A recombinogenic targeting method to modify large-inserts for cis-regulatory analysis in transgenic mice: construction and expression of a 100-kb, zebrafish Hoxa-11b-lacZ reporter gene.

The identification of cis-sequences responsible for spatiotemporal patterns of gene expression often requires the functional analysis of large genomic regions. In this study a 100-kb zebrafish Hoxa-11b-lacZ reporter gene was constructed and expressed in transgenic mice. PAC clone 10-O19, containing a portion of the zebrafish HoxA-b cluster, was captured into the yeast-bacterial shuttle vector, pPAC-ResQ, by recombinogenic targeting. A lacZ reporter gene was then inserted in-frame into exon 1 of the zfHoxa-11b locus by a second round of recombinogenic targeting. Expression of the zfHoxa-11b-lacZ reporter gene in 10.5 d.p.f. transgenic mouse embryos was observed only in the posterior portion of the A-P axis, in the paraxial mesoderm, neural tube, and somites. These findings demonstrate the utility of recombinogenic targeting for the modification and expression of large inserts captured from P1/PAC clones.

Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper.

We have developed a method to clone genomic DNA selectively into a yeast-bacterial shuttle vector, pClasper, by recombinogenic targeting in yeast. A gene-specific pClasper targeting vector was constructed with small recombinogenic ends (500 bp) derived from flanking sequences of the genomic region to be cloned. Linearized, recombinogenic pClasper targeting vector and native genomic DNA were cotransformed into yeast. The gene of interest is selectively cloned by recombination between the recombinogenic ends in the targeting vector and homologous regions in the genomic DNA. Here we demonstrate direct cloning of a stably integrated Hoxc8-LacZ-Ura3 reporter gene construct from a mouse embryo fibroblast cell line and single-copy genes from total human genomic DNA. The frequency of capture of the recombinant insert was 0.05-3% of transformants. In contrast to previous reports, we were able to clone genomic DNA directly with a vector containing yeast autonomous replicating sequences. This approach provides a powerful method with which to clone and modify genes precisely for functional analysis.

Mammalian Trithorax and polycomb-group homologues are antagonistic regulators of homeotic development.

Control of cell identity during development is specified in large part by the unique expression patterns of multiple homeobox-containing (Hox) genes in specific segments of an embryo. Trithorax and Polycomb-group (Trx-G and Pc-G) proteins in Drosophila maintain Hox expression or repression, respectively. Mixed lineage leukemia (MLL) is frequently involved in chromosomal translocations associated with acute leukemia and is the one established mammalian homologue of Trx. Bmi-1 was first identified as a collaborator in c-myc-induced murine lymphomagenesis and is homologous to the Drosophila Pc-G member Posterior sex combs. Here, we note the axial-skeletal transformations and altered Hox expression patterns of Mll-deficient and Bmi-1-deficient mice were normalized when both Mll and Bmi-1 were deleted, demonstrating their antagonistic role in determining segmental identity. Embryonic fibroblasts from Mll-deficient compared with Bmi-1-deficient mice demonstrate reciprocal regulation of Hox genes as well as an integrated Hoxc8-lacZ reporter construct. Reexpression of MLL was able to overcome repression, rescuing expression of Hoxc8-lacZ in Mll-deficient cells. Consistent with this, MLL and BMI-I display discrete subnuclear colocalization. Although Drosophila Pc-G and Trx-G members have been shown to maintain a previously established transcriptional pattern, we demonstrate that MLL can also dynamically regulate a target Hox gene.

Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice.

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of beta-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.

Evolution of chordate hox gene clusters.

In this article, we consider the role of the Hox genes in chordate and vertebrate evolution from the viewpoints of molecular and developmental evolution. Models of Hox cluster duplication are considered with emphasis on a threefold duplication model. We also show that cluster duplication is consistent with a semiconservative model of duplication, where following duplication, one daughter cluster remains unmodified, while the other diverges and assumes a new architecture and presumably new functions. Evidence is reviewed, suggesting that Hox gene enhancers have played an important role in body plan evolution. Finally, we contrast the invertebrates and vertebrates in terms of genome and Hox cluster duplication which are present in the latter, but not the former. We question whether gene duplication has been important in vertebrates for the introduction of novel features such as limbs, a urogenital system, and specialized neuromuscular interactions.

Phylogenetically conserved CK-II phosphorylation site of the murine homeodomain protein Hoxb-6.

