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Biomedical subjects

Frank H Ruddle

Publications and source records attributed to Frank H Ruddle.

At least 19 recordsLinked to original sources
PubMed2007

Mouse naked cuticle 2 (mNkd2) as a direct transcriptional target of Hoxc8 in vivo.

Mouse naked cuticle 2 (mNkd2), the mammalian homolog of the Drosophila segment polarity gene naked cuticle (nkd), encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. The transcription factor, Hoxc8, a member of the homeobox gene family, is vital for growth and differentiation. Chromatin immunoprecipitation (ChIP) assay, an electrophoretic mobility shift assay (EMSA), and a reporter assay demonstrated that endogenous Hoxc8 protein binds directly to the enhancer region of the mNkd2 gene, implying a Hoxc8-dependent transcriptional activity. Introduction of exogenous Hoxc8 into NIH3T3 cell lines lacking wild-type Hoxc8 dramatically reduced expression of mNkd2 mRNA. If, as the results suggest, mNkd2 is a direct target of Hoxc8, it represents a novel mechanism by which Hoxc8 might cross-talk with the Wnt signaling pathway by regulating mNkd2.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Identification of a Hoxc8-regulated transcriptional network in mouse embryo fibroblast cells.

The transcription factor, Hoxc8, is a member of the homeobox gene family that is vital for growth and differentiation. Previously, we identified 34 genes whose expression levels were changed at least 2-fold by forced expression of Hoxc8 in C57BL/6J mouse embryo fibroblast cells using a mouse 16,463-gene oligonucleotide microarray. In the present study, we used the combined power of microarray profiling, global Hoxc8 DNA-binding site analysis, and high-throughput chromatin immunoprecipitation assays to identify direct and biologically relevant targets of Hoxc8 in vivo. Here we show that 19 of the 34 responsive genes contain Hoxc8 consensus DNA-binding sequence(s) in their regulatory regions. Chromatin immunoprecipitation analysis indicated that Hoxc8-DNA interaction was detected in five of the 19 candidate genes. All of these five target genes have been implicated in oncogenesis, cell adhesion, proliferation, and apoptosis. Overall, the genes described here should aid in the understanding of global regulatory networks of Hox genes and to provide valuable insight into the molecular basis of Hoxc8 in development and carcinogenesis.

5' Flanking Region↗
PubMed2006

Multiple roles of hoxc8 in skeletal development.

We are interested in investigating the function of Hoxc8 in skeletogenesis during mouse development. Previous studies have shown that deregulation of Hoxc8 expression in the mouse leads to several skeletal defects, such as homeotic transformation in the thoracic vertebrae, abnormal development of the rib cage, and overproliferation of chondrocytes in the hypertrophic area. By deleting a crucial enhancer of Hoxc8 in vivo, we found that precise temporal expression of Hoxc8 is important for determining the correct identity of the vertebral column in early embryos. We also identified downstream targets of Hoxc8 relevant to osteoblast differentiation at later developmental stages.

Animals↗
PubMed2005

HnRNP A3 genes and pseudogenes in the vertebrate genomes.

The hnRNP A/B type proteins are abundant nuclear factors that bind to Pol II transcripts and are involved in numerous RNA-related activities. To date most data on the hnRNP A/B family have been obtained with recombinant proteins and cell cultures. Further characterization can result from an examination of the impact of various modifications in intact functional loci; however, such characterization is hampered by the presence of numerous and widely dispersed hnRNP A/B-related sequences in the mammalian genome. We have found hnRNP A3, a poorly recognized member of the hnRNP A/B family, among candidate transcription factors that interact with the regulatory region of the Hoxc8 gene and screened the human and mouse genomes for genes that encode hnRNP A3. We demonstrate that the sequence reported previously as the human hnRNP A3 gene (Accession number S63912) and located on 10p11.1 belongs to a processed pseudogene of the functional intron-containing locus HNRPA3, which we have identified on 2q31.2. We have also identified its murine orthologs on mouse chromosome 2D and rat chromosome 3q23. Alternative splices were revealed at the N-terminus and in the middle of hnRNP A3. 14 and 28 additional loci in the human and mouse genome, respectively, were mapped and identified as hnRNP A3 processed pseudogenes. In addition, we have found and compared hnRNP A3 orthologous genes in Gallus gallus, Xenopus tropicalis, and Danio rerio. The present in silico analysis serves as a necessary step toward a further functional characterization of hnRNP A3.

