33 results on '"Groudine M"'
Search Results
2. Rearrangement and Amplification of c-abl Sequences in the Human Chronic Myelogenous Leukemia Cell line K-562
- Author
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Collins, S. J. and Groudine, M. T.
- Published
- 1983
3. Activation of cellular genes by avian RNA tumor viruses.
- Author
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Groudine, M and Weintraub, H
- Abstract
We demonstrated previously that chicken embryo fibroblasts accumulate approximately 100 copies of embryonic globin RNA after transformation by Rous sarcoma virus. Here we demonstrate that the globin gene in chicken embryo fibroblasts is activated by infection with two other oncogenic retroviruses, avian erythroblastosis virus and strain MC-29 of avian myeloblastosis virus, which contain transforming genes unrelated in nucleotide sequence content to each other or to the Rous sarcoma virus src gene. In addition, we have measured the genetic complexity of transformation by using established techniques for determining the number of different RNA sequences in specific populations of cells. Our results indicate that transformation of chicken embryo fibroblasts by Rous sarcoma virus results in the accumulation of RNA from approximately 1000 average-sized new transcription units.
- Published
- 1980
- Full Text
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4. Asynchronous DNA replication within the human beta-globin gene locus.
- Author
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Epner, E, Forrester, W C, and Groudine, M
- Abstract
The timing of DNA replication of the human beta-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span greater than 120 kilobases across the human beta-globin gene locus, we show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-gamma-globin gene region and approximately 20 kilobases 5' to the epsilon-globin gene and 20 kilobases 3' to the beta-globin gene, replicate later and throughout S phase. A similar area is also present in the alpha-globin gene region in K562 cells. We suggest that these regions may represent sites of termination of replication forks.
- Published
- 1988
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5. Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast.
- Author
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Plon, S E, Leppig, K A, Do, H N, and Groudine, M
- Abstract
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.
- Published
- 1993
- Full Text
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6. Enhancers increase the probability but not the level of gene expression.
- Author
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Walters, M C, Fiering, S, Eidemiller, J, Magis, W, Groudine, M, and Martin, D I
- Abstract
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.
- Published
- 1995
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7. Transcription of three c-myc exons is enhanced in chicken bursal lymphoma cell lines.
- Author
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Linial, M and Groudine, M
- Abstract
The chicken c-myc gene, as defined by its homology to the v-myc gene of MC29 virus, is comprised of two exons. Using the techniques of runoff transcription, primer extension, and S1 nuclease protection, we demonstrate that there is a third c-myc exon of approximately equal to 345 base pairs (bp) located 0.7 kbp upstream of the 5' end of the v-myc homology. This first exon is transcribed and present in myc mRNA in normal chicken cells. We also examined RNA from five cell lines derived from avian leukosis virus-induced bursal lymphomas. In all these lines, the level of transcription of the 2.2- to 2.5-kbp myc mRNA is increased 30- to 60-fold over normal cells. The myc mRNA in four of these lines also contains increased levels of the first noncoding exon, and evidence is presented that the long terminal repeat (LTR) in the vicinity of c-myc is functioning as an enhancer of c-myc transcription rather than as a promoter in several of these cell lines. In two cell lines in which the viral LTR has integrated between the first and second exons in the proper orientation for downstream promotion of myc, the LTR does not exhibit promoter function. The pattern of c-myc transcription observed by others in a vast majority of avian leukosis virus-induced neoplasms is not observed in any of the five cell lines examined.
- Published
- 1985
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8. High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells.
- Author
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Novak, U, Harris, E A, Forrester, W, Groudine, M, and Gelinas, R
- Abstract
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.
- Published
- 1990
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9. Rous sarcoma virus activates embryonic globin genes in chicken fibroblasts.
- Author
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Groudine, M and Weintraub, H
- Abstract
Complementary DNA (cDNA) specific for chick globin mRNA sequences fails to hybridize to total RNA extracted from chicken fibroblasts. After infection by Rous sarcoma virus, RNA complementary to globin cDNA is detectable in 100-500 copies per cell. Infection of fibroblasts with the transformation defective (td) deletion mutant of Rous sarcoma virus leads to normal virus production, but not to host cell transformation or accumulation of RNA sequences complementary to globin cDNA. Our evidence shows that the globin genes activated by Rous sarcoma virus are those specified by embryonic chick red cells; adult-specific globin sequences were not detected.
