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Summary: Mitzi Kuroda is interested in chromatin organization, epigenetic regulation, sex chromosomes, and dosage compensation.

Gene regulation is generally thought to occur through the function of regulatory proteins. However, the discoveries of noncoding RNAs that associate along the length of dosage-compensated X chromosomes in mammals and in fruit flies suggest that RNAs can play an intriguing, unexpected role in the regulation of chromatin structure and gene expression.

Dosage compensation is a mechanism to make gene expression equivalent in males and females, despite their differences in X chromosome number. In humans and fruit flies (Drosophila melanogaster), the chromosomal basis for sex determination results in females with two X chromosomes, and males with one X and a Y. Early in development, compensation mechanisms are employed to make X chromosome expression similar in the two sexes. In mammals, one X chromosome is inactivated in each female nucleus, whereas in Drosophila, most X-linked genes are up-regulated in males. These unrelated solutions still provide a common theme for dosage compensation: In each case, the unique transcriptional state of the dosage-compensated X chromosome is the result of its unique chromatin composition. Specific molecules, including Xist RNA in female mammals and roX RNAs in male fruit flies, bind the X chromosomes to remodel chromatin structure in one sex, but not in the other.

	Spreading of roX1 RNA...

Dosage compensation in Drosophila requires a large male-specific ribonucleoprotein complex that binds the X chromosome along its length in a banded pattern. Binding of these complexes results in specific acetylation of nucleosomes and up-regulation of transcription on the male X. The known components are roX (RNA on X) RNAs and MSL (male-specific lethal) proteins. The RNAs, roX1 and roX2, are encoded by genes on the X chromosome. These male-specific RNAs lack significant protein-coding potential. They are retained in the nucleus, colocalize with the MSL complex on the male X, and coimmunoprecipitate with the MSL proteins from nuclear extracts. Males die with mislocalized MSL complexes if both roX1 and roX2 are mutant. Despite their functional redundancy, roX1 and roX2 RNAs show little primary sequence homology and are distinct in size (3.6 kb versus ~0.5 kb).

In addition to roX RNAs, MSL complexes contain at least five distinct proteins, collectively called the MSL proteins, as each is required for male viability. These include the MOF histone acetyltransferase and the MLE RNA helicase. The complex is not known to contain typical DNA-binding proteins. Thus, how the MSL complex is targeted specifically to the X chromosome has been a long-standing question.

X Chromosome Recognition and Spreading
The MSL complex strongly discriminates between the X chromosome and the autosomes, but the molecular basis for this recognition is poorly understood. Currently we have evidence for two distinct mechanisms for X targeting. Most pieces of X DNA, >30 kb, can attract the MSL complex even when translocated to autosomes. We are searching for clues in the DNA sequence that could account for this remarkable specificity. A second mechanism resembles the mammalian mode of X targeting. Unexpectedly, we discovered that the MSL complex has the ability to "spread" long distances from initial binding sites into flanking chromatin in cis. Complexes spread from the roX1 and roX2 genes, which encode the known RNA components of the complex. Thus, analogous to the properties of the X inactivation center that encodes the Xist RNA in mammals, chromatin modification in flies appears to begin at the sites of synthesis of untranslated RNAs. In both flies and mammals, these RNAs spread along with chromatin-modifying activities into neighboring chromatin in cis.

The ability of MSL complexes to spread in cis can be seen on autosomes when roX loci are inserted as transgenes. Spreading also can be seen on the native X chromosome by delaying normal MSL assembly until larval stages, when the giant polytene chromosomes make high-resolution analysis possible. When MSL complexes are induced late in development, they can be seen to accumulate locally at the sites of roX RNA transcription and spread into flanking X chromosome sequences. Once MSL proteins join roX RNAs, the complexes also become competent to bind distant target sites on the X.

The epigenetic aspects of X chromosome dosage compensation in mammals and fruit flies suggest a potential mechanism for rapid evolutionary change in the regulation of large chromatin domains. Such change could be facilitated through the selective localization of chromatin-remodeling machines that can then spread long distances in cis from their original sites of entry. Studies of dosage compensation in mammals and fruit flies suggest that RNAs can play a key role in chromosomal targeting of chromatin-modifying complexes. The mechanistic roles of the RNAs within such complexes remain to be understood.

Grants from the National Institutes of Health and the Welch Foundation provided partial support for the work described above.