Book of Abstracts: Albany 2003

category image Albany 2003
Conversation 13
Abstract Book
June 17-21 2003

Regulation of Promoter Activity by Sequence-Specific Minor Groove Binding Ligands

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone-and receptor-dependent gene activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the DNA-protein interactions following hormone activation. The strategy of artificial regulating of gene activity by sequence-specific minor groove binding ligands is very attractive. We have synthesized and studied the interaction with DNA of bis-linked netropsin (Nt) derivatives in which two monomers are attached via short linkers in head-to-head and tail-to-tail manners:

Lys→Gly→Py→Py-NH(CH2)5NH-Py←Py←Gly←Lys Bis-Lys-Nt
Dp-Py←Py←Gly-cis[Pt(NH3)2]2+-Gly→Py→Py-Dp Pt-Bis-Nt

Here Py is a N-propylpyrrole residue. Cis[Pt(NH3)2]2+ is a cis-diammineplatinum (II) residue. Dp is a dimethylaminopropylamino residue. Arrows indicate direction from the N-terminus to the C-terminus in each netropsin fragment. DNAase I footprinting and CD studies show that Bis-Lys-Nt in the extended conformation binds most strongly to a DNA site with the sequence 5'(-AAAATTTT-3', whereas Pt-Bis-Nt binds preferentially to DNA sites with sequences 5'-TTTTAAAA-3', 5'-TATTAATA-3' and 5'-TCTTAAAA-3'. The latter site within the MMTV promoter (positions – 166 to – 173) is overlapping in part with the binding site for glucorticoid receptor. Nt-derivatives were added directly to the oocyte media at concentration 1.2 μM and specific binding was assayed by in situ DNase I footprinting. We have found that Pt-Bis-Nt can penetrate cell and nuclear membrane and specifically bind to the 5'-TCTTAAAA-3' sequence within MMTV promoter in vivo. Now it can be considered as a "platform" for further design and synthesis of conjugates that could repress transcription of the MMTV promoter via interfering with glucocorticoid receptor binding or activate it in non-hormone dependent fashion.
S. V. Belikov1,2
S. L. Grokhovsky3,4
A. N. Surovaya3
G.V. Gursky3

1I. Ivanovsky Institute of Virology
123098 Moscow, Russia
2Dept. of Cell and Molecular Biology
The Medical Nobel Institute
Box 285
Karolinska Institutet
SE-17177 Stockholm, Sweden
3W. A. Engelhardt Institute of Molecular Biology
119991 Moscow, Russia
4University of Oslo
Centre for Medical Studies
Oslo, Norway
Fax: (7) (095) 135-1405

Supported by Swedish Academy of Sciences and Russian Foundation for Basic Research (grant 03-04-48903).

S. V. Belikov
S. L. Grokhovsky
A. N. Surovaya
G.V. Gursky