Gene Enhancers Are Important Despite Apparent Redundancy

Cell activity patterns of two gene enhancers (red and green cells). Cells in which both enhancers are active appear yellow. (Credit: Marco Osterwalder).

Every cell in the body has the same DNA and genes, so a cell’s properties and functions are determined by which genes are turned on. That’s why it is critical to understand enhancers, short sections of non-coding DNA that regulate the expression of specific genes.

An enhancer doesn’t have a one-to-one relationship with the gene it controls. Instead, there are many more enhancers than genes and their relationship is unclear. Do many enhancers regulate a given gene’s expression in a given tissue, providing redundancy? Researchers at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) investigated this question, and the overall importance of enhancers to development, in two recent studies.

The researchers answered a long-standing question about the role of enhancers. And by better linking the genomic complement of an organism with its expressed characteristics, their work offers new insights that further the growing field of systems biology, which seeks to gain a predictive understanding of living systems.

Perfect Conservation

In their new study published in Cell, the team investigated enhancers containing “ultraconserved elements,” which are at least 200 base pairs in length and are 100 percent identical in the genomes of humans, mice and rats. Ultraconserved elements have been perfectly conserved for over 80 million years since these mammals shared a common ancestor.

Previously, the group individually deleted four ultraconserved brain enhancers in the mouse genome. All four mouse lines were viable and fertile, which shocked the genomics and evolutionary biology research communities who thought these enhancers were critical for life since they have been so perfectly conserved.

“In our follow-up study, we wanted to dig deeper to test two possible explanations,” said Diane Dickel, a research scientist at Berkeley Lab’s Environmental Genomics and Systems Biology Division. “First, maybe there is some redundancy between these enhancer sequences, and losing two of them will cause the mice to be nonviable or infertile. Second, maybe the mice do have something wrong with them, but it’s more subtle.”

In the new study, the team used the gene-editing tool CRISPR-CAS9 to further investigate enhancers near the Arx gene that, when defective, cause neurological and sexual-development disorders in mice and humans. “We focused on Arx because it has an unusually large number of very long ultraconserved sites nearby,” said Dickel who co-led the studies with Len Pennacchio and Axel Visel, both senior scientists in the same division.

Specifically, they knocked out four of Arx’s brain enhancers, which are active in pairs in either the top or bottom of the forebrain. When these enhancers were individually deleted, all the mice were viable and fertile — confirming that ultraconserved enhancers aren’t essential to life within a given generation. When they were deleted in pairs with similar activity, the mice were still viable and fertile — ruling out redundancy as the primary explanation for the absence of major defects upon knockout of individual ultraconserved enhancers.

But were there more subtle brain defects? To answer this question, the researchers teamed up with John Rubenstein, a neurobiologist at UC San Francisco, who provided in-depth neurological phenotyping.

In three of the four cases with only one enhancer deletion, they found abnormalities —either in overall growth or brain development. In one case, the mice had a severe structural defect in the hippocampus. In another case, the mice had fewer cholinergic neurons.

“The changes to the brain in these mice are reminiscent of those seen in humans with seizure disorders or dementia,” Dickel said. “While we don’t know yet if these mice are affected by such problems, it’s likely that the physiological changes we found are selected against in the wild, and that’s why you maintain a high level of conservation at these sites.”

Pictures of a normal part of the mouse forebrain (left) compared with a mouse missing one ultraconserved enhancer (right). (Credit: Athena Ypsilanti /UCSF)

Protective redundancy

While there are only a few hundred ultraconserved sites in the human and mouse genomes, there are also approximately 100,000 other, less well-conserved enhancers. The defects observed upon deletion of individual ultraconserved enhancers raise the question if deletion of less well-conserved enhancers generally causes similar problems. Are defects the exception or the rule? This question was investigated in a second study, led by postdoctoral researcher Marco Osterwalder, which focused on enhancers for limbs. Limb enhancers were targeted because limbs are easy to assess, unlike neurological phenotyping.

