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M Groudine

Publications and source records attributed to M Groudine.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt's lymphoma cells.

In murine plasmacytoma and human Burkitt's lymphoma cells, one allele of c-myc is translocated into one of the immunoglobulin loci, resulting in a characteristic pattern of deregulated c-myc transcription. Translocation events between c-myc and the IgH locus segregate c-myc and the IgH intron enhancer to different reciprocal products in all plasmacytomas and in most Burkitt's lymphoma cells, suggesting that an additional element(s) capable of affecting c-myc expression over a large and variable distance must exist in the IgH locus. The region 3' of the IgH C alpha gene contains four tissue-specific and cell stage-specific DNase I hypersensitive sites (HSs), two of which map to the late B cell-specific 3' C alpha enhancer. We report here that DNA sequences comprising the two other 3' C alpha HSs contain potential protein-binding motifs for trans-activators commonly associated with immunoglobulin enhancers and that these sites can function as cell stage-specific enhancers in transient B cell assays. A DNA fragment containing all four HSs (HS1234) synergistically activates c-myc transcription in plasmacytoma and Burkitt's lymphoma cells in transient assays and induces high-level transcription, a promoter shift from P2 to P1, and an increase in readthrough transcription in stable transfections. Furthermore, plasmacytoma clones stably transfected with a HS1234-linked c-myc construct express c-myc in a position-independent, copy number-dependent manner. These results suggest that HS1234 may function as a locus control region (LCR), deregulating c-myc expression in t(15;12) plasmacytomas, as well as potentially contributing to aspects of normal IgH chain expression.

Animals↗
PubMed1994

Gene therapy of rat 9L gliosarcoma tumors by transduction with selectable genes does not require drug selection.

9L rat glioma cells have been used as a model for brain tumor therapies. It has been reported that in vivo infection of 9L cells with a replication-defective retrovirus expressing the herpes simplex thymidine kinase gene resulted in decreased tumor formation following treatment with the antiviral drug ganciclovir. In the study reported here, rats were injected either intracerebrally or subcutaneously with 9L glioma cells expressing a chimeric hygromycin phosphotransferase-thymidine kinase fusion protein or with unmodified 9L cells. Tumor formation was decreased in the rats receiving modified cells, even in the absence of treatment with ganciclovir. Suppression of tumor growth was also observed with cells modified to express the intracellular selectable marker neomycin phosphotransferase. These results indicate that an intracellular selectable marker, in the absence of pharmacologic selection, can inhibit tumor growth of 9L cells. The demonstration that intracellular marker genes can negatively influence the survival of transplanted cells has important implications for in vivo studies that use genetically modified cells.

Animals↗
PubMed1994

An alternative pathway of histone mRNA 3' end formation in mouse round spermatids.

During mammalian spermiogenesis, the post-meiotic stage of spermatogenesis, histones are replaced by protamines on the DNA. Despite this histone elimination, novel polyadenylated histone transcripts were detected in mouse round spermatids. Sequence analysis of a spermatid-specific H2a cDNA clone indicated that it was derived from a mRNA of a replication-dependent histone gene even though its transcript was not polyadenylated in somatic and earlier spermatogenic cells. In round spermatids, both the hairpin and purine-rich elements in the 3' untranslated region of the somatic pre-mRNA were retained in the mature poly(A)+ mRNA transcripts. Polyadenylation occurred downstream of the purine-rich element and was not preceded by the somatic AATAAA polyadenylation signal sequence. While polyadenylated histone transcripts from replication-dependent genes have been observed previously in somatic cells, characteristics of this type of 3'-end formation in mammalian round spermatids were unique. In particular, a specific replication-dependent H2a gene was transcribed either as a polyadenylated or non-polyadenylated transcript in these cells, suggesting that the type of transcript present was dependent on the RNA sequence. Finally, both poly(A)- and poly(A)+ mRNAs were found on polyribosomes from round spermatids, indicating that histones were being translated in these cells and that the polyadenylation status of these transcripts did not affect their translatability.

Animals↗
PubMed1994

Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts.

