Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes

Accumulative evidence suggests that more than 20 neuron-specific genes are regulated by a transcriptional cis-regulatory element known as the neural restrictive silencer (NRS). A trans-acting repressor that binds the NRS, NRSF [also designated RE1-silencing transcription factor (REST)] has been cloned, but the mechanism by which it represses transcription is unknown. Here we show evidence that NRSF represses transcription of its target genes by recruiting mSin3 and histone deacetylase. Transfection experiments using a series of NRSF deletion constructs revealed the presence of two repression domains, RD-1 and RD-2, within the N- and C-terminal regions, respectively. A yeast two-hybrid screen using the RD-1 region as a bait identified a short form of mSin3B. In vitro pull-down assays and in vivo immunoprecipitation-Western analyses revealed a specific interaction between NRSF-RD1 and mSin3 PAH1-PAH2 domains. Furthermore, NRSF and mSin3 formed a complex with histone deacetylase 1, suggesting that NRSF-mediated repression involves histone deacetylation. When the deacetylation of histones was inhibited by tricostatin A in non-neuronal cells, mRNAs encoding several neuronal-specific genes such as SCG10, NMDAR1, and choline acetyltransferase became detectable. These results indicate that NRSF recruits mSin3 and histone deacetylase 1 to silence neural-specific genes and suggest further that repression of histone deacetylation is crucial for transcriptional activation of neural-specific genes during neuronal terminal differentiation.

Interference with gene regulation in living sea urchin embryos: Transcription factor Knock Out (TKO), a genetically controlled vector for blockade of specific transcription factors

“TKO” is an expression vector that knocks out the activity of a transcription factor in vivo under genetic control. We describe a successful test of this concept that used a sea urchin transcription factor of known function, P3A2, as the target. The TKO cassette employs modular cis-regulatory elements to express an encoded single-chain antibody that prevents the P3A2 protein from binding DNA in vivo. In normal development, one of the functions of the P3A2 transcription factor is to repress directly the expression of the CyIIIa cytoskeletal actin gene outside the aboral ectoderm of the embryo. Ectopic expression in oral ectoderm occurs if P3A2 sites are deleted from CyIIIa expression constructs, and we show here that introduction of an αP3A2⋅TKO expression cassette causes exactly the same ectopic oral expression of a coinjected wild-type CyIIIa construct. Furthermore, the αP3A2⋅TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm and in the endoderm. αP3A2⋅TKO thus abrogates the function of the endogenous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Widespread expression of αP3A2⋅TKO in the endoderm has the additional lethal effect of disrupting morphogenesis of the archenteron, revealing a previously unsuspected function of SpP3A2 in endoderm development. In principle, TKO technology could be utilized for spatially and temporally controlled blockade of any transcription factor in any biological system amenable to gene transfer.

Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb βs-globin yeast artificial chromosome: A mouse model of sickle cell anemia

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine α- and β-globin genes and substitution with human α and βs transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human α-, γ-, and β-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human β-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the γ- and β-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb βs yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.

A model for spontaneous B-lineage lymphomas in IgHμ-HOX11 transgenic mice

HOX11, a divergent homeodomain-containing transcription factor, was isolated from the breakpoint of the nonrandom t(10;14)(q24;q11) chromosome translocation found in human T cell acute lymphoblastic leukemias. The translocation places the HOX11 coding sequence under the transcriptional control of TCR α/δ regulatory elements, resulting in ectopic expression of a normal HOX11 protein in thymocytes. To investigate the oncogenic potential of HOX11, we targeted its expression in lymphocytes of transgenic mice by placing the human cellular DNA under the transcriptional control of Ig heavy chain or LCK regulatory sequences. Only IgHμ-HOX11 mice expressing low levels of HOX11 were viable. During their second year of life, all HOX11 transgenic mice became terminally ill with more than 75% developing large cell lymphomas in the spleen, which frequently disseminated to thymus, lymph nodes, and other nonhematopoietic tissues. Lymphoma cells were predominantly clonal IgM+IgD+ mature B cells. Repopulation of severe combined immunodeficient mice with cells from hyperplastic spleens indicated that the HOX11 tumor phenotype was transplantable. Before tumor development, expression of the transgene did not result in perturbations in lymphopoiesis; however, lymphoid hyperplasia involving the splenic marginal zones was present in 20% of spleens. Our studies provide direct evidence that expression of HOX11 in lymphocytes leads to malignant transformation. These mice are a useful model system to study mechanisms involved in transformation from B-lineage hyperplasia to malignant lymphoma and for testing novel approaches to therapy. They represent a novel animal model for non-Hodgkin’s lymphoma of peripheral mature B cell origin.

Role of the Box C/D Motif in Localization of Small Nucleolar RNAs to Coiled Bodies and Nucleoli

Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.

