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  • Cold Spring Harbor Laboratory  (10)
  • Biology  (10)
  • 1
    Online Resource
    Online Resource
    Mechanisms of developmental control of transcription in the murine α- and β-globin loci
    Cold Spring Harbor Laboratory ; 1999
    In:  Genes & Development Vol. 13, No. 1 ( 1999-01-01), p. 112-124
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 13, No. 1 ( 1999-01-01), p. 112-124
    Abstract: We have characterized mRNA expression and transcription of the mouse α - and β-globin loci during development. S1 nuclease and primary transcript in situ hybridization analyses demonstrate that all seven murine globin genes ( ζ , α1 , α2 , εy , βH1 , βmaj , and βmin ) are transcribed during primitive erythropoiesis, however transcription of the ζ , εy , and βH1 genes is restricted to the primitive erythroid lineage. Transcription of the βmaj and βmin genes in primitive cells is EKLF-dependent demonstrating EKLF activity in embryonic red cells. Novel kinetic analyses suggest that multigene expression in the β locus occurs via alternating single-gene transcription whereas coinitiation cannot be ruled out in the α locus. Transcriptional activation of the individual murine β genes in primitive cells correlates inversely with their distance from the locus control region, in contrast with the human β locus in which the adult genes are only activated in definitive erythroid cells. The results suggest that the multigene expression mechanism of alternating transcription is evolutionarily conserved between mouse and human β globin loci but that the timing of activation of the adult genes is altered, indicating important fundamental differences in globin gene switching.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.13.1.112
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 1999
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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  • 2
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    Online Resource
    The genome-wide dynamics of the binding of Ldb1 complexes during erythroid differentiation
    Cold Spring Harbor Laboratory ; 2010
    In:  Genes & Development Vol. 24, No. 3 ( 2010-02-01), p. 277-289
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 24, No. 3 ( 2010-02-01), p. 277-289
    Abstract: One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex—in particular, the binding of the negative regulators Eto2 and Mtgr1—are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.551810
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2010
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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  • 3
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    Online Resource
    In vivo live imaging of RNA polymerase II transcription factories in primary cells
    Cold Spring Harbor Laboratory ; 2013
    In:  Genes & Development Vol. 27, No. 7 ( 2013-04-01), p. 767-777
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 27, No. 7 ( 2013-04-01), p. 767-777
    Abstract: Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as “transcription factories,” but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.216200.113
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2013
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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  • 4
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    Multiple interactions between regulatory regions are required to stabilize an active chromatin hub
    Cold Spring Harbor Laboratory ; 2004
    In:  Genes & Development Vol. 18, No. 12 ( 2004-06-15), p. 1495-1509
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 18, No. 12 ( 2004-06-15), p. 1495-1509
    Abstract: The human β-globin locus control region (LCR) is required for the maintenance of an open chromatin configuration of the locus. It interacts with the genes and the hypersensitive regions flanking the locus to form an active chromatin hub (ACH) transcribing the genes. Proper developmental control of globin genes is largely determined by gene proximal regulatory sequences. Here, we provide the first functional evidence of the role of the most active sites of the LCR and the promoter of the β-globin gene in the maintenance of the ACH. When the human β-globin gene promoter is deleted in the context of a full LCR, the ACH is maintained with the β-globin gene remaining in proximity. Additional deletion of hypersensitive site HS3 or HS2 of the LCR shows that HS3, but not HS2, in combination with the β-globin promoter is crucial for the maintenance of the ACH at the definitive stage. We conclude that multiple interactions between the LCR and the β-globin gene are required to maintain the appropriate spatial configuration in vivo.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.289704
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2004
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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  • 5
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    Online Resource
    The microtubule plus-end-tracking protein CLIP-170 associates with the spermatid manchette and is essential for spermatogenesis
    Cold Spring Harbor Laboratory ; 2005
    In:  Genes & Development Vol. 19, No. 20 ( 2005-10-15), p. 2501-2515
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 19, No. 20 ( 2005-10-15), p. 2501-2515
    Abstract: CLIP-170 is a microtubule “plus-end-tracking protein” implicated in the control of microtubule dynamics, dynactin localization, and the linking of endosomes to microtubules. To investigate the function of mouse CLIP-170, we generated CLIP-170 knockout and GFP-CLIP-170 knock-in alleles. Residual CLIP-170 is detected in lungs and embryos of homozygous CLIP-170 knockout mice, but not in other tissues and cell types, indicating that we have generated a hypomorphic mutant. Homozygous CLIP-170 knockout mice are viable and appear normal. However, male knockout mice are subfertile and produce sperm with abnormal heads. Using the knock-in mice, we followed GFP-CLIP-170 expression and behavior in dissected, live testis tubules. We detect plus-end-tracking GFP-CLIP-170 in spermatogonia. As spermatogenesis proceeds, GFP-CLIP-170 expression increases and the fusion protein strongly marks syncytia of differentiated spermatogonia and early prophase spermatocytes. Subsequently GFP-CLIP-170 levels drop, but during spermiogenesis (post-meiotic development), GFP-CLIP-170 accumulates again and is present on spermatid manchettes and centrosomes. Bleaching studies show that, as spermatogenesis progresses, GFP-CLIP-170 converts from a mobile plus-end-tracking protein to a relatively immobile protein. We propose that CLIP-170 has a structural function in the male germline, in particular in spermatid differentiation and sperm head shaping.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.344505
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2005
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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  • 6
