Submillimolar levels of calcium regulates DNA structure at the dinucleotide repeat (TG/AC)n

Submillimolar levels of calcium, similar to the physiological total (bound + free) intranuclear concentration (0.01–1 mM), induced a conformational change within d(TG/AC)n, one of the frequent dinucleotide repeats of the mammalian genome. This change is calcium-specific, because no other tested cation induced it and it was detected as a concentration-dependent transition from B- to a non-B-DNA conformation expanding from 3′ end toward the 5′ of the repeat. Genomic footprinting of various rat brain regions revealed the existence of similar non-B-DNA conformation within a d(TG/AC)28 repeat of the endogenous enkephalin gene only in enkephalin-expressing caudate nucleus and not in the nonexpressing thalamus. Binding assays demonstrated that DNA could bind calcium and can compete with calmodulin for calcium.

c-Jun interacts with the corepressor TG-interacting factor (TGIF) to suppress Smad2 transcriptional activity

The Sma and Mad related (Smad) family proteins are critical mediators of the transforming growth factor-β (TGF-β) superfamily signaling. After TGF-β-mediated phosphorylation and association with Smad4, Smad2 moves to the nucleus and activates expression of specific genes through cooperative interactions with DNA-binding proteins, including members of the winged-helix family of transcription factors, forkhead activin signal transducer (FAST)-1 and FAST2. TGF-β has also been described to activate other signaling pathways, such as the c-Jun N-terminal Kinase (JNK) pathway. Here, we show that activation of JNK cascade blocked the ability of Smad2 to mediate TGF-β-dependent activation of the FAST proteins. This inhibitory activity is mediated through the transcriptional factor c-Jun, which enhances the association of Smad2 with the nuclear transcriptional corepressor TG-interacting factor (TGIF), thereby interfering with the assembly of Smad2 and the coactivator p300 in response to TGF-β signaling. Interestingly, c-Jun directly binds to the nuclear transcriptional corepressor TGIF and is required for TGIF-mediated repression of Smad2 transcriptional activity. These studies thus reveal a mechanism for suppression of Smad2 signaling pathway by JNK cascade through transcriptional repression.

Stabilisation of TG- and AG-containing antiparallel DNA triplexes by triplex-binding ligands

We have used DNase I footprinting to examine the interaction of several triplex-binding ligands with antiparallel TG- and AG-containing triplexes. We find that although a 17mer TG-containing oligonucleotide on its own fails to produce a footprint at concentrations as high as 30 µM, this interaction can be stabilised by several ligands. Within a series of disubstituted amidoanthraquinones we find that the 2,7- regioisomer affords the best stabilisation of this TG triplex, though the 1,8- isomer also stabilises this interaction to some extent. By contrast the 1,5- and 2,6- regioisomers show no interaction with TG triplexes. Similar studies with a 13mer AG-containing oligonucleotide show the opposite pattern of stabilisation: the 2,6- and 1,5- isomers stabilise this triplex, but the 2,7- and 1,8-compounds do not. The polycyclic compound BePI strongly stabilises TG- but not AG-containing triplexes, while a substituted naphthylquinoline interacts with both antiparallel triplex motifs.