Yuh Hwa Wang

Expanded CTG triplet blocks from the myotonic dystrophy gene create the strongest known natural nucleosome positioning elements

Expanded blocks of repeating nucleotide triplets have been found in or near genes associated with several human diseases. In the case of myotonic dystrophy, a block of repeating CTG trinucleotides is located downstream of the gene, and expansions of this block to repeats of n = 100 or more are frequently found in afflicted individuals. Using electron microscopy, we recently demonstrated that these blocks form unusually stable nucleosomes. Here, competitive nucleosome reconstitution was employed to measure the energetics of nucleosome formation over CTG repeat blocks of n = 75 and n = 130. These values were compared to the Xenopus borealis somatic 5S RNA gene, previously one of the strongest known natural nucleosome positioning elements. It is shown that DNA fragments containing 75 and 130 CTG repeats are 6 and 9 times stronger in nucleosome formation, respectively, than the 5S RNA gene. These findings suggest that expanded CTG blocks may profoundly alter local chromatin structure.

Functional Analysis of the Subunits of the Chromatin Assembly Factor RSF

ABSTRACT The human ISWI-containing factor RSF (for remodeling and spacing factor) is composed of two subunits: the ATPase hSNF2H and p325 (Rsf-1), a protein encoded by a novel human gene. We previously showed that RSF mediates nucleosome deposition and generates regularly spaced nucleosome arrays. Here we report the characterization of the largest subunit of RSF, Rsf-1. We found that Rsf-1 is a highly acidic protein containing a plant homology domain. The present study includes the cloning of Rsf-1, the preparation of recombinant RSF, and the dissection of the role of each subunit in the chromatin assembly reaction. The sequence of the gene for Rsf-1 includes a recently characterized cDNA, HBXAP; postulated to be involved in the transcriptional regulation of the hepatitis B virus. HBXAP actually contains a 252-amino-acid truncation of the amino terminus of Rsf-1. Finally, comparison of HBXAP and Rsf-1 properties shows that they are functionally different.

Control of meiotic recombination and gene expression in yeast by a simple repetitive DNA sequence that excludes nucleosomes.

ABSTRACT Tandem repeats of the pentanucleotide 5′-CCGNN (where N indicates any base) were previously shown to exclude nucleosomes in vitro (Y.-H. Wang and J. D. Griffith, Proc. Natl. Acad. Sci. USA 93:8863–8867, 1996). To determine the in vivo effects of these sequences, we replaced the upstream regulatory sequences of the HIS4 gene ofSaccharomyces cerevisiae with either 12 or 48 tandem copies of CCGNN. Both tracts activated HIS4 transcription. We found that (CCGNN)12 tracts elevated meiotic recombination (hot spot activity), whereas the (CCGNN)48 tract repressed recombination (cold spot activity). In addition, a “pure” tract of (CCGAT)12 activated both transcription and meiotic recombination. We suggest that the cold spot activity of the (CCGNN)48 tract is related to the phenomenon of the suppressive interactions of adjacent hot spots previously described in yeast (Q.-Q. Fan, F. Xu, and T. D. Petes, Mol. Cell. Biol. 15:1679–1688, 1995; Q.-Q. Fan, F. Xu, M. A. White, and T. D. Petes, Genetics 145:661–670, 1997; T.-C. Wu and M. Lichten, Genetics 140:55–66, 1995; L. Xu and N. Kleckner, EMBO J. 16:5115–5128, 1995).

Relative orientation of RNA helices in a group I ribozyme determined by helix extension electron microscopy

The relative orientation of helical elements in a folded RNA molecule provides key information about its three‐dimensional architecture. We have developed a method that involves extending peripheral helices of an RNA, mounting for electron microscopy in the absence of protein and measuring interhelical angles. As a control, extended anticodon and acceptor stems of tRNA(Phe) were found to form a 92 +/− 20 degrees angle, consistent with the X‐ray structure. Single, double and triple extensions (50–80 bp) of helical elements P2.1, P6b and P8 of the Tetrahymena group I ribozyme did not alter its catalytic activity. The measured angle between P6b and P8 is consistent with the Michel‐Westhof structural model, while the P2.1‐P6b and P2.1‐P8 angles allow P2.1 to be positioned in the model. The angle distributions of the ribozyme are broader than those of the tRNA, which may reflect the dynamics of the RNA. Helix extension allows low‐resolution electron microscopy to provide much higher resolution information about the disposition of helical elements in RNA. It should be applicable to diverse RNAs and ribonucleoprotein complexes.

Visualizing nucleic acids and their complexes using electron microscopy.

Microscopic visualization of nucleic acid-protein complexes provides a means of obtaining structural information that is difficult to obtain in any other way, and of verifying conclusions derived from other approaches. The polymorphic, flexible, and irregular nature of these complexes presents particular problems in their analysis.