Paired Genes in Stem Cells Shed New Light On Gene Organization and Regulation

Feb. 4, 2013 — Whitehead Institute researchers have determined that DNA transcription, the process that produces messenger RNA (mRNA) templates used in protein production, also runs in the opposite direction along the DNA to create corresponding long noncoding RNAs (lncRNAs). Moreover, the mRNAs and lncRNAs are transcribed coordinately as stem cells differentiate into other cell types. This surprising finding could redefine our understanding of gene organization and its regulation.

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“It’s a surprise to me that genes come in pairs,” says Whitehead Member Richard Young, who is also a professor of biology at MIT. “At any one of the 20,000 protein-coding genes that are active in human stem cells, a lncRNA gene located upstream is also transcribed. So much effort has gone into studying protein-coding genes, and yet we have missed this concept that all protein-coding genes come in mRNA/lncRNA pairs. If you activate the mRNA gene, you’re going to activate the lncRNA gene.”

Young and his lab report their findings this week in theProceedings of the National Academy of Sciences (PNAS).

Until now, scientists thought transcription machinery attaches to DNA at certain points called promoters and moves just in one direction along the DNA to create mRNAs from protein-coding genes. Other RNAs that are not protein templates, including lncRNAs, are also created by transcription, but despite their important roles in in regulation of gene expression, development and disease, scientists knew little about how lncRNA transcription is initiated or where most lncRNA genes reside in the genome.

By looking at human and mouse embryonic stem cells, researchers in the Young lab found something astonishing — most lncRNA genes are located adjacent mRNA genes, and the transcription of about 65% of lncRNAs originates at active promoters associated with these mRNAs’ genes and runs “upstream” and in the opposite direction from the promoter.

When the transcription machinery attaches to a promoter, it seems that it is just as likely to move in one direction and transcribe the mRNA as it is to move in opposite direction and transcribe the neighboring lncRNA.

The researchers also noticed that as embryonic stem cells begin differentiating into other cell types, the mRNA/lncRNA pairs are regulated in the same way — the transcription of paired mRNAs and lncRNAs is upregulated or downregulated together. This further confirms the relationship between the transcription of mRNAs and lncRNAs.

“I think it’s quite a breakthrough,” says Alla Sigova, who is a postdoctoral researcher in the Young lab and a co-author of the paper in PNAS. “This provides a unifying principle for production of mRNAs and lncRNAs, and may lead to new insights into lncRNA misregulation in disease. For example, some mRNAs are up- or downregulated in cancer, so their lncRNA partners could potentially contribute to cancer.”

This work was supported by National Institutes of Health Grants HG002668, GM34277, and DK090122, the American Gastroenterological Association, the Damon Runyon Cancer Research Foundation, and National Cancer Institute Cancer Center Support (Core) Grant P30-CA14051.

 

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Story Source:

The above story is reprinted from materials provided byWhitehead Institute for Biomedical Research. The original article was written by Nicole Giese Rura.

Note: Materials may be edited for content and length. For further information, please contact the source cited above.

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Journal Reference:

1. Alla A. Sigova, Alan C. Mullen, Benoit Molinie, Sumeet Gupta, David A. Orlando, Matthew G. Guenther, Albert E. Almada, Charles Lin, Phillip A. Sharp, Cosmas C. Giallourakis, and Richard A. Young. Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. PNAS, 2013 DOI:10.1073/pnas.1221904110

Whitehead Institute for Biomedical Research (2013, February 4). Paired genes in stem cells shed new light on gene organization and regulation. ScienceDaily. Retrieved February 5, 2013, from http://www.sciencedaily.com/releases/2013/02/130204153608.htm

One Form of Neuron Turned Into Another in Brain

Jan. 20, 2013 — A new finding by Harvard stem cell biologists turns one of the basics of neurobiology on its head — demonstrating that it is possible to turn one type of already differentiated neuron into another within the brain.

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The discovery by Paola Arlotta and Caroline Rouaux “tells you that maybe the brain is not as immutable as we always thought, because at least during an early window of time one can reprogram the identity of one neuronal class into another,” said Arlotta, an Associate Professor in Harvard’s Department of Stem Cell and Regenerative Biology (SCRB).

The principle of direct lineage reprogramming of differentiated cells within the body was first proven by SCRB co-chair and Harvard Stem Cell Institute (HSCI) co-director Doug Melton and colleagues five years ago, when they reprogrammed exocrine pancreatic cells directly into insulin producing beta cells.

Arlotta and Rouaux now have proven that neurons too can change their mind. The work is being published on-line Jan. 20 by the journal Nature Cell Biology.

