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Biomedical subjects

F Grosveld

Publications and source records attributed to F Grosveld.

At least 91 records · Page 5Linked to original sources
PubMed1996

The POU factor Oct-6 and Schwann cell differentiation.

The POU transcription factor Oct-6, also known as SCIP or Tst-1, has been implicated as a major transcriptional regulator in Schwann cell differentiation. Microscopic and immunochemical analysis of sciatic nerves of Oct-6(-/-) mice at different stages of postnatal development reveals a delay in Schwann cell differentiation, with a transient arrest at the promyelination stage. Thus, Oct-6 appears to be required for the transition of promyelin cells to myelinating cells. Once these cells progress past this point, Oct-6 is no longer required, and myelination occurs normally.

Animals↗
PubMed1996

Zfp-37 is a member of the KRAB zinc finger gene family and is expressed in neurons of the developing and adult CNS.

The murine Zfp-37 gene encodes a protein with 12 zinc fingers at its C-terminus (Nelki et al., 1990, Nucleic Acids Res. 18: 3655; Burke and Wolgemuth, 1992, Nucleic Acids Res. 20: 2827-2834). Contrary to the published data, our Northern blot analysis demonstrates not only that the Zfp-37 gene is expressed as 2.3, 2.6, and 4.2 kb mRNAs in testis, but also that there is a 3.7-kb message in the adult mouse brain. Using a partial cDNA as a probe, we have isolated a brain-specific Zfp-37 cDNA clone of 3.3 kb, whose sequence was extended to full length using 5' end RACE. This revealed that the 3.7-kb mRNA is in fact a collection of transcripts with heterogenous 5' ends. Comparison of cDNA and genomic sequences shows that the Zfp-37 gene is spread over a region of approximately 20 kb and consists of six exons, the large 3' end exon containing the complete zinc finger domain, and 3' UTR. Our data show that the Zfp-37 gene utilizes different promoters, alternative splicing, and differential polyadenylation to generate the distinct transcripts of brain and testis. Several protein isoforms are encoded by these mRNAs, some of which contain a truncated form of a conserved domain (Krüppel-associated box) found in other zinc finger genes. In situ hybridization analysis of postnatal brain sections indicates that the Zfp-37 gene is expressed in all neurons of the central nervous system. Together, these results suggest that ZFP-37 is a transcriptional regulator predominantly present in postmitotic cells from two different lineages.

Amino Acid Sequence↗
PubMed1996

A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region.

Single-copy human beta-globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the locus control region (LCR). Here we show that the 6.5 kbp microlocus LCR cassette reproducibly directs full expression from independent single-copy beta-globin transgenes. By testing individual DNase I-hypersensitive sites (HS) present in the microlocus cassette, we demonstrate that the 1.5 kbp 5'HS2 enhancer fragment does not direct beta-globin expression from single-copy transgenes. In contrast, the 1.9 kbp 5'HS3 fragment directs beta-globin expression in five independent single-copy transgenic mouse lines. Moreover, the 5'HS3 core element and beta-globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5'HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5'HS2. We postulate that the different elements of the LCR form a 'holocomplex' that interacts with the individual globin genes.

Animals↗
PubMed1996

Role of the duplicated CCAAT box region in gamma-globin gene regulation and hereditary persistence of fetal haemoglobin.

Hereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly alleviate the symptoms of thalassaemia or sickle cell anaemia when co-inherited with these disorders. We have examined structure-function relationships in the -117 HPFH gamma promoter by analysing the effect of mutating specific promoter elements on the functioning of the wild-type and HPFH promoters. We find that CCAAT box mutants dramatically affect expression from the HPFH promoter in adult blood but have little effect on embryonic/fetal expression from the wild-type promoter. Our results suggest that there are substantial differences in the structure of the wild-type gamma promoter expressed early in development and the adult HPFH promoter. Together with previous results, this suggests that gamma silencing is a complex multifactorial phenomenon rather than being the result of a simple repressor binding to the promoter. We present a model for gamma-globin gene silencing that has significant implications for attempts to reactivate the gamma promoters in human adults by pharmacological means.

Animals↗
PubMed1996

A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection.