We have investigated the molecular basis of human immunodeficiency virus type 1 (HIV-1) latency in a tissue culture model and in HIV-infected people. We show that increased levels of Tat, but not Rev, can release the proviruses from latency in U1 cells. The absence of Tat in these cells is manifested by the accumulation of promoter-proximal viral transcripts, whereas the presence of Tat correlates with increased expression of viral proteins and an increase in promoter-distal transcripts. The presence of promoter-proximal transcripts also serves as a marker for latency in humans. We observed the exclusive presence of promoter-proximal viral transcripts in peripheral mononuclear cells from the majority (10/11) of asymptomatic HIV-infected individuals examined. Activation of these cells in vitro, and viremia in vivo, correlated with a switch from promoter-proximal transcription to promoter-distal transcription. These results suggest that the control between latency and replication of HIV in vivo is at the level of transcription elongation.

Base Sequence↗
PubMed1993

Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast.

In a screen designed to isolate human cDNAs that complement a yeast G2 phase checkpoint mutation (mec1), we isolated a cDNA homologous to the Saccharomyces cerevisiae CDC34 gene. The human CDC34 cDNA can functionally substitute for the yeast CDC34 gene and represents a mammalian homolog of the group of yeast genes required for the late G1-->S phase transition. The human CDC34 gene is expressed in multiple cell lines as a unique species and Southern blot analysis reveals evidence for a single gene that is highly conserved in higher eukaryotes. The human gene is located on the far telomeric region of 19p13.3 in a location that defines a region of homology between human chromosome 19p and mouse chromosome 11.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1993

In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T cells.

The IL-2 gene is a T cell specific gene that is expressed early during the activation-specific T lymphocyte development program. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assays have defined DNA/protein interactions at the IL-2 promoter cis-elements in vitro. To determine if the trans-activators documented in T cell nuclear extracts actually bind the IL-2 promoter in vivo, ligation mediated PCR (LMPCR) genomic footprinting was performed on the IL-2 promoter in both activated and non-activated T cells and HL60 promyelocytes, which do not express the IL-2 gene. The in vivo footprints indicate that the IL-2 gene transcription start site and TATA sequence are protected in both activated and resting T cells, prior to the appearance of detectable IL-2 steady state message. The distal NF-AT and the NF kappa B sites are each footprinted and the Oct/OAP site contains hypersensitive residues in the unstimulated T lymphocytes. Additional residues are protected in each of these sites after T cell activation. The proximal NF-AT site (NF-IL-2B) and the AP-1 site at -150 are protected in activated Jurkat T lymphocytes, but these two sites are not protected in activated Jurkat lymphocytes stably transfected a gene construct containing multiple NFAT binding sites.

Base Sequence↗
PubMed1993

An "in-out" strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: analysis of the beta-globin locus control region.

The human beta-globin locus control region (LCR) is a complex DNA regulatory element that controls the expression of the cis-linked beta-like globin genes located in the 55 kilobases 3' of the LCR. We have initiated the functional analysis of the LCR by homologous recombination in murine erythroleukemia cell somatic hybrids that carry a single copy of human chromosome 11 on which the beta-globin locus is situated. High-level expression of the human beta-globin gene normally occurs when these hybrid cells are induced to differentiate. We have reported that the insertion of an expressed selectable marker gene (driven by the Friend virus enhancer/promoter) into the LCR disrupts the LCR-mediated regulation of globin transcription. In these cells, beta-globin is no longer expressed when the cells differentiate; instead, expression of the selectable marker gene increases significantly after differentiation. Since present techniques for homologous recombination require the insertion of a selectable marker, further progress in using homologous recombination to analyze the LCR depends on deletion of the selectable marker and demonstration that the locus functions normally after the insertion, expression, and deletion of the selectable marker. Here we show that after precise deletion of the selectable marker by using the FLP recombinase/FRT (FLP recombinase target) system, the locus functions as it did before the homologous recombination event. These studies demonstrate the feasibility of using homologous recombination to analyze the LCR in particular, and other complex cis-regulatory DNA elements in general, in their normal chromosomal context.

Animals↗
PubMed1993

Common mechanisms for the control of eukaryotic transcriptional elongation.

Regulation of transcriptional elongation is emerging as an important control mechanism for eukaryotic gene expression. In this essay, we review the basis of the current view of the regulation of elongation in the human c-myc gene and discuss similarities in elongation control among the c-myc, Drosophila hsp70 and the HIV-1 genes. Based upon these similarities, we propose a model for control of expression of these genes at the elongation phase of transcription. This model suggests that distinct promoter elements direct the assembly of RNA polymerase II transcription complexes which differ in their elongation efficiency.

Animals↗
PubMed1993

Distinct properties of c-myc transcriptional elongation are revealed in Xenopus oocytes and mammalian cells and by template titration, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and promoter mutagenesis.

