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M Groudine

Publications and source records attributed to M Groudine.

At least 19 recordsLinked to original sources
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↗
PubMed1990

The block to transcription elongation is promoter dependent in normal and Burkitt's lymphoma c-myc alleles.

Aberrant c-myc expression patterns occur in human Burkitt's lymphoma cells, which consistently exhibit c-myc chromosomal translocations, mutations within and flanking the translocated allele, a loss of the block to transcription elongation in exon 1, and a promoter shift to use of the upstream P1 promoter. To define the mechanism responsible for the loss of transcription elongation blockage and resulting c-myc deregulation in Burkitt's lymphoma, we analyzed transcription patterns after transfer of normal and Burkitt's lymphoma c-myc alleles into murine cells and Xenopus oocyte germinal vesicles. We have determined that although the mutations within and surrounding several Burkitt's lymphoma c-myc alleles are not sufficient, in themselves, to abrogate the transcription elongation block, transcription initiation from the P2 promoter may be necessary to obtain the block to transcription elongation. To test directly the role of c-myc promoters in programming transcription elongation blockage, we analyzed transcription patterns from in vitro mutagenized c-myc genes containing deletions of either the P1 or P2 promoter. These data confirm that P1-initiated c-myc transcripts do not terminate at discrete sites near the 3' end of exon 1, whereas P2-initiated transcripts either terminate or read through the transcription block signals. Therefore, overexpression and/or constitutive expression from the c-myc P1 promoter may contribute to increased readthrough transcription in Burkitt's lymphoma cells and, hence, to aberrant expression patterns or levels of c-myc steady-state transcripts. In addition, the ability of normal cells to modulate c-myc P2-initiated transcription to either read through or to block elongation provides a fine control mechanism over c-myc steady-state RNA levels.

Alleles↗
PubMed1990

A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.

Naturally occurring deletions that remove sequences located approximately 60 kb upstream of the human adult beta-globin gene result in the failure to transcriptionally activate the cis-linked globin genes in erythroid cells. In addition, transfection, transgenic, and somatic cell hybrid studies have revealed that sequences within this region are essential for the developmentally regulated high-level expression of cis-linked globin genes. This regulatory region located at the 5' end of the beta-globin locus has been termed the locus activation region (LAR). Using somatic cell hybrids, we have studied the chromatin structure and timing of DNA replication of the normal human beta-globin locus and a locus containing a de novo 25-kb deletion that removes elements of the LAR. As a result of this deletion, the entire beta-globin locus and sequences approximately 100 kb 5' and 3' of the adult beta-globin gene are DNase I-resistant and do not form characteristic distant hypersensitive sites. These sequences also replicate late in S phase in an erythroid cell background. In contrast, the sequences of the normal locus are DNase I sensitive and early replicating. These results suggest that the LAR is required for both the erythroid-specific chromatin structure and timing of DNA replication over a large physical distance.

Chromatin↗
PubMed1990

Translocation of an erythroid-specific hypersensitive site in deletion-type hereditary persistence of fetal hemoglobin.

Hereditary persistence of fetal hemoglobin (HPFH) can involve large deletions which eliminate the 3' end of the beta-like globin gene cluster and more than 70 kilobases (kb) of flanking DNA. Blot hybridization revealed a DNase I-hypersensitive site extending from 1.1 to 1.4 kb downstream of the HPFH-1 3' deletion endpoint. The site was found in normal fetal and adult nucleated erythroid cells and in two erythroleukemia cell lines but not in nonerythroid cells and tissues. Simian virus 40 core enhancer-like sequences were found nonrandomly distributed within the boundaries of the site, which is contained in a fragment of known enhancer activity (E. A. Feingold and B. G. Forget, Blood, in press). A second hypersensitive site was found 0.5 kb upstream of the HPFH-1 3' deletion endpoint but was not erythroid specific. A third site, most prominent in fetal liver-derived erythroid cells, was found 1 kb upstream of the HPFH-2 deletion endpoint. As predicted by the locations of the deletion endpoints, the first two sites were translocated to within 12 kb of the A gamma gene in erythroid colonies derived from an HPFH-2 heterozygote and in hybrid mouse-human erythroid cells carrying the HPFH-2 deletion chromosome. Further analysis of this region showed that it was DNase I sensitive in erythroid and myeloid cells, indicating that it resides in an open chromatin domain. These observations suggest that alterations of chromatin structure flanking the fetal globin genes may contribute to abnormal gene regulation in deletion-type HPFH.

Adult↗
PubMed1990

Transcription elongation and eukaryotic gene regulation.

