The sea urchin sns5 insulator protects retroviral vectors from chromosomal position effects by maintaining active chromatin structure.
Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders.
Unità di Ricerca P. Cutino, U.O.C. Ematologia II, A.O. V. Cervello, Palermo, Italy.
Danilo
D
D'Apolito
Elena
E
Baiamonte
Mariella
M
Bagliesi
Rosalba
R
Di Marzo
Roberta
R
Calzolari
Leda
L
Ferro
Vito
V
Franco
Giovanni
G
Spinelli
Aurelio
A
Maggio
Santina
S
Acuto
D000818:Animals; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D055012:Chromosomal Position Effects; D050982:GATA1 Transcription Factor / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D038101:Insulator Elements / Q000235:genetics / Q000502:physiology*; D051379:Mice; D041681:NIH 3T3 Cells; D050811:Octamer Transcription Factor-1 / Q000378:metabolism; D011485:Protein Binding; D012190:Retroviridae / Q000235:genetics*; D012617:Sea Urchins / Q000235:genetics*
D002843:Chromatin; D050982:GATA1 Transcription Factor; C494689:Gata1 protein, mouse; D050811:Octamer Transcription Factor-1; C494166:Pou2f1 protein, mouse