Tag Archives: mouse models

A heart-healthy microRNA

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A paper published June 1st in Nature Genetics began by asking how a defect in micro RNA regulation might affect the heart. The result is the surprising finding that deregulation of a single micro RNA (or miRNA), miR208-a, in heart cells severely affects the ability of heart muscles to contract.

Heart and its blood vessels

Heart and its blood vessels{credit}Leonardo da Vinci{/credit}

The authors, Jian Ding et al., deleted the gene Trbp specifically in mouse cardiomyocytes, the muscle cells of the heart. Trbp encodes an important protein for the production and regulation of miRNAs, which in turn are thought to regulate the expression levels of target genes. Because miRNAs can each have many target genes, and genes can each have many miRNAs that target them, it is thought that, generally, the effect of losing any one miRNA will be very small.

Trbp is too important to be deleted from the whole mouse. This would lead to a very early death. However, when Trbp was deleted only in cardiomyocytes, mice were able to live up to 8 months (though most died before 4 months). Immediately after birth, there were no obvious defects in the hearts of mice with the Trbp deletion, but by 3 weeks after birth there were signs of trouble. The heart chambers of these mice were dilated and the heart couldn’t contract normally. Eventually, this led to cardiomyopathy and heart failure.

Gross pathology of idiopathic cardiomyopathy. Opened left ventricle of heart shows a thickened, dilated left ventricle with subendocardial fibrosis manifested as increased whiteness of endocardium. Autopsy.

Gross pathology of idiopathic cardiomyopathy. Opened left ventricle of heart shows a thickened, dilated left ventricle with subendocardial fibrosis manifested as increased whiteness of endocardium. Autopsy.{credit}CDC/Dr. Edwin P. Ewing, Jr. {/credit}

 

To find out how deletion of a gene for general miRNA regulation could have such a specific effect on heart muscle function, the authors looked for genes that were aberrantly expressed in the deletion mice. They found that the levels of genes for slow and fast twitch muscle fibers were not expressed at their normal ratios due to increased expression of a gene called Sox6. They also noticed that several miRNAs were not processed normally in Trbp deletion mice. One miRNA in particular, miR-208a, was found to be responsible for misregulation of Sox6. Adding normally-processed miR-208a back into the Trbp deletion mice completely reversed the heart defects.

We asked the senior author of the study, Da-Zhi Wang from Harvard Medical School, to tell us a little more about the background of this fascinating discovery.

What motivated you to choose Trbp for this study? Why did you specifically choose to knock this gene out in cardiomyocytes?

We have been interested in microRNA biogenesis [the creation of miRNAs from longer RNA precursors] and the underlying functional mechanisms for the past ten years. It has been generally thought that most (if not all) microRNAs are produced and processed in a similar manner, where they are transcribed by RNA polymerase II and processed by the microprocessor (Drosha/DGCR8 complex) in the nucleus.  In the cytoplasm, they are further processed by Dicer and Dicer co-factors.  Trbp is interesting because, unlike Dicer, Drosha and Ago2, which were primarily characterized as regulators of miRNA pathway, Trbp was originally identified as an RNA binding protein binding to HIV RNA, suggesting it may play a distinct role in RNA biology. Interestingly, several previous studies, primarily conducted in vitro, suggested that Trbp could function as a Dicer binding partner to modulate miRNA biogenesis. However, few studies have directly linked the biochemical properties of this RNA binding protein to any physiological function.

Our lab studies molecular mechanism of the cardiac system. We have previously knocked out Dicer in mice and we found that cardiac-specific deletion of Dicer resulted in the blocking of miRNA biogenesis. As a consequence, we found that cardiac function was impaired in Dicer mutant mice (Chen et al., PNAS 2008). In order to have our hands on Trbp, we thought that we would first generate cardiac-specific Trbp knockout mice, which will enable us to examine its biological function as well as to define the molecular mechanism in a defined biological system.

Were you surprised to see such a significant cardiac defect in these mice?

Not really. As we predicted that Trbp is functionally important in the heart, we actually expected to see the cardiac defects. On the other hand, we were aware of the presence of a Trbp homologous gene, Pact, which potentially could have resulted in functional redundancy.

What was your initial reaction when you learned that only 60 miRNA transcripts were affected by Trbp deletion? Was this expected? Why or why not?

We were indeed surprised to see that only 60 miRNA transcripts were affected in Trbp-KO hearts.  As indicated earlier, prior studies have suggested that Trbp functions as an obligate co-factor of Dicer to regulate miRNA biogenesis globally.  Our study, on the other hand, clearly demonstrated that the effect of Trbp on miRNA expression is context-dependent.  We would like to emphasize that most previous Trbp-related studies were conducted biochemically in vitro, whereas we took a mouse genetic approach to delete the Trbp gene in vivo. These apparently inconsistent observations further underscore the importance of genetic studies in vivo.

What do you see as the most significant aspect of this study?

