The genetic expansion of internal polyglutamine sequences in several proteins renders them toxic to neurons in unexplained ways. Two new papers tie this polyGlu toxicity to the process of gene transcription in slightly different ways. In the first, compelling biochemical studies point to transcriptional repression as a key factor in the toxicity of polyGlu-expanded huntingtin protein (htt), the cause of Huntington’s disease. The work, coming from the labs of Robert Tjian at the University of California in Berkeley and Dimitri Krainc at Massachusetts General Hospital in Boston, shows that soluble polyGlu-containing fragments of htt bind to key components of the transcription machinery and interfere with gene expression. And just as it appears that polyGlu repeats are bad for transcription, a second paper reports that transcription might be bad for polyGlu expansions, by giving the DNA repair machinery a window of opportunity to shrink the long trinucleotide repeat tracts that encode them.
In a paper published December 29 in Cell, first author Weiguo Zhai and colleagues developed a defined, in-vitro assay of transcription driven by the transcription factor Sp1 to probe the effects of mutant htt on RNA synthesis. In previous work, Krainc and coworkers had shown that mutant htt repressed Sp1-dependent transcription of the dopamine D2 receptor gene through direct interaction with Sp1 and its coactivator TAF4 in neurons (see ARF related news story). In cells, proteolytic processing of mutant htt produces an N-terminal fragment containing the expanded polyglutamine tract. This fragment enters the nucleus, where it appears to mimic glutamine-rich transcriptional activation domains and disrupt protein-protein interactions important for assembly of the large polymerase complex.
To determine unequivocally which components of the transcription complex htt targeted, the researchers added recombinant htt protein to their purified transcription machinery. Transcription in this system was sensitive to soluble, recombinant fragments of htt containing 120 glutamine repeats (i.e., mutant) but not a 25-glutamine stretch (i.e., wild-type). The repression was specific to Sp1-driven transcription, as neither protein inhibited a Gal4-driven system. Adding excess Sp1 or the TAF4-containing TFIID complex reversed the repression, and this is consistent with previous demonstrations that these proteins are targets of mutant htt. This approach of adding excess factors identified the TFIIF complex as an additional target for htt, and further experiments showed that mutant htt bound specifically to the RAP30 subunit of this complex. Htt binds all three proteins in a similar, polyGlu-dependent way.
Transcription repression in vitro correlated closely with htt toxicity when the researchers tested a number of recombinant proteins containing 25, 46, 97, or 120 Glu residues. Just as increasing length of the polyGlu tract correlates with disease onset and severity, it also correlated with the extent to which transcription was inhibited in vitro. In all cases, adding excess Sp1, TFIID or TFIIF reversed the inhibition.
Transcriptional repression was associated with toxicity in neurons in culture, as well. In cultures of primary striatal neurons from mutant htt-transgenic mice, transfection of RAP30 reversed repression of D2 dopamine receptor expression, similar to previous results for Sp1 and TAF4. Further, coexpression of RAP30 blocked the death of neurons from normal mice induced by transfecting in mutant htt. To demonstrate physiological significance by another method, the investigators used chromatin immunoprecipitation to show that there was less RAP30 localized to the D2 promoter in Huntington mice compared to wild-type animals.
These results demonstrate that mutant htt interferes with gene transcription by targeting a transcriptional activator (Sp1) as well as components of the general transcription machinery. While there is some evidence that global transcription may be decreased in htt cells, the results allow for selective gene inhibition as well, given the differential requirements for Sp1, TFIID, and TFIIF for different genes to be active. Curiously, Sp1 expression is implicated in Alzheimer disease and related tauopathies as well (see, e.g., Santpere et al., 2005; Christensen et al., 2004; Prinzen et al., 2005).
Tjian and Krainc’s studies were all carried out with truncated htt protein, and further work will be required to determine if this faithfully imitates what happens with the endogenously processed form of htt. But if this is confirmed, then the results clearly point to transcriptional repression as an early deleterious effect of soluble mutant htt in neurons.
Cells do not take lightly attacks on necessary gene expression, so it may come as no surprise that they would try to reverse the effects of polyglutamine expansion. Just how much or whether cells can repair this kind of pathogenic repeat has been open to debate, and new data from John Wilson and colleagues at Baylor College of Medicine in Houston, Texas, may give some reason for encouragement.
Their paper, published online January 1 in Nature Structural Molecular Biology, uses a TET-inducible expression system to assess the ability of cells to contract CAG repeats introduced into an HPRT reporter gene. In their experiments, a (CAG)95 repeat placed in an intron of the HPRT gene prevents protein expression. Only when the repeat contracts to 39 triplets or less do cells become HPRT-positive, as measured by survival under selection. In this system, the investigators only saw contraction of the CAG repeats after transcription of the reporter gene was switched on with doxycycline. In contrast, DNA replication did not affect the frequency of CAG contractions, as dividing cells and non-dividing cells showed the same contraction frequency as long as the gene was being transcribed.
Experiments using siRNA knockdown of a number of DNA repair proteins demonstrated that both mismatch repair and nucleotide excision repair components were involved in shrinking the repeats. It remains to be seen if transcription or faulty DNA repair is involved in the instability of CAG repeats in humans, which tend to increase, rather than decrease with time. But understanding this novel pathway opens up the possibility of therapy aimed at shrinking pathologic repeats in patients with polyGlu diseases.—Pat McCaffrey
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