- Published
- 1975
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10. 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.
- Author
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Fiering, S, Kim, C G, Epner, E M, and Groudine, M
- Abstract
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.
- Published
- 1993
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11. A developmentally stable chromatin structure in the human beta-globin gene cluster.
- Author
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Forrester, W C, Thompson, C, Elder, J T, and Groudine, M
- Abstract
The DNase I-hypersensitive sites in the human embryonic beta-globin gene region have been mapped in erythroid-enriched fractions of disaggregated fetal livers, in adult nucleated red blood cells, and in fetal brain tissue. Our analysis of a region extending 11 kilobases (kb) 5' of the epsilon-globin gene reveals many minor nuclease-hypersensitive sites and one major site located 6.1 kb upstream of the epsilon-globin gene. All of these hypersensitive sites are erythroid-specific, and the major site is stable throughout erythroid development. As assayed by nuclear runoff transcription, little or no epsilon-globin gene expression is detectable in fetal or adult erythroid cells. Thus, the presence of the major hypersensitive site 5' of the epsilon-globin gene in both fetal and adult erythroid cells demonstrates that this site is not specifically correlated with transcription of the gene or with a particular stage of development. Rather, this site may reflect an early event in erythroid differentiation. In addition, DNase I has been used to probe the overall sensitivity of epsilon-globin chromatin in fetal erythroid cells. Our findings indicate that the epsilon-globin gene as well as the other genes in the beta-globin cluster reside within the chromatin domain that is more DNase I-sensitive than "bulk" chromatin.
- Published
- 1986
- Full Text
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12. The modulation of Hb F synthesis in adult erythroid progenitor (burst-forming unit) cultures reflects changes in gamma-globin gene transcription and chromatin structure.
- Author
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Groudine, M, Peretz, M, Nakamoto, B, Papayannopoulou, T, and Stamatoyannopoulos, G
- Abstract
Fetal hemoglobin production can be reactivated in vivo in adult persons with various hemoglobinopathies and other hemopoietic disorders, and in cultures of adult erythroid progenitors. We show that the activation of Hb F in adult cells is transcriptional in nature and is accompanied by the appearance of DNase I-hypersensitive sites and undermethylation of Hpa II sites 5' to the gamma-globin genes. Production of Hb F in culture is strongly modulated by the environment, and the repression of Hb F synthesis by specific culture conditions has been reported. By nuclear runoff, chromatin, and methylation analyses, we show that this inhibition of Hb F production in vitro is at the level of transcription with the concomitant loss of characteristic gamma hypersensitive sites and methylation of gamma Hpa II sites. These data indicate, first, that the organization of globin chromatin of adult cells that produce fetal hemoglobin resembles that of fetal erythroid cells and, second, that this organization switches from a fetal to an adult pattern in response to changes in the environment of the erythroid cells.
- Published
- 1986
- Full Text
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13. Molecular analysis of the human beta-globin locus activation region.
- Author
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Forrester, W C, Novak, U, Gelinas, R, and Groudine, M
- Abstract
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.
- Published
- 1989
- Full Text
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14. Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts.
- Author
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Adams, M, Sharmeen, L, Kimpton, J, Romeo, J M, Garcia, J V, Peterlin, B M, Groudine, M, and Emerman, M
- Abstract
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.
- Published
- 1994
- Full Text
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15. Human fetal to adult hemoglobin switching: changes in chromatin structure of the beta-globin gene locus.
