Double Strand Breaks For The Win

 

By Rebecca Delker, PhD

The blueprint of an organism is its genome, the most fundamental code necessary for life. The carefully ordered – and structured – composition of As, Ts, Cs and Gs provides the manual that each cell uses to carry out its diverse function. As such, unintended alterations to this code often produce devastating consequences, manifesting themselves in disease phenotypes. From mutations to insertions and deletions, changes in the sequence of nucleotides alter the cell’s interpretation of the genome, like changing the order of words in a sentence. However, arguably one of the most threatening alterations is the double-strand break (DSB), a fracture in the backbone of the helical structure, splitting a linear piece of DNA in two, as if cut by molecular scissors. While the cell has a complex set of machinery designed to repair the damage, this process can be erroneous generating deletions, or even worse, translocations – permanently reordering the pages of the manual and ultimately transforming the cell. Given the central role translocations can play in oncogenic transformation, DSBs have understandably received a bad rap; but, as can be expected, not all is black and white and it’s worth asking whether there is an upside to DSBs.

 

One such commendable pursuit of the DSB serves to expand the capabilities of our genome. While it is true that the genome is the most basic code necessary for life, many of the processes within a cell actually require changes to the code. These can occur at all levels of the Central Dogma – modifications of proteins, RNA, and even DNA. B- and T-lymphocytes, cells that provide a good amount of heft to our immune system, are notable for their DNA editing skills. Tasked with protecting an organism from billions of potential pathogens, B- and T-cells must generate receptors specific for each unique attack. Rather than encoding each of these receptors in the genome – an impossibility due to size restrictions – B- and T-lymphocytes use DSBs to cut and paste small gene fragments to build a myriad of different receptor genes, each with a unique sequence and specificity (reviewed here). For immune cells, and for the survival of the organism, these DSBs are essential. Although tightly controlled, DNA rearrangements in immune cells are mechanistically similar to the sinister DSB-induced translocations that promote cancer formation; however, rather than causing disease, they help prevent it.

 

New research published this summer points to exciting, and even more unusual uses of DSBs in the regulation of gene expression. In a quest to understand the molecular effects of DSBs that are causally linked to a variety of neurological disorders, Ram Madabhushi, Li-Huei Tsai and colleagues instead discovered a necessary role for DSBs in the response of neurons to external stimulus. To adapt to the environment and generate long-term memories, changes in the “morphology and connectivity of neural circuits” occur in response to neuron-activation. This synaptic plasticity relies on a rapid increase in gene expression of a select set of early-response genes responsible for initiating the cascade of cellular changes needed for synaptogenic processes. In their paper published in Cell this summer, the authors reveal that the formation of DSBs in the promoter of early-response genes induces gene expression in response to neuron stimulation.

 

By treating neuronal cells with etoposide, an inhibitor of type-II topoisomerase enzymes (TopoII) that causes DSB formation, the researchers expected to find that DSBs interfere with transcription. In fact, most genes found to be differentially expressed in cells treated with the drug showed a decrease in expression; however, a small subset of genes, including the early-response genes, actually increased. Through a series of in vivo and ex vivo experiments, the researchers showed that even in the absence of drug treatment, DSB formation in the promoters of early-response genes is critical for gene expression – beautifully weaving a connection between neuronal activation, DSB formation and the rapid initiation of transcription in this subset of genes.

 

The serendipitous discovery of the positive effect of etoposide on gene expression lead the researchers to focus in on the role of topoisomerases, the guardians of DNA torsion, in DSB formation. As a helical structure composed of intertwined strands, nuclear processes like replication and transcription cause the over- or under-twisting of the DNA helix, leading the DNA molecule to twist around itself to relieve the torsional stress and form a supercoiled structure. Topoisomerases return DNA to its relaxed state by generating breaks in the DNA backbone – single-strand breaks by type I enzymes and DSBs by type II – untwisting the DNA and religating the ends. While etoposide can artificially force sustained DSBs, physiological TopoII-induced breaks are typically too transient to allow recognition by DNA repair proteins. The finding that TopoIIb-induced DSBs at the promoters of neuronal early-response genes are persistent and recognized by DNA repair machinery suggests a non-traditional role for TopoII enzymes, and DSBs, in transcription initiation and regulation.

