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Researchers Use Sequencing to Tackle X-Chromosome Dosage Compensation Questions

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – An American research team reported online today in Nature that it has uncovered new clues about how gene expression patterns are tweaked on the X chromosome in species where males have one copy of the sex chromosome and females have two.

The team used a combination of global run-on sequencing and chromatin immunoprecipitation to look at X chromosome-related RNA polymerase II activity in male Drosophila cells. Compared with the autosomal chromosomes, they found that the polymerase was more concentrated on the X chromosome and remained active even on regions of X-linked genes where expression typically tapers off.

"Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome," senior author Mitzi Kuroda, a genetics researcher affiliated with Harvard Medical School and Brigham and Women's Hospital, and co-authors wrote.

Past research has shown that a protein known as male-specific lethal, or MSL, contributes to dosage compensation in men by bumping up the expression of genes on the X chromosome, the researchers explained. But the protein's specific mechanism of action and related X chromosome gene characteristics were not as well understood, they added.

To explore this in more detail, the researchers used the Illumina GAIIx to do global run-on sequencing in a Drosophila cell line known as SL2, which has well studied MSL protein function. To simplify their analyses, they focused on genes larger than 2,500 bases, including 822 X-linked and 3,420 autosomal genes.

From their sequencing experiments the team concluded that active RNA polymerase II enzyme is more concentrated in and around X-linked genes — especially on gene bodies — than it is on genes found on the autosomal chromosomes. For instance, they detected some 1.4 times as many polymerase tags on X-linked gene bodies as on autosomal gene bodies.

As expected, the researchers' RNA polymerase II chromatin immunoprecipitation experiments in the same cell line also pointed to excess polymerase occupancy on the X-linked genes.

When they further scrutinized their GRO-sequencing data to look at RNA polymerase elongation and pausing patterns across specific gene regions, the team found evidence that RNA polymerase II occupancy and, subsequently, activity remains unusually high along X-linked bodies — apparently due to interactions mediated by proteins in the MSL complex.

"These results confirm that MSL-dependent changes in steady-state RNA levels reflect differences in active transcription on the X chromosome," the researchers noted.

"In summary, we propose that the MSL complex functions on the male X chromosome to promote progression and processivity of [RNA polymerase II] through the nucleosomal template," they concluded. "Improving transcriptional output downstream of typical gene-specific regulation makes biological sense when compensating the diverse set of genes found along an entire chromosome."