Search PubMed⌕ Search

Biomedical subjects

M Groudine

Publications and source records attributed to M Groudine.

At least 37 records · Page 2Linked to original sources
PubMed2000

Dynamic analysis of proviral induction and De Novo methylation: implications for a histone deacetylase-independent, methylation density-dependent mechanism of transcriptional repression.

Methylation of cytosines in the CpG dinucleotide is generally associated with transcriptional repression in mammalian cells, and recent findings implicate histone deacetylation in methylation-mediated repression. Analyses of histone acetylation in in vitro-methylated transfected plasmids support this model; however, little is known about the relationships among de novo DNA methylation, transcriptional repression, and histone acetylation state. To examine these relationships in vivo, we have developed a novel approach that permits the isolation and expansion of cells harboring expressing or silent retroviruses. MEL cells were infected with a Moloney murine leukemia virus encoding the green fluorescent protein (GFP), and single-copy, silent proviral clones were treated weekly with the histone deacetylase inhibitor trichostatin A or the DNA methylation inhibitor 5-azacytidine. Expression was monitored concurrently by flow cytometry, allowing for repeated phenotypic analysis over time, and proviral methylation was determined by Southern blotting and bisulfite methylation mapping. Shortly after infection, proviral expression was inducible and the reporter gene and proviral enhancer showed a low density of methylation. Over time, the efficacy of drug induction diminished, coincident with the accumulation of methyl-CpGs across the provirus. Bisulfite analysis of cells in which 5-azacytidine treatment induced GFP expression revealed measurable but incomplete demethylation of the provirus. Repression could be overcome in late-passage clones only by pretreatment with 5-azacytidine followed by trichostatin A, suggesting that partial demethylation reestablishes the trichostatin-inducible state. These experiments reveal the presence of a silencing mechanism which acts on densely methylated DNA and appears to function independently of histone deacetylase activity.

Animals↗
PubMed1999

A functional enhancer suppresses silencing of a transgene and prevents its localization close to centrometric heterochromatin.

To explore the mechanism by which enhancers maintain gene expression, we have assessed the ability of an enhancer and derivative mutants to influence silencing and nuclear location of a transgene. Using site-specific recombination to place different constructs at the same integration sites, we find that disruption of core enhancer motifs impairs the enhancer's ability to suppress silencing. FISH analysis reveals that active transgenes linked to a functional enhancer localize away from centromeres. However, enhancer mutations that result in increased rates of transgene silencing fail to localize the transgene away from centromeric heterochromatin, even when the transgene is in an active state. These mutations thus dissociate transcriptional activity and subnuclear location. Together, our results suggest that the functional enhancer antagonizes gene silencing by preventing localization of a gene near centromeric heterochromatin.

Enhancer Elements, Genetic↗
PubMed1999

Conservation of sequence and structure flanking the mouse and human beta-globin loci: the beta-globin genes are embedded within an array of odorant receptor genes.

In mouse and human, the beta-globin genes reside in a linear array that is associated with a positive regulatory element located 5' to the genes known as the locus control region (LCR). The sequences of the mouse and human beta-globin LCRs are homologous, indicating conservation of an essential function in beta-globin gene regulation. We have sequenced regions flanking the beta-globin locus in both mouse and human and found that homology associated with the LCR is more extensive than previously known, making up a conserved block of approximately 40 kb. In addition, we have identified DNaseI-hypersensitive sites within the newly sequenced regions in both mouse and human, and these structural features also are conserved. Finally, we have found that both mouse and human beta-globin loci are embedded within an array of odorant receptor genes that are expressed in olfactory epithelium, and we also identify an olfactory receptor gene located 3' of the beta-globin locus in chicken. The data demonstrate an evolutionarily conserved genomic organization for the beta-globin locus and suggest a possible role for the beta-globin LCR in control of expression of these odorant receptor genes and/or the presence of mechanisms to separate regulatory signals in different tissues.

Animals↗
PubMed1999

Analysis of mammalian cis-regulatory DNA elements by homologous recombination.

The use of homologous recombination to modify and thereby functionally analyze cis-regulatory DNA elements in mammalian cells has become an important approach in mammalian gene expression research. We have emphasized the necessity of designing a system that allows the removal of selectable markers used in targeting and facilitates the further modification of the region under study. To perform these tasks, we presently favor making an initial HR-mediated replacement of the entire element under study with an active positive selectable marker in combination with either an inactive second positive selectable marker or an active negative selectable marker. The plug and socket system, in which an inactive selectable marker is complemented by HR, is the most dependable and well-characterized option for making secondary modifications. However, the double-replacement system has certain advantages, and the recently developed RMCE approach, which allows replacement of a negative selectable marker by site-specific recombinase-mediated insertion without using a positive selectable marker, will likely prove very valuable in future experiments. Each of the systems, or combinations thereof, should be considered in light of the specifics of any given experiment to select the most appropriate option. Although the emphasis of this article has been the analysis of cis-acting regulatory elements involved in transcription, these same approaches can be used to analyze other regulatory elements (e.g., origins of replication) and to make multiple subtle mutations in polypeptides.