Alternative Splicing↗
PubMed2005

The identification of Hoxc8 target genes.

Hox genes encode transcription factors that control spatial patterning during embryogenesis. To date, downstream targets of Hox genes have proven difficult to identify. Here, we describe studies designed to identify target genes under the control of the murine transcription factor Hoxc8. We used a mouse 16,463 gene oligonucleotide microarray to identify mRNAs whose expression was altered by the overexpression of Hoxc8 in C57BL/6J mouse embryo fibroblasts (MEF) in cell culture (in vitro). We identified a total of 34 genes whose expression was changed by 2-fold or greater: 16 genes were up-regulated, and 18 genes were down-regulated. The majority of genes encoded proteins involved in critical biological processes, such as cell adhesion, migration, metabolism, apoptosis, and tumorigenesis. Two genes showed high levels of regulation: (i) secreted phosphoprotein 1 (Spp1), also known as osteopontin (OPN), was down-regulated 4.8-fold, and (ii) frizzled homolog 2 (Drosophila) (Fzd2) was up-regulated 4.4-fold. Chromatin immunoprecipitation (ChIP) analysis confirmed the direct interaction between the OPN promoter and Hoxc8 protein in vivo, supporting the view that OPN is a direct transcriptional target of Hoxc8.

Animals↗
PubMed2004

GTF2IRD2 is located in the Williams-Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix-loop-helix repeats.

Williams-Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I, located within this critical region, encode proteins of the TFII-I family with multiple helix-loop-helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2, which has sequence and structural similarity to the GTF2I and GTF2IRD1 paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that GTF2IRD2 is more closely related to GTF2I than to GTF2IRD1 and apparently is derived from the GTF2I sequence. The comparison of GTF2I and GTF2IRD2 genes revealed two distinct regions of homology, indicating that the helix-loop-helix domain structure of the GTF2IRD2 gene has been generated by two independent genomic duplications. We speculate that GTF2I is derived from GTF2IRD1 as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding GTF2IRD2 genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams-Beuren syndrome critical region.

Amino Acid Sequence↗
PubMed2004

The shark HoxN cluster is homologous to the human HoxD cluster.

The statistical analysis of phylogenetic footprints in the two known horn shark Hox clusters and the four mammalian clusters shows that the shark HoxN cluster is HoxD-like. This finding implies that the most recent common ancestor of jawed vertebrates had at least four Hox clusters, including those which are orthologous to the four mammalian Hox clusters.

Amino Acid Sequence↗
PubMed2004

Evidence for independent Hox gene duplications in the hagfish lineage: a PCR-based gene inventory of Eptatretus stoutii.

Hox genes code for transcription factors that play a major role in the development of all animal phyla. In invertebrates these genes usually occur as tightly linked cluster, with a few exceptions where the clusters have been dissolved. Only in vertebrates multiple clusters have been demonstrated which arose by duplication from a single ancestral cluster. This history of Hox cluster duplications, in particular during the early elaboration of the vertebrate body plan, is still poorly understood. In this paper we report the results of a PCR survey on genomic DNA of the pacific hagfish Eptatretus stoutii. Hagfishes are one of two clades of recent jawless fishes that are an offshoot of the early radiation of jawless vertebrates. Our data provide evidence for at least 33 distinct Hox genes in the hagfish genome, which is most compatible with the hypothesis of multiple Hox clusters. The largest number, seven, of distinct homeobox fragments could be assigned to paralog group 9, which could imply that the hagfish has more than four clusters. Quartet mapping reveals that within each paralog group the hagfish sequences are statistically more closely related to gnathostome Hox genes than with either amphioxus or lamprey genes. These results support two assumptions about the history of Hox genes: (1) The association of hagfish homeobox sequences with gnathostome sequences suggests that at least one Hox cluster duplication event happened in the stem of vertebrates, i.e., prior to the most recent common ancestor of jawed and jawless vertebrates. (2) The high number of paralog group 9 sequences in hagfish and the phylogenetic position of hagfish suggests that the hagfish lineage underwent additional independent Hox cluster/-gene duplication events.

