Functional characterization of the sea urchin sns chromatin insulator in erythroid cells
Introduction
Insulators are genetic elements present in a wide range of eukaryotic organisms capable of attenuating the activity of enhancers or other regulative elements in a directional manner. They maintain transcription fidelity by interfering with the communication between enhancer and promoter only when interposed between the two [1], [2]. Insulators, by restricting enhancer and silencer function, may impart functional independence to transcription units in the eukaryotic genome [3], [4], [5], [6], [7], [8], [9], [10]. It has been suggested that some insulators, such as gypsy and c-HS4, with their bound proteins, may be tethered at the nuclear periphery forming loop domains [11], [12]. In addition, insulators may act as barriers to the spreading of heterochromatin to euchromatic region [13], [14], [15], [16], [17]. Protection from chromosomal silencing requires the constitutive recruitment by the insulator binding proteins of histone modifying enzymes, such as H3 lysine 4 methyltransferase and histone acetylases, to counteract the propagation of condensed chromatin structures [18], [19], [20]. Because of the barrier function, an insulator placed in flanking location can buffer a transgene from extinction of expression and also block the passage of regulatory signals between the transgene and the chromatin domains at the genomic insertion site.
We have previously described the characterization of the sea urchin chromosomal insulator, termed sns, localized at the 3′ end of the α-H2A histone gene. 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]. Recent evidence indicates that sns is part of a DNA fragment, defined as sns 5, needed for silencing the α-H2A histone gene at gastrula stage [23]. This result suggests that the sea urchin insulator, in the natural environment of the histone gene sequences, is involved in the mechanism of repression. Remarkably, sns shielded a promoter from the CMV enhancer in two different human cell lines using plasmid and retroviral vectors, suggesting that sns maintains the directional enhancer-blocking function in a human chromatin context [24].
Because of the potential use of sns in gene transfer experiments in human cells, we carried out functional tests and DNA binding studies in erythroid cell lines. 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 5′HS2 of β-globin LCR in the interaction with the γ-globin promoter in human (K562) and mouse (MEL) cell lines. In addition, by electrophoretic mobility shift assays (EMSA), we show that the three cis-acting elements, essential for the enhancer-blocking activity [22], bind specific nuclear proteins, both erythroid and ubiquitous transcription factors.
Section snippets
Plasmid constructions
The DNA construct pAγNEO that was used for the assembly of the plasmids for the enhancer-blocking assays and displayed in Fig. 1 was previously described [25]. To generate plasmid A, we cloned, upstream to the 1.3 Kb Aγ promoter, the 435 bp EcoRI–EcoRI fragment, containing the sequence essential for the enhancer activity of 5′ hypersensitive site-2 (5′HS2) of the human β-globin LCR. The enhancer-less plasmids (E and F of Fig. 1) contain two tandem copies of the sea urchin insulator sns that
The sea urchin insulator sns attenuates the globin enhancer in erythroid cell lines in a directional manner
By colony assay [26], we tested whether the sea urchin sns insulator displays enhancer blocker activity in erythroid environment. The DNA constructs depicted in Fig. 1 were derived from the previously described pγNEO plasmid [25] which contains the neomycin (G418)-resistant gene transcribed by the 1.3 Kb human Aγ-globin promoter. These constructs were transfected by electroporation in human (K562) and mouse (MEL) erythroleukemia cell lines, and G418-resistant clones were counted after 14 days
Discussion
The results presented in this paper demonstrate that the sea urchin chromosomal insulator sns interferes with the interaction of the human β-globin 5′HS2 with the γ-globin promoter in stable transfected erythroid cells. Attenuation of enhancer function occurs only when sns is interposed between the two regulatory elements, indicating that the sea urchin sequence behaves as a positional enhancer blocker also with tissue-specific transcription sequence elements in an erythroid milieu.
It is of
Acknowledgments
This work was supported by grants from the University of Palermo (ex 60%) MIUR (Programmi di Ricerca Scientifica di Interesse Nazionale), Assessorato Sanità Regione Sicilia (Project n. T11/3). Associazione per la Ricerca “P. Cutino” ONLUS, and Ditta Zappalà SpA, Zafferana Etnea. Many thanks are due to D. D'Apolito and V. Motta for helpful discussion.
References (33)
- et al.
Stopped at the border: boundaries and insulators
Curr. Opin. Genet. Dev.
(1999) - et al.
Boundary and insulator elements in chromosomes
Curr. Opin. Genet. Dev.
(1996) - et al.
Setting the boundaries of chromatin domains and nuclear organization
Cell
(2002) - et al.
CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species
Mol. Cell
(2004) - et al.
A chromatin insulator determines the nuclear localization of DNA
Mol. Cell
(2000) - et al.
A Drosophila protein that imparts directionality on a chromatin insulator is an enhancer of position-effect variegation
Cell
(1995) - et al.
Heterochromatin: silence is golden
Curr. Biol.
(2003) - et al.
Recruitment of histone modifications by USF proteins at a vertebrate barrier element
Mol. Cell
(2004) - et al.
Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences
J. Mol. Biol.
(2000) - et al.
Down-regulation of early sea urchin histone H2A gene relies on cis regulative sequences located in the 5′ and 3′ regions and including the enhancer blocker sns
J. Mol. Biol.
(2004)
The sea urchin sns insulator blocks CMV enhancer following integration in human cells
Biochem. Biophys. Res. Commun.
The role of human globin gene promoters in the expression of hybrid genes in erythroid and non-erythroid cells
Biochem. Biophys. Res. Commun.
A 5′ element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila
Cell
A region upstream of the human delta-globin gene shows a stage-specific interaction with globin promoters in erythroid cell lines
Blood Cells, Mol. Dis.
The protein CTCF is required for the enhancer blocking activity of vertebrate insulators
Cell
A group of scs elements function as domain boundaries in an enhancer-blocking assay
Mol. Cell. Biol.
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These authors contributed equally.