In their experiments, Arlotta targeted callosal projection neurons, which connect the two hemispheres of the brain, and turned them into neurons similar to corticospinal motor neurons, one of two populations of neurons destroyed in Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease. To achieve such reprogramming of neuronal identity, the researchers used a transcription factor called Fezf2, which long as been known for playing a central role in the development of corticospinal neurons in the embryo.

What makes the finding even more significant is that the work was done in the brains of living mice, rather than in collections of cells in laboratory dishes. The mice were young, so researchers still do not know if neuronal reprogramming will be possible in older laboratory animals — and humans. If it is possible, this has enormous implications for the treatment of neurodegenerative diseases.

“Neurodegenerative diseases typically effect a specific population of neurons, leaving many others untouched. For example, in ALS it is corticospinal motor neurons in the brain and motor neurons in the spinal cord, among the many neurons of the nervous system, that selectively die,” Arlotta said. “What if one could take neurons that are spared in a given disease and turn them directly into the neurons that die off? In ALS, if you could generate even a small percentage of corticospinal motor neurons, it would likely be sufficient to recover basic functioning,” she said.

The experiments that led to the new finding began five years ago, when “we wondered: in nature you never seen a neuron change identity; are we just not seeing it, or is this the reality? Can we take one type of neuron and turn it into another?” Arlotta and Rouaux asked themselves.

Over the course of the five years, the researchers analyzed “thousands and thousands of neurons, looking for many molecular markers as well as new connectivity that would indicate that reprogramming was occurring,” Arlotta said. “We could have had this two years ago, but while this was a conceptually very simple set of experiments, it was technically difficult. The work was meant to test important dogmas on the irreversible nature of neurons in vivo. We had to prove, without a shadow of a doubt, that this was happening.”

The work in Arlotta’s lab is focused on the cerebral cortex, but “it opens the door to reprogramming in other areas of the central nervous system,” she said.

Arlotta, an HSCI principal faculty member, is now working with colleague Takao Hensch, of Harvard’s Department of Molecular and Cellular Biology, to explicate the physiology of the reprogrammed neurons, and learn how they communicate within pre-existing neuronal networks.

“My hope is that this will facilitate work in a new field of neurobiology that explores the boundaries and power of neuronal reprogramming to re-engineer circuits relevant to disease,” said Paola Arlotta.

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Story Source:

The above story is reprinted from materials provided byHarvard University, via EurekAlert!, a service of AAAS.

Note: Materials may be edited for content and length. For further information, please contact the source cited above.

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Journal Reference:

1. Caroline Rouaux, Paola Arlotta. Direct lineage reprogramming of post-mitotic callosal neurons into corticofugal neurons in vivo. Nature Cell Biology, 2013; DOI: 10.1038/ncb2660

Harvard University (2013, January 20). One form of neuron turned into another in brain.ScienceDaily. Retrieved January 27, 2013, from http://www.sciencedaily.com/releases/2013/01/130120150031.htm

Neuroscientists Find Brain Stem Cells That May Be Responsible for Higher Functions, Bigger Brains

ScienceDaily (Aug. 7, 2012) — Scientists from The Scripps Research Institute have identified a new stem cell population that may be responsible for giving birth to the neurons responsible for higher thinking. The finding also paves the way for scientists to produce these neurons in culture — a first step in developing better treatments for cognitive disorders, such as schizophrenia and autism, which result from disrupted connections among these brain cells.

Scientists from The Scripps Research Institute have identified a new stem cell population that may be responsible for giving birth to the neurons responsible for higher thinking. (Credit: © rolffimages / Fotolia)

Published in the August 10, 2012 issue of the journal Science, the new research reveals how neurons in the uppermost layers of the cerebral cortex form during embryonic brain development.

“The cerebral cortex is the seat of higher brain function, where information gets integrated and where we form memories and consciousness,” said the study’s senior author Ulrich Mueller, a professor and director of the Dorris Neuroscience Center at Scripps Research. “If we want to understand who we are, we need to understand this area where everything comes together and forms our impression of the world.”

In the new study, Mueller’s team identified a neural stem cell in mice that specifically gives rise to the neurons that make up the upper layers of the cerebral cortex. Previously, it was thought that all cortical neurons — those making up both the lower and upper layers — came from the same type of stem cell, called a radial glial cell, or RGC. A neuron’s fate was thought to be determined by the timing of its birth date. The Scripps Research team, however, showed that there is a distinct stem cell progenitor that gives rise to upper layer neurons, regardless of birth date or place.

“Advanced functions like consciousness, thought, and creativity require a lot of different neuronal cell types and a central question has been how all this diversity is produced in the cortex,” said Santos Franco, a senior research associate in Mueller’s laboratory and first author of the paper. “Our study shows this diversity already exists in the progenitor cells.”

