Biomedical subjects

D I Martin

Publications and source records attributed to D I Martin.

At least 37 records · Page 2Linked to original sources

PubMed1996

Age-dependent silencing of globin transgenes in the mouse.

Variegation of transgene expression, a heterocellular or mosaic pattern of expression seen in all mice in a given transgenic line, is a frequently observed but unexplained phenomenon. We have encountered variegation with globin transgenes; when lacZ expression is driven by globin control elements a proportion of erythrocytes express beta-galactosidase (beta-gal), while the remaining erythrocytes express none. The percentage of expressing cells is constant within each line (at any particular developmental stage), but varies between lines. Such variation may account for much of the line-to-line variability which has been reported in the expression of a transgene construct. We have now extended these observations by studying expression of several globin/lacZ transgenes with increasing age. Expression of beta-gal is variegated in all lines in adult mice, including those made with a beta-globin promoter and locus control region driving lacZ. The extent of variegation differs widely between lines, but in all lines there is a marked decline in the number of erythrocytes expressing beta-gal with increasing age. Progression of silencing continues long past the point at which globin switching is complete, suggesting that it is not related to this process. We observe that age-dependent silencing is most severe in high copy number animals. Increasing variegation of transgene expression with ageing of mice is likely to complicate interpretation of the developmental regulation of transgenes. We speculate that it reflects a general mechanism of epigenetic regulation.

Aging↗

PubMed1996

Transcriptional enhancers act in cis to suppress position-effect variegation.

We have examined the basis of enhancer effects on gene expression by altering the action of enhancers on expression of a stably integrated reporter gene. We used two distinct experimental approaches: recombinase-mediated deletion of an enhancer and modulation of the activity of another enhancer composed of downstream metal response elements (MREs). The flp recombinase was used to delete the 5'HS2 globin enhancer from a site downstream of beta-geo at nine separate integration sites in K562 erythroleukemia cells. In no case does deletion of 5'HS2 have a significant effect on the level of expression; however, the deletion does increase dramatically the rate at which expression of beta-geo is silenced. Zinc stimulation of a metallothionein enhancer has no effect on the level of reporter expression, but slows the rate of silencing. Silencing in both cases is highly site dependent, and resembles position-effect variegation (PEV). These results strongly support a binary mode of enhancer action, as in both cases the enhancer maintains reporter expression without a strong effect on the level of expression. Taken together, these findings suggest that transcriptional activators have a direct interaction with repressive chromatin structures, which is independent of an effect on the rate of transcription. We propose that cis-acting transcriptional control elements may act primarily through this mechanism.

Enhancer Elements, Genetic↗

PubMed1996

The vagaries of variegating transgenes.

Expression of transgenes in mice, when examined with assays that can distinguish individual cells, is often found to be heterocellular, or variegated. Line-to-line variations in expression of a transgene may be due largely to differences in the proportion of cells in which it is expressed. Variegated silencing by centromeric heterochromatin is well described, but other factors may also affect transgene silencing in mice. Tandem arrays of transgenes themselves form heterochromatin, and some cell lineages may tend to silence transgenes because of extensive facultative heterochromatin in their nuclei. The cis-acting transcriptional control elements within a transgene inhibit silencing, and strain-specific differences in chromatin proteins may strongly influence the extent of variegation. The accessibility of multiple differentiated cell lineages in mice suggests that they may provide a tool for dissecting the role of chromatin-mediated silencing in cell differentiation and tissue-specific gene expression.

Animals↗

PubMed1996

Regulation of beta-globin gene expression: straightening out the locus.

A casual examination of the globin literature would leave most readers with the impression that all aspects of beta-globin gene regulation are controlled by the upstream locus control region (LCR). There is no clear evidence, however, that the LCR affects transcription in the beta-globin locus other than by altering its topology to maintain it in a state permissive for expression of the globin genes. Developmental switching of the globin genes may be independent of the LCR, relying only on elements close to the genes and the arrangement of the genes with respect to each other.

Animals↗

PubMed1996

Erythroid AP-1/NF-E2 elements vary in their response to NF-E2.

AP-1/NF-E2 motifs found in erythroid transcription control elements are associated with powerful transcription activation and thought to be regulated by the erythroid transcription factor NF-E2. We have studied AP-1/NF-E2 motifs from three different erythroid control elements (5'HS2 of the human beta-globin locus control region [LCR], the porphobilinogen deaminase [PBGD] promoter, and the mouse Band 3 promoter). We find that these AP-1/NF-E2 elements differ both in their ability to bind NF-E2 and their activity in transient assays. Each of the elements is bound by AP-1, but only the 5'HS2 and PBGD sites are bound by NF-E2. We examined the activity of these sites in minimal promoter constructs in transient assays. In erythroid cells, activity of duplicated NF-E2 motifs is positively correlated with binding by NF-E2; however, the Band 3 element not bound by NF-E2 is also active in some contexts. In HeLa cells, all sites were active and duplicated sites were most active. In F9 mouse teratocarcinoma cells, which express neither NF-E2 nor AP-1, the elements' activity parallels that in erythroid cells. While these finding are consistent with other evidence that NF-E2 is an important regulator of erythroid transcription, they suggest that some sites that resemble NF-E2 elements are actually regulated by other factors; we speculate that other tissue-specific and/or generally expressed factors may act on these sites.