- Author
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Groudine, M, Kohwi-Shigematsu, T, Gelinas, R, Stamatoyannopoulos, G, and Papayannopoulou, T
- Abstract
We have investigated the chromatin structure of the chromosomal DNA regions containing the human G gamma-, A gamma-, delta-, and beta-globin structural genes in both fetal and adult erythropoietic tissues and in two human erythroleukemia cells lines before and after induction. Our results indicate that DNase I introduces specific cuts into the beta-globin gene cluster in erythroid cells but not in leukocytes. The predominant sites are located at the 5' sides of the G gamma-, A gamma-, delta-, and beta-globin genes, within 200 base pairs of the respective cap sites. Examination of fetal liver cells has revealed the presence of hypersensitive sites at the 5' side of all four genes, whereas analysis of adult bone marrow has revealed the characteristic sites near the delta- and beta-globin genes but no hypersensitive sites at the 5' termini of the G gamma- or A gamma-globin genes. The presence of delta and beta hypersensitive sites in fetal cells suggests that the increment in expression of the delta and beta genes during development most likely involves the modulation of another pathway to gene expression. Using isolated nuclei from HEL and K562 cells, we have found that the G gamma, A gamma, delta, and beta genes are preferentially sensitive [relative to the pro-alpha2(I) collagen gene] to mild digestion with DNase I, whereas these genes are as resistant as collagen genes in cells that do not express globin. These findings are discussed within the context of chromatin structural correlates of hemoglobin switching.
- Published
- 1983
- Full Text
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16. Tat-dependent adenosine-to-inosine modification of wild-type transactivation response RNA.
- Author
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Sharmeen, L, Bass, B, Sonenberg, N, Weintraub, H, and Groudine, M
- Abstract
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.
- Published
- 1991
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17. Helix-loop-helix transcription factors E12 and E47 are not essential for skeletal or cardiac myogenesis, erythropoiesis, chondrogenesis, or neurogenesis.
- Author
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Zhuang, Y, Kim, C G, Bartelmez, S, Cheng, P, Groudine, M, and Weintraub, H
- Abstract
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.
- Published
- 1992
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18. Olfactory receptor genes expressed in distinct lineages are sequestered in different nuclear compartments.
- Author
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Yoon KH, Ragoczy T, Lu Z, Kondoh K, Kuang D, Groudine M, and Buck LB
- Subjects
- Alleles, Animals, Cell Lineage, Chromosomes, Artificial, Bacterial, Crosses, Genetic, Female, Gene Expression Regulation, Heterochromatin metabolism, In Situ Hybridization, In Situ Hybridization, Fluorescence, Lamin Type A metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Odorants, Olfactory Mucosa metabolism, Olfactory Receptor Neurons physiology, Sensory Receptor Cells metabolism, Smell physiology, Cell Nucleus metabolism, Receptors, Odorant genetics
- Abstract
The olfactory system translates a vast array of volatile chemicals into diverse odor perceptions and innate behaviors. Odor detection in the mouse nose is mediated by 1,000 different odorant receptors (ORs) and 14 trace amine-associated receptors (TAARs). ORs are used in a combinatorial manner to encode the unique identities of myriad odorants. However, some TAARs appear to be linked to innate responses, raising questions about regulatory mechanisms that might segregate OR and TAAR expression in appropriate subsets of olfactory sensory neurons (OSNs). Here, we report that OSNs that express TAARs comprise at least two subsets that are biased to express TAARs rather than ORs. The two subsets are further biased in Taar gene choice and their distribution within the sensory epithelium, with each subset preferentially expressing a subgroup of Taar genes within a particular spatial domain in the epithelium. Our studies reveal one mechanism that may regulate the segregation of Olfr (OR) and Taar expression in different OSNs: the sequestration of Olfr and Taar genes in different nuclear compartments. Although most Olfr genes colocalize near large central heterochromatin aggregates in the OSN nucleus, Taar genes are located primarily at the nuclear periphery, coincident with a thin rim of heterochromatin. Taar-expressing OSNs show a shift of one Taar allele away from the nuclear periphery. Furthermore, examination of hemizygous mice with a single Taar allele suggests that the activation of a Taar gene is accompanied by an escape from the peripheral repressive heterochromatin environment to a more permissive interior chromatin environment.
- Published
- 2015
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19. Molecular basis for chromatin binding and regulation of MLL5.
