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Environmental Factor, November 2012

Exploring regulatory mechanisms in the fruit fly genome

By Sheila Yong

Hemakumara Mutra, Ph.D., and Kami Ahmad, Ph.D.

Shown with his host, visiting fellow Hemakumara Mutra, Ph.D., left, Ahmad said the goal of his research is to identify new regulatory elements and dissect the epigenomic changes that occur throughout the genome during development. “We are excited about these observations, because they imply that our purification technique can isolate proteins bound to the DNA fragments, based on proximity, even if they do not make direct contact with one another,” he told the audience. (Photo courtesy of Steve McCaw)

“People generally view the nucleosomes as static structures of histone core proteins in the center with DNA wrapped around them,” said Kami Ahmad, Ph.D., at the beginning of his talk at NIEHS Oct. 2. Nucleosomes are the fundamental units of the chromatin in a eukaryotic cell that facilitate the packing of large genomes into the nucleus while still allowing ample access to the DNA. “However, nucleosomes are in fact dynamic structures and their histone core proteins are frequently replaced,” he explained.

Ahmad, (http://www.hms.harvard.edu/dms/BBS/fac/ahmad.php)  an assistant professor at Harvard Medical School, uses the fruit fly, Drosophila melanogaster, as a model system for his research on histone variants and the biological properties of chromatin. During his talk, he discussed his group’s findings on the mechanism of histone replacement and efforts to identify regulatory elements in the Drosophila genome.

Histone H3.3 variant participates in replication-independent histone replacement

The first part of Ahmad’s talk focused on histone H3.3, a histone H3 variant that differs from histone H3 by only four amino acids. This histone variant is evolutionarily conserved in many species, highlighting its importance in chromatin structure maintenance. Ahmad pointed out that, unlike canonical histones, which are mainly used for nucleosome assembly during different cell cycle phases, histone H3.3 participates in DNA replication-independent histone replacement, which occurs outside of S-phase of the cell cycle. In particular, histone H3.3 plays a role in facilitating nucleosome assembly at sites of transcription where DNA has to unwind to allow RNA polymerase II access.

Ahmad and his team employ a temperature-controlled heat shock protein Hsp70 expression system that enables them to activate transcription of the Hsp70 gene and observe histone H3.3 replacement. Their studies revealed that chromatin remodeler Xnp, which is normally dispersed throughout the genome, colocalized with histone H3.3 during transcription activation.

Besides Xnp, several histone chaperones such as Hira and ASF1 were also localized at the transcription bulbs. Upon recovery, these factors were released and the nucleosomes were repackaged. However, when they knocked down H3.3 expression, the nucleosome repackaging efficiency was markedly reduced. Moreover, while most of the proteins involved in Hsp70 transcription were released upon recovery, Xnp and Hira remained at the transcription sites.

One interesting question that was raised during the presentation was if histone H3 can rescue the phenotype observed in H3.3-deficient flies. “Yes, it can,” Ahmad responded. “Therefore, it remains to be determined why the cells choose to use H3.3 instead of H3 for histone replacement during transcription.”

Identifying regulatory elements in the Drosophila genome

In the second part of his talk, Ahmad discussed his group’s involvement with the Drosophila model organism ENCyclopedia of DNA Elements (modENCODE) (http://www.modencode.org/)  which aims to unravel the transcriptome, as well as various properties of the chromatin and nucleosomes. Ahmad’s group uses a modified micrococcal nuclease digestion and salt fractionation protocol to detect and measure nucleosome and chromatin dynamics.

To illustrate this point, Ahmad used the fruit fly Hsp26 promoter as an example. Ahmad pointed out that the purification method not only confirmed that the heat shock factor (HSF) was bound to the promoter, but also revealed that the Drosophila GAGA factor (GAF) was also present at the site. “In this case, we discovered that GAF and HSF form a complex, although they bind to adjacent sites on the promoter,” Ahmad noted.

Ahmad concluded that this isolation method can potentially be used to identify clusters of small chromatin particles, and subsequently lead to the discovery of novel regulatory elements in the genome and proteins that bind to them. Data obtained from these studies can then be analyzed using a systems biology approach, to illustrate the architecture of regulatory elements in the eukaryotic genome and the relationship between different protein factors that regulate gene expression.

(Sheila Yong, Ph.D., is a visiting fellow in the NIEHS Laboratory of Signal Transduction.)




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