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ChIP-Seq Study IDs Transcription Factor Binding Pattern Divergence in Vertebrates

By Andrea Anderson

NEW YORK (GenomeWeb News) – Genome-wide transcription factor binding patterns vary dramatically from one vertebrate lineage to the next, according to a paper appearing in the online version of Science today.

Using chromatin immunoprecipitation combined with high-throughput sequencing, a UK-based research team mapped genome-wide binding patterns for the transcription factor CEBPA in liver samples from five vertebrate species. In the process, they found a slew of binding pattern differences between species — results that they verified by looking at a second transcription factor, HNF4A.

Together, the researchers' findings add to a growing body of research hinting at rapid shifts in transcription factor dynamics during vertebrate evolution.

"Most people's operating assumption five years ago was that most transcription factor binding would actually be conserved," co-corresponding author Duncan Odom, a systems biology and oncology researcher affiliated with Cancer Research UK and the University of Cambridge, told GenomeWeb Daily News.

Since then, though, there have been clues that that isn't the case, he added. For instance, in 2002 a team of researchers led by Emmanouil Dermitzakis, then at Pennsylvania State University and now at the University of Geneva, found binding site differences in humans and rats. And in 2007, Odom and his co-workers reported on tissue-specific transcription factor binding site discrepancies in mice and humans.

Meanwhile, a pair of recent papers in Science revealed inter-individual transcription factor binding differences in humans and yeast.

In contrast, a recent PLoS Biology study found that binding patterns for six transcription factors were very similar in two different Drosophila species, though the levels of binding varied.

In an effort to explore the nature and extent of transcription factor binding differences in vertebrates, Odom and his colleagues used chromatin immunoprecipitation combined with Illumina GAII sequencing to map CEBPA binding patterns in healthy liver tissue from five species, each from a different vertebrate order: humans, mice, dogs, short-tailed opossum, and chickens.

They then used an algorithm to map parts of the genome bound by the transcription factor and Ensembl species alignment to detect overlapping binding patterns between species.

In their subsequent experiments, the researchers used a similar approach to look at binding patterns for a second transcription factor, HNF4A, in liver tissue from humans, mice, and dogs.

In general, the team noted, the transcription factors bound 16,000 to 30,000 spots in the mammalian genomes and about half as many sites in the chicken genome.

But while binding patterns were similar between individuals from the same species, the team found numerous differences between the species. Within the mammalian species, the team found that ten to 22 percent of binding sites overlapped between any two placental animals.

Because animals in the placental lineage are thought to have a shared common ancestor going back some 80 million years, the researchers explained, the findings suggest transcription factor binding patterns can shift quite quickly.

The differences were even more pronounced when they compared the placental mammals or "eutherians" with the opossum, a member of the marsupial or "metatherian" group. For example, the team found that in the liver samples tested the opossum shared six to eight percent of binding sites for CEBPA with mice, dogs, or humans.

The overlap dropped to just two percent when they compared CEBPA binding sites in chickens and humans.

"What shocked us was the rapid loss along each lineage," Odom said.

The team did find a few dozen "ultra-shared" CEBPA binding sites in all five species. These tended to fall near genes involved in liver function, Odom noted, including some that may be involved in early embryogenesis.

"[S]hared regions represent examples of deeply conserved regulatory architecture featuring multiple motifs at specific sequence locations maintained through vertebrate evolution," the researchers noted. "The most conserved of these, the five-way ultra-shared sites, also exhibit the strongest sequence constraint."

Even so, as many as 40 percent of motifs did not show significant changes even when binding patterns did, leading them to speculate these areas "may represent cases of epigenetic redirection, yet-to-be characterized SNPs or indels, or loss of nearby genomic binding partners."

"Taken together," they added, "the steady accumulation of small changes in the genetic sequence appears to rapidly remodel thousands of transcription factor binding sites."

In their subsequent experiments, the team found that there was no more sequence constraint in regions near genes whose expression relies on the transcription factors than there is at other binding sites.

"Despite the rapid gain and loss of [transcription factor] binding events in mammals, tissue-specific gene regulation seems to be maintained by identifiable regulatory architectures that can be independent of sequence constraint," the team concluded.

In addition, when binding sites are lost in one species, the researchers reported, other nearby regions only seem to be in a position to pick up the slack some of the time: roughly half of the transcription factor binding sites missing in one of the lineages fell near other binding sites.

Of these, about half of the alternative binding sites were totally different from those detected in the other species tested, Odom explained, while the other half were shared with one or more of the other species.

Down the road, the team plans to not only investigate transcription factor binding patterns in other tissues, but also at various stages of development. They also intend to investigate binding patterns for sets of multiple transcription factors.

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