- Author
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Ali M, Rincón-Arano H, Zhao W, Rothbart SB, Tong Q, Parkhurst SM, Strahl BD, Deng LW, Groudine M, and Kutateladze TG
- Subjects
- Amino Acid Sequence, DNA-Binding Proteins chemistry, Humans, Models, Molecular, Molecular Sequence Data, Phosphorylation, Protein Binding, Protein Conformation, Sequence Homology, Amino Acid, Chromatin metabolism, DNA-Binding Proteins metabolism
- Abstract
The human mixed-lineage leukemia 5 (MLL5) protein mediates hematopoietic cell homeostasis, cell cycle, and survival; however, the molecular basis underlying MLL5 activities remains unknown. Here, we show that MLL5 is recruited to gene-rich euchromatic regions via the interaction of its plant homeodomain finger with the histone mark H3K4me3. The 1.48-Å resolution crystal structure of MLL5 plant homeodomain in complex with the H3K4me3 peptide reveals a noncanonical binding mechanism, whereby K4me3 is recognized through a single aromatic residue and an aspartate. The binding induces a unique His-Asp swapping rearrangement mediated by a C-terminal α-helix. Phosphorylation of H3T3 and H3T6 abrogates the association with H3K4me3 in vitro and in vivo, releasing MLL5 from chromatin in mitosis. This regulatory switch is conserved in the Drosophila ortholog of MLL5, UpSET, and suggests the developmental control for targeting of H3K4me3. Together, our findings provide first insights into the molecular basis for the recruitment, exclusion, and regulation of MLL5 at chromatin.
- Published
- 2013
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20. Dynamics and control of state-dependent networks for probing genomic organization.
- Author
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Rajapakse I, Groudine M, and Mesbahi M
- Subjects
- Animals, Cell Differentiation physiology, Feedback, Physiological, GATA1 Transcription Factor genetics, GATA1 Transcription Factor metabolism, Hematopoiesis genetics, Hematopoiesis physiology, Humans, Models, Biological, MyoD Protein genetics, MyoD Protein metabolism, Cell Differentiation genetics, Gene Regulatory Networks, Models, Genetic
- Abstract
A state-dependent dynamic network is a collection of elements that interact through a network, whose geometry evolves as the state of the elements changes over time. The genome is an intriguing example of a state-dependent network, where chromosomal geometry directly relates to genomic activity, which in turn strongly correlates with geometry. Here we examine various aspects of a genomic state-dependent dynamic network. In particular, we elaborate on one of the important ramifications of viewing genomic networks as being state-dependent, namely, their controllability during processes of genomic reorganization such as in cell differentiation.
- Published
- 2011
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21. The emergence of lineage-specific chromosomal topologies from coordinate gene regulation.
- Author
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Rajapakse I, Perlman MD, Scalzo D, Kooperberg C, Groudine M, and Kosak ST
- Subjects
- Cell Differentiation genetics, Cell Lineage genetics, Chromosomes genetics, Gene Expression Regulation
- Abstract
Although the importance of chromosome organization during mitosis is clear, it remains to be determined whether the nucleus assumes other functionally relevant chromosomal topologies. We have previously shown that homologous chromosomes have a tendency to associate during hematopoiesis according to their distribution of coregulated genes, suggesting cell-specific nuclear organization. Here, using the mathematical approaches of distance matrices and coupled oscillators, we model the dynamic relationship between gene expression and chromosomal associations during the differentiation of a multipotential hematopoietic progenitor. Our analysis reveals dramatic changes in total genomic order: Commitment of the progenitor results in an initial increase in entropy at both the level of gene coregulation and chromosomal organization, which we suggest represents a phase transition, followed by a progressive decline in entropy during differentiation. The stabilization of a highly ordered state in the differentiated cell types results in lineage-specific chromosomal topologies and is related to the emergence of coherence-or self-organization-between chromosomal associations and coordinate gene regulation. We discuss how these observations may be generally relevant to cell fate decisions encountered by progenitor/stem cells.
- Published
- 2009
- Full Text
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22. Bivalent domains enforce transcriptional memory of DNA methylated genes in cancer cells.
