Functional Characterization of the Enhancer Blocking Element of the Sea Urchin Early Histone Gene Cluster Reveals Insulator Properties and Three Essential cis-acting Sequences

Melfi, Raffaella; Palla, Franco; Simone, Paola Di; Alessandro, Claudia; Calı̀, Larissa; Anello, Letizia; Spinelli, Giovanni

doi:10.1006/jmbi.2000.4273

Cited by 25 publications

(38 citation statements)

References 51 publications

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“…We have previously described three cis-acting sequence elements, Box A, Box B, and Box C + T, that are each required for the enhancer-blocking function of sns in sea urchin embryos and are binding sites for specific protein complexes [22,23]. Recent evidence indicates that all three sequence elements are needed for the enhancer-blocking function of sns in transfected human H1299 cell line (unpublished observation).…”

Section: Resultsmentioning

confidence: 95%

“…3A and B). The Box B is a direct repeat of AT-rich sequence [22] and contains a putative binding site for the Oct-1 transcription factor. As expected, full inhibition of the most retarded complex was observed when the nuclear extracts were preincubated with molar excess of Box B (lanes 2 and 8 in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Protein concentration was determined by Bradford's method. EMSA were performed by incubating 1 ng of 32 P-labeled Box A, Box B oligonucleotides, and Box C + T fragment obtained by PCR as described [22], with 5 to 10 Ag of total nuclear extracts on ice for 20 min and then fractionated on 5% polyacrylamide gel in 0.5Â TBE. For competition experiments, nuclear extracts were preincubated with 20 or 40 ng of unlabeled homologous or heterologous oligonucleotides.…”

Section: Nuclear Extracts and Emsa Analysismentioning

confidence: 99%

“…Here, we show, by colony assays, that sns maintains its insulator activity in erythroid milieu, in that it directionally blocks the human erythroid-specific enhancer 5VHS2 of h-globin LCR in the interaction with the g-globin promoter in human (K562) and mouse (MEL) cell lines. In addition, by electrophoretic mobility shift assays (EMSA), we show that the three cisacting elements, essential for the enhancer-blocking activity [22], bind specific nuclear proteins, both erythroid and ubiquitous transcription factors.…”

Section: Introductionmentioning

confidence: 98%

“…When included in artificial constructs, sns displays directional enhancer-blocking activity in either orientation, both at early and late developmental stages [21,22]. In addition, if sns intervenes between two enhancers, only the enhancer located distally from the promoter with respect to the site of insertion is attenuated [22].…”

Section: Introductionmentioning

confidence: 99%

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Functional characterization of the sea urchin sns chromatin insulator in erythroid cells

Acuto

Marzo

Calzolari

et al. 2005

Blood Cells, Molecules, and Diseases

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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

Section: Nuclear Extracts and Emsa Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells

Acuto

Marzo

Calzolari

et al. 2005

Blood Cells, Molecules, and Diseases

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The modulator is a constitutive enhancer of a developmentally regulated sea urchin histone H2A gene

Spinelli

Birnstiel

2002

BioEssays

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Going back to the late 1970s and early 1980s, we trace the Xenopus oocyte microinjection experiments that led to the emergence of the concept of "modulator". The finding that the modulator could transactivate transcription from far upstream and in either orientation suggested that a new genetic element, different from the classical prokaryotic promoter sequences, had been discovered. This particular enhancer transactivates transcription of the sea urchin early (alpha) histone H2A gene which is regulated in early sea urchin development. We summarise the data from sea urchin microinjection experiments that confirm and extend the results obtained with Xenopus oocytes. We conclude that the H2A enhancer is bipartite, is located approx. 100 bp upstream of the TATAAATA box in the H2A gene of two sea urchin species and enhances transcription when placed at a position far upstream or far downstream of the gene unless an insulator intervenes between enhancer and promoter. Evidence from microinjection experiments with sea urchin embryos suggests that the developmental control of H2A expression resides not with the enhancer, which is constitutively active, but with a striking chromatin structure with two positioned nucleosomes near the 3' end of the gene. Within this structure, there is an insulator element.

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Conservative segregation of maternally inherited CS histone variants in larval stages of sea urchin development

Oliver¹,

Rodríguez²,

Bustos³

et al. 2002

J of Cellular Biochemistry

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Three sets of histone variants are coexisting in the embryo at larval stages of sea urchin's development: the maternally inherited cleavage stage variants (CS) expressed during the two initial cleavage divisions, the early histone variants, which are recruited into embryonic chromatin from middle cleavage stages until hatching and the late variants, that are fundamentally expressed from blastula stage onward. Since the expression of the CS histones is confined to the initial cleavage stages, these variants represent a very minor proportion of the histones present in the plutei larvae, whereas the late histone variants are predominant. To determine the position of these CS in the embryonic territories, we have immunolocalized the CS histone variants in plutei larvas harvested 72 h post-fertilization. In parallel, we have pulse labeled the DNA replicated during the initial cleavage cycle with bromodeoxyuridine (BrdU) and its position was further determined in the plutei larvas by immunofluorescence. We have found that the CS histone variants were segregated to specific territories in the plutei. The position in which the CS histone variants were found to be segregated was consistent with the position in which the DNA molecules that were replicated during the initial cleavage divisions were localized. These results strongly suggest that a specification of embryonic nuclei occurs at the initial cleavage divisions which is determined by a chromatin organized by CS histone variants.

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Functional Characterization of the Enhancer Blocking Element of the Sea Urchin Early Histone Gene Cluster Reveals Insulator Properties and Three Essential cis-acting Sequences

Cited by 25 publications

References 51 publications

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells

The modulator is a constitutive enhancer of a developmentally regulated sea urchin histone H2A gene

Conservative segregation of maternally inherited CS histone variants in larval stages of sea urchin development

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