Animals↗

PubMed1995

HMG-I binds to GATA motifs: implications for an HPFH syndrome.

We have examined binding of the nuclear protein HMG-I to the human gamma-globin promoter. We find that HMG-I binds preferentially to the more 3' of a pair of GATA motifs in the gamma-globin promoter; this paired motif is bound by the erythroid factor GATA-1. A naturally occurring mutation (-175 T-C) in the area bound by HMG-I results in overexpression of gamma-globin in adult red blood cells (HPFH) and up-regulation of the gamma-globin promoter in in vitro expression assays; HMG-I does not bind to this mutant sequence. A survey of GATA motifs from other globin cis-elements demonstrates HMG-I binding to most of them. These findings implicate HMG-I in the HPFH phenotype; we speculate that it may participate in the formation of multiprotein complexes that regulate globin gene expression.

Adult↗

PubMed1995

Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus.

The beta-globin locus control region (LCR) is a complex regulatory element that is essential for the appropriate red cell-specific expression of all cis-linked beta-globin genes. Of the five hypersensitive sites that define the LCR, only 5'HS2 has been shown to augment gene expression in vitro in both transient and stable assays, as well as in transgenic mice. Thus, 5'HS2 has been assumed to be an important element for the function of the LCR in vivo. We have utilized homologous recombination in murine embryonic stem (ES) cells and phenotypic analysis in derived mice to investigate the function of 5'HS2 in its normal chromosomal position in the murine beta-globin locus. Replacement of 5'HS2 with a selectable marker gene (delta HS2 + neo) causes a 2-5-fold reduction in expression of all of the genes in the locus, and a more pronounced effect (10-12-fold) on the most 5' embryonic globin gene, Ey, when expression of this gene is first detectable during embryogenesis. The mutation produces no alterations in the developmental timing of expression of the globin genes. When homozygous, the deletion/replacement mutation is lethal in utero, with the embryos dying during the stage of yolk sac and early fetal liver erythropoiesis. To distinguish phenotypic effects resulting from the deletion of 5'HS2 from those attributable to insertion of the selectable marker, the selectable marker was removed by expressing the FLP site-specific recombinase in ES cells harboring the homologous recombination event. Mice derived from these ES cells (delta HS2 delta neo) demonstrated nearly full expression of all the beta-like globin genes on the mutated chromosome. These results indicate that although 5'HS2 demonstrates significant regulatory activities in a variety of assays, deletion of this element from the endogenous beta-globin locus has no significant effect on the timing or extent of expression of the locus. In addition, this result emphasizes that when using homologous recombination to analyze complex regulatory elements in vivo, the inserted selectable marker must be removed to avoid influencing the phenotype of the mutation.

Animals↗

PubMed1995

Enhancers increase the probability but not the level of gene expression.

We have studied enhancer function in transient and stable expression assays in mammalian cells by using systems that distinguish expressing from nonexpressing cells. When expression is studied in this way, enhancers are found to increase the probability of a construct being active but not the level of expression per template. In stably integrated constructs, large differences in expression level are observed but these are not related to the presence of an enhancer. Together with earlier studies, these results suggest that enhancers act to affect a binary (on/off) switch in transcriptional activity. Although this idea challenges the widely accepted model of enhancer activity, it is consistent with much, if not all, experimental evidence on this subject. We hypothesize that enhancers act to increase the probability of forming a stably active template. When randomly integrated into the genome, enhancers may affect a metastable state of repression/activity, permitting expression in regions that would not permit activity of an isolated promoter.

Blotting, Southern↗

PubMed1995

Generation of hematopoietic colony-forming cells from embryonic stem cells: synergy between a soluble factor from NIH-3T3 cells and hematopoietic growth factors.

