Retrovirus silencer blocking by the cHS4 insulator is CTCF independent

Abstract

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRbeta-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.

Prospects and implications of using chromatin insulators in gene therapy and transgenesis

“…(61) As a means of combating these negative viral elements, it has recently been demonstrated that the cHS4 insulator is able to block retroviral silencing in a murine stem cell virus (MSCV) vector in transgenic mice. (60) Interestingly, the results obtained with this MSCV retrovirus are in agreement with the data obtained previously in chicken stably transfected cell lines. (33,60) First, when two copies of the cHS4 core element are placed on each side of an LCR-b-globin transgene (Fig.…”

“…(60) Interestingly, the results obtained with this MSCV retrovirus are in agreement with the data obtained previously in chicken stably transfected cell lines. (33,60) First, when two copies of the cHS4 core element are placed on each side of an LCR-b-globin transgene (Fig. 3), the highest protection against silencing is obtained, an effect that is translated to the most-elevated copy number and with homogeneous gene expression, again confirming that two copies of the 250 bp core insulator are as good as the two copies of the original 1.2 kb element (Fig.…”

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

“…11,16 Previous attempts to incorporate insulators into recombinant viral vectors intended for gene therapy yielded reduced transgene expression variegation and limited chromosomal position effect. [17][18][19][20][21] Most of these studies were performed on gamma-retroviral and lentiviral vectors with the cHS4 insulator in various mouse and human cell types. 22 Implementation of the full-length, 1.2-kb element in gammaretroviral vectors was associated with reduced genotoxicity, but also with important constraints concerning vector design and titer of infectious particles, mainly owing to the substantial size of this sequence.…”

Chromatin Insulator Elements: Establishing Barriers to Set Heterochromatin Boundaries

“…CTCF remains the only insulator-binding protein known to direct enhancer blocking in vertebrates. Despite CTCF's pivotal role in enhancer blocking, removal of the CTCF binding site has no effect on the barrier activity of HS4 [42,60]. The enhancer blocking and barrier to silencing properties are separable functions, and therefore employ different mechanisms.…”

“…A role for CTCF elements in heterochromatin boundary formation is frequently posited, but conclusive evidence in support of this particular function is not available at present. The CTCF binding site of the chicken HS4 insulator is not required for its barrier activity [42,60]. Furthermore, the well characterized CTCF-mediated human H19 ICR and TCRA/D BEAD-1 enhancer-blocking elements also lack barrier activity [42].…”