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Technique could be useful for quantifying response to therapies

BioPhotonics
Jul 2008
With high-throughput microscopy, researchers reveal estrogen receptor regulation.

Hank Hogan

By exploiting high-throughput microscopy and a new cell line, researchers have uncovered how the transcription regulator estrogen receptor-α can control gene expression. A group from Baylor College of Medicine in Houston and from the University of Texas Health Science Center in San Antonio has shown that the unwinding, or decondensation, of chromatin from a compact form may not be tightly linked to the transcription rate of messenger RNA, or mRNA, by the estrogen receptor.

BREstrogen_Fig-1_pastedGraphic.jpg

High-throughput microscopy combined with a new cell line leads to better understanding of how the estrogen receptor-α controls gene expression. Here a high-resolution image shows the colocalization of a fusion green fluorescent protein with the receptor (GFP-ER in green) and immunofluorescence labeling of the active form of RNA polymerase II (red) in the PRL-HeLa cell line. The line contains a stable multicopy integration of a reporter gene based on the prolactin promoter/enhancer. Two hours after treatment with the sex hormone estradiol, there was a marked decondensation of the gene locus and an increased recruitment of polymerase to the activated promoter/enhancers. Images courtesy of Michael Mancini, Baylor College of Medicine.

The finding could have implications for understanding biochemistry and the disease process. What’s more, the technique that uncovered this fact could prove useful in quantifying the response to drugs or other therapy. The ultimate result of such methods could be personalized medicine.

Surprising results

The uncoupling of decondensation to mRNA accumulation was initially surprising, noted team leader Michael A. Mancini. “The fact that the condensation happened relatively quickly, got to maximal size and did not fluctuate over time but the message did — that was the big surprise.”

Mancini is an associate professor of molecular and cellular biology at Baylor College of Medicine. He noted that some biochemical data from previous tissue studies suggested that there could be rising and falling levels of mRNA. However, there had been no cellular context to this data and, hence, no knowledge of the state of the chromatin during these fluctuations.

As reported in the May 28, 2008, issue of PLoS One, the researchers provided that context thanks to two enabling technologies. One was a cell line, PRL-HeLa, containing a multicopy visible prolactin enhancer/promoter array integrated into its genome. For easy visualization, the investigators had introduced a green fluorescent fusion protein of estrogen receptor-α into the cell line. This made analysis of the response to stimuli simpler and made it easy to follow the response both spatially and temporally. Likewise, this made it feasible to optically track large-scale chromatin modification and the accumulation of mRNA at the array.

BREstrogen_Fig-2_pastedGraphic2.jpg
High-resolution colocalization of fusion green fluorescent protein with the estrogen receptor-α (GFP-ER in green) and dsRED2 reporter gene mRNA (red) in the new cell line PRL-HeLa is shown. Two hours after treatment with the sex hormone estradiol, there was a marked chromatin decondensation of the locus and increased reporter gene mRNA (roughly fivefold over the control).

Turning this information into quantitative data required the second enabling technology: high-throughput microscopy. For this, the researchers employed an imaging cytometer from Beckman Coulter of Fullerton, Calif., using one channel to automatically find focus on nuclei stained with the DNA-binding fluorescent dye DAPI. When excited by ultraviolet light, the dye has a blue emission and so was spectrally distinct from the green emission of the fluorescent fusion protein. The investigators monitored the fusion protein with a second channel of the instrument.

The importance of imaging

They used a proprietary algorithm provided by the instrument maker to identify and quantify the PRL array targeted by the fusion protein. The image acquisition and processing were almost completely automated, an important advantage given the requirements of the research. “Valeria Berno, the lead postdoctoral associate, needed to take a lot of pictures and have them in focus at high magnification,” Mancini said.

The research, he added, involved thousands of images. With the automated setup, the data from dozens of coverslips and hundreds of wells in multiwell plates could be collected in a few hours or overnight. With manual techniques, the process would have taken days or weeks.

In their study the researchers used these enabling technologies to better understand the two modes of estrogen receptor activity regulation. One of the modes is dependent on ligands such as the sex hormone estradiol. Such ligands have a direct effect on receptor activity. The other mode is ligand-independent and indirect, arising from triggers such as epidermal growth factor. The investigators treated cells transiently, expressing the fusion protein with either direct or indirect stimuli for different times and under various conditions. They then fixed and DAPI-stained the cells, following that with high-throughput image acquisition.

They extracted measurements from those images and analyzed the data by determining various metrics, such as the size of the PRL array. Analysis of large-scale chromatin dynamics and the accumulation of mRNA over 24 h showed that the response to the growth factor consisted of a single pulse of new reporter mRNA. At the same time, there was a transient increase in the array decondensation. Use of estradiol, on the other hand, showed a waxing and waning of mRNA accumulation but a sustained increase in array decondensation.

Together, these findings indicated a stimuli-specific response pattern involving chromatin and transcript-level changes, the researchers concluded. The results also showed, somewhat surprisingly, that the production of mRNA might not be as tightly linked with the degree of chromatin condensation as had been thought.

Work in this area continues, with increased data gathering now possible because of technology improvements. During the study, the investigators had to intervene manually in the data processing to ensure accurate array measurements. Subsequent to the study, they implemented new software that can automatically deal with images at different depths, or Z-stacks, in multiple colors. That and the advent of a graphical programming language to control data handling, image analysis, statistics and reporting, have enabled complete automation of the process.

Enhancements to the software make it possible to perform hundreds of measurements per cell or fluorescent channel. These may, for example, involve the nuclear area, shape or texture. In a sense, researchers now can quantify what pathologists have traditionally visualized, Mancini said.

However, this is not a totally unmixed blessing. There are challenges involved in storing and analyzing these large data sets. The latter involves automated preparation to organize the data prior to analysis, and that currently is a problem.

“That’s the bottleneck right now,” Mancini said.


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