Animals↗
PubMed2003

Homeoprotein DLX-1 interacts with Smad4 and blocks a signaling pathway from activin A in hematopoietic cells.

In the transforming growth factor beta (TGF-beta) superfamily, activin A, TGF-beta1, and bone morphogenic protein 4 (BMP-4) have various effects on hematopoiesis, including early mesodermo-hematogenesis. After these cytokines bind to their respective receptor, a regulatory Smad is phosphorylated and becomes associated with Smad4, the common Smad, and the resulting complex translocates to the nucleus to regulate transcription. DLX1 is the product of a member of the distal-less homeobox gene family, which is known to have important roles in embryogenesis, particularly in craniofacial development, and in GABAergic neurogenesis. DLX1 has been reported to be temporally and spatially coexpressed with BMP-4 during embryogenesis in selected contexts. We report here that, in addition to the previously reported regions/cells, DLX1 is expressed in hematopoietic cells in a lineage-dependent manner and that DLX1 interacts with Smad4 through its homeodomain. We show that it blocks multiple signals from TGF-beta superfamily cytokines such as activin A, TGF-beta1, and BMP-4, including differentiation of a hematopoietic cell line by activin A. Taken together, these data suggest that DLX1 may function as a regulator of multiple signals from TGF-beta superfamily members in broad biological contexts during blood production.

Activins↗
PubMed2003

Homez, a homeobox leucine zipper gene specific to the vertebrate lineage.

This work describes a vertebrate homeobox gene, designated Homez (homeodomain leucine zipper-encoding gene), that encodes a protein with an unusual structural organization. There are several regions within Homez, including three atypical homeodomains, two leucine zipper-like motifs, and an acidic domain. The gene is ubiquitously expressed in human and murine tissues, although the expression pattern is more restricted during mouse development. Genomic analysis revealed that human and mouse genes are located at 14q11.2 and 14C, respectively, and are composed of two exons. The zebrafish and pufferfish homologs share high similarity to mammalian sequences, particularly within the homeodomain sequences. Based on homology of homeodomains and on the similarity in overall protein structure, we delineate Homez and members of ZHX family of zinc finger homeodomain factors as a subset within the superfamily of homeobox-containing proteins. The type and composition of homeodomains in the Homez subfamily are vertebrate-specific. Phylogenetic analysis indicates that Homez lineage was separated from related genes >400 million years ago before separation of ray- and lobe-finned fishes. We apply a duplication-degeneration-complementation model to explain how this family of genes has evolved.

Alternative Splicing↗
PubMed2003

Enhancer timing of Hox gene expression: deletion of the endogenous Hoxc8 early enhancer.