- Author
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Rodriguez J, Muñoz M, Vives L, Frangou CG, Groudine M, and Peinado MA
- Subjects
- Base Sequence, Cell Transformation, Neoplastic genetics, Chromatin metabolism, Colonic Neoplasms pathology, Colonic Neoplasms therapy, Genetic Therapy, Humans, Promoter Regions, Genetic, Repressor Proteins metabolism, Transcription, Genetic, Chromosomes, Human, Pair 5 genetics, Colonic Neoplasms genetics, DNA Methylation drug effects, Gene Expression Regulation, Neoplastic, Gene Silencing, Genes, Neoplasm
- Abstract
Silencing of multiple cancer-related genes is associated with de novo methylation of linked CpG islands. Additionally, bivalent histone modification profiles characterized by the juxtaposition of active and inactive histone marks have been observed in genes that become hypermethylated in cancer. It is unknown how these ambiguous epigenetic states are maintained and how they interrelate with adjacent genomic regions with different epigenetic landscapes. Here, we present the analysis of a set of neighboring genes, including many frequently silenced in colon cancer cells, in a chromosomal region at 5q35.2 spanning 1.25 Mb. Promoter DNA methylation occurs only at genes maintained at a low transcriptional state and is characterized by the presence of bivalent histone marks, namely trimethylation of lysines 4 and 27 in histone 3. Chemically induced hyperacetylation and DNA demethylation lead to up-regulation of silenced genes in this locus yet do not resolve bivalent domains into a domain-wide active chromatin conformation. In contrast, active genes in the region become down-regulated after drug treatment, accompanied by a partial loss of chromatin domain boundaries and spreading of the inactive histone mark trimethylated lysine 27 in histone 3. Our results demonstrate that bivalent domains mark the promoters of genes that will become DNA methylated in adult tumor cells to enforce transcriptional silence. These bivalent domains not only remain upon drug induced gene reactivation, but also spread over adjacent CpG islands. These results may have important implications in understanding and managing epigenetic therapies of cancer.
- Published
- 2008
- Full Text
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23. DNA replication-timing analysis of human chromosome 22 at high resolution and different developmental states.
- Author
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White EJ, Emanuelsson O, Scalzo D, Royce T, Kosak S, Oakeley EJ, Weissman S, Gerstein M, Groudine M, Snyder M, and Schübeler D
- Subjects
- Cell Line, DNA biosynthesis, DNA genetics, Gene Expression Profiling, Humans, Karyotyping, Lung cytology, Lung metabolism, Organ Specificity, RNA, Messenger biosynthesis, RNA, Messenger genetics, Time Factors, Transcription, Genetic genetics, Cell Differentiation, Chromosomes, Human, Pair 22 genetics, Chromosomes, Human, Pair 22 metabolism, DNA Replication Timing
- Abstract
Duplication of the genome during the S phase of the cell cycle does not occur simultaneously; rather, different sequences are replicated at different times. The replication timing of specific sequences can change during development; however, the determinants of this dynamic process are poorly understood. To gain insights into the contribution of developmental state, genomic sequence, and transcriptional activity to replication timing, we investigated the timing of DNA replication at high resolution along an entire human chromosome (chromosome 22) in two different cell types. The pattern of replication timing was correlated with respect to annotated genes, gene expression, novel transcribed regions of unknown function, sequence composition, and cytological features. We observed that chromosome 22 contains regions of early- and late-replicating domains of 100 kb to 2 Mb, many (but not all) of which are associated with previously described chromosomal bands. In both cell types, expressed sequences are replicated earlier than nontranscribed regions. However, several highly transcribed regions replicate late. Overall, the DNA replication-timing profiles of the two different cell types are remarkably similar, with only nine regions of difference observed. In one case, this difference reflects the differential expression of an annotated gene that resides in this region. Novel transcribed regions with low coding potential exhibit a strong propensity for early DNA replication. Although the cellular function of such transcripts is poorly understood, our results suggest that their activity is linked to the replication-timing program.
- Published
- 2004
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24. Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network.