BACKGROUND: In recent years, interest has been revived in the possibility of transplanting organs into humans from a phylogenetically disparate species such as the pig (xenotransplantation). Such discordant xenografts, however, are subject to hyperacute rejection (HAR) and activation of host complement plays a major role in this rejection. This problem may be solved through the use of transgenic technology by providing the grafted tissue with molecules that down-regulate the action of host complement. RESULTS: Transgenesis with a yeast artificial chromosome (YAC) was used to produce transgenic mice with the complete genomic gene of the human complement regulator membrane cofactor protein (MCP). Transgenic mice were obtained that exhibit full regulation of MCP as normally observed in humans. Hearts from these mice were shown to be significantly protected from HAR caused by human serum in an in vivo experimental procedure. CONCLUSIONS: We conclude that MCP can protect discordant xenografts from HAR caused by human serum and that transgenic mice can be used effectively as in vivo models for the study of the role of human complement regulatory molecules in xenotransplantation.

Animals↗
PubMed1996

The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene.

The beta-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5'HS1-5. A fully active 6.5 kb version of the LCR, called the muLCR, has been described. Expression of the beta-like globin genes is absolutely dependent on the presence of the LCR. The developmental expression pattern of the genes in the cluster is achieved through competition of the promoters for the activating function of the LCR. Transgenic mice experiments suggest that subtle changes in the transcription factor environment lead to the successive silencing of the embryonic epsilon-globin and fetal gamma-globin promoters, resulting in the almost exclusive transcription of the beta-globin gene in adult erythropoiesis. In this paper, we have asked the question whether the LCR and its individual hypersensitive sites 5'HS1-4 can activate a basic promoter in the absence of any other globin sequences. We have employed a minimal promoter derived from the mouse Hsp68 gene driving the bacterial beta-galactosidase (lacZ) gene. The results show that the muLCR and 5'HS3 direct erythroid-specific, embryonic expression of this construct, while 5'HS1, 5'HS2 and 5'HS4 are inactive at any stage of development. Expression of the muLCR and 5'HS3 transgenes is repressed during fetal stages of development. The transgenes are in an inactive chromatin conformation and the lacZ gene is not transcribed, as shown by in situ hybridization. These data are compatible with the hypothesis that the LCR requires the presence of an active promoter to adopt an open chromatin conformation and with models proposing progressive heterochromatization during embryogenesis. The results suggest that the presence of a beta-globin gene is required for LCR function as conditions become more stringent during development.

Animals↗
PubMed1996

Neurocristopathy resembling neurofibromatosis type 1 in an NGF-SV40 transgenic line.

BACKGROUND: Animal models of carcinogenesis have been produced in transgenic mice by directing the expression of oncogenes such as SV40 T antigen and myc to different tissues by creating fusions with promoter/enhancer elements of various mammalian or viral genes. RESULTS: A transgenic mouse line was created in which SV40 T antigen is under the control of the mouse nerve growth factor (NGF) promoter. While the oncogene is expressed in a wide range of NGF producing tissues, it specifically causes the development of either neurofibromas or neurofibrosarcomas similar to those found in the human disease neurofibromatosis type 1 (NF1). These tumours are completely penetrant and appear after a mean latency of about 8 months. In contrast to the previously reported neurofibromatosis mouse model HTLV-1 tax, the tumours in these transgenic mice arise in Schwann cells rather than perineural fibroblasts and have a very restricted tissue distribution. In a cell line cloned from a neurofibroma from these mice, NGF was detected in the culture medium at levels similar to those produced by cultured primary Schwann cells. CONCLUSION: As all animal model for a heritable neurocristopathy resembling NF1, this mouse should allow study of the pathology and treatment of this disease.

Animals↗
PubMed1995

The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion.

Although the CD34 antigen is widely used in the identification and purification of hemopoietic stem and progenitor cells, its function within hemopoiesis is unknown. We have investigated this issue by ectopically expressing human (hu) CD34 on the surface of murine hemopoietic cells. Forced expression of hu-CD34 in the thymocytes of transgenic mice did not appear to affect the development, maturation, or distribution of murine T cells but did significantly increase their ability to adhere to bone marrow stromal layers of human but not mouse origin. Ectopic expression of hu-CD34 on murine 416B cells, a multipotential progenitor that expresses murine CD34, yielded similar results. In both cases hu-CD34-dependent adhesion was enhanced by molecular engagement of the hu-CD34 protein using anti-CD34 antibodies. These results provide evidence that CD34 promotes the adhesive interactions of hemopoietic cells with the stromal microenvironment of the bone marrow thereby implicating CD34 in regulation and compartmentalization of stem cells. We propose that CD34 regulates these processes in part via an indirect mechanism, signaling changes in cellular adhesion in response to molecular recognition of an as yet unidentified stromal CD34 counterreceptor or ligand.

