Left-handedly curved DNA regulates accessibility to cis-DNA elements in chromatin

Abstract

There is little information on chromatin structure that allows access of trans-acting transcription factors. Logically, the target DNA elements become accessible by either exposing themselves towards the environment on the surface of the nucleosome, or making the regulatory region free of the nucleosome. Here, we demonstrate that curved DNA that mimics a negative supercoil can play both roles in the promoter region. By constructing 35 reporter plasmids and using in vivo assay systems, we scrutinized the relationships between upstream DNA geometry, nucleosome positioning and promoter activity. When the left-handedly curved DNA was linked to the herpes simplex virus thymidine kinase (HSV tk) promoter at a specific rotational phase and distance, the curved DNA attracted the nucleosome and the TATA box was thereby left in the linker DNA with its minor groove facing outwards, which led to the activation of transcription. Neither planar curving, nor right-handedly curved DNA nor straight DNA had this effect. Our results seem to provide a clue for solving the problem of why curved DNA is often located near transcriptional control regions.

Transient activation of transgene expression by hydrodynamics-based injection may cause rapid decrease in plasmid DNA expression

“…For example, the location of a left-handedly curved sequence with high histone affinity affects the transgene expression. 38,39 Thus, the control of the histone interactions is crucial for controlling the intranuclear disposition of the exogenous DNA.…”

Transgene expression efficiency from plasmid DNA delivered as a complex with histone H3

“…Therefore, histones could be a key factor for the intranuclear disposition of the exogenous DNA. In agreement with this hypothesis, transgene expression is affected by the introduction of DNA sequences with high and low histone affinities into plasmid DNAs (Nishikawa et al, 2003;Sumida et al, 2006;Kamiya et al, 2007Kamiya et al, , 2009.…”

“…The intranuclear disposition of the delivered plasmid is an important factor in efficient transgene expression (Kamiya et al, 2003). Nucleosomes are formed on non-integrated plasmid DNAs (Reeves et al, 1985), and the introduction of high and low histone-affinity DNA sequences into plasmid DNAs influences transgene expression (Nishikawa et al, 2003;Sumida et al, 2006;Kamiya et al, 2007Kamiya et al, , 2009. Moreover, decreased expression efficiency (silencing) seems to be accompanied by increased histone binding, not by histone modification (Ochiai et al, 2007).…”

A designed curved DNA sequence remarkably enhances transgene expression from plasmid DNA in mouse liver

“…The pLHC20/TLN-6 (T20), pLHC24/TLN-6 (T24), pLHC28/TLN-6 (T28), pLHC32/TLN-6 (T32), pLHC36/TLN-6 (T36), pLHC40/TLN-6 (T40) and pST0/TLN-7 (ST) plasmids, containing the herpes simplex virus tk promoter and the luciferase (luc) gene (Figure 1), 9,12 were amplified in Escherichia coli (JM109) and purified with a Qiagen (Hilden, Germany) EndoFree Plasmid Mega kit.…”

“…MNase digestion-based analysis of chromatin structure Seventy-two h after the injection, the liver was excised from the mouse and nuclei were prepared, as described by Nishikawa et al 9 Aliquots of 100 ml nuclei suspension (1 Â 10 7 nuclei ml…”