Each step in the synthesis of functional transcript by RNA polymerase II provides a level at which gene expression can be regulated. Control over the elongation phase of transcription is a recognized regulatory mechanism in prokaryotes; however, only recently have examples of conditional transcription elongation blockage been reported in eukaryotic cellular genes. In several cases, control over transcription elongation clearly contributes to the regulated expression of these genes. Indeed, reports that transcription by RNA polymerase II is initiated and paused on many Drosophila promoters, prior to induction of gene expression, suggests that release of an arrested polymerase, as opposed to polymerase recruitment to a disengaged promoter, may be the key regulatory step for many genes thought to be controlled by transcription initiation (Rougvie & Lis, 1988). RNA polymerase II undergoes modifications, such as association with ancillary elongation factors and phosphorylation of its large subunit carboxy terminal domain (CTD), at stages subsequent to recruitment to a promoter and establishment of a pre-initiation complex (Reinberg & Roeder, 1987; Rappaport et al., 1987; Payne et al., 1989; Laybourn & Dahmus, 1989). It is possible that modifications such as these, or others occurring prior to, during or following transcription initiation, may alter the holoenzyme's transcription elongation properties, to allow recognition or read-through of elongation block signals within a transcription unit. In this review, we will present features of transcription elongation blockage in several eukaryotic cellular genes in the context of our understanding of attenuation and premature transcription termination in prokaryotic and viral genes. We will also present evidence supporting the model that modifications to the RNA polymerase II transcription complex are pivotal to the control of transcriptional at the level of elongation.

Animals↗
PubMed1989

Expression of a novel histone 2B during mouse spermiogenesis.

During mammalian spermiogenesis transitional proteins and protamines replace histones on the DNA as the chromatin condenses. While previous studies suggested that histone genes are inactive postmeiotically, we have shown both by steady-state RNA analysis and nuclear run-off transcription assays that histone 2b (H2b) transcription occurs in mouse round spermatids. In addition, a novel H2b cDNA clone has been isolated from an adult mouse testes cDNA library. The sequence of this cDNA clone predicts a protein that is extremely similar to other mouse H2b proteins, except at the carboxyl-terminus where the testes H2b contains an additional 12 amino acids, seven of which are hydrophobic. In contrast to the replication-dependent histone mRNAs, the 3' untranslated region of this cDNA contains the poly(A) addition sequence (AAUAAA) upstream of a poly(A) tract. Furthermore, the conserved hairpin structure immediately upstream of replication-dependent histone mRNA termini is not present. Northern blot analysis of RNA from embryonic, ovarian, spermatogenic, and a variety of somatic tissues reveals that this novel H2b transcript is spermatid specific. The H2b mRNA is in polyribosomes isolated from spermatogenic cells, strongly suggesting that it is translated during spermiogenesis.

Amino Acid Sequence↗
PubMed1989

Molecular analysis of the human beta-globin locus activation region.

Recently, DNA sequences containing four erythroid-specific DNase I hypersensitive sites within 20 kilobases 5' of the human epsilon-globin gene have been identified as an important cis-acting regulatory element, the locus activation region (LAR). Subfragments of the LAR, containing either all or only the two 5' or two 3' hypersensitive sites were linked to the human beta-globin gene and analyzed for their effect on globin gene expression in stably transformed mouse erythroleukemia (MEL) cells. Constructs containing all four of the hypersensitive sites increase beta-globin mRNA levels 8- to 13-fold, while constructs with only the 5' or 3' sites increase globin expression to a lesser extent. No effect was seen when the constructs were assayed in 3T3 fibroblasts. All of the LAR derivatives form hypersensitive sites at the corresponding sequence position in MEL cells prior to and after induction of MEL cell differentiation. However, in 3T3 fibroblasts only the hypersensitive site corresponding to the previously described erythroid-specific -10.9 site was formed.

Animals↗
PubMed1989

Polyadenylation and U7 snRNP-mediated cleavage: alternative modes of RNA 3' processing in two avian histone H1 genes.

The six chicken histone H1 genes have 3'-processing sequences typical of replication-dependent histone genes, which are expressed as poly(A)- mRNAs. However, by Northern analysis of RNA from several adult chicken tissues, as well as from embryonal skeletal muscle in vivo and in vitro, we have observed histone H1 transcripts longer than those predicted on the basis of the published genomic sequences. These RNAs are polyadenylated transcripts of the genes H1.01 and H1.10, which encode the 'c fraction' H1 protein subtypes. Both transcripts contain an internal stem-loop and purine-rich box associated with the 3' processing of poly(A)- histone mRNAs. The 2-kb poly(A)+ H1.01 transcript is present at high steady-state levels in tissues with low rates of DNA synthesis, has a longer half-life than the poly(A)- mRNA from the same gene, and is polyribosomal in embryonal skeletal muscle. The 1-kb poly(A)+ H1.10 RNA is the major H1.10 transcript in adult skeletal muscle. The properties of these RNAs suggest that they may contribute to the relaxed replication dependence of c fraction subtype expression. The polyadenylation signals of both genes are unusual in their association with processed (nonhistone) pseudogene-like elements, an arrangement with possible implications for the mechanism of alternative 3'-end formation in these genes.

