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N Raich

Publications and source records attributed to N Raich.

17 recordsLinked to original sources
PubMed1995

GATA1 and YY1 are developmental repressors of the human epsilon-globin gene.

The human epsilon-globin gene is transcribed in erythroid cells only during the embryonic stages of development. Expression of epsilon-globin gene, however, can be maintained in adult transgenic mice following removal of DNA positioned between -467 and -182 bp upstream of the epsilon-globin cap site. We have identified three protein binding regions within this silencer; a CCACC motif around -379, two overlapping motifs for YY1 and GATA around -269 and a GATA motif around -208 and we have analyzed their function during development by studying several mutants in transgenic mice. Mutation of the -208 GATA motif allows high epsilon-globin transgene expression in the adult suggesting that, in addition to its positive effects on transcription, GATA-1 also plays a negative role in the regulation of globin gene expression during development. Repression of epsilon gene expression in the adult also requires a functional YY1 binding site at position -269. Finally, mutation of the -379 CCACC site results in a small but detectable level of epsilon expression in adult erythroid cells. Thus, multiple proteins, including GATA-1, participate in the formation of the epsilon gene repressor complex that may disrupt the interaction between the proximal epsilon-promoter and the locus control region (LCR) in definitive erythroid cells.

Animals↗
PubMed1993

Erythroid regulatory elements.

Erythroid differentiation leads to the production of red blood cells that contain a high level of hemoglobin. This level is mainly regulated by globin gene transcription during development and differentiation. Although numerous cis-acting sequences are involved in transcriptional activity of globin genes, combinations of three motifs, CCACC, SP1 and GATA represent the core elements of their regulatory sequences. These combinations are also found in promoters and/or enhancers of non-globin genes specifically expressed in the late stages of erythroid differentiation. The CCACC and SP1 sequences bind proteins that do not display erythrocytic specificity, and the GATA sequences bind a family of transacting factors recently cloned. The GATA family members are distinctive for a highly homologous DNA binding domain that exists in two zinc fingers reminiscent of those of the glucocorticoid receptor. None of the GATA family members displays only erythroid specificity, but gene disruption followed by rescue indicates that GATA-1 is necessary for terminal erythroid differentiation throughout development. The GATA/SP1 and GATA/CCACC associations are present in positive, negative or inducible regulatory sequences suggesting that other elements control the fine tuning of erythroid gene expression. NF-E2, which is a major transcriptional activator, members of the ets family which are implicated in the early stages of erythropoiesis and finally c-erbA which directly regulates a set of erythroid-specific genes are proteins that bind these latter regulatory motifs.

Amino Acid Sequence↗
PubMed1992

Demonstration of a human epsilon-globin gene silencer with studies in transgenic mice.

The human epsilon-globin gene displays normal developmental regulation in transgenic mice; it is expressed only in embryonic and in definitive erythroid cells. We show here that deletion of a negative element located between -182 and -467 bp upstream of the epsilon-globin gene cap site results in continuation of epsilon gene expression in the definitive erythroblasts of the fetal liver and in the red blood cells of adult transgenic mice. These data provide direct in vivo evidence that cis acting silencing elements are involved in the developmental control of the epsilon-globin gene.

Animals↗
PubMed1990

Autonomous developmental control of human embryonic globin gene switching in transgenic mice.

The mechanisms by which expression of the beta-like globin genes are developmentally regulated are under intense investigation. The temporal control of human embryonic (epsilon) globin expression was analyzed. A 3.7-kilobase (kb) fragment that contained the entire human epsilon-globin gene was linked to a 2.5-kb cassette of the locus control region (LCR), and the developmental time of expression of this construct was studied in transgenic mice. The human epsilon-globin transgene was expressed in yolk sac-derived primitive erythroid cells, but not in fetal liver or bone marrow-derived definitive erythroid cells. The absence of epsilon gene expression in definitive erythroid cells suggests that the developmental regulation of the epsilon-globin gene depends only on the presence of the LCR and the epsilon-globin gene itself (that is, an autonomous negative control mechanism). The autonomy of epsilon-globin gene developmental control distinguishes it from the competitive mechanism of regulation of gamma and beta-globin genes, and therefore, suggests that at least two distinct mechanisms function in human hemoglobin switching.

Animals↗
PubMed1990

Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice.