Animals↗
PubMed1998

Description and targeted deletion of 5' hypersensitive site 5 and 6 of the mouse beta-globin locus control region.

The most upstream hypersensitive site (HS) of the beta-globin locus control region (LCR) in humans (5' HS 5) and chickens (5' HS 4) can act as an insulating element in some gain of function assays and may demarcate a beta-globin domain. We have mapped the most upstream HSs of the mouse beta-globin LCR and sequenced this region. We find that mice have a region homologous to human 5' HS 5 that is associated with a minor HS. In addition we map a unique HS upstream of 5' HS 5 and refer to this novel site as mouse 5' HS 6. We have also generated mice containing a targeted deletion of the region containing 5' HS 5 and 6. We find that after excision of the selectable marker in vivo, deletion of 5' HS 5 and 6 has a minimal effect on transcription and does not prevent formation of the remaining LCR HSs. Taken together these findings suggest that the most upstream HSs of the mouse beta-globin LCR are not necessary for maintaining the beta-globin locus in an active configuration or to protect it from a surrounding repressive chromatin environment.

Animals↗
PubMed1998

Long-distance transcriptional enhancement by the histone acetyltransferase PCAF.

Enhancers are defined by their ability to stimulate gene activity from remote sites and their requirement for promoter-proximal upstream activators to activate transcription. Here we demonstrate that recruitment of the p300/CBP-associated factor PCAF to a reporter gene is sufficient to stimulate promoter activity. The PCAF-mediated stimulation of transcription from either a distant or promoter-proximal position depends on the presence of an upstream activator (Sp1). These data suggest that acetyltransferase activity may be a primary component of enhancer function, and that recruitment of polymerase and enhancement of transcription are separable. Transcriptional activation by PCAF requires both its acetyltransferase activity and an additional activity within its N terminus. We also show that the simian virus 40 enhancer and PCAF itself are sufficient to counteract Mad-mediated repression. These results are compatible with recent models in which gene activity is regulated by the competition between deacetylase-mediated repression and enhancer-mediated recruitment of acetyltransferases.

Acetyltransferases↗
PubMed1998

Requirement of the mouse I-mfa gene for placental development and skeletal patterning.

The bHLH-repressor protein I-mfa binds to MyoD family members, inhibits their activity, and blocks their nuclear import and binding to DNA. In situ hybridization analysis demonstrated that mouse I-mfa was highly expressed in extraembryonic lineages, in the sclerotome, and subsequently within mesenchymal precursors of the axial and appendicular skeleton, before chondrogenesis occurs. Targeted deletion of I-mfa in a C57Bl/6 background resulted in embryonic lethality around E10.5, associated with a placental defect and a markedly reduced number of trophoblast giant cells. Overexpression of I-mfa in rat trophoblast (Rcho-1) stem cells induced differentiation into trophoblast giant cells. I-mfa interacted with the bHLH protein Mash2, a negative regulator of trophoblast giant cell formation, and inhibited its transcriptional activity in cell culture. In contrast, I-mfa did not interfere with the activity of the bHLH protein Hand1, a positive regulator of giant cell differentiation. Interestingly, I-mfa-null embryos on a 129/Sv background had no placental defect, generally survived to adulthood, and exhibited delayed caudal neural tube closure and skeletal patterning defects that included fusions of ribs, vertebral bodies and abnormal formation of spinous processes. Our results indicate that I-mfa plays an important role in trophoblast and chondrogenic differentiation by negatively regulating a subset of lineage-restricted bHLH proteins.

Animals↗
PubMed1998

Identification of Bach2 as a B-cell-specific partner for small maf proteins that negatively regulate the immunoglobulin heavy chain gene 3' enhancer.

Maf family transcription factors are important regulators in various differentiation systems. Putative Maf recognition elements (MAREs) are found in the 3' enhancer region of the immunoglobulin heavy chain (IgH) gene. These elements are bound in B-cell extracts by a heterodimeric protein complex containing both Bach2 and a small Maf protein. Analysis of normal hematopoietic cells revealed that Bach2 is specifically expressed in B cells. Bach2 is abundantly expressed in the early stages of B-cell differentiation and turned off in terminally differentiated cells. Bach2 acts together with MafK as a negative effector of the IgH 3' enhancer and binds to the co-repressor SMRT (silencing mediator of retinoid and thyroid receptor). Hence the Bach2-small-Maf heterodimer may represent the first example of a B-cell lineage, and of a developmental stage-restricted negative effector of the MARE in the IgH 3' enhancer region.