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    Online Resource
    CTCF mediates long-range chromatin looping and local histone modification in the β-globin locus
    Cold Spring Harbor Laboratory ; 2006
    In:  Genes & Development Vol. 20, No. 17 ( 2006-09-01), p. 2349-2354
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 20, No. 17 ( 2006-09-01), p. 2349-2354
    Abstract: CTCF (CCCTC-binding factor) binds sites around the mouse β -globin locus that spatially cluster in the erythroid cell nucleus. We show that both conditional deletion of CTCF and targeted disruption of a DNA-binding site destabilize these long-range interactions and cause local loss of histone acetylation and gain of histone methylation, apparently without affecting transcription at the locus. Our data demonstrate that CTCF is directly involved in chromatin architecture and regulates local balance between active and repressive chromatin marks. We postulate that throughout the genome, relative position and stability of CTCF-mediated loops determine their effect on enhancer–promoter interactions, with gene insulation as one possible outcome.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.399506
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2006
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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  • 7
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    Online Resource
    The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development
    Cold Spring Harbor Laboratory ; 2003
    In:  Genes & Development Vol. 17, No. 11 ( 2003-06-01), p. 1380-1391
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 17, No. 11 ( 2003-06-01), p. 1380-1391
    Abstract: The genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during development and tissue regeneration in adults following damage. In this report we demonstrate the involvement of a third transcription factor, the POU domain factor Brn-2. We show that Schwann cells express Brn-2 in a developmental profile similar to that of Oct-6 and that Brn-2 gene activation does not depend on Oct-6. Overexpression of Brn-2 in Oct-6-deficient Schwann cells, under control of the Oct-6 Schwann cell enhancer (SCE), results in partial rescue of the developmental delay phenotype, whereas compound disruption of both Brn-2 and Oct-6 results in a much more severe phenotype. Together these data strongly indicate that Brn-2 function largely overlaps with that of Oct-6 in driving the transition from promyelinating to myelinating Schwann cells.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.258203
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2003
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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  • 8
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    Context-dependent EKLF responsiveness defines the developmental specificity of the human ɛ-globin gene in erythroid cells of YAC transgenic mice
    Cold Spring Harbor Laboratory ; 2000
    In:  Genes & Development Vol. 14, No. 21 ( 2000-11-01), p. 2778-2794
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 14, No. 21 ( 2000-11-01), p. 2778-2794
    Abstract: We explored the mechanism of definitive-stage ɛ-globin transcriptional inactivity within a human β-globin YAC expressed in transgenic mice. We focused on the globin CAC and CAAT promoter motifs, as previous laboratory and clinical studies indicated a pivotal role for these elements in globin gene activation. A high-affinity CAC-binding site for the erythroid krüppel-like factor (EKLF) was placed in the ɛ-globin promoter at a position corresponding to that in the adult β-globin promoter, thereby simultaneously ablating a direct repeat (DR) element. This mutation led to EKLF-independent ɛ-globin transcription during definitive erythropoiesis. A second 4-bp substitution in the ɛ-globin CAAT sequence, which simultaneously disrupts a second DR element, further enhanced ectopic definitive erythroid activation of ɛ-globin transcription, which surprisingly became EKLF dependent. We finally examined factors in nuclear extracts prepared from embryonic or adult erythroid cells that bound these elements in vitro, and we identified a novel DR-binding protein (DRED) whose properties are consistent with those expected for a definitive-stage ɛ-globin repressor. We conclude that the suppression of ɛ-globin transcription during definitive erythropoiesis is mediated by the binding of a repressor that prevents EKLF from activating the ɛ-globin gene.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.822500
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2000
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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  • 9
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    Online Resource
    The active spatial organization of the β- globin locus requires the transcription factor EKLF
    Cold Spring Harbor Laboratory ; 2004
    In:  Genes & Development Vol. 18, No. 20 ( 2004-10-15), p. 2485-2490
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 18, No. 20 ( 2004-10-15), p. 2485-2490
    Abstract: Three-dimensional organization of a gene locus is important for its regulation, as recently demonstrated for the β- globin locus. When actively expressed, the cis -regulatory elements of the β- globin locus are in proximity in the nuclear space, forming a compartment termed the Active Chromatin Hub (ACH). However, it is unknown which proteins are involved in ACH formation. Here, we show that EKLF, an erythroid transcription factor required for adult β- globin gene transcription, is also required for ACH formation. We conclude that transcription factors can play an essential role in the three-dimensional organization of gene loci.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.317004
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2004
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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  • 10
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    Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the β-globin locus control region in vivo
    Cold Spring Harbor Laboratory ; 1998
    In:  Genes & Development Vol. 12, No. 18 ( 1998-09-15), p. 2863-2873
    Details
    In: Genes & Development, Cold Spring Harbor Laboratory, Vol. 12, No. 18 ( 1998-09-15), p. 2863-2873
    Abstract: The locus control region of the β-globin cluster contains five DNase I hypersensitive sites (5′HS1–5) required for locus activation. 5′HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5′HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5′HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5′HS3.
    Type of Medium: Online Resource
    ISSN: 0890-9369 , 1549-5477
    URL: Article
    DOI: 10.1101/gad.12.18.2863
    RVK:
    WA 15000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 1998
    detail.hit.zdb_id: 806684-X
    detail.hit.zdb_id: 1467414-2
    SSG: 12
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