Monthly Archives: December 2014

A piggyBac ride to pancreatic cancer genes

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A cluster of pancreatic cancer cells. Scanning electron micrograph

A cluster of pancreatic cancer cells. Scanning electron micrograph{credit} Anne Weston, LRI, CRUK. https://wellcomeimages.org{/credit}

Pancreatic cancer is a highly heterogeneous disease that often has a poor prognosis. Development of drugs or treatment strategies to target cancers, including pancreatic cancer, depends on identifying the drivers of disease. These are the genes that promote carcinogenesis and coordinate development of the cancer. But by the time a patient is diagnosed, it can often be very difficult to tell which of the many mutations present in the tumor are actually disease drivers, and which are just along for the ride.

A new paper published in Nature Genetics describes a strategy for finding the genetic drivers in pancreatic cancer. The authors used a forward genetic screen in mice that targets a particular transposable element, the piggyBac transposon, to the pancreas. When the transposon inserts itself into the genome, it disrupts genes, causing mutations that may then lead to cancer. By using the screen in “sensitized” mice (i.e., mice with particular mutations that will accelerate disease progression), the authors were able to cause pancreatic tumors to form in the mice. The genetic changes in these tumors were then examined to identify which genes are most often targeted by the transposon.

Other studies have been published recently that use a similar approach to find drivers of other types of cancer. Neal Copeland, Nancy Jenkins and colleagues pioneered the use of Sleeping Beauty transposon mutagenesis to screen for genes important in cancer, including a recent study in liver cancer associated with hepatitis B. Rama Khokha and colleagues recently used the Sleeping Beauty mutagenesis method to identify driver genes responsible for the formation of sarcomas.

These screens have been very successful; there have even been Sleeping Beauty screens for pancreatic cancer driver genes (here and here). However, Roland Rad and colleagues found that a Sleeping Beauty transposon screen was not ideal for studying certain types of pancreatic cancer. In addition, Sleeping Beauty and piggyBac have different insertion preferences, so the tools complement one another. This means that, while some sets of genes identified with the two methods do overlap, there are other genes that can only be found by using one or the other method. Importantly, Dr. Rad and colleagues observed different histological subtypes of pancreatic cancer in mice when using piggyBac, which were not observed using Sleeping Beauty.

We asked Dr. Rad, one of the lead authors of the study, to tell us a little more about the paper.

For readers unfamiliar with insertional mutagenesis screens, could you tell us what a piggyBac transposon is and how it was discovered?

Transposons are mobile DNA segments that can move around the genome. They were first discovered by Barbara McClintock more than 50 years ago. The DNA transposon piggyBac encodes a transposase, which moves the transposon from one genomic locus to another by a cut-and-paste mechanism. Transposable elements, which have been widely used for genetic screening in bacteria, yeast, arthropodes and nematodes, had been inactivated during vertebrate evolution and were hence not available as genetic tools in higher organisms until recently. Successful efforts over the past ten years to make piggyBac work in mammalian cells motivated us to target it to the mouse genome and test its applicability for somatic mutagenesis in mice.

Lifecycle_of_the_Piggybac_Transposon_System

The PB transposase recognizes the specific inverted terminal repeats (ITRs) at each end of the transposon. PB then “cuts” the transposon out of its original location and moves it to a new, random location in the genome with a TTAA sequence. {credit}Transposagenbio via Wikimedia Commons{/credit}

How do screens like this (performed in mice) inform us about human cancer? What is the advantage of this approach over direct sequencing of patient tumors?

Genetic screening and cancer genome sequencing are highly complementary approaches. Sequencing and array-based analyses of patient tumors can very accurately identify all classes of somatic alterations in cancer. However, many of these changes are difficult to interpret. For example, hundreds or even thousands of genes are found to be transcriptionally or epigenetically dysregulated within a single patient´s tumor, meaning that they are not mutated but just being turned on or off. Pinpointing the few cancer-causing events among these large gene sets is extremely difficult. Likewise, copy number variation in cancer often affects large chromosomal segments, and for 75% of commonly amplified or deleted regions in human cancer, the cancer-causing genes have not yet been identified.

PiggyBac screening can tremendously facilitate this “search for the needle in the haystack” because transposons jump directly into the relevant genes. Even if a cancer gene is unequivocally identified through sequencing (for example based on its mutation), understanding downstream complexity can be difficult. Many cancer genes (e.g. methyltransferases, histone modifying enzymes, DNA repair genes) have large numbers of targets. Others (e.g. Ras) have many effector pathways that are used differently in various cancer types or have numerous interaction partners. Here again, unbiased genetic screening can identify ‘players’ at all levels of these cascades and can directly pinpoint important downstream effectors. Moreover, genetic screening provides a first level of biological validation of cancer genes and functional insights at an organismal level. These are some examples, which show that transposon-based screening can answer biological questions that cannot be systematically addressed by other approaches to cancer genome analysis.

What was the most surprising aspect of this study?