- Author
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Sun J, Brand M, Zenke Y, Tashiro S, Groudine M, and Igarashi K
- Subjects
- Acetylation, Animals, Base Sequence, Basic-Leucine Zipper Transcription Factors, Binding, Competitive, DNA Primers, Histones metabolism, Methylation, Mice, NIH 3T3 Cells, Heme physiology, Trans-Activators metabolism, Transcription Factors metabolism
- Abstract
Small Maf proteins serve as dual-function transcription factors through an exchange of their heterodimerization partners. For example, as heterodimers with hematopoietic cell-specific p45 NF-E2 or NF-E2-related factors (Nrf), they activate the beta-globin or antioxidative stress enzyme heme oxygenase 1 (HO-1) genes, respectively. In contrast, together with Bach1, they repress these same genes. However, the signals leading to this partner exchange are not known. Using chromatin immunoprecipitation assays in NIH 3T3 cells, we show that heme, an inducer of ho-1, promotes displacement of Bach1 from the MafK-occupied ho-1 enhancers, which is followed by Nrf2 binding to these elements. Whereas histone H3 at the ho-1 enhancers and promoter is hyperacetylated irrespective of gene activity, exposure of cells to heme results in de novo hyperacetylation and hypermethylation of histone H3 in the transcribed region. These data indicate that, under normal conditions, the chromatin structure of ho-1 is in a preactivation state, but transcription is repressed by Bach1. Heme induces switching of Maf dimers, resulting in ho-1 expression. Heme also promotes displacement of Bach1 from the beta-globin locus control region without affecting MafK binding in murine erythroleukemia cells. Thus, heme functions as a signaling molecule for gene expression in higher eukaryotes.
- Published
- 2004
- Full Text
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25. Chromatin insulation by a transcriptional activator.
- Author
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Sutter NB, Scalzo D, Fiering S, Groudine M, and Martin DI
- Subjects
- Cell Line, Cell Separation, DNA-Binding Proteins, Dose-Response Relationship, Drug, Flow Cytometry, Genes, Reporter, Green Fluorescent Proteins, Humans, K562 Cells, Luminescent Proteins metabolism, Plasmids metabolism, Protein Structure, Tertiary, Time Factors, Transcription Factors metabolism, Transgenes, Transcription Factor MTF-1, Chromatin metabolism, Transcriptional Activation
- Abstract
In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression.
- Published
- 2003
- Full Text
- View/download PDF
26. Nuclear relocation of a transactivator subunit precedes target gene activation.
- Author
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Francastel C, Magis W, and Groudine M
- Subjects
- Active Transport, Cell Nucleus physiology, Animals, Cell Differentiation, Cell Line, Centromere, Dimerization, Erythroid-Specific DNA-Binding Factors, Globins metabolism, Heterochromatin metabolism, MafK Transcription Factor, Mice, NF-E2 Transcription Factor, NF-E2 Transcription Factor, p45 Subunit, Protein Isoforms metabolism, Tumor Cells, Cultured, Cell Nucleus metabolism, DNA-Binding Proteins metabolism, Trans-Activators metabolism, Transcription Factors metabolism, Transcriptional Activation
- Abstract
Murine erythroleukemia (MEL) cells are a model system to study reorganization of the eukaryotic nucleus during terminal differentiation. Upon chemical induction, MEL cells undergo erythroid differentiation, leading to activation of globin gene expression. Both processes strongly depend on the transcriptional activator NF-E2. Before induction of differentiation, both subunits of the NF-E2 heterodimer are present, but little DNA-binding activity is detectable. Using immunofluorescence microscopy, we show that the two NF-E2 subunits occupy distinct nuclear compartments in uninduced MEL cells; the smaller subunit NF-E2p18 is found primarily in the centromeric heterochromatin compartment, whereas the larger subunit NF-E2p45 occupies the euchromatin compartment. Concomitant with the commitment period of differentiation that precedes globin gene activation, NF-E2p18, along with other transcriptional repressors, relocates to the euchromatin compartment. Thus, relocation of NF-E2 p18 may be a rate-limiting step in formation of an active NF-E2 complex. To understand the mechanisms of NF-E2 localization, we show that centromeric targeting of NF-E2p18 requires dimerization, but not with an erythroid-specific partner, and that the transactivation domain of NF-E2p45 may be necessary and sufficient to prevent its localization in centromeric heterochromatin. Finally, using fluorescence in situ hybridization, we show that, upon differentiation, the beta-globin gene loci relocate away from heterochromatin compartments to euchromatin. This relocation correlates with both transcriptional activation of the globin locus and relocation of NF-E2p18 away from heterochromatin, suggesting that these processes are linked.
- Published
- 2001
- Full Text
- View/download PDF
27. The murine beta-globin locus control region regulates the rate of transcription but not the hyperacetylation of histones at the active genes.