Animals↗
PubMed1995

Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress.

Glucose 6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme encoded in mammals by an X-linked gene. It has important functions in intermediary metabolism because it catalyzes the first step in the pentose phosphate pathway and provides reductive potential in the form of NADPH. In human populations, many mutant G6PD alleles (some present at polymorphic frequencies) cause a partial loss of G6PD activity and a variety of hemolytic anemias, which vary from mild to severe. All these mutants have some residual enzyme activity, and no large deletions in the G6PD gene have ever been found. To test which, if any, function of G6PD is essential, we have disrupted the G6PD gene in male mouse embryonic stem cells by targeted homologous recombination. We have isolated numerous clones, shown to be recombinant by Southern blot analysis, in which G6PD activity is undetectable. We have extensively characterized individual clones and found that they are extremely sensitive to H2O2 and to the sulfydryl group oxidizing agent, diamide. Their markedly impaired cloning efficiency is restored by reducing the oxygen tension. We conclude that G6PD activity is dispensable for pentose synthesis, but is essential to protect cells against even mild oxidative stress.

Animals↗
PubMed1995

Transcription complex stability and chromatin dynamics in vivo.

Distant regulatory sequences affect transcription through long-range chromatin interactions. Visualization of transcriptional activity of genes that compete for distant elements, using the globin locus as a model, has revealed the dynamics of chromatin interactions in vivo. Multiple genes appear to be transcribed alternately rather than at the same time to generate several messenger RNAs in one cell. The regulator may stably complex with one gene at a time and switch back and forth between genes in a flip-flop mechanism.

Animals↗
PubMed1995

TBP binding and the rate of transcription initiation from the human beta-globin gene.

DNA-protein interaction studies in vitro revealed several factors binding over the TATA box and the region of transcription initiation (cap) site of the human beta-globin promoter; TATA binding protein TBP at -30, Sp1 at -19, GATA-1 at -12 and +5, YY1 at -9 and a novel factor C1 over the site of initiation (-4 to +7). Point mutants which specifically abolish the binding of each of these proteins were tested in a beta-globin locus control region (LCR) construct which allows quantitative comparisons at physiological levels of transcription. Only mutants which drastically affect the binding of TBP resulted in decreased levels of transcription. A threshold value of TBP binding of 15-30% of wild type was sufficient to give normal levels of transcription. This indicates that the association of TF IID with the TATA box is not limiting in the rate of initiation of transcription.

Animals↗
PubMed1995

The acute promyelocytic leukaemia-associated PML gene is induced by interferon.

The PML protein concentrates within discrete nuclear structures known as nuclear bodies, also called NDs or PODs, which contain several proteins including the interferon (IFN)-inducible SP100 product. The function of these structures remains elusive. We and others have shown recently that they represent specific targets for adenovirus and herpes simplex virus. This prompted us to investigate whether PML, like SP100, might be induced by IFN and to explore the role of PML in viral infection. Here we report that PML mRNA levels increase rapidly in response to interferon treatment. This accumulation of PML transcripts is a primary IFN response since it does not require de novo protein synthesis. The IFN-induced activation of the PML gene is accompanied by enhanced protein expression as revealed by immunolabelling. Both the intensity of the staining and the number of labelled structures increased upon interferon exposure. To probe the role of PML in IFN action, we compared the antiviral state established by alpha-interferon in embryonic fibroblasts (EFs) derived from null mutant mice for PML and from wild-type control mice. The resistance to viral infection conferred by IFN-alpha was identical in both PML+/+ and PMLm/m fibroblasts indicating that PML is not an essential mediator of the antiviral effect of interferon. We also noted that DNA-binding factors are normally activated by IFN in PMLm/m cells.

Base Sequence↗
PubMed1995

Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.

Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of retrovirus-mediated gene transfer.