 

In fact, the contribution of TopoII and DSBs in the regulation of neuronal genes may not be so niche. Another study published recently found a similar relationship between transcriptional activation and Topo-mediated DSB formation. Using the primordial cells of the germline in C. elegans as a model system, Melina Butuči, W. Matthew Michael and colleagues found that the abrupt increase in transcription as embryonic cells switch from a dependence on maternally provided RNA and protein to activation of its own genome induced widespread DSB formation. Amazingly, TOP-2, the C. elegans ortholog of TopoII is required for break formation; but, in contrast to neuronal activation, these DSBs occur in response to transcription rather than as a causative agent.

 

These recent studies build upon a growing recognition of a potentially intimate relationship between DSBs, torsion and transcription. DNA repair proteins, as well as topoisomerase enzymes have been shown to physically interact with transcription factors and gene regulatory elements; topoisomerase I and II facilitate the transcription of long genes; and, as in neuronal cells, studies of hormone-induced gene expression in cell culture reveal an activation mechanism by which TopoIIb induces DSBs selectively in the promoters of hormone-sensitive genes. Thus, DSBs may constitute a much broader mechanism for the regulation of gene-specific transcription than previously thought.

 

Given the grave danger associated with creating breaks in the genome, it is curious that the use of DSBs evolved to be an integral component of the regulation of transcription – an inescapable and ubiquitously employed process; however, as we expand our understanding of transcription to include the contribution of the higher-order structure of DNA, the utility of this particular evolutionary oddity comes into focus. Genomic DNA is not naked, but rather wrapped around histone proteins and packaged in the 3D space of the nucleus such that genomic interactions influence gene expression. Changes in the torsion and supercoiling of DNA have been associated with histone exchange, as well as changes in the affinity of DNA-binding proteins for DNA. In addition, the necessity of topoisomerase for the transcription of long genes occurs early as RNA polymerase transitions from initiation to elongation, suggesting that the role of TopoI and II is not to relieve transcription-induced torsion, but rather to resolve an inhibitory, likely 3D, genomic structure that is specific to genes of longer length. A similar mechanism may be involved at the neuronal early-response genes. In these cells, genomic sites of TopoIIb-binding and DSB-formation significantly overlap binding sites of CTCF – a crucial protein involved in genomic looping and higher-order chromatin structure – and, again, DNA breaks may function to collapse a structure constraining gene activation. Whatever the exact mechanisms at play here, these results inspire further inquiry into the relationship between DSBs, genome topology and transcription.

 

A cell’s unique interpretation of the genome via distinct gene expression programs is what generates cell diversity in multicellular organisms. Immune cells, like B- and T-lymphocytes, are different from neurons, which are different from skin cells, despite working from the same genomic manual. In B- and T-cells, DSBs are essential to piece together DNA fragments in a choose-your-own-adventure fashion to produce a reorganization of the manual necessary for cell function. And, as is emphasized in this growing body of research, DSBs function along with a variety of other molecular mechanisms to highlight, underline, dog-ear, and otherwise mark-up the genome in a cell-specific manner to facilitate the activation and repression of the correct genes at the correct time. Here, DSBs may not reorder the manual, but, nevertheless, play an equally important role in promoting proper cell function.

 

East of Eden: The Suboptimal State of Funding in the Natural Sciences

By Asu Erden

“Don’t stay here, go to the U.S. if you can.” I heard my fair share of invaluable insight into the world of scientific research during my time at the Pasteur Institute in Paris, but this one really stuck with me. “The difference between Europe and the United States is that, if there are about ten hypotheses you can formulate to address a specific question, in France we have to choose the three or four more likely ones to test. In the U.S., they can test all ten in a heartbeat without worrying about funding.” This romanticized view of scientific research in the U.S. held some truth to it when I heard it back in 2005. But while the U.S. seemed to be the Eden of scientific funding in the early 2000s, funding cuts have had a tremendous impact on the state of research in the natural sciences on this side of the pond too.

 

A historical overview of public science funding

 

The National Institutes of Health (NIH) is the United States government’s medical research agency and the largest source of funding for medical research in the world. However over the last decade, it has not been able to fund as many projects as it used to. The funding for research project grants by the NIH – including the much coveted R01 grants which determine a lot of the tenure track positions in the natural sciences – increased steadily between 1995 and 2003, but has decreased by over 20% since 2004. “With shrinking government funding (or flat-lined, which is the same as shrinking), labs have to look for alternative sources, it’s just a fact of the situation,” admitted Dr. Heather Marshall, a former postdoctoral researcher in the Immunobiology Department at Yale University.