Murine embryonic stem cells are able to differentiate into embryoid bodies (EBs) in vitro in the absence of leukemia-inhibitory factor with the formation of different types of hematopoietic precursors within these EBs. With the aim of determining the in vitro requirements for the continued development of hematopoietic colony-forming cells (CFCs) and their progeny from embryonic stem-derived cells, cells from EBs disrupted after 9 days of formation in the absence of leukemia-inhibitor factor were cultured under different conditions. Low numbers of day-9 EB cells (5 x 10(5) or less) cultured in the presence of several growth factors (interleukin-3 [IL-3], IL-1, c-kit ligand, basic fibroblast growth factor, insulin growth factor-1, IL-6, granulocyte colony-stimulating factor, fetal liver kinase-2 ligand) develop few or no CFCs after 1 week of culture. When these cells are plated on irradiated NIH-3T3 with IL-3 or c-kit ligand or combinations containing these and other growth factors, they are able to generate CFCs for at least 3 weeks. These cultures were found to include granulocytic, monocytic, erythrocytic, and megakaryocytic cells. Transwell cultures in which NIH-3T3 cells were separated from the EB cells and cultures in which cells were replaced by NIH-3T3 conditioned medium showed that the interaction between EB-derived cells and NIH-3T3 is via a soluble factor(s). These studies show that maximal generation of hematopoietic CFCs from precursors present in day-9 EBs is stimulated by a combination of known hematopoietic growth factors and a soluble factor(s) produced by NIH-3T3 cells.

3T3 Cells↗

PubMed1994

A human homologue of the Drosophila developmental gene, Notch, is expressed in CD34+ hematopoietic precursors.

Members of the Notch gene family have been shown to mediate cell-fate decisions by multipotent precursors in a number of different systems. To determine whether members of this family might play a similar role in hematopoiesis, we asked if homologues of the Notch gene are expressed in human hematopoietic precursors. Using degenerate oligonucleotides corresponding to conserved amino acid sequences in known Notch homologues as primers for the polymerase chain reaction (PCR), we demonstrated that at least one Notch homologue is expressed in human bone marrow CD34+ cells, a population enriched for hematopoietic precursors. Cloning and sequencing of the PCR products identified this Notch homologue as TAN-1, a member of the Notch family previously cloned from a T-cell leukemia with a translocation involving this gene. Subsequent evaluation of bone marrow hematopoietic cells for TAN-1 expression using a reverse transcription-PCR assay confirmed the expression of TAN-1 in CD34+ hematopoietic precursors, including the immature subset that lacks expression of lineage-associated antigens (CD34+lin-). These findings, together with the known role of Notch homologues in other systems, suggest that members of the Notch family, including TAN-1, may be involved in mediating cell-fate decisions during hematopoiesis.

Animals↗

PubMed1994

GATA-1 is expressed in acute erythroblastic leukaemia.

Previous studies have demonstrated the expression of GATA-1 (a DNA-binding nuclear protein) in erythrocytes, megakaryocytes, eosinophils, basophils, mast cells, early marrow progenitor cells and in mouse and human erythroid leukaemia cell lines. We studied 31 bone marrow specimens from patients with acute myeloid leukaemia (AML) for GATA-1 expression by reverse transcriptase/polymerase chain reaction (RT/PCR) analysis. GATA-1 expression was detected in all of the patients with erythroleukaemia, and in one of nine patients with megakaryoblastic leukaemia, but absent from 17 patients with French-American-British (FAB) M1-5 leukaemia. In AML, GATA-1 expression is indicative of differentiation to the erythroid and possibly megakaryocytic lineages, analogous to its expression in normal haemopoiesis.

Bone Marrow↗

PubMed1993

Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34+ bone marrow cells.

Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.

Adenosine Deaminase↗

PubMed1992

Functional erythroid promoters created by interaction of the transcription factor GATA-1 with CACCC and AP-1/NFE-2 elements.

We have investigated interactions between the erythroid transcription factor GATA-1 and factors binding two cis-acting elements commonly linked to GATA sites in erythroid control elements. GATA-1 is present at all stages of erythroid differentiation, is necessary for erythropoiesis, and binds sites in all erythroid control elements. However, minimal promoters containing GATA-1 sites are inactive when tested in erythroid cells. Based on this observation, two erythroid cis elements, here termed CACCC and AP-1/NFE-2, were linked to GATA sites in minimal promoters. None of the elements linked only to a TATA box created an active promoter, but GATA sites linked to either CACCC or AP-1/NFE-2 elements formed strong erythroid promoters. A mutation of T to C at position -175 in the gamma-globin promoter GATA site, associated with hereditary persistence of fetal hemoglobin (HPFH), increased expression of these promoters in both fetal and adult cells. A construct bearing the beta-globin CACCC element was more active in adult and less active in fetal erythroid cells, when compared with the gamma-globin CACCC element. These studies suggest that erythroid control elements are formed by the interactions of at least three transcription factors, none of which functions alone.

Animals↗

PubMed1990

Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages.

