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Conservation and divergence of animal and fungal cell cycle regulators after gene duplication

All living beings have important regulatory proteins to help control genes and guide cells through the cell cycle. The Buchler lab recently published two papers building on their expertise on the evolution of regulatory proteins involved in the animal and fungal cell cycles.

The cell cycle allows parent cells to duplicate all their components, most importantly their genome, and physically partition these components into two complete daughter cells. One checkpoint during this process is regulated by E2F and DP proteins in animal cells to ensure that the conditions are right for division. Without these checkpoints, cell division can lead to cancer, detrimental mutations and diseases.

E2F proteins are a family of transcription factors that have both activators and repressors. The activating E2Fs are bound to DNA, and when conditions are right, they activate genes to promote entry to the cell cycle. The Retinoblastoma (Rb) protein binds to activating E2Fs, essentially pumping the brakes on the cell cycle. Rb is one of three pocket proteins and the one most likely to mutate and lead to cancer. “It’s a potent tumor suppressor,” said Edgar Medina, a fifth-year grad student in the Buchler lab. “It interacts more strongly with activating E2Fs than other E2Fs.” The other pocket proteins, p107 and p130 interact with a broader set of E2Fs including the repressing E2Fs.

Researchers at the University of California – Santa Cruz were investigating why Rb prefers to bind to activators and not repressors and when Rb became so potent, so they reached out to the Buchler lab for help with a genomic and evolutionary perspective on the problem.

“We reconstructed the evolution of E2F and DP transcription factors and their pocket protein inhibitors across animal lineages to find the evolutionary points at which these families changed how they interact with each other,” Medina said. “We wanted to find that moment in time when Rb evolved to become so potent.”

In a study published in PNAS in May, the Buchler lab set out to find all of the E2Fs, DPs and pocket proteins from 50 different animals and pre-animals, everything from single cell amoebas, jelly fish, sharks and humans. They looked at the three pocket proteins and six important E2Fs and found that once jawed fish, like sharks, came along, there was a huge expansion in transcription factors. “The changes that made Rb a better tumor suppressor all started happening when the lineage of jawed fish branched off from eels and lamprey,” Medina said.

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This study is important because it identifies the time and the protein-protein interactions that make Rb interact more strongly with activating E2Fs. This evolutionary blueprint could be useful for understanding similarities and differences in cell cycle and the genetics of cancer in different animals.

Not only has the Buchler lab been tracing the evolution of animal transcription networks, they have also been hard at work researching regulatory proteins in the cell cycles of fungi.

Most fungi don’t have E2F transcription factors. Instead, they have SBFs, probably due to a virus they contracted over a billion years ago. SBF, though, works like E2F. It, too, controls important genes for activating the cell cycle.

Yeast, unlike any other fungi, have two transcription factors: SBF and MBF. Both regulate genes upon entering the cell cycle, but researchers aren’t sure why yeast have two or what roles the transcription factors play.

Researchers from Ben Gurion University and University College London wanted to know what the differences are in the genes regulated by SBF as compared to MBF, and what happened during evolution to result in having two transcription factors to begin with, so they sought out the Buchler lab’s expertise in genomics. Their research findings were published in PLOS Genetics in May.

The Buchler lab worked with the genomes of different yeasts. “Researchers have already sequenced these genomes,” Medina said, “so we had budding yeast, its close cousin, cousins 100 million years out, 300 million years out, and on down the line.” The lab was able to follow the SBF and MBF proteins all the way back to a yeast genome with only one transcription factor.

The research team at Ben-Gurion University made evolutionary chimeras of the SBF or MBF transcription factor of the budding yeast. They took a cousin’s SBF or MBF and plucked out the DNA binding domain and grafted it with the budding yeast, effectively making a Franken-transcription factor. They tested the ability of these chimeras to regulate SBF genes in the budding yeast.  Then repeated the process, examining different chimeric transcription factor complexes, until they worked back far enough to find yeast with only one binding domain.

These experiments provided insight on what the transcription factors like to bind to. They used RNA-sequencing to see which genes were turned on by the chimeras and which weren’t. From there, they separated the genes that turned on from those that didn’t and looked for certain DNA motifs. “Our data suggests,” said Medina, “that whilst SBF is the likely ancestral regulatory complex, the ancestral DNA binding element is more MCB-like.”

Together, these two publications give researchers more insights the evolution of cell cycle networks and how gene duplication rewired these networks and the genes controlled by them.

Story was originally published on the Duke Center for Genomic and Computational Biology website on June 7, 2017. View original

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