 

The percentage of successful research grant applications to the NIH was of 26.8% in 1995, reached 32.0% in 1999, before decreasing to 17.5% by 2013. “The state of funding most definitely shifted during my postdoc and it was most evident when discussing with PIs [ED: principal investigators], successful ones at that.  The attitude was depressing and demoralizing.  The funding percentiles of postdoctoral fellowships went down each year I was a postdoc and it became evident that attaining a fellowship was mostly out of your control,” shared Dr. Marshall. The success rate for new applications, which new faculty members rely on to start up their labs and research, has fluctuated between 18.6% in 1995, reached approximately 22% by 1999, before plummeting to 13.4% in 2013. According to the latest numbers, this means that investigators are 37.8% less likely to obtain an R01 or equivalent award today than they were in 2003. This decrease is partially explained by the NIH budget being cut by over one fifth of what it was in 1995.

 

Most of the time, it is the task of PIs – i.e. the professors running labs – to write grants lauding the importance of the research carried out in their laboratories in order to ensure the future of their line of research and that of their trainees. Graduate students and postdoctoral researchers also apply for fellowships and grants but the pecuniary benefits at stake only really affect their own work, not that of the lab overall. With the success rates for grants – new or continued – decreasing over the last decade and a half, grant applications have become particularly stressful endeavors. While PIs usually serve as a protective shield from this reality, it seems to be increasingly hard to cut the stress out…”I’ve been in research since 2003. PIs are communicating their stress more,” said Dr. Smita Gopinath, a postdoctoral researcher immunology at Yale University. The situation is not as dire in Ivy League or top tier universities. But for those PIs that do not work for private universities, loss in funding means laying off personnel or closing their lab altogether. This hinders research and in the long run biomedical advances.

 

With this decrease in funding for research in the natural sciences comes a sense of lack of job security for the extremely skilled highly educated workers that scientific researchers are. “Funding directly impacts the number of jobs available, so in that sense the state of funding absolutely played a major role in my decision to leave academia,” said Dr. Marshall. “In addition to that, I was worried that I wouldn’t get enough grants to fund the projects I wanted to take on, which would also impact my ability to train students and postdocs.  That caused a lot of anxiety and I didn’t even have a job yet!”

 

The budget fluctuations in the NIH and other science funding agencies accompany political changes in the White House, the Senate, and the Congress. During the Bush presidency, the NIH funding initially increased between 2001 and 2003 but suffered a big decrease from then onwards. Entire fields of research were completely defunded such as stem cell research. In 2009, Obama refunded these fields but the NIH has not seen an increase in budget to match inflation. Over the last few years, Republicans have increasingly criticized a number of national agencies such as the National Science Foundation (NSF) and the NIH for the type of research they fund. “Sometimes these dollars they go to projects having little or nothing to do with the public good. Things like fruit fly research in Paris, France. I kid you not!” exclaimed McCain’s running buddy, ever-entertaining Sarah Palin, during the 2008 presidential campaign.

 

The importance of funding basic sciences

 

You may argue that basic research does not always lead to biomedical advances that can be translated to the treatment, cure, or prevention of infectious or non-infectious diseases in humans. “It’s like the mosquito bed net problem. I have so many friends that work on malaria but stop and think, “My stipend can buy 200 bed nets, what am I doing with my life when I could save people directly?” This is the eternal struggle,” shared Dr. Gopinath. “Why fund basic science? Because at some point bed nets will not be effective enough.” There lies the central problem of basic science funding. It can take years between the research and its putative application for humans. This time lag has affected the mentality of the funding agencies. There is a growing gap between what the NSF, which has traditionally funded more basic research in the natural sciences, and the NIH fund, which now funds more translatable research endeavors.

 

But you never know where the next thing is going to come from. The nature of basic research is that while fuelled first by scientific curiosity, it also aims to develop our understanding of the world surrounding us in order to potentially make translational contributions. Let’s take Sarah Palin’s insightful comments on fruit fly research. In 1933, Dr. Thomas Hunt Morgan received the Nobel Prize for his research on the inheritance of physical traits. His animal model? The fruit fly. Since then, fruit fly research has led to the identification of genes as the unit of biological inheritance, to understanding how organismal ontology works, and to the now growing field of epigenetics. Working on fruit flies, scientists have also been able to identify key components of the immune system, which in the long run increased our power to medically reduce human suffering. Drosophila melanogaster – one of the more studied fruit fly species – has provided much insight into the role of genes in neurological behavior including human genes involved in autism. The irony…I kid you not Mrs. Palin!