The screen produced numerous unexpected results. This is the beauty of a hypothesis-free forward genetic approach. We have discovered a large set of novel transcription factors involved in pancreatic cancer and shown that transposons can be used to identify cancer-relevant non-coding regulatory regions in the genome. The study also showed that insertional mutagenesis can induce different subtypes of pancreatic cancer and can dissect underlying genetic causes.

What was the biggest challenge your group faced during the course of the study?

The biggest challenge was to make the system work in mice. PiggyBac originates from Trichoplusia ni, the Cabbage moth. We modified PiggyBac and introduced it into the mouse genome. Naturally, we did not have a priori knowledge as to how the system would behave in the mouse. Will it be efficient enough to achieve transposition? How many transposons per cell will we need to achieve tumor induction in individual tissues? Do high transposon copies induce toxicity? How will the genetic elements (enhancers, gene trapping elements etc.) affect the phenotype? We addressed these questions by developing many different transposon mouse lines and systematically exploring PiggyBac’s characteristics in vivo.

How do you see your results being used in the future by other researchers or clinicians working with pancreatic cancer?

The study has produced rich biological insights and large sets of putative novel “players” in pancreatic cancer. Researchers will use this knowledge and take individual aspects further, e.g. perform in depth analysis of individual genes discovered in our screen or test whether they are targetable. Our genome-wide screen adds further pieces to pancreatic cancer´s “puzzle” in order to better understand the complexity of the biological processes driving tumorigenesis. We hope that this will ultimately help guide the development of novel therapeutic strategies.

You can find the paper describing this study here. More information about Dr. Rad and the piggyBac transposon system can be found here. 

Mentoring for success in science

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Success_imageOn November 14, the Junior Faculty at Karolinska Institutet in Stockholm, together with Nature Genetics, hosted a workshop for early-career researchers about mentoring in the sciences. The goal of the workshop was to identify what postdocs and new faculty members wanted from a potential mentor and how the institute could go about establishing a formal mentoring program. The workshop was a direct result of a previous workshop at KI, also co-organized by Nature Genetics. A commentary about that workshop can be found here.

Formal mentoring programs, while rare, do exist at other institutions. For example, one of the day’s speakers, Pam Ohashi, spoke about the mentorship program at the University of Toronto. Professor Ohashi spoke about the need to convince the institute that mentoring is important and will benefit the institute in the long run. In addition, it is important to provide incentives for mentors, such as including mentoring outcomes in annual performance reviews. In a formal mentoring program, an official within the institute or department (such as the department or division chair) will pair mentors and mentees. Together with the mentee, the mentor should outline an implementation plan so that specific goals can be set and progress toward them monitored. Professor Ohashi also emphasized that mentoring needs to be flexible and tailored to the specific individual. Common questions mentors had for mentees, in her experience, were related to personnel management, how to navigate the promotion process, how to write successful grant applications and what expectations should be set for trainees.

We also heard from two previous recipients of the Nature awards for mentorship in science: Barbara Demeneix and Andrew McMichael. Professor Demeneix also emphasized the point that mentoring should be a part of the career assessment for professors. This is because both the institute and mentor benefit from mentoring, not just the mentee. She also noted that mentors should be mindful of particular difficulties faced by women when mentoring young female colleagues. Professor McMichael pointed out that scientists can have many mentors, both formal and informal, throughout their careers, and that networking (such as at conferences) is crucial especially for identifying potential informal mentors. He also made an important point in that mentees have duties to their mentors, not just the other way around. You shouldn’t only contact your mentor when you need something from them.

The need for incentives for mentors was emphasized by nearly all of the speakers. Although, as one speaker noted, mentoring future scientists is an essential part of the scientific system, professors are busy and may see it only as an added burden. What kind of incentives, and how they might be implemented, was a topic of discussion. 

Telemachus and Mentor in the Odyssey

Telemachus and Mentor in the Odyssey{credit}Wikipedia{/credit}

Another issue discussed was how to maintain a mentoring program once one was established. There should be a specific person in charge of pairing mentors and mentees and making sure that incoming faculty and postdocs are assigned mentors as early as possible. There was also a general consensus that there should be regular meetings between mentors and mentees and that progress of the relationship should be formally evaluated, though what criteria should be used for evaluation was an open question.

Finally, many junior faculty members noted that the number one thing they wanted from a mentor (in addition to general career advice) was access to the mentor’s network. For example, young faculty may not know who is the best person to contact for help with a specific problem, but the mentor (a more established faculty member) will likely be able to point to the right contacts.

Based on the information from the speakers and feedback from participants and department officials present at the workshop, the Junior Faculty will likely implement a pilot program to determine the best model for an institute-wide mentoring program.

Mentorship can be an important part of each stage of an academic career, and we applaud the Junior Faculty for taking this first step toward a formal mentorship program at KI. We look forward to hearing the feedback from the workshop and seeing how the program unfolds.

Uncovering the secrets of the orchid

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It seems that every day, another species of plant or animal is being sequenced. How do scientists choose which species should have its genome sequenced?