The proper expression of Hox genes is necessary for the accurate patterning of the body plan. The elucidation of the developmental genetic basis of transcriptional regulation of Hox genes by the study of their cis-regulatory elements provides crucial information regarding the establishment of axial specification. In this report, we investigate the role of the early enhancer (EE) of the murine Hoxc8 gene to better understand its role in pattern formation. Previous reports show that knockouts of the endogenous Hoxc8 coding region result in a combination of neural, behavioral and skeletal phenotypes. In this report, we limit ourselves to a consideration of the skeletal abnormalities. Early reports from our laboratory based on exogenous transgenic reporter constructs implicate a 200 bp non-coding element 3 kb upstream of the Hoxc8 promoter as a crucial enhancer that regulates the transcription of Hoxc8. In the present work, we have deleted this regulatory region from the endogenous genome using embryonic stem cell technology. Our results show that the deletion of the EE results in a significant delay in the temporal expression of Hoxc8. We also show that the deletion of the EE does not eliminate the expression of the Hoxc8 protein, but delays the attainment of control levels of expression and anterior and posterior boundaries of expression on the AP axis. The temporal delay in Hoxc8 expression is sufficient to produce phenocopies of many of the axial skeletal defects associated with the complete absence of Hoxc8 gene product as previously reported for the Hoxc8-null mutation. Our results are consistent with emerging evidence that the precise temporal expression of Hox genes is crucial for the establishment of regional identities. The fact that the EE deletion does not eliminate Hoxc8 expression indicates the existence of a Hoxc8 transcriptional regulatory apparatus independent to some degree of the Hoxc8 EE. In a comparison of our results with those reported previously by others investigating temporal control of Hox gene expression, we have discovered a structural similarity between the Hoxc8 EE reported here and a transcriptional control element located in the Hoxd11 region. We speculate that a distributed system of expression timing control may exist that is similar the one we propose for Hoxc8. Last, our data is consistent with the position that disparate regulatory pathways are responsible for the expression of Hoxc8 in the organogenesis of somites, neural tube and limb bud.

Animals↗
PubMed2003

Regulation of Dlx3 gene expression in visceral arches by evolutionarily conserved enhancer elements.

The mammalian Distal-less (Dlx) clusters (Dlx1-2, Dlx5-6, and Dlx3-7) have a nested expression pattern in developing visceral (branchial) arches. Genetic regulatory mechanisms controlling Dlx spatial expression within the visceral arches have not yet been defined. Here we show that an enhancer in the Dlx3-7 cluster can regulate the visceral arch specific expression pattern of the Dlx3 gene. We have used a 79-kb transgene construct containing the entire Dlx3-7 bigene cluster with a LacZ reporter inserted in frame in the first exon of the Dlx3 gene. Visceral arch expression is absent when a 4-kb element located within the Dlx3-7 intergenic region is deleted. A 245-bp element (I37-2) whose DNA sequence is highly conserved between human and mouse located within the 4kb-deleted region can drive visceral arch expression when fused to a hsp68-lacZ reporter transgene construct. Reporter expression is detected in 9.5 and 10.5 days postcoitum transgenic embryos in a manner consistent with the endogenous Dlx3 expression pattern in the mesenchyme of the first and second visceral arches. Thus the I37-2 element is both necessary and sufficient for Dlx3 expression. The I37-2 element contains several putative binding sites for several transcription factors including Dlx and other homeodomain proteins within the evolutionarily conserved region. Significantly, the I37-2 element shows a sequence-match including a Dlx binding site to a cis-element in the Dlx5-6 intermediate region designated mI56i [Zerucha, T., Stuhmer, T., Hatch, G., Park, B. K., Long, Q., Yu, G., Gambarotta, A., Schultz, J. R., Rubenstein, J. L. & Ekker, M. (2000) J. Neurosci. 20, 709-721], despite distant phylogenetic relationship between these clusters. Our results provide evidence for a concerted role for DLX auto- and cross-regulation in the establishment of a nested expression pattern for Dlx3-7 and Dlx5-6 clusters within the visceral arches.

Animals↗
PubMed2003

The role of gene duplication in the evolution and function of the vertebrate Dlx/distal-less bigene clusters.

The Dlx gene family controls developmental patterning principally in the pharyngeal and cranial regions. We review the structure and function of these genes in the vertebrates and relate these properties to their evolution. We particularly focus on the Dlx3-7 bigene cluster which we postulate to be more derived phylogenetically and functionally than the other two bigene clusters, Dlx1-2 and Dlx5-6. We stress the transcriptional control of the Dlx3-7 bigene cluster, and postulate its control by Dlx1-2.