- Author
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Schübeler D, Groudine M, and Bender MA
- Subjects
- Acetylation, Alleles, Animals, Mice, Polymerase Chain Reaction, Probability, Gene Expression Regulation, Globins genetics, Histones genetics, Locus Control Region, Transcription, Genetic
- Abstract
Locus control regions (LCRs) are defined by their ability to confer high-level tissue-specific expression to linked genes in transgenic assays. Previously, we reported that, at its native site, the murine beta-globin LCR is required for high-level beta-globin gene expression, but is not required to initiate an open chromatin conformation of the locus. To further investigate the mechanism of LCR-mediated transcriptional enhancement, we have analyzed allele-specific beta-globin expression and the pattern of histone acetylation in the presence and absence of the LCR. In single cells from mice heterozygous for a deletion of the LCR, beta-globin expression from the LCR-deleted allele is consistently low ( approximately 1-4% of wild type). Thus, the endogenous LCR enhances globin gene expression by increasing the rate of transcription from each linked allele rather than by increasing the probability of establishing transcription per se. Furthermore, in erythroid cells from mice homozygous for the highly expressing wild-type beta-globin locus, hyperacetylation of histones H3 and H4 is localized to the LCR and active genes. In mice homozygous for the LCR deletion reduced histone hyperacetylation is observed in LCR proximal sequences; however, deletion of the LCR has no effect on the localized hyperacetylation of the genes. Together, our results suggest that, in its native genomic context, the LCR follows the rate model of enhancer function, and that the developmentally specific hyperacetylation of the globin genes is independent of both the rate of transcription and the presence of the LCR.
- Published
- 2001
- Full Text
- View/download PDF
28. Activation of beta-major globin gene transcription is associated with recruitment of NF-E2 to the beta-globin LCR and gene promoter.
- Author
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Sawado T, Igarashi K, and Groudine M
- Subjects
- Acetylation, Animals, Bacterial Proteins genetics, Cell Line, Erythroid-Specific DNA-Binding Factors, Gene Expression Regulation, Histones metabolism, Leukemia, Erythroblastic, Acute genetics, Leukemia, Erythroblastic, Acute metabolism, Locus Control Region, MafK Transcription Factor, Mice, Models, Biological, Multigene Family, NF-E2 Transcription Factor, NF-E2 Transcription Factor, p45 Subunit, Promoter Regions, Genetic, Transcription, Genetic, Tumor Cells, Cultured, DNA-Binding Proteins metabolism, Globins genetics, Transcription Factors metabolism
- Abstract
The mouse beta-globin gene locus control region (LCR), located upstream of the beta-globin gene cluster, is essential for the activated transcription of genes in the cluster. The LCR contains multiple binding sites for transactivators, including Maf-recognition elements (MAREs). However, little is known about the specific proteins that bind to these sites or the time at which they bind during erythroid differentiation. We have performed chromatin immunoprecipitation experiments to determine the recruitment of the erythroid-specific transactivator p45 NF-E2/MafK (p18 NF-E2) heterodimer and small Maf proteins to various regions in the globin gene locus before and after the induction of murine erythroleukemia (MEL) cell differentiation. We report that, before induction, the LCR is occupied by small Maf proteins, and, on erythroid maturation, the NF-E2 complex is recruited to the LCR and the active globin promoters, even though the promoters do not contain MAREs. This differentiation-coupled recruitment of NF-E2 complex correlates with a greater than 100-fold increase in beta-major globin transcription, but is not associated with a significant change in locus-wide histone H3 acetylation. These findings suggest that the beta-globin gene locus exists in a constitutively open chromatin conformation before terminal differentiation, and we speculate that recruitment of NF-E2 complex to the LCR and active promoters may be a rate-limiting step in the activation of beta-globin gene expression.
- Published
- 2001
- Full Text
- View/download PDF
29. C(m)C(a/t)GG methylation: a new epigenetic mark in mammalian DNA?
- Author
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Lorincz MC and Groudine M
- Subjects
- Animals, Base Sequence, DNA chemistry, DNA genetics, DNA metabolism, Mammals metabolism, Methyltransferases genetics, Methyltransferases metabolism, Models, Genetic, DNA Methylation, Mammals genetics
- Published
- 2001
- Full Text
- View/download PDF
30. Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse beta-globin gene clusters.
