Domesticated transposons help feng shui DNA

Science Spotlight

Domesticated transposons help feng shui DNA

From Malik (Basic Sciences Division, Fred Hutch) and the Yao (Academia Sinica, Taiwan) Laboratories

March 20, 2017

Tetrahymena thermophila cell. The micronucleus is labeled in green and the macronucleus appears blue. Microtubules are labeled in red, outlining the many cilia on the cell wall.

Image courtesy of Chao-Yin Cheng (Yao Laboratory, Academia Sinica of Taiwan)

Click for high-res version

Multicellular organisms have two general types of cells: germline and somatic.  Germline cells carry a "master copy" of all of our genes, the genome, while somatic cells have altered the structure or composition of their genome to perform their unique functions.  In the human immune system, lymphocytes rearrange their genomes to generate armies of genetically unique T and B cells to fight pathogens.  In the single-celled eukaryote Tetrahymena thermophila, which lives in freshwater ponds, there are two copies of the genome: the full, master copy is kept silent in the micronucleus while an edited version is made, copied many times and used in the macronucleus.  Thus, this unicellular organism has both a germline and a somatic copy of its genome.  When Tetrahymena reproduce, they degrade their old copies of the genome kept in the macronucleus and then make new copies from the micronucleus.  Presumably for optimization purposes, the cell cuts out sequences from the copies that do not need to be expressed in the macronucleus.  These sequences are called "internal eliminated sequences" (IESs) and they make up 34% of the Tetrahymena genome. 

While the excision of these sequences has been found to depend upon an enzyme very similar to a "mobile genetic element" called a transposon (also known as a "jumping gene"), the sequences that are actually excised do not appear to depend upon recognition sequences which transposons are known to recognize.  Instead, the snipping of the Tetrahymena genome by transposon-like enzymes appears to be directed largely by signals in the way the DNA is packaged together, which can be called its epigenetic state.  While previous analysis identified a transposon-like gene TPB2 (Tetrayhymena piggyBac-like 2, where piggyBac is a type of transposon) responsible for excising internal eliminated sequences in Tetrahymena depending on their epigenetic state, the trail of breadcrumbs between transposon-like excision and excision depending upon the epigenetic state of the DNA was missing.  Scientists in Meng-Chao Yao's Laboratory (Institute of Molecular Biology, Taipei, Taiwan) and their collaborators in the Malik Laboratory (Basic Sciences Division, Fred Hutch) hypothesized that studying the additional genes in the Tetrahymena genome that resemble transposons may shed light on how transposons were domesticated by the cell to recognize internal eliminated sequences defined by epigenetic, not genetic states of the DNA.  

To this end, they focused on two other genes in Tetrahymena that resemble transposons: TPB1 and TBP6.  They found that the gene TPB1 is not responsible for the majority of excision events but that, without TPB1, cells retain 20 internal eliminated sequences in their macronucleus, 12 of which disrupt the expression of genes that make protein ("coding" sequence or genes).  One of these 12 disrupted genes, DOP1, is required for the integrity of the vacuole, a structure that regulates osmotic pressure.  If the cell can't respond well to differences in osmotic pressure, it is less able to cope with environmental changes. Lead author Chao-Yin and his colleagues found that artificially re-introducing functional DOP1 into TPB1-deficient Tetrahymena rescued many of the vacuole defects. 

Wild-type Tetrahymena cells viewed using a light microscope.

Image courtesy of Chao-Yin Cheng (Yao Laboratory, Academia Sinica of Taiwan)

Click for high-res version

DOP1-deficient cells have large vacuoles.

Image courtesy of Chao-Yin Cheng (Yao Laboratory, Academia Sinica of Taiwan)

Click for high-res version

Tetrahymena piggyBac-like 1 (TPB1) is responsible for the excision of 12 internal elimated sequences (IESs) that are found in genes. (Left) Schematic of an IES, shown as a gray box, being excised in the macronuclear (MAC) copy of the Tetrahymena genome but remaining in the "master" micronuclear (MIC) copy of the genome. In cells lacking TPB1 (bottom), the IES stays in the macronuclear copy. (Right) Sequencing reads from three different wild-type (normal) and TPB1-deficient strains of Tetrahymena. All wild-type strains lack the IES sequence in the macronucleus while the TBP1-deficient strains retain the IES boxed in red.

Image courtesy of Janet Young (Malik Laboratory, Basic Sciences Division)

Click for high-res version

Janet Young, staff scientist in the Malik Laboratory (Basic Sciences), along with members of the Yao and Malik Labs analyzed the sequences surrounding the 12 internal eliminated sequences deleted by TPB1 that are found inside coding regions.  Fortuitously, they found a short (12bp) motif on either side of this select subset of gene-interrupting internal eliminated sequences, which is very similar to typical transposon-signal motifs called TIRs, terminal inverted repeats.  They searched the genome for these motifs, properly oriented and spaced, and found 127 total copies of it in the Tetrahymena genome but 118 of those were not associated with deleted sequence under any condition.  This indicates that the TIR-like motifs are not sufficient for excision. 

The scientists confirmed that the TIR-like motifs they found are required for excision of one of the 12 internal elimated sequences they identified as depending upon TPB1.  When they deleted one of the two TIR-like motifs on an artificial piece of DNA with an internal eliminated sequence, no excision occurred.  Similarly, moving the location of the motif did not change the length of the excised DNA and did not direct removal of the internal eliminated sequence.  Genetic analysis identified that a 45-60bp sequence on either side of the internal eliminated sequence, including one of the 12-bp motifs, is required for recognition and removal by TPB1. 

            Said staff scientist Janet Young, " Our paper adds to previous studies that showed that the mechanism of removing the 10,000 IESs evolved from piggyBac transposons (selfish genetic elements). In this study we now also show that the IESs themselves are most likely derived from piggyBac transposons."  Additionally, their results identify a "fossil record" of the domestication of transposon enzymes to rearrange DNA in host cells.  TPB1 and TPB6 recognize inverted repeat sequences similar to typical transposons while TPB2 recognizes epigenetically defined internal eliminated sequences.  Who knows what other mechanisms have been inherited by "domesticating" transposons?

 

Cheng C, Young JM, Lin CG, Chao J, Malik HS, Yao M.  2016.  "The piggyBac transposon-derived genes TPB1 and TPB6 mediate essential transposon-like excision during the developmental rearrangement of key genes in Tetrahymena thermophila."  Genes & Development. 30:2724-2736.  

This research was funded by the Howard Hughes Medical Institute, the National Institutes of Health, the Ministry of Science and Technology of Taiwan, and the Institute of Molecular Biology, Academia Sinica of Taiwan.