Amino Acid Sequence↗
PubMed1989

Accurate, TATA box-dependent polymerase III transcription from promoters of the c-myc gene in injected Xenopus oocytes.

We have investigated the factors that permit a gene normally transcribed by RNA polymerase II to be transcribed by RNA polymerase III. It was shown previously that the human c-myc gene could be transcribed in vitro and in Xenopus oocytes by both alpha-amanitin-sensitive and alpha-amanitin-resistant polymerases, probably corresponding to polymerase II and polymerase III. We confirmed this observation in microinjected oocytes and showed that the alpha-amanitin-resistant transcription of c-myc was competed by known polymerase III genes. Polymerase III transcription of c-myc was very inefficient compared to other polymerase III genes, however, and was observed only when large amounts of template DNA were injected. At lower DNA concentrations the gene was transcribed, exclusively by polymerase II. In contrast, the adenovirus major late promoter was not transcribed by polymerase III. The 5' ends of polymerase III RNAs were almost indistinguishable from those of polymerase II RNAs initiating at the P1 and P2 promoters of the human and mouse c-myc genes. Furthermore, point mutations in the TATA box of the human P2 promoter greatly reduced polymerase III activity. At this promoter, therefore, polymerase II and polymerase III recognize a common element, the TATA box, which probably plays an important role in specifying the start site of transcription for both polymerases. We suggest that the highly accurate though inefficient mimicry of polymerase II by polymerase III at the c-myc promoters reflects the common evolutionary origin of these two enzymes.

Animals↗
PubMed1989

Tissue-specific lability and expression of avian leukosis virus long terminal repeat enhancer-binding proteins.

Avian leukosis virus (ALV) induces bursal lymphomas in chickens, after proviral integration next to the cellular myc proto-oncogene, and subsequent c-myc hyperexpression. Our previous work suggested that labile or short-lived cellular proteins interact with the viral long terminal repeat (LTR) enhancer, and binding of these proteins appeared to be essential for high rates of LTR-enhanced transcription (A. Ruddell, M. Linial, W. Schubach, and M. Groudine, J. Virol. 62:2728-2735, 1988). This lability is specific for B-lymphoid cell types, since T cells and fibroblasts show stable high rates of LTR-enhanced transcription and stable LTR-binding activity. Moreover, the lability of these proteins may be important in determining susceptibility to bursal lymphoma. In this study, we separated and characterized the labile and stable LTR-binding proteins and examined their lability and expression in different cell types. Gel shift and DNase I footprinting analyses indicated that at least five proteins interact with the 140-base-pair LTR enhancer region. These proteins were distinct by several criteria, including lability or stability after inhibition of protein synthesis, resistance to heat denaturation, chromatographic behavior, and expression in different cell types. Two binding proteins were present in many cell types and were specifically labile in B cells. A third binding protein showed hematopoietic-cell-type-specific expression and was also labile in B cells. These findings indicate that there is tissue-specific modulation of the lability and expression of ALV LTR-binding proteins, which may be important for regulation of LTR transcription enhancement and ALV bursal lymphomagenesis.

Animals↗
PubMed1989

Expression of replication-dependent histone genes in avian spermatids involves an alternate pathway of mRNA 3'-end formation.

In somatic cells the expression of replication-dependent histone genes is coupled to the S phase of the cell cycle. However, we have found a number of novel H2a, H2b, and H3 poly(A)+ RNA species in avian haploid round spermatids. The spermatid-specific H2a and H2b 0.8-kilobase RNAs are transcribed from a subset of the replication-dependent H2a and H2b gene families. Two cDNAs derived from the spermatid-specific H2b transcripts were isolated and sequenced. The structures of these cDNAs reveal that the spermatid-specific RNAs are identical to the 0.5-kilobase poly(A)- H2b mRNAs expressed in proliferating somatic cells, except for the addition of poly(A) at the 3' ends. The site of poly(A) addition in the spermatid-specific RNAs is located 26 to 28 nucleotides 3' of the poly(A)- H2b mRNA terminus. Thus, the hairpin structures and purine-rich elements required for the U7 small nuclear ribonucleoprotein-mediated cleavage reaction that generates the 3' ends of poly(A)- H2b mRNAs are not utilized in spermatids and are retained in the poly(A)+ H2b RNAs.

Amino Acid Sequence↗