As reported today in Nature, the team deleted ten limb enhancers near genes essential for limb development. They expected to see some anomalies but all ten mouse lines had perfectly normal-looking limbs.

However, the team also observed some genes with two enhancers that appeared to be active at the same time during limb development. When the team knocked out such pairs of limb enhancers with similar activity, they saw characteristics like extra digits or differences in bone length — indicating that these enhancers functioned redundantly. “It’s like the pilot and copilot having redundant control sticks in the cockpit,” explained Visel. “Either one can control the plane, but you’re in trouble if you get rid of both control sticks.”

To determine whether or not it’s common to have such a regulatory back-up system, the team computationally analyzed genomic datasets from many different tissues. They determined that genes that control central processes in embryonic development are commonly equipped with sets of enhancers that are likely redundant. In more than 1000 extreme cases, they found sets of five or more enhancers with similar activity patterns controlling the same gene.

Despite the redundancy, these enhancers are evolutionarily conserved, which leads the scientists to surmise that disrupting these enhancers may cause some sort of decrease in fitness in the wild, even if it is so small it can’t be readily detected in the lab.

“We’re not saying these enhancers are perfectly redundant in that one or the other isn’t important, but rather there is a mechanism of protecting against deleterious effects on the order of a given generation,” Pennacchio summarized. “Selection happens over many generations.”

Taken together, these studies demonstrate a varied importance of enhancer redundancy. “The genome is a big place,” Dickel said. “It’s hard to fit every single locus in the genome into one specific model of gene regulation. Some loci have more redundancy than others.”

Broader implications

Complex questions such as these are addressed by biological systems science, where these results can be used to understand the effects of genetic perturbations on inherited and expressed characteristics in a larger context.

Ultimately, the team is interested in whether enhancer mutations contribute to human disease. “With whole human genome sequencing now a reality, we are focusing on studying how human mutations impact health and development in vivo,” Pennacchio said.

The research, funded by the National Institutes of Health, was performed at Berkeley Lab and is a natural evolution of the work that was begun by the DOE and became the Human Genome Project.

The following Berkeley Lab researchers also contributed to the studies: Iros Barozzi, Yoko Fukuda-Yuzawa, Brandon Mannion, Sarah Afzal, Elizabeth Lee, Yiwen Zhu, Ingrid Plajzer-Frick, Catherine Pickle, Momoe Kato, Tyler Garvin, Quan Pham, Anne Harrington, Jennifer Akiyama and Veena Afzal. Also participating in the research were scientists from the University of California San Francisco, University of Basel, and the Centro Andaluz de Biología del Desarrollo.

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Lawrence Berkeley National Laboratory addresses the world’s most urgent scientific challenges by advancing sustainable energy, protecting human health, creating new materials, and revealing the origin and fate of the universe. Founded in 1931, Berkeley Lab’s scientific expertise has been recognized with 13 Nobel Prizes. The University of California manages Berkeley Lab for the U.S. Department of Energy’s Office of Science. For more, visit www.lbl.gov.

DOE’s Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit science.energy.gov.

This is a reposting of my news release, courtesy of Berkeley Lab.

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“Fierce, empowered… and less alone”: A 32-year-old cancer patient reflects on her new film

Photo by Kerith Lemon Productions of writer Becky Hall, left, and director Kerith Lemon.

As someone who battled against Hodgkin’s lymphoma when I was in my 20s, I can relate to Becky Hall, a young breast cancer patient at Stanford. I recently spoke with Hall about her cancer experiences and her short film, bare, which describes the night her friends shaved her head before starting chemotherapy.

When were you diagnosed with cancer?

