Functional characterization of the sea urchin sns chromatin insulator in erythroid cells.
Chromatin insulators are regulatory elements that determine domains of genetic functions. We have previously described the characterization of a 265 bp insulator element, termed sns, localized at the 3' end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C + T) all needed for the enhancer-blocking activity in both sea urchin and human cells. The goal of this study was to further characterize the sea urchin sns insulator in the erythroid environment. We employed colony assays in human (K562) and mouse (MEL) erythroid cell lines. We tested the capability of sns to interfere with the communication between the 5'HS2 enhancer of the human beta-globin LCR and the gamma-globin promoter. We found that the sns sequence displays directional enhancer-blocking activity. By the use of antibodies against known DNA binding proteins, in electrophoretic mobility shift assays, we demonstrated the binding of the erythroid-specific GATA-1 and the ubiquitous Oct-1 and Sp1 transcription factors. These factors bind to Box A, Box B, and Box C + T, respectively, in both K562 and MEL nuclear extracts. These results may have significant implications for the conservation of insulator function in evolutionary distant organisms and may prove to be of practical benefit in gene transfer applications for erythroid disorders such as hemoglobinopathies and thalassemias.
Acuto
Santina
S
Unità di Ricerca "P. Cutino", Ematologia II, A.O. "V. Cervello", via Trabucco n. 180-90146, Palermo, Italy. acutosanti@virgilio.it
Di Marzo
Rosalba
R
Calzolari
Roberta
R
Baiamonte
Elena
E
Maggio
Aurelio
A
Spinelli
Giovanni
G
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000235:genetics / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics; D041905:Erythroid Cells / Q000378:metabolism*; D050982:GATA1 Transcription Factor / Q000502:physiology; D018014:Gene Transfer Techniques; D005914:Globins / Q000235:genetics; D006801:Humans; D038101:Insulator Elements / Q000235:genetics / Q000502:physiology*; D019901:Locus Control Region / Q000502:physiology; D051379:Mice; D050811:Octamer Transcription Factor-1 / Q000502:physiology; D047328:Paracentrotus / Q000235:genetics; D012617:Sea Urchins / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000502:physiology
D002843:Chromatin; D050982:GATA1 Transcription Factor; D050811:Octamer Transcription Factor-1; D016329:Sp1 Transcription Factor; D005914:Globins