Animals↗
PubMed1995

Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene.

Erythroid Krüppel-like factor (EKLF) was originally isolated from erythroid cell RNA by differential screening and shown to be erythroid-specific, although a low level of EKLF was found in mast cell lines. EKLF contains three zinc-fingers homologous to those found in the Krüppel family of transcription factors. Because it binds the sequence CCACACCCT, EKLF may affect erythroid development as a result of its ability to bind to the CAC box in the promoter of the beta-globin gene. Mutation of this element leads to reduced beta-globin expression and it appears to mediate the effect of the globin locus control region on the promoter. Here we inactivate the EKLF gene through insertion of a lacZ reporter gene by homologous recombination in embryonic stem (ES) cells. Heterozygous EKLF+/- mice show that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-/- mice appear normal during the embryonic stage of haematopoiesis in the yolk sac, but develop a fatal anaemia during early fetal life when haematopoiesis has switched to the fetal liver. Enucleated erythrocytes are formed but these do not contain the proper amount of haemoglobin. We conclude that the transcription factor EKLF is essential for the final steps of definitive erythropoiesis in fetal liver.

Animals↗
PubMed1995

Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer.

Gene therapy approaches for beta-thalassemia and sickle cell anemia focus on the transfer of a human beta-globin gene into the patient's hematopoietic stem cells (HSC). Expression of the transferred sequences should be erythroid specific and match the expression of the endogenous alpha-globin genes in adult erythropoiesis. Here we explore the potential of recombinant adeno-associated virus (AAV) vectors for human beta-globin gene transfer. We have constructed a recombinant AAV-vector containing a human beta-globin gene together with the DNasel hypersensitive sites 4, 3 and 2 of the human beta-globin locus control region. The vector replicates to high titers and can efficiently transduce hematopoietic and non-hematopoietic cells. In transduced and G418 selected murine erythroleukemia (MEL) cell clones, human beta-globin gene expression was regulated and reached levels comparable to endogenous murine beta maj. These data show that AAV-vectors are promising tools in gene therapy approaches for the haemoglobinopathies.

Animals↗
PubMed1994

Transcriptional activation by hypersensitive site three of the human beta-globin locus control region in murine erythroleukemia cells.

In this paper we describe a complete deletional analysis of hypersensitive site three (HS3) of the human beta-globin Locus Control Region (LCR). The previously defined core fragment consists of 6 footprinted regions, with multiple binding sites for the erythroid-specific factor GATA-1 and G-rich motifs that can interact with ubiquitous factors such as Sp1 and TEF-2. We show in this paper that the 5' half of this fragment is the most important for activity in murine erythroleukemia (MEL) cells. A fragment containing footprints 1-4 can stimulate transcription of a linked human beta-globin gene to levels of about 40% of that obtained with footprints 1-6. Constructs containing either footprints 1-3 or 3-6 cannot be distinguished from the beta-globin gene alone. We further show that binding sites for the erythroid-specific factor NF-E2 can co-operatively interact with parts of the HS3 core fragment, and that HS3 requires elements upstream from -103 in the human beta-globin promoter for full activity. The importance of these results is discussed in the context of the regulation of the genes in the human beta-globin cluster.

Animals↗
PubMed1994

Dissection of the locus control function located on the chicken lysozyme gene domain in transgenic mice.

The entire chicken lysozyme gene locus including all known cis-regulatory sequences and the 5' and 3' matrix attachment sites defining the borders of the DNase I sensitive chromatin domain, is expressed at a high level and independent of its chromosomal position in macrophages of transgenic mice. It was concluded that the lysozyme gene locus carries a locus control function. We analysed several cis-regulatory deletion mutants to investigate their influence on tissue specificity and level of expression. Position independent expression of the gene is lost whenever one of the upstream tissue specific enhancer regions is deleted, although tissue specific expression is usually retained. Deletion of the domain border fragments has no influence on copy number dependency of expression. However, without these regions an increased incidence of ectopic expression is observed. This suggests that the domain border fragments may help to suppress transgene expression in inappropriate tissues. We conclude, that position independent expression of the lysozyme gene is not controlled by a single specific region of the locus but is the result of the concerted action of several tissue specific upstream regulatory DNA elements with the promoter.

Animals↗