- Author
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Bulger M, Bender MA, van Doorninck JH, Wertman B, Farrell CM, Felsenfeld G, Groudine M, and Hardison R
- Subjects
- Animals, Base Sequence, DNA, Complementary, Exons, Humans, Locus Control Region, Mice, Molecular Sequence Data, Globins genetics, Multigene Family, Receptors, Odorant genetics
- Abstract
By sequencing regions flanking the beta-globin gene complex in mouse (Hbbc) and human (HBBC), we have shown that the beta-globin gene cluster is surrounded by a larger cluster of olfactory receptor genes (ORGs). To facilitate sequence comparisons and to investigate the regulation of ORG expression, we have mapped 5' sequences of mRNA from olfactory epithelium encoding beta-globin-proximal ORGs. We have found that several of these genes contain multiple noncoding exons that can be alternatively spliced. Surprisingly, the only common motifs found in the promoters of these genes are a "TATA" box and a purine-rich motif. Sequence comparisons between human and mouse reveal that most of the conserved regions are confined to the coding regions and transcription units of the genes themselves, but a few blocks of conserved sequence also are found outside of ORG transcription units. The possible influence of beta-globin regulatory sequences on ORG expression in olfactory epithelium was tested in mice containing a deletion of the endogenous beta-globin locus control region, but no change in expression of the neighboring ORGs was detected. We evaluate the implications of these results for possible mechanisms of regulation of ORG transcription.
- Published
- 2000
- Full Text
- View/download PDF
31. 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.
- Author
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Bulger M, van Doorninck JH, Saitoh N, Telling A, Farrell C, Bender MA, Felsenfeld G, Axel R, and Groudine M
- Subjects
- Animals, Gene Expression Regulation, Humans, Mice, Molecular Sequence Data, Phylogeny, Sequence Analysis, DNA, Sequence Homology, Nucleic Acid, Conserved Sequence, Globins genetics, Receptors, Odorant genetics
- Abstract
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.
- Published
- 1999
- Full Text
- View/download PDF
32. Long-distance transcriptional enhancement by the histone acetyltransferase PCAF.
- Author
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Krumm A, Madisen L, Yang XJ, Goodman R, Nakatani Y, and Groudine M
- Subjects
- Animals, Histone Acetyltransferases, Mice, Transcriptional Activation, Tumor Cells, Cultured, Acetyltransferases genetics, Promoter Regions, Genetic genetics, Saccharomyces cerevisiae Proteins, Transcription, Genetic
- Abstract
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.
- Published
- 1998
- Full Text
- View/download PDF
33. An upstream activator of transcription coordinately increases the level and epigenetic stability of gene expression.
- Author
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Magis W, Fiering S, Groudine M, and Martin DI
- Subjects
- Animals, Cell Line, Gene Expression Regulation drug effects, Humans, Leukemia, Erythroblastic, Acute, Mice, Recombinant Proteins biosynthesis, Transfection, Tumor Cells, Cultured, Zinc pharmacology, beta-Galactosidase biosynthesis, Metallothionein biosynthesis, Metallothionein genetics, Promoter Regions, Genetic, Regulatory Sequences, Nucleic Acid, Transcription, Genetic
- Abstract
The mouse metallothionein-I (mMT-I) promoter is activated by the metal response element-binding transcription factor (MTF), which binds metal response elements (MREs) when stimulated with heavy metals. We analyzed eight K562 erythroleukemia cell clones, each carrying a single integrated copy of an mMT-I/beta-geo construct, using a system that can independently assess the level of beta-geo expression and the rate at which it is silenced. In these clones, basal expression and rate of silencing vary widely and independently with integration site. This implies that the rates of transcription and of silencing are separate properties determined by interaction of the regulatory elements of the transgene with the site of integration. Induction of the mMT-I promoter with zinc both increases expression level and strongly retards silencing of beta-geo expression. At a given integration site, expression level and silencing are affected coordinately by induction. Taken together with earlier studies of distant metal-responsive elements, these results suggest that distance from the promoter may determine whether a factor can increase transcription rate. Stimulation of an MRE can both increase transcription and overcome repressive effects of chromatin; we suggest that these functions are linked.
- Published
- 1996
- Full Text
- View/download PDF
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