B-Lymphocytes↗
PubMed1998

Reduced beta-globin gene expression in adult mice containing deletions of locus control region 5' HS-2 or 5' HS-3.

To gain insights into the functions of individual DNA'se hypersensitive sites within the beta globin locus control region (LCR), we deleted the endogenous 5' HS-2 and HS-3 regions from the mouse germline using homologous recombination techniques. We demonstrated that the deletion of either murine 5' HS-2 or 5' HS-3 reduced the expression of the embryonic epsilon y and beta h1 globin genes minimally in yolk sac-derived erythrocytes, but that both knockouts reduced the output of the adult beta (beta-Major + beta-Minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-Neo cassette was retained within either the HS-2 or HS-3 region, a much more severe reduction in globin gene expression was observed at all developmental stages. PGK-Neo was shown to be expressed in an erythroid-specific fashion when it was retained in the HS-3 position. These results show that neither 5' HS-2 nor HS-3 is required for the activity of embryonic globin genes, nor are these sites required for correct developmental switching. However, each site is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult red blood cells. Each site therefore contains some non-redundant information that contributes to adult globin gene function.

Animals↗
PubMed1998

DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs.

The repeated modification of a genomic locus is a technically demanding but powerful strategy to analyze the function of a particular gene product or the role of cis-regulatory DNA elements in mammalian cells. The initial step is "tagging" a site with a selectable marker which is done by homologous recombination (HR) to modify a known locus or by random integration to study cis-regulatory elements at a reproducibly accessible genomic location. The tag is then used to target the construct of choice during an exchange step. Presented here is a novel technique in which the exchange is independent of HR and does not introduce vector sequences. It relies on our previous studies on the replacement of DNA cassettes by FLP-recombinase, whereby some common limitations can be overcome. To this end, the tag, a hygtk positive/negative selection marker, is integrated into the genome of embryonic stem (ES) cells. This marker is flanked by a wild-type Flp-recognition target (FRT) site on one end and by a modified heterospecific FRT site on the other. Successful Flp-mediated replacement of the hygtk cassette is enriched by ganciclovir (GANC) selection for cells that lack the encoded fusion protein. Thereby, the hygtk gene can be exchanged for virtually any sequence in a single efficient step without the need of introducing a positive selectable marker. The system can hence be used to analyze the function of either a gene product or regulatory sequences in ES cells or the transgenic mice derived thereof.

Animals↗
PubMed1998

Cell cycle perturbations in acute myeloid leukemia samples following in vitro exposures to therapeutic agents.

Cell cycle checkpoints establish the timing and strength of arrest, repair and apoptosis responses to damaging treatments. We designed flow cytometric assays to measure cell cycle arrest and apoptosis in acute myeloid leukemia (AML) samples treated in vitro with relevant therapeutic agents so as to functionally characterize checkpoints in these samples and to ask if checkpoint abnormalities are common in AML and contribute to therapeutic failures. We show here that cell cycle responses to daunomycin (DNR), cytosine arabinoside (ARA-C) and gamma irradiation (RAD) were reproducibly treatment agent- and dose-dependent and distinct in different myeloid cell lines. DNR treatments differentially induced cell accumulations in the gap 2 and mitosis (G2/M) phases of the cell cycle and/or in the gap 1 (G1) phase, as did RAD, while ARA-C induced accumulations in the DNA synthesis (S) phase or in the G1 phase. Flow cytometric gates were devised to exclude lymphocytes and mature neutrophils in analyses of primary myeloid cell samples. Cell subsets in bone marrow samples from normal donors were thus enriched for myeloid constituents and used as normal myeloid cell controls. Proliferating cell nuclear antigen (PCNA) immunostaining was used to further identify actively dividing cell subpopulations in primary cell samples. AML samples were similarly analyzed and the majority showed lower DNA synthesis cell cycle phase (S) fractions and lower PCNA-positive fractions than normal myeloid cells, suggesting that AMLs are generally less proliferative in these culture conditions. Exceptional AML samples with high S phase fractions had cytogenetic abnormalities associated with poor prognosis. Most AML samples mounted weak cell cycle responses relative to normal myeloid cells, while a minority showed robust, agent-specific cell cycle arrests. This non-responsiveness was not simply associated with lower cycling indices-neither the response patterns nor the degrees of response were correlated with untreated S phase fractions or with PCNA-positive fractions. Cell cycle responses were also not associated with clinical parameters including patient age, FAB class, or white blood cell count, nor with immunophenotypic features including CD34 status, nor with specific cytogenetic markers. This suggests that functional cell cycle response assays could provide unique diagnostic information in AML. These assays might also have prognostic value as ARA-C induced G1 arrests and DNR-specific G2/M arrests tended to be associated with failure to achieve clinical remission. In addition, G1 arrests after ARA-C and G2/M accumulations after DNR treatments tended to be more robust in samples that had previously been shown to be more highly immunopositive for bcl-2 expression. This data suggests that the association of bcl-2 expression with particular cell cycle responses to therapeutic agents may contribute to the association of bcl-2 with poor clinical responses in AML. These data provide the basis for further laboratory studies aimed at examining specific cell cycle arrests as mechanisms of therapeutic resistance and prospective studies aimed at rigorously assessing the prognostic utility of in vitro assays of checkpoint function.