A transgenic mouse model with an inducible skin blistering disease phenotype

One of the current limitations of gene transfer protocols involving mammalian genomes is the lack of spatial and temporal control over the desired gene manipulation. Starting from a human keratin gene showing a complex regulation as a template, we identified regulatory sequences that confer inducible gene expression in a subpopulation of keratinocytes in stratified epithelia of adult transgenic mice. We used this cassette to produce transgenic mice with an inducible skin blistering phenotype mimicking a form of epidermolytic hyperkeratosis, a keratin gene disorder. Upon induction by topical application of a phorbol ester, the mutant keratin transgene product accumulates in the differentiating layers of epidermis, leading to keratinocyte lysis after application of mechanical trauma. This mouse model will allow for a better understanding of the complex relationship between keratin mutation, keratinocyte cytoarchitecture, and hypersensitivity to trauma. The development of an inducible expression vector showing an exquisite cellular specificity has important implications for manipulating genes in a spatially and temporally controlled fashion in transgenic mice, and for the design of gene therapy strategies using skin as a tissue source for the controlled delivery of foreign substances.

Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes

The regulatory regions surrounding many genes may be large and difficult to study using standard transgenic approaches. Here we describe the use of bacterial artificial chromosome clones to rapidly survey hundreds of kilobases of DNA for potential regulatory sequences surrounding the mouse bone morphogenetic protein-5 (Bmp5) gene. Simple coinjection of large insert clones with lacZ reporter constructs recapitulates all of the sites of expression observed previously with numerous small constructs covering a large, complex regulatory region. The coinjection approach has made it possible to rapidly survey other regions of the Bmp5 gene for potential control elements, to confirm the location of several elements predicted from previous expression studies using regulatory mutations at the Bmp5 locus, to test whether Bmp5 control regions act similarly on endogenous and foreign promoters, and to show that Bmp5 control elements are capable of rescuing phenotypic effects of a Bmp5 deficiency. This rapid approach has identified new Bmp5 control regions responsible for controlling the development of specific anatomical structures in the vertebrate skeleton. A similar approach may be useful for studying complex control regions surrounding many other genes important in embryonic development and human disease.

Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca2+ entry through voltage-sensitive Ca2+ channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca2+. Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca2+ response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca2+-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.

An SmtB-like repressor from Synechocystis PCC 6803 regulates a zinc exporter

ORF slr0798, now designated ziaA, from Synechocystis PCC 6803 encodes a polypeptide with sequence features of heavy metal transporting P-type ATPases. Increased Zn2+ tolerance and reduced 65Zn accumulation was observed in Synechococcus PCC 7942, strain R2-PIM8(smt), containing ziaA and upstream regulatory sequences, compared with control cells. Conversely, reduced Zn2+ tolerance was observed following disruption of ziaA in Synechocystis PCC 6803, and ziaA-mediated restoration of Zn2+ tolerance has subsequently been used as a selectable marker for transformation. Nucleotide sequences upstream of ziaA, fused to a promoterless lacZ gene, conferred Zn2+-dependent β-galactosidase activity when introduced into R2-PIM8(smt). The product of ORF sll0792, designated ZiaR, is a Zn2+-responsive repressor of ziaA transcription. Reporter gene constructs lacking ziaR conferred elevated Zn2+-independent expression from the ziaA operator–promoter in R2-PIM8(smt). Gel retardation assays detected ZiaR-dependent complexes forming with the zia operator–promoter and ZiaR–DNA binding was enhanced by treatment with a metal-chelator in vitro. Two mutants of ZiaR (C71S/C73S and H116R) bound to, and repressed expression from, the ziaA operator–promoter but were unable to sense Zn2+. Metal coordination to His-imidazole and Cys-thiolate ligands at these residues of ZiaR is thus implicated in Zn2+-perception by Synechocystis PCC 6803.

Mouse Deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix–loop–helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.

Aldehyde dehydrogenase class 3 expression: Identification of a cornea-preferred gene promoter in transgenic mice

Aldehyde dehydrogenase class 3 (ALDH3) constitutes 20–40% of the total water-soluble proteins in the mammalian cornea. Here, we show by Northern blot analysis that ALDH3 expression in the mouse is at least 500-fold higher in the cornea than in any other tissue examined, with very low levels of expression detected in the stomach, urinary bladder, ocular lens, and lung. Histochemical localization reveals that this exceptional level of expression in the mouse cornea occurs in the anterior epithelial cells and that little ALDH3 is present in the keratocytes or corneal endothelial cells. A 13-kbp mouse ALDH3 promoter fragment containing >12 kbp of the 5′ flanking sequence, the 40-bp untranslated first exon, and 29 bp of intron 1 directed cat reporter gene expression to tissues that express the endogenous ALDH3 gene, except that transgene promoter activity was higher in the stomach and bladder than in the cornea. By contrast, when driven by a 4.4-kbp mouse ALDH3 promoter fragment [1,050-bp 5′ flanking region, exon 1, intron 1 (3.4 kbp), and 7 bp of exon 2] expression of the cat reporter gene was confined to the corneal epithelial cells, except for very low levels in the liver, effectively reproducing the corneal expression pattern of the endogenous ALDH3 gene. These results indicate that tissue-specific expression of ALDH3 is determined by positive and negative elements in the 5′ flanking region of the gene and suggests putative silencers located in intron 1. We demonstrate regulatory sequences capable of directing cornea-specific gene expression, affording the opportunity for genetic engineering in this transparent tissue.