A block to c-myc transcription elongation has been observed in Xenopus oocytes and mammalian cells. Here, we show that the distribution of RNA polymerase II transcription complexes in the c-myc promoter proximal region in Xenopus oocytes is different from that observed previously in mammalian cells. Thus, there are major differences in the c-myc elongation block observed in the two systems. In addition, as first reported for a Xenopus tubulin gene (K. M. Middleton and G. T. Morgan, Mol. Cell. Biol. 10:727-735, 1990). c-myc template titration experiments reveal the existence of two classes of RNA polymerase II transcription complexes in oocytes: one (at low template concentration) that is capable of reading through downstream sites of premature termination, and another (high template concentration) that does not. We show that these classes of polymerases are distinct from those previously identified by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which distinguishes transcription complexes on the basis of transcribed distance, rather than on the basis of differential elongation through sites of premature termination. We also show that mutations that affect the efficiency of initiation of transcription from the c-myc P2 promoter can influence premature termination by at least two mechanisms: TATA box mutations function by the titration effect (decrease in transcription initiation results in a relative decrease in premature termination), while an upstream activator (E2F) site functions by contributing to the assembly of polymerase complexes competent to traverse the downstream sites of premature termination.

Animals↗
PubMed1992

Helix-loop-helix transcription factors E12 and E47 are not essential for skeletal or cardiac myogenesis, erythropoiesis, chondrogenesis, or neurogenesis.

E12 and E47 are two non-tissue-specific helix-loop-helix (HLH) transcription factors encoded by the E2A gene. Previous studies suggested that they are involved in regulation of differentiation in many tissue types including muscle, blood, and nerve through direct heterodimer interactions with tissue-specific HLH proteins. To gain further genetic insight into the functions of E12 and E47 during cell differentiation, we mutated both copies of the E2A gene in mouse embryonic stem (ES) cells and then tested the effect on differentiation in vitro. We find that the ES cells lacking functional E12 and E47 are capable of differentiating into both skeletal and cardiac muscle, erythrocytes, neurons, and cartilage that the same extent as wild-type cells. These results indicate that the E2A gene is not essential for differentiation of these cell types and suggest that redundant genes may control these developmental pathways.

Alternative Splicing↗
PubMed1992

The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region.

A conditional block to transcriptional elongation is an important mechanism for regulating c-myc gene expression. This elongation block within the first c-myc exon was defined originally in mammalian cells by nuclear run-on transcription analyses. Subsequent oocyte injection and in vitro transcription analyses suggested that sequences near the end of the first c-myc exon are sites of attenuation and/or premature termination. We report here that the mapping of single stranded DNA in vivo with potassium permanganate (KMnO4) and nuclear run-on transcription assays reveal that polymerase is paused near position +30 relative to the major c-myc transcription initiation site. Deletion of 350 bp, including the sites of 3'-end formation and intrinsic termination defined in oocyte injection and in vitro transcription assays does not affect-the pausing of polymerase in the promoter-proximal region. In addition, sequences upstream of +47 are sufficient to confer the promoter-proximal pausing of polymerases and to generate the polarity of transcription farther downstream. Thus, the promoter-proximal pausing of RNA polymerase II complexes accounts for the block to elongation within the c-myc gene in mammalian cells. We speculate that modification of polymerase complexes at the promoter-proximal pause site may determine whether polymerases can read through intrinsic sites of termination farther downstream.

Base Sequence↗
PubMed1992

Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region.

The human beta-globin locus control region (LCR) is a complex regulatory element that controls the erythroid-specific expression of all cis-linked globin genes. The LCR is composed of five DNase I hypersensitive sites (HS) spanning 16 kb and located greater than 50 kb upstream of the beta-globin gene on chromosome 11. Constructs containing all or some of these HS have been shown to produce high-level erythroid-specific expression of linked genes in transgenic mice and transfected cells. In all transgenic and transfection experiments reported to date, however, the spatial relationships between the LCR and globin genes have been disrupted. We have used homologous recombination (HR) as an approach to gain insights into the potential interactions between the LCR and globin genes in their native locations. A hygromycin B resistance (hygro(R)) gene was inserted into the human beta-globin LCR on chromosome 11 in a mouse/human hybrid erythroid cell line that expresses the human beta-globin gene after the induction of differentiation. As a consequence of this targeted insertion, the beta-globin gene is transcriptionally inactive and not inducible. In contrast, the hygro(R) gene within the LCR is inducible, whereas randomly integrated hygro(R) genes are not inducible in these cells. The chromatin structure of the targeted locus is also altered. A new DNase I HS is present in the enhancer/promoter of the hygro(R) gene inserted into the LCR, whereas a HS normally present in the LCR 3' to the insertion is lost and the beta-globin gene promoter HS is not detectable. These results are consistent with the promoter/enhancer competition model for LCR function and globin gene switching.