Animals↗
PubMed2003

Phylogenetic analysis of the mammalian Hoxc8 non-coding region.

The non-coding intergenic regions of Hox genes are remarkably conserved among mammals. To determine the usefulness of this sequence for phylogenetic comparisons, we sequenced an 800-bp fragment of the Hoxc9-Hoxc8 intergenic region from several species belonging to different mammalian clades. Results obtained from the phylogenetic analysis are congruent with currently accepted mammalian phylogeny. Additionally, we found a TC mini satellite repeat polymorphism unique to felines. This polymorphism may serve as a useful marker to differentiate between mammalian species or as a genetic marker in feline matings. This study demonstrates usefulness of a comparative approach employing non-coding regions of Hox gene complexes.

Animals↗
PubMed2003

Impact of transgenic technologies on functional genomics.

Gene transfer technologies in mammals are the focus of renewed interest owing to the recent emphasis on analyzing gene function in the postgenomic era. Three important developments in this area include transgenics, gene targeting and nuclear transfer or animal cloning. These technological innovations have enhanced our ability to analyze gene function at the level of the whole organism and have provided the means to modify gene expression. This review discusses the origins and current status of transgenic technologies. Various applications and technologies including chromosome engineering, stem cells, gene traps and modification of livestock are presented. The impact of mouse technologies and genomics on functional analyses is also discussed.

Animals↗
PubMed2002

Physical and functional interactions of histone deacetylase 3 with TFII-I family proteins and PIASxbeta.

TFII-I family proteins are characterized structurally by the presence of multiple reiterated I-repeats, each containing a putative helix-loop-helix domain. Functionally, they behave as multifunctional transcription factors that are activated by a variety of extracellular signals. In studying their subcellular localization, we noticed that these transcription factors frequently reside in subnuclear domains/dots. Because nuclear dots are believed often to harbor components of histone deacetylase enzymes (HDACs), we investigated whether TFII-I family proteins colocalize and interact with HDACs. Here, we show that TFII-I and its related member hMusTRD1/BEN physically and functionally interact with HDAC3. The TFII-I family proteins and HDAC3 also show nearly identical expression patterns in early mouse development. Consistent with our earlier observation that TFII-I family proteins also interact with PIASxbeta, a member of the E3 ligase family involved in the small ubiquitin-like modifier (SUMO) pathway, we show further that PIASxbeta physically and functionally interacts with HDAC3 and relieves the transcriptional repression exerted by HDAC3 upon TFII-I-mediated gene activation. These results suggest a complex interplay between two posttranslational pathways-histone modification and SUMOylation-brokered in part by TFII-I family proteins.

Animals↗
PubMed2002

Genomic analysis of Hox clusters in the sea lamprey Petromyzon marinus.

The sea lamprey Petromyzon marinus is among the most primitive of extant vertebrates. We are interested in the organization of its Hox gene clusters, because, as a close relative of the gnathostomes, this information would help to infer Hox cluster organization at the base of the gnathostome radiation. We have partially mapped the P. marinus Hox clusters using phage, cosmid, and P1 artificial chromosome libraries. Complete homeobox sequences were obtained for the 22 Hox genes recovered in the genomic library screens and analyzed for cognate group identity. We estimate that the clusters are somewhat larger than those of mammals (roughly 140 kbp vs. 105 kbp) but much smaller than the single Hox cluster of the cephalochordate amphioxus (at more than 260 kb). We never obtained more than three genes from any single cognate group from the genomic library screens, although it is unlikely that our screen was exhaustive, and therefore conclude that P. marinus has a total of either three or four Hox clusters. We also identify four highly conserved non-coding sequence motifs shared with higher vertebrates in a genomic comparison of Hox 10 genes.

Amino Acid Sequence↗