In an effort to characterize the signal transduction mechanisms that operate to regulate homeodomain protein function, we have analyzed the phosphorylation state of two homeodomain proteins, Hoxb-6 and Hoxc-8, in vitro and in vivo. The baculovirus expression system was employed to demonstrate that Hoxb-6 is phosphorylated in Sf9 cells while Hoxc-8 is not. Using two-dimensional tryptic phosphopeptide mapping and purified protein kinases, we demonstrate that Hoxb-6 is phosphorylated in vitro by casein kinase II and cAMP-dependent protein kinase. The casein kinase II phosphorylation site was mapped to serine-214. Two-dimensional tryptic phosphopeptide mapping of immunoprecipitated Hoxb-6 from mouse embryonic spinal cords demonstrates that the same peptide phosphorylated in vitro and in Sf9 cells by casein kinase II is also phosphorylated in vivo. The conservation of this site in several homeodomain proteins from various species is discussed.

pPAC-ResQ: A yeast-bacterial shuttle vector for capturing inserts from P1 and PAC clones by recombinogenic targeted cloning.

We have developed a method to capture inserts from P1 and P1 artificial chromosome (PAC) clones into a yeast-bacteria shuttle vector by using recombinogenic targeting. We have engineered a vector, pPAC-ResQ, a derivative of pClasper, which was previously used to capture inserts from yeast artificial chromosome clones. pPAC-ResQ contains DNA fragments flanking the inserts in P1 and PAC vectors as recombinogenic ends. When linearized pPAC-ResQ vector and P1 or PAC DNA are cotransformed into yeast, recombination between the two leads to the transfer of inserts into pPAC-ResQ. pPAC-ResQ clones thus obtained can be further modified in yeast for functional analysis and shuttled to Escherichia coli to produce large quantities of cloned DNA. This approach provides a rapid method to modify P1/PAC clones for functional analysis.

Recombinogenic targeting: a new approach to genomic analysis--a review.

Currently, recombinational cloning procedures based upon methods developed for yeast, Saccharomyces cerevisiae, are being exploited for targeted cloning and in-vivo modification of genomic clones. In this review, we will discuss the development of large-insert vectors, homologous recombination-based techniques for cloning and modification, and their application towards functional analysis of genes using transgenic mouse model systems.

Comparative studies on mammalian Hoxc8 early enhancer sequence reveal a baleen whale-specific deletion of a cis-acting element.

Variations in regulatory regions of developmental control genes have been implicated in the divergence of axial morphologies. To find potentially significant changes in cis-regulatory regions, we compared nucleotide sequences and activities of mammalian Hoxc8 early enhancers. The nucleotide sequence of the early enhancer region is extremely conserved among mammalian clades, with five previously described cis-acting elements, A-E, being invariant. However, a 4-bp deletion within element C of the Hoxc8 early enhancer sequence is observed in baleen whales. When assayed in transgenic mouse embryos, a baleen whale enhancer (unlike other mammalian enhancers) directs expression of the reporter gene to more posterior regions of the neural tube but fails to direct expression to posterior mesoderm. We suggest that regulation of Hoxc8 in baleen whales differs from other mammalian species and may be associated with variation in axial morphology.

Multiple phases of expression and regulation of mouse Hoxc8 during early embryogenesis.

Hox genes are expressed in dynamic patterns during embryogenesis consistent with their role in axial specifications. To study the distribution of mouse Hoxc8, a homeodomain containing protein, we raised monoclonal antibodies against the least conserved portion of Hoxc8. Using these antibodies, we have examined early and mid-gestation embryos for the distribution of the protein. At the end of gastrulation Hoxc8 is expressed in the caudal portion of the embryo. In the neural tube, an early phase when all cells express Hoxc8 is distinguished from a late phase with predominant expression in differentiating neurons. A comparison of this expression pattern with that of a reporter gene under the control of the early Hoxc8 enhancer demarcates three separate regulatory components: (1) initiation and establishment; (2) maintenance; and (3) downregulation. We propose that Hoxc8 expression during embryogenesis is established in multiple phases. Possible regulatory mechanisms involved in generating such a complex domain of Hox gene expression are discussed.

Modification of expression and cis-regulation of Hoxc8 in the evolution of diverged axial morphology.

Differential Hox gene expression between vertebrate species has been implicated in the divergence of axial morphology. To examine this relationship, we have compared expression and transcriptional regulation of Hoxc8 in chicken and mouse. In both species, expression of Hoxc8 in the paraxial mesoderm and neural tube is associated with midthoracic and brachial identities, respectively. During embryogenesis, there is a temporal delay in the activation of Hoxc8 in chicken compared with mouse. As a result, chicken Hoxc8 expression in the paraxial mesoderm is at a posterior axial level, extending over a smaller domain compared with mouse Hoxc8 expression. This finding is consistent with a shorter thoracic region in chicken compared with mouse. In addition, the chicken Hoxc8 early enhancer, differing from its mouse counterpart in only a few specific nucleotides, directs a reporter gene expression to a more posterior domain in transgenic mouse embryos. These findings are consistent with the concept that the diversification of axial morphology has been achieved through changes in cis-regulation of developmental control genes.