Transgenic mice containing a human fetal (gamma-) or adult (beta-) globin gene linked to the beta-globin gene locus activation region (LAR) express the gene throughout development. By contrast, transgenic mice containing LAR linked to both a fetal and an adult globin gene display the normal developmental switch from fetal to adult gene expression. This suggests that the human fetal-to-adult globin gene switch is controlled through a mutually exclusive interaction between LAR and either the gamma- or beta-globin gene, resulting in the expression of only one gene at any given moment.

Animals↗
PubMed1990

Butyrate induces expression of transfected human fetal and endogenous mouse embryonic globin genes in GM 979 erythroleukemia cells.

We have analyzed the expression of endogenous murine genes and of transfected human fetal A gamma globin gene in GM 979, a mouse erythroleukemia line which produces adult as well as embryonic globins. Optimal induction of the endogenous murine adult globin genes was obtained with DMSO or HMBA while the epsilon y and beta h1 embryonic genes were preferentially induced by butyrate. Similarly, the transferred human A gamma-globin gene was preferentially induced by butyrate. These results as well as previous observations in vivo or in erythroid cell cultures suggest that butyrate preferentially induces the expression of fetal globin genes.

Acetamides↗
PubMed1989

Regulated expression of the overlapping ubiquitous and erythroid transcription units of the human porphobilinogen deaminase (PBG-D) gene introduced into non-erythroid and erythroid cells.

The human gene coding for porphobilinogen deaminase (PBG-D) is transcribed into two distinct transcription units giving two mRNAs. These units originate from two adjacent promoters distant of 3 kilobase pairs. The upstream promoter is active in all cell types, whereas the downstream promoter is active only in erythroid cells. We have studied the expression of this gene either after introduction of the corresponding human chromosome into murine erythroid cells using somatic hybrids or after transfection into both erythroid and non-erythroid cells. Using somatic hybrids, we showed that activation of the erythroid-specific promoter of the PBG-D gene did not reduce the rate of initiation of the ubiquitous promoter. Transfection experiments in erythroid cells showed that the PBG-D erythroid transcription unit, controlled by the PBG-D erythroid promoter, was correctly transcribed and regulated. Furthermore, we found that the PBG-D erythroid promoter alone was sufficient for correct expression and regulation of a reporter gene during erythroid differentiation. When the human PBG-D gene was transfected into non-erythroid cells, only the ubiquitous promoter was active. Deletion of the ubiquitous promoter did not lead to any activation of the erythroid promoter, suggesting that its inactivity in non-erythroid cells was not due to promoter occlusion but to a strict erythroid specificity.

Ammonia-Lyases↗
PubMed1989

Cis- and trans-acting elements involved in the regulation of the erythroid promoter of the human porphobilinogen deaminase gene.

Two cis-acting sequences, recognized by two erythroid-specific trans-acting factors, are involved in the regulation of the erythroid promoter of the human gene coding for porphobilinogen deaminase (PBGD). The first region, located at -70, binds the erythroid factor NF-E1, and point mutations within this region abolish the induction of transcription of this promoter during murine erythroleukemia (MEL) cell differentiation. The second region, located at -160, binds the erythroid-specific factor NF-E2 and the ubiquitous factor AP1. Using UV cross-linking, we show that NF-E2 has a higher molecular weight than AP1, demonstrating that NF-E2 is not an erythroid-specific degradation product of AP1. By point mutagenesis of the NF-E2/AP1 binding site, we define mutations that abolish binding of either NF-E2 alone or AP1 and NF-E2 together. Regulation of transcription of the PBGD erythroid promoter is abolished by those mutations, suggesting that NF-E2 but not AP1 is necessary for correct regulation of this promoter in erythroid cells.

Ammonia-Lyases↗
PubMed1988

Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression.

Porphobilinogen deaminase [PBGD; porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] is a cytosolic enzyme involved in the heme biosynthetic pathway. Two isoforms of PBGD, encoded by two mRNAs differing solely in their 5' end, are known: one is found in all cells and the other is present only in erythroid cells. We have previously shown that the human PBGD is encoded by a single gene and have now cloned and characterized this gene, which is split into 15 exons spread over 10 kilobases of DNA. We demonstrate that the two mRNAs arise from two overlapping transcription units. The first one (upstream) is active in all tissues and its promoter has some of the structural features of a housekeeping promoter; the second, located 3 kilobases downstream, is active only in erythroid cells and its promoter displays structural homologies with the beta-globin gene promoters.