“I was a 25-year-old graduate student in a veterinary program at UC Davis. I wanted to work with horses. I was a Stanford graduate and was all about school. Then I went for a run one Saturday and laid down on my chest to stretch — a stretch that I’ve done every day for years — and it felt like I was laying on a golf ball. The nurse at the health center said I was too young for breast cancer and didn’t have a family history so I didn’t need to worry, but she sent me to get a mammogram just to be 100 percent sure. I completely panicked, because that’s what I do. The mammogram showed lumps in my breast and lymph nodes, so they biopsied right then and everything spiraled. A week after I found the lump, I was a stage 3 breast cancer patient.

I couldn’t do such a demanding academic program during chemotherapy. So I dropped out of school and moved back in with my parents in Santa Cruz — really losing all my independence, privacy and social interactions for a year as I went through chemotherapy, surgery, radiation and biotherapies. I went into remission for about two years and then I found out my cancer had metastasized three weeks after my wedding. So I’m now a terminal, stage 4 patient. Since August 2014, I’ve been in continual treatment to keep up with and out smart my cancer.”

What was that like?

“Having cancer is always going to be traumatic and challenging, but there are additional challenges when you’re young. My 25-year-old friends were busy going out drinking, so we couldn’t relate to each other’s interests any more. And they didn’t have enough life experience and skills to cope with being a friend to someone with a major illness. So many of my friends just disappeared, because they didn’t know how to handle it. It was very isolating.

There was also the whole element of being single, because dating was terrifying after I’d been through breast cancer. I had a reconstructed breast. I had scars all over my body. I knew there was a chance that it was going to metastasize. And I had to explain all of this to someone.

And finally there was the fertility issue. I had cancer, and suddenly they ripped away this huge element of my life that I’d always envisioned for myself. When I was stage 3, I was hopeful and a little delusional, thinking that I’d still be able to have kids after chemo. When I was diagnosed stage 4, I was married to an amazing teacher who loves kids. On our honeymoon, we’d talked about how many kids we wanted and even about names. And then we got home and were told we could not have kids. And it just destroyed me. I would never be able to carry a child. Adoption is exceedingly difficult for someone with stage 4 disease, and surrogacy is pretty cost prohibitive. It’s just a whole other layer of heartbreak that cancer throws at a young woman.”

What motivated you to capture the experience of shaving your head in a short film?

“The idea of watching my hair fall out slowly was absolutely terrifying. And I was facing so many changes physically that I had no control over, but I could control when and how I lost my hair. It turned out shaving my head before chemo really helped me process some of my emotions and biggest fears — potentially dying, being unattractive, and losing a big part of my identity.

I wanted other women to feel what I felt when I looked in the mirror after shaving my head — fierce, empowered and like I could take on anything. I also wanted to help people feel less alone. In mainstream media, so many of the stories told about cancer are very rosy and falsely upbeat. I wanted to show a more honest slice of what it’s like — that you can be in the darkness, but you can emerge from it if friends are willing to just sit in the darkness with you. I also wanted to shed some light on metastatic disease and the disparities in research between early stage and late stage breast cancer.

Before the project, I felt like all I did was go to doctor appointments. The film reminded me that I’m still capable of creating and contributing. I co-wrote the script with my childhood friend Kerith Lemon and helped with the project while recovering from brain surgery and brain radiation — I’m really proud of that.”

Do you have any advice on living with cancer?

“When living with cancer, you’re constantly dealing with treatments and side effects. Everything is always up and down — one scan is good and the next is bad. It’s really, really draining. In an effort to help, people often encourage you to stay positive and upbeat. But my advice is to feel whatever you feel: sad, angry, scared or whatever.

It’s also important to find other metastatic cancer patients who understand on a deep level what you’re going through because they are going through it too. Find people to reach out to on those dark days, who can help you process and get through it when you’re ready.”

What are you doing now?

“I’m living in Santa Cruz with my husband and dog. In addition to being a full time cancer patient, I’m writing and getting involved in metastatic advocacy. My advocacy work started a couple of months ago after I participated in a panel during a screening of the film. I really enjoyed public speaking and talking about the needs of metastatic patients to a room full of people that can actually effect change. It felt like I found my purpose beyond just surviving cancer. And I give credit for this to the film.”