Molecular evolution of Hox gene regulation: cloning and transgenic analysis of the lamprey HoxQ8 gene.

The mammalian Hox clusters arose by duplication of a primordial cluster. The duplication of Hox clusters created redundancy within cognate groups, allowing for change in function over time. The lamprey, Petromyzon marinus, occupies an intermediate position within the chordates, both in terms of morphologic complexity and possibly cluster number. To determine the extent of divergence among Hox genes after duplication events within vertebrates, we analyzed Hox genes belonging to cognate group 8. Here we report characterization of the HoxQ8 gene, which shows conservation with mammalian genes in its amino-terminal, homeobox and hexapeptide sequences, and in the position of its splice sites. A beta-galactosidase reporter gene was introduced in the HoxQ8 genomic region by targeted recombinational cloning using a yeast-bacteria shuttle vector, pClasper. These reporter gene constructs were tested for their ability to direct region-specific expression patterns in transgenic mouse embryos. Lamprey enhancers direct expression to posterior neural tube but not to mesoderm, suggesting conservation of neuronal enhancers. In the presence of the mouse heat shock promoter, lamprey enhancers could also direct expression to the posterior mesoderm suggesting that there has been some divergence in promoter function. Our results suggest that comparative studies on Hox gene structure and analysis of regulatory elements may provide insights into changes concomitant with Hox cluster duplications in the chordates.

Phylogenetic reconstruction of vertebrate Hox cluster duplications.

In vertebrates and the cephalochordate, amphioxus, the closest vertebrate relative, Hox genes are linked in a single cluster. Accompanying the emergence of higher vertebrates, the Hox gene cluster duplicated in either a single step or multiple steps, resulting in the four-cluster state present in teleosts and tetrapods. Mammalian Hox clusters (designated A, B, C, and D) extend over 100 kb and are located on four different chromosomes. Reconstructing the history of the duplications and its relation to vertebrate evolution has been problematic due to the lack of alignable sequence information. In this study, the problem was approached by conducting a statistical analysis of sequences from the fibrillar-type collagens (I, II, III, and IV), genes closely linked to each Hox cluster which likely share the same duplication history as the Hox genes. We find statistical support for the hypothesis that the cluster duplication occurred as multiple distinct events and that the four-cluster situation arose by a three-step sequential process.

Selective expression of mRNA coding for the truncated form of erythropoietin receptor in hematopoietic cells and its decrease in patients with polycythemia vera.

The mRNA encoding full-length erythropoietin (EPO) receptor (EPOR-F) comprises exons I through VIII. Another membrane-bound EPOR (EPOR-T) isoform has a truncated cytoplasmic region and is encoded by the mRNA containing unspliced intron VII (EPOR-T mRNA). EPOR-T is believed to have a dominantly negative function against EPOR-F. We show that EPOR-T mRNA is markedly decreased in the blood cells of patients with polycythemia vera (PV). We also show that EPOR-T mRNA is not detected in erythroid/megakaryocytic leukemia cell lines, but is expressed in nonerythroid/nonmegakaryocytic lines, suggesting the presence of a cell type-specific system by which intron VII of the EPOR transcript is spliced. Deregulation of this splicing system in early hematopoietic progenitors possibly explains the profound decrease in EPOR-T mRNA and consequent pathophysiology of PV.

Inhibition of DLX-7 homeobox gene causes decreased expression of GATA-1 and c-myc genes and apoptosis.

The DLX gene family is a family of divergent homeobox genes which are related to the Drosophila distal-less (Dll) gene and has been reported to be expressed primarily in the forebrain and craniofacial structures. We have previously identified a new member of this family, DLX-7. We now report that this gene is expressed in normal hematopoietic cells and leukemia cell lines with erythroid characteristics. We used an antisense oligonucleotide targeted against the translation start site of DLX-7 mRNA to inhibit its expression in a human erythroleukemia cell line K562, which expresses DLX-7 at a high level. The antisense oligonucleotide efficiently reduced the DLX-7 mRNA, while control oligonucleotides, including a mutant oligonucleotide identical to the antisense sequence except for four nucleotide mismatches, had no effect on DLX-7 mRNA level. Inhibition of DLX-7 expression decreased the plating efficiency by approximately 70% compared with control. The antisense treatment caused apoptosis, as shown by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) method. Down-regulation of DLX-7 expression by antisense treatment was associated with a reduction in GATA-1 and c-myc mRNA levels. Thus, we conclude that DLX-7 is expressed in hematopoietic cells and that the inhibition of its expression results in the decreased levels of GATA-1 and c-myc genes, with an accompanying induction of apoptosis.