Ammonia-Lyases↗
PubMed1986

Molecular cloning and complete primary sequence of human erythrocyte porphobilinogen deaminase.

We have cloned and sequenced a cDNA clone coding for human erythrocyte porphobilinogen deaminase. It encompasses the translated region, part of the 5' and the 3' untranslated regions. The deduced 344 amino acid sequence is consistent with the molecular weight and the partial amino-acid sequence of the NH2 terminal of the purified erythrocyte enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. Quantitative determinations indicate that the amount of PBG-D mRNA is modulated both by the erythroid nature of the tissue and by cell proliferation, probably at the transcriptional level.

Amino Acid Sequence↗
PubMed1986

Molecular cloning and nucleotide sequence of a complete human uroporphyrinogen decarboxylase cDNA.

We have cloned and sequenced a full-length cDNA coding for human uroporphyrinogen decarboxylase. The deduced 367-amino acid sequence is consistent with the molecular weight, the partial amino acid sequence of cyanogen bromide peptides, and the total amino acid composition of the purified enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome, and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. We have also demonstrated that the level of uroporphyrinogen decarboxylase mRNA is markedly increased in tissues or cell lines of erythroid origin and that this is due to a tissue-specific transcriptional activation of the uroporphyrinogen decarboxylase gene.

Amino Acid Sequence↗
PubMed1986

Assignment of human uroporphyrinogen decarboxylase (URO-D) to the p34 band of chromosome 1.

A cDNA probe corresponding to mRNA encoding human uroporphyrinogen decarboxylase (URO-D) was used to determine the chromosomal localization of the URO-D gene in the human genome. In agreement with previous studies, we have found that the locus for URO-D is located on chromosome 1 in hybrid cell mapping panels. The use of in situ hybridization allowed us to map the URO-D locus to band 1p34.

Animals↗
PubMed1986

Molecular analysis of uroporphyrinogen decarboxylase deficiency in a family with two cases of hepatoerythropoietic porphyria.

In order to determine the molecular basis of uroporphyrinogen (URO) decarboxylase deficiency responsible for hepatoerythropoietic porphyria (HEP) and familial porphyria cutanea tarda, we used a human URO decarboxylase cDNA to analyze the organization and expression of the URO decarboxylase gene in lymphoblastoid cells from normal individuals and from two patients with HEP. We could detect neither deletions nor rearrangements in the URO decarboxylase gene. Synthesis, processing, and cell-free translation of the specific transcripts appeared to be normal. The half-life of the abnormal protein was 12 times shorter than that of the normal enzyme. The results indicate that the enzyme defect is due to a rapid degradation of the protein in vivo. This study is the first to provide information regarding the molecular mechanism responsible for the URO decarboxylase deficiency in HEP.

Carboxy-Lyases↗
PubMed1984

Molecular cloning of a cDNA sequence complementary to porphobilinogen deaminase mRNA from rat.

A cDNA clone containing sequences complementary to the mRNA coding for anemic rat spleen porphobilinogen deaminase (EC 4.3.1.8) has been isolated. A cDNA library was prepared from partially purified mRNA (1% purity). This library was then screened by colony hybridization, using a cDNA probe derived from porphobilinogen deaminase mRNA further enriched (10-20% purity) by gel electrophoresis in the presence of methylmercury hydroxide. Colonies hybridizing with the probe were analyzed by hybrid-selected translation using anemic rat spleen mRNA. Four recombinant plasmids containing porphobilinogen deaminase cDNA sequences were identified by specific immunoprecipitation of the translational product from hybrid-selected mRNA. Porphobilinogen deaminase mRNA was shown to contain 1800 bases by blot hybridization analysis. The cloned cDNA sequence consists of 1500 bases. Hybridization analysis of poly(A)+ RNA from uninduced and induced mouse erythroleukemic cells indicated that induction to erythroid differentiation by dimethyl sulfoxide results in a 10-fold increase of porphobilinogen deaminase mRNA. The rat cDNA clones hybridize to the corresponding sequences encoding human porphobilinogen deaminase. This property will be useful for isolation of human gene(s) and further characterization of the molecular lesion(s) responsible for acute intermittent porphyria.

Ammonia-Lyases↗