 

 

Many of the drugs and treatments we use today are derived from such discoveries in the basic natural sciences. 40% of the medical drugs we use target a protein family known as G protein-coupled receptors (GPCRs), which translate signals external to the cell into intracellular signaling. Hormones, neurotransmitters in the brain, and even light can activate these receptors leading to biological processes such as vision, taste, smell, mood regulation, and that of the immune system. Drs. Brian Kobilka and Robert Lefkowitz won the Nobel Prize in Chemistry – not Physiology and Medicine – for solving the crystal structure of this class of transmembrane proteins. Their work focused on the chemical structure of GPCRs. This had immense ramifications in understanding how these receptors transduce signal from outside the cell by interacting with components inside the cell. Eventually, it led to the better understanding of the cellular and physiological processes these receptors are involved in which allowed the scientific community to recognize their central importance in drug targeting. From crystals we reached therapy.

 

Other examples of basic science research leading to translational advances abound. Dr. Jennifer Doudna moved to the University of California Berkeley in 2002 where she started studying how bacteria can defend themselves against viruses that infect them, also known as bacteriophages. In particular, she was interested in the clustered regularly interspaced short palindromic repeats – CRISPR – in bacterial genomes that enable these microbes to kill off bacteriophages that previously infected them. With help from collaborators, Dr. Doudna was able to identify Cas9 as the protein allowing for this viral DNA editing. Thus was born the CRISPR-Cas9 system. Since its discovery in 2012, this system has allowed the genome editing of multiple cell lines commonly used in research, but also organ-specific genetic editing in mice. The method allows scientists to make mouse lines with permanent gene silencing – also known as knock-outs – in a matter of a few weeks where it previously took them years to breed the gene of interest out.  Moreover, CRISPR-Cas9 allows researchers to delete genes of interest in fully developed mice, as opposed to embryonic deletions of genes, which can prove fatal if they are required during development. The technique is set to allow for great medical advances especially if applied to the genome editing of hematopoietic cells to cure blood disorders such as sickle cell anemia, primary immunodeficiencies (such as AIDS), and cancer. When Dr. Doudna’s research was funded, no one knew the implications it would have. It took over ten years to go from better understanding bacterial defenses against viruses to developing an incredibly potent tool that will potentiate the cure of many human woes.

 

Better tailored funding for the natural sciences involves better communication

 

There is a mismatch between the public’s understanding of the importance their taxes play in funding fundamental scientific advances and scientists pleading for politicians not to further cut their funding. As Dr. Marshall pointed out “We can’t really expect all government officials to have strong science backgrounds if they are also expected to have strong backgrounds in law, history, economics etc., but we absolutely need to have our representatives surrounded by scientists.  So from that perspective, [better science funding] does start with the general public.” The nature of scientific funding, as with all funding, is that it is limited. “We can’t rely on our achievements alone. We need to put it out there and communicate the importance of scientific research,” shared Dr. Gopinath. “We need to be managers and communicators. We do get training to collaborate with other scientists and communicate on that level. Communicating science to non-scientists is not at all on our radar!”

 

It is usually the University Office, which is in charge of what gets or does not get communicated about the scientific research carried out on campuses. While the impetus should not be on scientists to carry out science communication by themselves, additional training of PhD students, postdoctoral fellows, and PIs is required. When a journalist picks up the phone to talk about the latest advance in stem cell research, she should not face a public relations wall. Perhaps unbeknownst to the public, researchers have to meet an astounding number of training requirements annually to be able to continue carrying out their research: radiation safety, animal care and use, biosafety, laboratory chemical safety, medical surveillance for animal handlers, blood-borne pathogens and so on and so forth. To these requirements we should add science communication.  The ramifications are countless!

Neurodevelopment and the Health-Wealth Gap

 

By Danielle Gerhard

 

The famous Roman poet Virgil said that the greatest wealth is health. But what if your actual wealth affects your access to health?

 

It is estimated that more than 45 million Americans, or 14.5% of the population, live below the poverty line, according to the most recent Census Bureau survey. Although slightly lower than previous years, the poverty rate for children under 18 is still startlingly high: 19.9%. Poverty dictates how an individual lives their life and most importantly, what resources they have easy access to. Proper nutrition, environmental stimulation, basic healthcare, and family nurturing are all resources shown to aid healthy development yet are lacking in low-income communities.