The nuclear factor GF-1 (also known as NF-E1, Eryf-1; refs 1-3 respectively) is important in regulation of the transcription of globin and other genes that are specifically expressed in erythroid cells. We have previously shown that GF-1 of both mouse and human origin is a 413-amino-acid polypeptide with two novel zinc-finger domains whose expression is restricted to erythroid cells. Using in situ hybridization of mouse bone marrow cells and northern blot analysis of purified cell populations and permanent cell lines, we show here that GF-1 is expressed in two other hematopoietic lineages, megakaryocytes and bone marrow-derived mast cells. Our findings are consistent with results from hematopoietic progenitor culture which suggest a relationship between erythroid, megakaryocytic and mast cell lineages, and imply that GF-1 is expressed in committed multipotential cells and their progeny. Hence, the mere presence of this transcription factor is unlikely to be sufficient to programme differentiation of a single haematopoietic lineage. GF-1 may regulate the transcription of not only erythroid genes, but also many genes characteristic of megakaryocytes and mast cells, or genes shared among these lineages.

Animals↗

PubMed1990

One year follow-up of epikeratoplasty for myopia.

Eighteen surgeons performed 166 consecutive epikeratoplasty procedures to correct myopia in 103 patients. Of this total, 147 (89%) were first surgeries and 19 (11%) were repeat surgeries. First surgery patients were divided into three subpopulations based on preoperative spherical equivalents (low -5.00 diopters [D] to -9.49 D, medium -9.50 D to -14.00 D, and high greater than -14.00 D). The percentage of eyes with 20/40 or better best corrected visual acuity before surgery was 93% for the low group, 89% for the medium group, and 57% for the high group. The percentage of eyes with 20/40 or better uncorrected visual acuity at 12 months after surgery was 58% for the low group, 42% for the medium group, and 17% for the high group. The average and median spherical equivalent after one year was -1.40 D (2.45 SD) and -1.00 D, respectively; 41% of the eyes were within one diopter of emmetropia, 65% within two diopters, and 76% within three diopters. Clinically significant regression was only noted in low and medium myope groups under 25 years of age. Repeat surgery outcomes were equivalent to those in first surgery patients who had a single procedure.

Adolescent↗

PubMed1990

Transcriptional activation and DNA binding by the erythroid factor GF-1/NF-E1/Eryf 1.

The murine, erythroid DNA-binding protein GF-1 (also known as NF-E1, Eryf 1), a 413-amino acid polypeptide with two novel finger domains of the Cx-Cx variety, recognizes a consensus GATA motif present in cis elements of the majority of erythroid-expressed genes. We have performed a structure-function analysis of this protein to evaluate its potential as a transcriptional activator and to examine the role of the finger domains in DNA binding. Using a cotransfection assay, we find that GF-1 is a potent transcriptional activator with several activation domains but that this is revealed only in heterologous cells and with reporters containing minimal promoters onto which either a single or multiple GATA-binding sites are placed. The two fingers of GF-1 are functionally distinct and cooperate to achieve specific, stable DNA binding. The amino finger is necessary only for full specificity and stability of binding, whereas the carboxyl finger is required for binding. The role of each finger is more pronounced with some GATA-binding sites than with others, suggesting a diversity of interactions between GF-1 and different target sites. The complex activation and DNA-binding properties of GF-1 are likely to contribute to the ability of this single protein to participate widely in gene expression throughout erythroid development.

Amino Acid Sequence↗

PubMed1989

Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells.

Genes expressed in erythroid cells contain binding sites for a cell-specific factor believed to be an important regulator for this haematopoietic lineage. Using high-level transient expression in mammalian cells, we have identified complementary DNA encoding the murine protein. The factor, a new member of the zinc-finger family of DNA-binding proteins, is restricted to erythroid cells at the level of RNA expression and is closely homologous between mouse and man.

Amino Acid Sequence↗

PubMed1989

Increased gamma-globin expression in a nondeletion HPFH mediated by an erythroid-specific DNA-binding factor.

In man, a shift from gamma- to beta-globin gene expression in erythroblasts underlies a switch from fetal to adult haemoglobin during development. In hereditary persistence of fetal haemoglobin (HPFH), inappropriately high gamma-globin expression in adult life is associated with deletions in the beta-globin cluster or with single-base changes upstream of the gamma-globin genes. To account for enhanced gamma-gene expression in HPFH of the non-deletion type, we tested the nuclear proteins of human erythroleukaemia cells that bind gamma-promoter sequences in vitro by correlating specific mutations in their binding sites with promoter activity. An erythroid-specific factor (GF-1) binds as a single molecule to the -195 to -170 region and contacts two TATCT(AGATA) motifs, but not the conserved octamer (ATGCAAAT) that separates them. We observe that a single change (at -175, T----C) found in HPFH leads to increased promoter activity only in erythroid cells. This effect is mediated by GF-1, the human counterpart of the chicken erythroid factor Eryf 1. The form of HPFH we studied here is an inherited disorder which can be ascribed to the action of a cell-specific DNA-binding factor on a mutant promoter.

DNA-Binding Proteins↗