Base Sequence↗
PubMed1992

Sequences in the human c-myc P2 promoter affect the elongation and premature termination of transcripts initiated from the upstream P1 promoter.

A conditional block to transcription elongation provides one mechanism for controlling the steady-state levels of c-myc RNA in mammalian cells. Although prematurely terminated c-myc RNAs are not detectable in mammalian cells, truncated c-myc RNAs with 3' ends that map near the end of the first exon are transcribed from human c-myc templates injected into Xenopus oocytes germinal vesicles. A series of linker scanner and deletion mutants within the c-myc P2 promoter was tested in the Xenopus oocyte injection assay to determine the potential contribution of promoter elements to the elongation or premature termination of c-myc transcription. Although this analysis failed to identify sequences in the P2 promoter that significantly affect the elongation or termination of P2-initiated transcripts, our results suggest that sequences within the P2 promoter contribute to the premature termination of transcripts initiated at the upstream P1 promoter. A subset of these sequences is essential for the efficient elongation of P1-initiated transcripts through intrinsic sites of termination at the end of exon 1. These sequences affect P1 elongation when they are downstream of the site of initiation, and we hypothesize that they may be analogous to a class of prokaryotic elements required for antitermination.

Animals↗
PubMed1991

Tat-dependent adenosine-to-inosine modification of wild-type transactivation response RNA.

Tat is a potent activator of gene expression in human immunodeficiency virus type 1 (HIV-1). Activation by Tat requires a cis-acting element, the transactivation response (TAR) site, located in the viral long terminal repeat and the 5' end of all viral mRNAs. Sequences in TAR RNA can fold into a specific stem-loop structure, and certain features of the stem-loop are essential for Tat-mediated transactivation. In Xenopus oocytes, TAR sequences can inhibit the translation of 3' cis-linked mRNAs. However, coinjection of Tat and the TAR-containing RNA into oocyte nuclei relieves this translational inhibition [Braddock, M., Chambers, A., Wilson, W., Esnout, M. A., Adams, S.E. & Kingsman, S.M. (1989) Cell 58, 269-279]. We report here that the intramolecular TAR stem-loop structure is a substrate for the double-stranded RNA (dsRNA)-modifying activity, which converts adenosines to inosines. This activity is located in the nuclei of Xenopus oocytes. The specificity and extent of modification of adenosines in TAR is dependent on Tat. We propose that the dsRNA-modifying activity may be one of the cellular proteins that interacts with TAR in the nucleus. The possible role of TAR RNA modification in the expression of HIV-1 is discussed.

Adenosine↗
PubMed1990

High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells.

The locus activation region (LAR) of the human beta-globin-like gene cluster is characterized by a group of four DNase I hypersensitive sites, which arise specifically in erythroid tissues and are required for a normal pattern of beta-globin-like gene expression. The hypersensitive sites are found at positions 6.1, 10.9, 14.7, and 18 kilobase pairs (kbp) 5' of the epsilon-globin gene. Recently functional assays of the LAR that tested determinants for all four hypersensitive sites showed that expression of the human beta-globin gene was increased to normal or near-normal levels in both transgenic mice and erythroid cells. We constructed retroviral vectors with a human beta-globin gene and the determinant for a single hypersensitive site and measured beta-globin gene expression after retroviral infection of murine erythroleukemia cells. Fragments for the hypersensitive sites at -18 or -10.9 kbp increased human beta-globin RNA levels respectively to 35% or 132% of the endogenous mouse beta maj-globin RNA level. In addition, greater expression was also observed for the neomycin phosphotransferase RNA, which was transcribed from the retroviral LTR, showing that the LAR fragments activated expression from a heterologous promoter. In the context of gene-transfer experiments ultimately aimed at gene therapy, our results show that LAR determinants lead to an increased level of human beta-globin RNA expression after retroviral transfer into erythroid cells. But inclusion of LAR determinants in retroviral vectors also entails the potential risk of activating the expression of nonglobin genes in erythroid cells.

Animals↗