 

An individual’s zip code is considered to be as much of a risk to one’s health as their genetics. Dr. Melody Goodman of Washington University in St. Louis researches the contribution of social risk factors to health disparities in local communities. One particular area in St. Louis, known as the Delmar Divide, is a stark example of how location is predictive of education and health. To the south of Delmar Boulevard is a largely white community with an average income of $47,000 and 67% of residents having a bachelor’s degree. Directly north of Delmar Boulevard is a predominantly African American community with a lower average income of $22,000 and only 5% of residents have a bachelor’s degree. In addition to income and education following the so-called Delmar Divide, health is also negatively affected. Higher rates of cancer, heart disease and obesity are only a few of the diseases plaguing these neglected, low-income neighborhoods.

 

Because our brains are rapidly developing during childhood, this leaves them more vulnerable to stress and environmental changes. Recently scientists have extended their efforts to better understand the long-lasting effects of income and environment on the brain and behavior. There have been a number of studies that look at the behavioral consequences of growing up in disadvantaged families, including increased risk for behavioral disorders, developmental delays, and learning disabilities. Fewer human studies have looked into the long-lasting effects of childhood poverty on brain regions known to be critical for executive function, attention and memory. Two studies published recently attempt to investigate this very question using a large-scale, longitudinal design in children between 3 and 20 years of age coming from different socioeconomic backgrounds.

 

One longitudinal, multi-site study published in JAMA Pediatrics investigated whether or not childhood poverty caused significant structural impairments in brain regions known to be important for academic performance. Key regions targeted in the study include the frontal lobes, involved in behavioral inhibition and emotion regulation, the temporal lobes, important for language and memory, and the hippocampus, a region shown to be critical for long-term memory as well as spatial and contextual memory. Demographic information and neuroimaging data was collected from nearly 400 economically diverse participants who were controlled for potential confounding factors such as health problems during or after pregnancy, complicated medical histories, familial history of psychiatric disorders, and behavioral deficits.

 

As hypothesized, children raised in low-income families had lower scores on the Wechsler Abbreviated Scale of Intelligence (WASI), which measures intelligence via verbal and performance IQ, and the Woodcock-Johnson III Tests of Achievement (WJ-III), a test for math skills and reading comprehension. Anatomically, children raised in low-income families showed reductions in gray matter (or volume – where most of the brain’s cells are housed), in the frontal and temporal lobes as well as in the hippocampus, with the largest deficits seen in children living well below the federal poverty line.

 

Another study recently published in Nature Neuroscience reported similar findings. The authors investigated whether poverty, defined by a parent’s education level and income, is predictive of neurodevelopmental deficits in key brain regions. As hypothesized, income is related to structural impairments in brain regions important for reading, language, and other executive skills. Similar to the study published in JAMA Pediatrics, this study found the strongest interaction in children from the poorest families.

 

These studies highlight the importance of access to beneficial resources during childhood and adolescence and how income and environment can drastically affect the trajectory of health and development of brain regions key to success into adulthood. A number of different programs for social change that are guided by empirical data and public policy are being implemented in disadvantaged communities. Sending healthcare workers out of the clinic and into these communities is a step in the right direction. However, some clinicians argue that this is unsustainable and instead advocate taking further steps towards training individuals who live in these communities and/or have healthcare providers move into these communities.

 

Furthermore, initiatives focusing on children and adolescents, in particular, could prevent more problems, possibly irreversible ones, from occurring down the road. Interventions directed towards reducing income inequality, improving nutrition, and increasing access to educational opportunities could drastically redirect a child’s trajectory into adulthood. Early education programs targeting children aged 3-5 years of age have been shown to improve future education attainment and earnings as well as reduce crime and adult poverty.

 

An unhealthy, broken social support system nurses an unhealthy, broken environment in disadvantaged regions lacking basic resources. Scientific knowledge can help direct public policy initiatives towards programs that could have greater impacts on society. A continued dialogue among scientists, politicians, and community activists is vital to the health not only of the children growing up in low-income communities but arguably to the health of our society as a whole. Solely placing funds and resources towards ameliorating adult poverty is akin to placing a band-aid on the problem. Today’s children are tomorrow’s adults, thus helping today’s children help’s tomorrow’s adults.