This is a reposting of my Scope blog story, courtesy of Stanford School of Medicine.

Living with cancer: A Q&A with comedian Fred Reiss

Photo by Ben Moon

Cancer isn’t what comes to mind when I think of stand-up comedy, but Fred Reiss may change that. A three-time cancer survivor, Reiss shares at Comedy Day 37 what it’s like to go for a follow-up PET scan to find out if your cancer is back, also joking that they found a computer chip in his neck and returned him to his original owner. I recently spoke with Reiss, who is also an inspirational wellness speaker and novelist, to learn more. 

When were you diagnosed and treated with cancer?

“I was diagnosed with testicular cancer when I was 28 in 1982. Then about 4 years ago, I was diagnosed again with testicular cancer and treated at Stanford — I should have gone for the 2-for-1 deal on testicular removal and saved myself some money. And then a year ago when going for a routine endoscope, I found out I had esophageal cancer. Fortunately Stanford caught it early, and it’s gone now with treatment. So I’ve had cancer three times. I guess I’ll keep on doing this until I get it right.

I started wearing boxing gloves to chemo during my second bout of testicular cancer. I thought of the scene in “Rocky” when he throws the first punch and knocks Apollo down in the first round. I kept thinking: I need to be in shape to throw that punch. I need to fight for my life. So I decided to wear boxing gloves. Why should I be self-conscious? And the first day, I had someone take a photo in a fighting stance with the gloves. The nurses loved that.”

Why do you do stand-up comedy?

“The genesis was when I was 28 with testicular cancer. I was lying there with an IV in my soft blue vein and I thought: If I’m here again, who will I be? Who will be lying in this bed? And I decided to move from the East Coast to California, go do stand-up comedy and write books.

Later, after I had cancer again, no one wanted to hire a two-time cancer survivor in his 50s to do comedy, radio, journalism, public relations or administration. I had to become Fred 2.0. So I thought, what do I have to say about going through all of this? And I headed back to the stage.

I started going to open mics to use the gravitas of my own mortality to help other people and explore myself. People in the audience wondered why I was there, because I’ve been on national TV, but you have to develop material at smaller clubs — the only way to find out if something works is by saying it. I’ve been on a billboard on Times Square to promote the film, “This is Living with Cancer.” That was the result of two to three years of going to open mics and working on material. I know I’m betraying myself if I don’t go out and perform. I’m betraying the person that I vowed to be.”

How did you become a cancer advocate?

“My cancer advocacy grew out of my suffering and watching other people suffering — it alters you. When I first had cancer, I went through self-actualization to figure out what I wanted to do. The second time, I thought about what I was going to do and what I’d done. And the third time, my ego was completely gone and I thought about other people.

So I decided to travel two tracks — comedy and cancer patient advocacy. I started doing “Fred talks” (my brand of motivational Ted-style talks) and speaking to hospital groups, offering myself to people. In comedy, the audience wants jokes. But if I’m speaking to groups, the audience wants to know how I feel; it’s enormously satisfying. If they can help me out financially when I speak, that’s great. But if they can’t afford it, I don’t mind speaking for free.

During my talks, I use jokes, personal anecdotes and photographs to tell my story of being diagnosed and at the end overcoming cancer. I stress how to draw on your personality, passion, humor and the character of your life to overcome it. I also give practical tips on how to reduce your suffering. My main message is that you can’t let cancer define you; you have to let the spirit that enabled you to overcome it be given to others to help them prevail over the disease too.

In that spirit, I still visit Stanford when I can, giving out my books, CDs and food. I can’t do it every day, but it’s a temple that I have to respect. It’s a way to pay homage and show that I haven’t forgotten the oncologists, nurses and all the people that made a difference. It’s not out of a sense of duty. These people did great things for me, so I’m trying to propel that toward the other people around me.”

This is a reposting of my Scope blog story, courtesy of Stanford School of Medicine.