Acute Disease↗
PubMed1998

The beta-globin LCR is not necessary for an open chromatin structure or developmentally regulated transcription of the native mouse beta-globin locus.

The murine beta-globin locus control region (LCR) was deleted from its native chromosomal location. The approximately 25 kb deletion eliminates all sequences and structures homologous to those defined as the human LCR. In differentiated ES cells and erythroleukemia cells containing the LCR-deleted chromosome, DNasel sensitivity of the beta-globin domain is established and maintained, developmental regulation of the locus is intact, and beta-like globin RNA levels are reduced 5%-25% of normal. Thus, in the native murine beta-globin locus, the LCR is necessary for normal levels of transcription, but other elements are sufficient to establish the open chromatin structure, transcription, and developmental specificity of the locus. These findings suggest a contributory rather than dominant function for the LCR in its native location.

Animals↗
PubMed1998

The locus control region is necessary for gene expression in the human beta-globin locus but not the maintenance of an open chromatin structure in erythroid cells.

Studies in many systems have led to the model that the human beta-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the beta-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous beta-globin locus in an erythroid background, transcription of all beta-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the beta-globin locus are not affected by the deletion. In contrast, deletion of 5'HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on beta-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an "open" conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the beta-like globin genes.

Adult↗
PubMed1998

The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes.

In chromosome translocations characteristic of Burkitt lymphomas (BL) and murine plasmacytomas, c-myc genes become juxtaposed to immunoglobulin heavy-chain (IgH) sequences, resulting in aberrant c-myc transcription. Translocated c-myc alleles that retain the first exon exhibit increased transcription from the normally minor c-myc promoter, P1, and increased transcriptional elongation through inherent pause sites proximal to the major c-myc promoter, P2. We recently demonstrated that a cassette derived from four DNase I-hypersensitive sites (HS1234) in the 3'Calpha region of the IgH locus functions as an enhancer-locus control region (LCR) and directs a similar pattern of deregulated expression of linked c-myc genes in BL and plasmacytoma cell lines. Here, we report that the HS1234 enhancer-LCR mediates a widespread increase in histone acetylation along linked c-myc genes in Raji BL cells. Significantly, the increase in acetylation was not restricted to nucleosomes within the promoter region but also was apparent upstream and downstream of the transcription start sites as well as along vector sequences. Histone hyperacetylation of control c-myc genes, which was induced by the deacetylase inhibitor trichostatin A, mimics the effect of the HS1234 enhancer on expression from the c-myc P2 promoter, but not that from the P1 promoter. These results suggest that the HS1234 enhancer stimulates transcription of c-myc by a combination of mechanisms. Whereas HS1234 activates expression from the P2 promoter through a mechanism that includes increased histone acetylation, a general increase in histone acetylation is not sufficient to explain the HS1234-mediated activation of transcription from P1.

Acetylation↗
PubMed1997

A new member of the cationic amino acid transporter family is preferentially expressed in adult mouse brain.

We have isolated and characterized a novel member (CAT3) of the cationic amino acid transporter (CAT) family. In oocyte injection assays, CAT3 cRNA exhibited a saturable, sodium ion-independent transport activity with high affinity for L-arginine and L-lysine (Km = 40-60 and 115-165 microM, respectively). Transport of L-arginine was effectively competed only by cationic amino acids in L-form: arginine, lysine, ornithine, and 2,4-diamino-n-butyric acid but not by 2,3-diaminopropionic acid. The presence of L-arginine in the incubation medium stimulated the efflux rate of L-arginine, indicating that CAT3 is subject to trans-stimulation. All these results are consistent with the idea that CAT3, along with CAT1 and CAT2, constitutes the transport activity originally assigned to system y+. Like CAT2, but unlike CAT1, the expression of CAT3 is regulated in a highly tissue-specific manner; when various adult tissues were examined, significant levels of CAT3 transcript were detectable only in brain. In situ hybridization on brain sections revealed that CAT3 transcripts were localized predominantly along the midbrain-thalamus-hypothalamus axis, whereas neither CAT1 nor CAT2 transcripts demonstrated a similar localization. In contrast to its highly localized expression during the primitive streak stage and in the adult stage, CAT3 expression was detected more widely in 13.5 day post-coitum mouse embryos.

Amino Acid Sequence↗