Glutamine expansion under control

Expansion of the CAG triplet coding for glutamine (Q) is a key characteristic of human spinocerebellar ataxia type 3 (SCA3), an inherited disorder leading to loss of muscle movement. Mechanisms underlying CAG repeat instability upon transmission to the next generation have now been studied in a Drosophila model system by Jung and Bonini. Transgenic flies expressing human polyQ SCA3 (SCA3-Q78) showed a wide range of repeat expansions and reductions similar to those detected in human SCA3 patients. Expanded trinucleotides fold into unusual DNA structures that are detected by DNA repair pathways. The authors tested the role of transcription-coupled repair (TCR) in polyQ instability by crossing SCA3-Q78 transgenic flies with a mutant fly line lacking TCR. Loss of TCR correlated with reduced CAG repeat instability. Previous work has shown that DNA repair is regulated by the histone acetyltransferase CREB-binding protein (CBP) and that CBP activity is impaired by polyQ proteins. Reduction of CBP levels further expanded polyQ repeats and, conversely, treatment with the histone deacetylase inhibitor trichostatin A (TSA) blocked polyQ repeat expansion in flies. These data underline the value of Drosophila as a model for human polyQ diseases and point to CAG repeat instability as a process influenced by TCR and the polyQ protein itself. Further research is needed to understand whether TSA treatment could be applied to polyQ diseases in general and whether somatic polyQ repeat instability also depends on TCR. (Science, published online 1 March 2007, doi:10.1126/science.1139517) APD

Getting a membrane protein's number

Many membrane proteins act as multimeric complexes that require proper assembly to function in their target membranes. Determination of the subunit architecture and stoichiometry of membrane proteins is often done indirectly, for example by channel conductance measurements for ion channels, but such estimates can be inaccurate. Ulbrich and Isacoff have developed a relatively straightforward technique to define subunit number in membrane complexes using single-molecule fluorescent imaging in live cells. A green fluorescent protein (GFP)-tagged protein of interest was expressed in Xenopus oocytes and total internal reflection fluorescent microscopy was then used to count the photobleaching steps of the GFP tag. They tested their technique on proteins of known subunit stoichiometry, including a Ca2+ channel, a cyclic nucleotide-gated (CNG) channel comprised of three CNGA1 subunits and one CNGB1 subunit, and the glutamate-sensitive NMDA receptors, which have two NR1 and two NR2B subunits. They then used the technique on the glycine-gated NMDA receptor, a tetrameric channel consisting of NR1 and NR3B subunits of unknown stoichiometry. Unlike NR2B, which binds glutamate, NR3B binds glycine. The authors found that, like the glutamate-sensitive receptors, the glycine-gated NMDA receptors have an NR1/NR3B subunit stoichiometry of 2:2. Thus, while the ligand-binding properties of NR2B and NR3B differ, their interactions with NR1 subunits are similar. The method of GFP tagging with photobleaching may be applied to other channels and receptors of fixed stoichiometry, and the authors propose that it could also be used on accessory receptor subunits, as well as GPCRs and some signaling complexes. (Nature Methods, advance online publication 18 March 2007, doi:10.1038/nmeth1024) MM

piRNAs dance flamenco

Piwi proteins have been implicated in transposon regulation and bind a specific class of small RNAs (Piwi-interacting RNAs, or piRNAs). Uncontrolled transposon activity has the potential to disrupt genome integrity, but the precise mechanism of transposon suppression has been unclear. Hannon and colleagues have now analyzed piRNAs bound to the three Drosophila Piwi family proteins Piwi, Aubergine and Ago3, finding that the majority of piRNA sequences represent portions of annotated transposon sequences. Further analyses suggest that the piRNA sequences tend to originate from discrete heterochromatic loci, including the flamenco and X-TAS loci, long implicated as major transposon regulatory regions. The flamenco locus is a >180-kilobase region encompassing essentially only defunct transposons. flamenco alleles known to lead to transposon derepression show decreased piRNA expression from the entire locus, suggesting a model where an initial long transcript that traverses this locus is processed to generate primary piRNAs. A second amplification phase is suggested by the observation that Piwi- and Aubergine-bound piRNAs represent antisense transposon sequences, whereas complementary sequences associate with Ago3. Thus, Ago3 and Aubergine may participate in a positive feedback loop, where Ago3-cleaved targets form Aubergine piRNAs, which then guide cleavage to form new Ago3 piRNAs, and so on. This is consistent with data from the Zamore and Siomi groups, respectively suggesting a distinct piRNA biogenesis pathway and analyzing Piwi protein enzymatic activity. Hannon and colleagues outline a number of new testable models for piRNA biogenesis. Furthermore, they suggest models for the function of 'transposon graveyard' loci, such as flamenco, as potential sequence repositories ready to be processed into piRNAs that can target rogue mobile elements. (Cell, published online 7 March, doi:10.1016/j.cell.2007.01.043) SL

Opening the DNA gate

Type II topoisomerases (Topo II) are essential for cell viability: they relieve or introduce supercoiling, resolve knots and decatenate DNA. These activities are accomplished by making a transient double-stranded break in a DNA segment, passing another stretch of DNA (called the T segment) through this break and finally religating the ends. Thus, a DNA gate opens to allow passage of the T segment, then closes to restore chromosomal integrity. Hsieh and colleagues have recently reported measurements of DNA gate changes by fluorescence resonance energy transfer (FRET). The authors attached donor and acceptor fluorophores to a DNA segment, in positions that allowed efficient FRET. In the presence of eukaryotic Topo II, Mg2+ and ATP, there was a decrease in bulk FRET signal. When observed at the single-molecule level, the FRET signal oscillated between high and low, corresponding to a closed and open DNA gate, respectively. Because the DNA ends are held by the enzyme, these movements reflect large conformational changes in Topo II. Under conditions of steady-state ATP hydrolysis, the rates of interconversion between the closed and the open states were similar. Future experiments with a T segment present should provide insights into the reaction mechanism of strand passage during the Topo II catalytic cycle. (Proc. Natl. Acad. Sci. USA 104, 4840–4845, 2007) IC

Research Highlights written by Inês Chen, Alexander P. Dorr, Sabbi Lall and Michelle Montoya.