Peeling Back the Onion Layers

In mammals, the cortex is made up of six distinct anatomic layers holding different types of excitatory neurons. They are not the uniform layers of a cake, but rather, they are more like the layers wrapped around an onion. The smaller lower layers, on the inside, host neurons that connect to the brain stem and spinal cord to help regulate essential functions such as breathing and movement. The larger upper layers, closer to the outer surface of the brain, contain neurons that integrate information coming in from the senses and connect across the two halves of the brain.

The upper layers are a “relatively young invention,” evolutionarily speaking, having been greatly expanded during primate evolution, said Mueller. They give humans in particular the unique abilities to think abstractly, plan for the future and problem-solve.

For the last two decades, scientists have believed that the fate of cerebral cortex neurons was determined by their birth date because each layer is formed in a time-dependent manner. The lower layer neurons form in the center of the “ball” first, and then the cells that will become the upper layers form last, migrating through the lower layers.

“So the model was that there is a stem cell in the center of the ball that generates the different types of neurons in successive waves,” said Mueller. “What we now show is that there are at least two different populations of RGCs and potentially more.”

Following Fate

Franco first created a line of mice in which he could track upper-layer neurons as they were born and migrated. The team followed a marker gene called Cux2, which was previously reported to be expressed only by upper-layer neurons. By linking a gene for an enzyme called Cre to the Cux2 gene, the scientists could watch any cell expressing Cux2 under the microscope, because the Cre enzyme flips on another gene that glows fluorescent red.

Surprisingly, the team observed Cux2 already turned on in some of the RGCs, even at the earliest points in brain development — embryonic day nine or ten — before any upper-layer neurons exist. Following this population of glowing stem cells through development, the team showed that the cells almost exclusively generated upper-layer neurons. In contrast, the subgroup of RGCs not expressing Cux2 became lower-layer neurons.

Next, the team removed these Cux2-positive precursor cells from their niche in the embryonic brain to see how they would develop in a lab dish. When they cultured both types of RGCs, again only Cux2-expressing RGCs developed into upper-layer neurons.

In developing brains, these Cux2-positive stem cells first self-renew and proliferate before differentiating later into neurons. So, the team wanted to know if a neuron’s birth date determined its fate. To test this, the researchers delivered a TCF4 molecule in utero that forced the Cux2-positive RGCs to prematurely differentiate. Even though it was too early in normal development, the Cux2-positive RGCs still produced upper-layer neurons.

In other words, regardless of position or timing, the Cux2-positive RGCs are destined to become upper-layer neurons. Mueller and colleagues concluded that these stem cells have some intrinsic property that determines their fate from the start.

The work also shows that this RGC subset is responsible for the huge proliferation of cells necessary to create the larger upper-layer cortex found in primate brains. “If we want to understand how the human brain evolved, how we are different from an amphibian, then this one precursor cell may have been important,” said Mueller.

But, bigger brains came with a risk, making humans more prone to disorders when upper-layer neurons don’t form connections properly. Up until now, researchers trying to reproduce human cortical neurons in the lab from stem cells have only generated lower-layer-type neurons. “This opens a door now to try to make the upper-layer neurons, which are frequently affected in psychiatric disorders,” said Mueller.

In addition to Mueller and Franco, authors of the paper, “Fate-restricted neural progenitors in the mammalian cerebral cortex,” were Cristina Gil-Sanz, Isabel Martinez-Garay, Ana Espinosa, Sarah R. Harkins-Perry, and Cynthia Ramos of Scripps Research. Martinez-Garay is now at the University of Oxford.

This research was supported by the Dorris Neuroscience Center, US National Institutes of Health (grant award numbers NS060355, NS046456, MH078833), and California Institute for Regenerative Medicine, and conducted in affiliation with the NIH Blueprint-funded Cre Driver Network.

Story Source:

The above story is reprinted from materials provided byThe Scripps Research Institute. 

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Journal Reference:

1. Santos J. Franco, Cristina Gil-Sanz, Isabel Martinez-Garay, Ana Espinosa, Sarah R. Harkins-Perry, Cynthia Ramos, and Ulrich Müller. Fate-Restricted Neural Progenitors in the Mammalian Cerebral Cortex. Science, 10 August 2012: 746-749 DOI: 10.1126/science.1223616

Citation:

The Scripps Research Institute (2012, August 7). Neuroscientists find brain stem cells that may be responsible for higher functions, bigger brains. ScienceDaily. Retrieved August 11, 2012, from http://www.sciencedaily.com/releases/2012/08/120809141505.htm

Entire Genetic Sequence of Individual Human Sperm Determined

ScienceDaily (July 19, 2012) — The entire genomes of 91 human sperm from one man have been sequenced by Stanford University researchers. The results provide a fascinating glimpse into naturally occurring genetic variation in one individual, and are the first to report the whole-genome sequence of a human gamete — the only cells that become a child and through which parents pass on physical traits.

