We assess the role of intrinsic histone-DNA interactions by mapping nucleosomes assembled in vitro on genomic DNA. Nucleosomes strongly prefer yeast DNA over E. coli DNA, indicating that the yeast genome evolved to favor nucleosome formation. Many yeast promoter and terminator regions intrinsically disfavor nucleosome formation, and nucleosomes assembled in vitro display strong rotational positioning. Nucleosome arrays generated by the ACF assembly factor display fewer nucleosome-free regions, reduced rotational positioning, and less translational positioning than obtained by intrinsic histone-DNA interactions. Importantly, in vitro assembled nucleosomes display only a limited preference for specific translational positions and do not show the pattern observed in vivo. Our results argue against a genomic code for nucleosome positioning, and they suggest that the nucleosomal pattern in coding regions arises primarily from statistical positioning from a barrier near the promoter that involves some aspect of transcriptional initiation by RNA polymerase II.

The roX1 and roX2 genes of Drosophila produce malespeci®c non-coding RNAs that co-localize with the Male-Speci®c Lethal (MSL) protein complex. This complex mediates up-regulation of the male X chromosome by increasing histone H4 acetylation, thus contributing to the equalization of X-linked gene expression between the sexes. Both roX genes overlap two of~35 chromatin entry sites, DNA sequences proposed to act in cis to direct the MSL complex to the X chromosome. Although dosage compensation is essential in males, an intact roX1 gene is not required by either sex. We have generated¯ies lacking roX2 and ®nd that this gene is also non-essential. However, simultaneous removal of both roX RNAs causes a striking male-speci®c reduction in viability accompanied by relocation of the MSL proteins and acetylated histone H4 from the X chromosome to autosomal sites and heterochromatin. Males can be rescued by roX cDNAs from autosomal transgenes, demonstrating the genetic separation of the chromatin entry and RNA-encoding functions. Therefore, the roX1 and roX2 genes produce redundant, male-speci®c lethal transcripts required for targeting the MSL complex.

SUMMARY The high mobility group N (HMGN) proteins are abundant nonhistone chromosomal proteins that bind specifically to nucleosomes at two high affinity sites. Here we report that purified recombinant human HMGN1 (HMG14) and HMGN2 (HMG17) potently repress ATP-dependent chromatin remodeling by four different molecular motor proteins. In contrast, mutant HMGN proteins with double Ser to Glu mutations in their nucleosome-binding domains are unable to inhibit chromatin remodeling. The HMGN-mediated repression of chromatin remodeling is reversible and dynamic. With the ACF chromatin remodeling factor, HMGN2 does not directly inhibit the ATPase activity, but rather appears to reduce the affinity of the factor to chromatin. These findings suggest that HMGN proteins serve as a counterbalance to the action of the many ATP-dependent chromatin remodeling activities in the nucleus.

Dosage compensation equalizes the expression of sex-linked genes between males and females. Most genes on the X chromosome of male Drosophila are transcribed at an increased level, contributing to compensation. The roX1 and roX2 genes produce non-coding transcripts that localize along the X-chromosome of male flies. Although lacking sequence similarity, they are necessary but redundant components of a system that up-regulates gene expression. Simultaneous mutation of both roX genes disrupts the X-limited distribution of proteins that modify chromatin to enhance gene expression. We have generated and characterized loss of function roX1 alleles that display a continuum of activity. Those that support intermediate male survival have strikingly reduced RNA accumulation, while alleles with minor contributions to male viability typically lack detectable transcript accumulation. Severely mutated roX1 alleles retain some ability to direct modifying proteins to the X chromosome. This ability predicts the level of male survival that each allele supports. This points to a peripheral or transient role for roX in the RNA and protein complex that binds to and regulates the X chromosome.

The MSL complex of Drosophila upregulates transcription of the male X chromosome, equalizing male and female X-linked gene expression. Five male-specific lethal proteins and at least one of the two noncoding roX RNAs are essential for this process. The roX RNAs are required for the localization of MSL complexes to the X chromosome. Although the mechanisms directing targeting remain speculative, the ratio of MSL protein to roX RNA influences localization of the complex. We examine the transcriptional regulation of the roX genes and show that MSL2 controls male-specific roX expression in the absence of any other MSL protein. We propose that this mechanism maintains a stable MSL/roX ratio that is favorable for localization of the complex to the X chromosome.