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Whole-Body Regeneration in Worms Directed by Gene Regulatory Network

NEW YORK (GenomeWeb) – A team led by investigators at Harvard University has uncovered gene regulatory shifts contributing to whole-body regeneration in the acoel worm Hofstenia miamia, or three-banded panther worm — a creature capable of re-growing its body when lopped in half.

The researchers found that the H. miamia genome "is very dynamic and really changes during regeneration as different parts are opening and closing," first author Andrew Gehrke, a post-doctoral researcher in the group of senior author Mansi Srivastava, an assistant professor of organismic and evolutionary biology at Harvard, said in a statement. 

As they reported in Science this week, the researchers began by putting together a 950 Mb draft genome assembly for H. miamia using short-read sequencing, a series of assembly methods, and nanopore long-read assembly validation, identifying more than 22,600 protein-coding genes with the help of tissue transcriptome data.

By incorporating chromatin accessibility profiles, transcription factor binding patterns, RNA interference on candidate genes, and other regulatory clues from regenerating three-banded panther worms, the team proposed a model in which an early growth response (EGR) protein orchestrates changes in chromatin openness across thousands of regeneration-related regions, shifting accessibility of transcription factor motifs involved in regeneration.

"Combining [EGR gene] inhibition with chromatin profiling suggests that [the early growth response protein] functions as a pioneer factor to directly regulate early wound-induced genes," the authors reported, adding that "genetic connections inferred by this approach allowed the construction of a gene regulatory network for whole-body regeneration, enabling genomics-based comparisons of regeneration across species."

The team noted that prior studies — including research focused on candidate genes or gene expression profiling alone — have revealed some of the processes at play during wound healing in general and tissue regeneration in particular. Even so, a complete picture of the genetic, epigenetic, and regulatory features that prompt regeneration and ensure appropriate tissue repatterning remains more mysterious, prompting the current three-banded panther worm analyses.

Starting with DNA from up to five three-banded panther worms grown in the lab from a wild worm population, the researchers used Illumina paired-end and mate pair sequencing to generate sequences spanning the H. miamia genome to an average depth of nearly 90-fold. They applied a series of SOAPdenovo, PRICE, SSPACE, and Dovetail Chicago assembly methods to come up with a 950 Mb draft genome, evaluating its contiguity with long read data from Oxford Nanopore Technologies.

The team turned to transcriptome data to identify 22,632 predicted protein-coding genes, including most of the metazoan genes described in the Benchmarking Universal Single-Copy Orthologs (BUSCO) database, along with a large proportion of repetitive sequences.

From there, the researchers dug into regulatory processes involved in whole-body regeneration using ATAC-seq (assay for transposase-accessible chromatin sequencing) and RNA-seq on wound sites from bisected three-banded panther worms at the wounding time and after three, six, 12, 24, and 48 hours of regeneration.

Along the approximately 18,000 chromatin peaks, corresponding to changes in open or closed chromatin regions during regeneration, the investigators saw related alterations in accessibility of transcription factor binding motifs. Based on the binding motifs that became more accessible in this process, and on results from subsequent RNA interference experiments targeting the EGR gene, they suggested that these dynamics are influenced by EGR protein activity.

"Basically, what's going on is, the noncoding regions are telling the coding regions to turn on or off," Gehrke said. "A lot of those very tightly packed portions of the genome actually physically become more open, because there are regulatory switches in there that have to turn genes on or off."

The team saw potentially related variability in the accessibility of early growth response transcription factor motifs in new ATAC-seq data for a planarian called Schmidtea mediterranea that is capable of tissue regeneration.

"By viewing whole-body regeneration through an epigenomic lens, we uncovered a regulatory mechanism underlying dynamic gene transcription during regeneration," the authors concluded, noting that the same strategy "can be applied to established and emerging model systems to gain a deeper mechanistic understanding of the transcriptional cascades that underlie the phenomenon of regeneration."