Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication.

Abstract

Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin‐promoter‐enhancer region of SV40, when introduced into CV‐1 or Cos‐1 monkey cells by DEAE‐dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin‐specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non‐replicating and post‐replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein‐DNA interactions during viral infection or upon cell differentiation.

LCR-mediated, long-term tissue-specific gene expression within replicating episomal plasmid and cosmid vectors

“…[33][34][35] This difference in the activity of ␤LCR elements when assayed by transient transfection or within REVs, may be due to the fact that REVs adopt a well-ordered, nucleosomal configuration akin to that of native chromatin. 14,15 Since most (HS1, 3 and 4) ␤LCR elements enhance transcription only when integrated within the host cell genome, the chromatin configuration adopted by REVs would appear to provide the necessary environment for their function. In support of this idea is the report that HS2 can remodel chromatin on REVs 36 in K562 cells, although this study addressed neither tissue-specificity, the cumulative effect of combining ␤LCR HS sites nor the maintenance of the open remodelled chromatin state upon prolonged periods of culture.…”

“…The silencing of transgene expression in K562 cells containing human ␤-globin under ␤LCR HS3 control (3b; Figure 3a), suggests changes in the chromatin structure of these REV molecules 13,15,16 to a transcriptionally nonpermissive state. We therefore determined the general DNaseI sensitivity of the 3␤ REV episomes in K562 cells after 30 days of continuous culture in the presence of drug-selective pressure, during which time ␤-globin gene expression is silenced, and compared this with cells containing the ␤LCR HS234-driven construct (432␤), where gene expression is retained over the same period ( Figure 3a).…”

“…[3][4][5] REVs based on viral origins of replication such as those from EBV, 6 human papovavirus BK, 7,8 mammalian chromosomal origins of replication have also been found to improve nuclear retention of the episomes. 11,12 It has been demonstrated that both non-replicating, transiently transfected plasmids, 13,14 as well as REVs, 13,15,16 assemble nucleosomes. Assembly on REVs is more organised and resembles native chromatin, whereas nucleosomes on transient plasmids are less well ordered allowing greater access of transcription factors to target sequences.…”

Domain structure of the simian virus 40 core origin of replication.

“…These elements are T-antigen-binding region I, the 21-base pair (bp) repeats, and the 72-bp enhancers. Each ancillary region is a component of the early promoteroperator that overlaps the core origin, and each may play a role in the organization of the open region of SV40 chromatin (4,13,14,17,19,33). A single viral protein, T antigen, binds to the core origin of replication to initiate each round of DNA synthesis (8,18,23,31,37).…”

CpG Islands from the α-Globin Gene Cluster Increase Gene Expression in an Integration-Dependent Manner

“…It is known that nonreplicating circular DNA molecules are readily assembled into nucleosomes upon transient transfection (9,35). However, at most 60 to 70% of the transfected plasmids reaching nuclei are assembled into chromatin and at the concentration of transfecting DNA typical of gene expression analysis, an even smaller fraction of the transfected templates are assembled (35).…”