Identification and dissection of an enhancer controlling epithelial gene expression in skin

Keratins 14 and 5 are the structural hallmarks of the basal keratinocytes of the epidermis and outer root sheath (ORS) of the hair follicle. Their genes are controlled in a tissue-specific manner and thus serve as useful tools to elucidate the regulatory mechanisms involved in keratinocyte-specific transcription. Previously we identified several keratinocyte-specific DNase I hypersensitive sites (HSs) in the 5′ regulatory sequences of the K14 gene and showed that a 700-bp regulatory domain encompassing HSs II and III can confer epidermal and ORS-specific gene expression in transgenic mice in vivo. Although HS II harbored much of the transactivation activity in vitro, it was not sufficient to restrict expression to keratinocytes in vivo. We now explore the HS III regulatory element. Surprisingly, this element on its own confers gene expression to the keratinocytes of the inner root sheath (IRS) of the hair follicle, whereas a 275-bp DNA fragment containing both HSs II and III shifts the expression from the IRS to the basal keratinocytes and ORS in vivo. Electrophoretic mobility-shift assays and mutational studies of HSs III reveal a role for CACCC-box binding proteins, Sp1 family members, and other factors adding to the list of previously described factors that are involved in keratinocyte-specific gene expression. These studies highlight a cooperative interaction of the two HSs domains and strengthen the importance of combinatorial play of transcription factors that govern keratinocyte-specific gene regulation.

Sequence conservation at human and mouse orthologous common fragile regions, FRA3B/FHIT and Fra14A2/Fhit

It has been suggested that delayed DNA replication underlies fragility at common human fragile sites, but specific sequences responsible for expression of these inducible fragile sites have not been identified. One approach to identify such cis-acting sequences within the large nonexonic regions of fragile sites would be to identify conserved functional elements within orthologous fragile sites by interspecies sequence comparison. This study describes a comparison of orthologous fragile regions, the human FRA3B/FHIT and the murine Fra14A2/Fhit locus. We sequenced over 600 kbp of the mouse Fra14A2, covering the region orthologous to the fragile epicenter of FRA3B, and determined the Fhit deletion break points in a mouse kidney cancer cell line (RENCA). The murine Fra14A2 locus, like the human FRA3B, was characterized by a high AT content. Alignment of the two sequences showed that this fragile region was stable in evolution despite its susceptibility to mitotic recombination on inhibition of DNA replication. There were also several unusual highly conserved regions (HCRs). The positions of predicted matrix attachment regions (MARs), possibly related to replication origins, were not conserved. Of known fragile region landmarks, five cancer cell break points, one viral integration site, and one aphidicolin break cluster were located within or near HCRs. Thus, comparison of orthologous fragile regions has identified highly conserved sequences with possible functional roles in maintenance of fragility.

Evolutionary conservation of sequence elements controlling cytoplasmic polyadenylylation.

Cytoplasmic polyadenylylation is an evolutionarily conserved mechanism involved in the translational activation of a set of maternal messenger RNAs (mRNAs) during early development. In this report, we show by interspecies injections that Xenopus and mouse use the same regulatory sequences to control cytoplasmic poly(A) addition during meiotic maturation. Similarly, Xenopus and Drosophila embryos exploit functionally conserved signals to regulate polyadenylylation during early post-fertilization development. These experiments demonstrate that the sequence elements that govern cytoplasmic polyadenylylation, and hence one form of translational activation, function across species. We infer that the requisite regulatory sequence elements, and likely the trans-acting components with which they interact, have been conserved since the divergence of vertebrates and arthropods.

Mouse model of human beta zero thalassemia: targeted deletion of the mouse beta maj- and beta min-globin genes in embryonic stem cells.

beta zero-Thalassemia is an inherited disorder characterized by the absence of beta-globin polypeptides derived from the affected allele. The molecular basis for this deficiency is a mutation of the adult beta-globin structural gene or cis regulatory elements that control beta-globin gene expression. A mouse model of this disease would enable the testing of therapeutic regimens designed to correct the defect. Here we report a 16-kb deletion that includes both adult beta-like globin genes, beta maj and beta min, in mouse embryonic stem cells. Heterozygous animals derived from the targeted cells are severely anemic with dramatically reduced hemoglobin levels, abnormal red cell morphology, splenomegaly, and markedly increased reticulocyte counts. Homozygous animals die in utero; however, heterozygous mice are fertile and transmit the deleted allele to progeny. The anemic phenotype is completely rescued in progeny derived from mating beta zero-thalassemic animals with transgenic mice expressing high levels of human hemoglobin A. The beta zero-thalassemic mice can be used to test genetic therapies for beta zero-thalassemia and can be bred with transgenic mice expressing high levels of human hemoglobin HbS to produce an improved mouse model of sickle cell disease.