Re-expression of a single miRNA (miR-208a) or knockdown of a single mRNA target (Sox6) could fully rescue the loss of Trbp phenotype. This may make us rethink the functional mechanisms of miRNAs and their targets.

How do you envision others using this study in the future?

Although our study clearly demonstrated that the function of Trbp is mediated by miR-208a and Sox6 in the heart, it remains to be determined of how Trbp specifically regulates the biogenesis/processing of miR-208a. In other words, how is the specificity of Trbp conferred/determined?

Another important finding of our study is that the Trbp-miRNA-mediated fast- and slow-twitch myofiber gene program is correlated with cardiac function.  The fast- and slow-twitch gene regulation has been extensively studied in skeletal muscle (and linked to skeletal muscle function), but not in heart.  It will be important to investigate whether human cardiomyopathy patients are linked to dysregulation or mutation of the fast- and slow-twitch myofiber program.

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. 

Cancer: what’s Down syndrome got to do with it?

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A Wright's stained bone marrow aspirate smear of patient with precursor B-cell acute lymphoblastic leukemia.

A Wright’s stained bone marrow aspirate smear of patient with precursor B-cell acute lymphoblastic leukemia. {credit}VashiDonsk via Wikipedia{/credit}

Trisomy 21 (having 3 copies of chromosome 21) is most well known as the cause of Down syndrome. But as you can imagine, having an entire extra copy of a chromosome has other negative consequences as well. For one, people with Down syndrome are 20 times more  likely than the average person to develop a severe form of leukemia, B cell acute lymphoblastic leukemia (B-ALL). Two recent studies have helped further our understanding of the molecular disturbances that take place in trisomy 21.

In Nature, Audrey Letourneau et al. took advantage of a rare situation to identify the genes that are misregulated in Down syndrome. The researchers profiled the transcriptomes of identical twins that differed in one crucial aspect: one twin had 3 copies of chromosome 21, while the other had a normal complement of chromosomes. (The samples were collected post-mortem from the fetuses, with the permission of the parents). This approach allowed the researchers to avoid any noise from irrelevant differences, since the genes of both twins would be identical. (Read the article in The Scientist about this study here).

Not surprisingly, they found that trisomy 21 causes gene regulation problems on all chromosomes. Misregulated genes are organized along the chromosomes in domains, and these domains were defined by changes to the chromatin methylation patterns. Importantly for future research efforts, they also showed that the corresponding genomic regions in the mouse model for trisomy 21 were similarly modified compared to control mice.

In a second paper published online this week by Andrew Lane et al. in Nature Genetics looked specifically at the relationship between Down syndrome and B-ALL.  The authors identified two genomic events as the drivers behind Down syndrome-related B cell acute lymphoblastic leukemia (B-ALL): overexpression of the nucleosome remodeling protein HMGN1 and changes in histone methylation marks. (You can read the Dana-Farber Cancer Institute’s press release about the study here).

Through a very meticulous set of experiments, they first show that just having an extra copy of a small region of chromosome 21 (or in this case, the corresponding mouse chromosome, 16) with 31 genes is sufficient for giving B cell precursors the ability to self renew indefinitely—the first step to cancer formation. From there, they identify and confirm a single driver gene on chromosome 21, HMGN1, as being expressed at unusually high levels. This high expression of HMGN1 causes a decrease in one type of methylation (H3K27me3), leading to overexpression of genes usually carrying both H3K27me3 and another histone mark, H3K4me3.

Trisomy 21 karyotype. All the normal chromosomes + 1.

Trisomy 21 karyotype. All the normal chromosomes + 1. {credit}Wikipedia{/credit}

Interestingly, the authors of Letourneau et al. mention HMGN1 as a good candidate for regulating the genome-wide chromatin modifications they found. The accompanying News & Views article by Benjamin Pope and David Gilbert note that HMNG1 should be a target of future study in Down syndrome. Looks like the authors of Lane et al. got the message far in advance!

So a pattern emerges: changes in chromatin methylation patterns are a key event in trisomy 21 overall and in Down syndrome-associated B-ALL specifically. Dr. Lane, lead author of the paper in Nature Genetics  wonders “Could this [chromatin modifications] be a unifying theme for phenotypes (not only cancer) associated with DS?” As I mentioned earlier, having 3 copies of this chromosome is bad for a number of reasons: higher risk of B-ALL and testicular cancer, vision and hearing problems, thyroid issues, higher risk of type I diabetes, gastrointestinal issues, low or no fertility and the more widely known neurocognitive isssues. Future studies on epigenetic changes in Down syndrome, and the regualtion of HMNG1, should be able to unravel the mechanisms underlying these different aspects of Down syndrome.

Outside of Down syndrome, these 2 studies may also lead to a better understanding of (and hopefully new treatments for) cancers caused by epigenetic changes. As the article by Lane et al. showed, changes to the chromatin landscape allowed B cell precursors to make that first step toward leukemia. By understanding how this happens, we can start to find ways to prevent it.