For some, such as African rice, the main consideration is whether the genome sequence will allow for improvement of agriculturally important crops. For others, including the marmoset, the interest lies mainly in the connection to human evolution.

Phalaenopsis_equestris_var._leucaspis_small

{credit}Wikipedia{/credit}

Now, Zhong-Jian Liu at the National Orchid Conservation Center of China and colleagues from around the world have sequenced the genome of the orchid Phalaenopsis equestris. Besides being a popular ornamental plant (and therefore a commercially important plant) with gorgeous flowers, the orchid has another unique claim to fame. This species uses a type of photosynthesis that is different from all other plant species sequenced to date.

Orchids use a photosynthesis strategy called crassulacean acid metabolism (CAM). CAM plants make up approximately 7% of plant species. Other notable CAM plants include cacti (such as the saguaro—a native of my home state, Arizona), agave (where tequila comes from), aloe vera and pineapple.

Most plants use the C3 metabolic pathway to turn carbon dioxide (CO2) into energy (there is also a third pathway, called C4, used by about 3% of plant species). All plants use sunlight and water to incorporate the carbons from CO2 into sugar, producing oxygen as a byproduct. When it is very hot or dry, C3 plants are at a disadvantage because they cannot efficiently use carbon due to a process called photorespiration. CAM plants are specifically adapted to these extreme environments. Their specialized leaves chemically store the carbon from CO2 acquired during the night and use it for photosynthesis during the day (when their stomata are closed, to prevent water loss) .

Many orchids, such as the species sequenced in the new paper, are epiphytes, meaning that they do not get their water from roots in the soil, but rather from the air or rain. They would therefore need to budget their water supply. This adaptation is likely related to their use of CAM instead of C3 metabolism.

In the genome paper, the authors identified genes important for CAM and analyzed their evolutionary history. They also analyzed genes involved in flower development, to better understand how orchids develop their spectacular flowers. The paper is certain to be an important resource for future studies of plant evolution and adaptation.

We asked one of the senior authors of the paper, Zhong-Jian Liu, to tell us a little bit more about the background of this study.

Can you tell us a little about the National Orchid Conservation Center of China?

The National Orchid Conservation Center of China was established in 2006 and is located beside Wutong Shan Mountain and Shenzhen reservoir, which is a very good location for the growth of orchids. The center is aimed at conducting the conservation of Orchidaceae germplasm, improving the level of orchid protection and advancing the cause of orchid conservation in China.

The center now owns the most endangered orchid species in China and there are more than 1,000 Chinese orchids belonging to international and national first and second-class protective orchids. There is a herbarium, tissue culture room and special library for orchids at the center. The herbarium has 3,835 specimens and 110 type specimens of orchids, and 243 animal and plant fossil specimens related to orchid evolution, which is the most in China. More than 1,800 books and 20,000 audio-visual documents are stored in the library. In academic research, 187 papers and 14 monographs have been published in China and abroad. “Pollination: Self-fertilization strategy in an orchid” was published in Nature and summarized by Year in Review 2006 and included in Book of the Year 2007 by Encyclopaedia Britannica.

The orchid genome represents the first genome sequence of a CAM plant. Why do you think this is so significant and how will it affect plant research in the future?

The CAM pathway for photosynthesis is indeed of importance. It not only leads to more efficient power conversion, but also strengthens the adaptation to harsh environments, especially drought, in comparison with C3 plants. Meanwhile, research of CAM can provide new directions for breeding programs to produce neo-species with drought resistance.

In our manuscript, we found gene duplication and loss events in four of the six key gene families in the CAM pathway. These events are important to the adaptation and evolution of orchids.

What was the most surprising result of the study and why?

We consider the finding that Orchidaceae has undergone an orchid-specific whole-genome duplication (WGD) event to be the most intriguing result. WGD can trigger a tremendous burst in gene diversification within quite a short period, which provides extensive gene material for neo-functionalization, sub-functionalization or dosage strengthening. All of these outcomes can give rise to diversity in morphology, metabolism, live style, etc. that can finally result in tremendous species radiation. We think the WGD event may be linked to the success of the orchid family. There are more than 20,000 species of orchid within 880 genera.

What was the most difficult part of the study?

The unexpectedly high heterozygosity rate in the orchid genome was the most challenging aspect for us. It is extremely difficult to assemble its genome using the raw reads. But we finally overcame this difficulty via the use of diverse assembly software packages, optimization of their core parameters and verification with the complement of the BAC sequences. Finally, we accomplished a very accurate, complete genome assembly.

There have been other genomes published with a similar level of heterozygosity to our genome, but we  were able to achieve a much more accurate and complete assembly than was the case with those genomes.

Paphiopedilum Armeniacum

Paphiopedilum armeniacum flowers {credit}Stefano via Flickr.com{/credit}

Do you have a personal favorite type or color of orchid?

I love all the species and colors of orchids very much. If there was one for me to choose, it would be Paphiopedilum armeniacum. I like its beautiful pale yellow flowers, which I have sometimes thought symbolize a yellow Chinese dream.