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“This represents the culmination of nearly a decade of work in my lab,” said Stephen Quake, PhD, the Lee Otterson Professor in the School of Engineering and professor of bioengineering and of applied physics. “We now have devices that will allow us to routinely amplify and sequence to a high degree of accuracy the entire genomes of single cells, which has far-ranging implications for the study of cancer, infertility and many other disorders.”

Quake is the senior author of the research, published July 20 in Cell. Graduate student Jianbin Wang and former graduate student H. Christina Fan, PhD, now a senior scientist at ImmuMetrix, share first authorship of the paper.

Sequencing sperm cells is particularly interesting because of a natural process called recombination that ensures that a baby is a blend of DNA from all four of his or her grandparents. Until now, scientists had to rely on genetic studies of populations to estimate how frequently recombination had occurred in individual sperm and egg cells, and how much genetic mixing that entailed.

“Single-sperm sequencing will allow us to chart and understand how recombination differs between individuals at the finest scales. This is an important proof of principle that will allow us to study both fundamental dynamics of recombination in humans and whether it is involved in issues relating to male infertility,” said Gilean McVean, PhD, professor of statistical genetics at the Wellcome Trust Centre for Human Genetics. McVean was not involved in the research.

The Stanford study showed that the previous, population-based estimates were, for the most part, surprisingly accurate: on average, the sperm in the sample had each undergone about 23 recombinations, or mixing events. However, individual sperm varied greatly in the degree of genetic mixing and in the number and severity of spontaneously arising genetic mutations. Two sperm were missing entire chromosomes. The study has long-ranging implication for infertility doctors and researchers.

“For the first time, we were able to generate an individual recombination map and mutation rate for each of several sperm from one person,” said study co-author Barry Behr, PhD, HCLD, professor of obstetrics and gynecology and director of Stanford’s in vitro fertilization laboratory. “Now we can look at a particular individual, make some calls about what they would likely contribute genetically to an embryo and perhaps even diagnose or detect potential problems.”

Most cells in the human body have two copies of each of 23 chromosomes, and are known as “diploid” cells. Recombination occurs during a process called meiosis, which partitions a single copy of each chromosome into a sperm (in a man) or egg (in a woman) cell. When a sperm and an egg join, the resulting fertilized egg again has a full complement of DNA.

To ensure an orderly distribution during recombination, pairs of chromosomes are lined up in tight formation along the midsection of the cell. During this snug embrace, portions of matching chromosomes are sometimes randomly swapped. The process generates much more genetic variation in a potential offspring than would be possible if only intact chromosomes were segregated into the reproductive cells.

“The exact sites, frequency and degree of this genetic mixing process is unique for each sperm and egg cell,” said Quake, “and we’ve never before been able to see it with this level of detail. It’s very interesting that what happens in one person’s body mirrors the population average.”

Major problems with the recombination process can generate sperm missing portions or even whole chromosomes, making them incapable of or unlikely to fertilize an egg. But it can be difficult for fertility researchers to identify potential problems.

“Most of the techniques we currently use to assess sperm viability are fairly crude,” said Quake.

To conduct the research, Wang, Quake and Behr first isolated and sequenced nearly 100 sperm cells from the study subject, a 40-year-old man. The man has healthy offspring, and the semen sample appeared normal. His whole-genome sequence (obtained from diploid cells) has been previously sequenced to a high level of accuracy.

They then compared the sequence of the sperm with that of the study subject’s diploid genome. They could see, by comparing the sequences of the chromosomes in the diploid cells with those in the haploid sperm cells, where each recombination event took place. The researchers also identified 25 to 36 new single nucleotide mutations in each sperm cell that were not present in the subject’s diploid genome. Such random mutations are another way to generate genetic variation, but if they occur at particular points in the genome they can have deleterious effects.

It’s important to note that individual sperm cells are destroyed by the sequencing process, meaning that they couldn’t go on to be used for fertilization. However, the single-cell sequencing described in the paper could potentially be used to diagnose male reproductive disorders and help infertile couples assess their options. It could also be used to learn more about how male fertility and sperm quality change with increasing age.

“This could serve as a new kind of early detection system for men who may have reproductive problems,” said Behr, who also co-directs Stanford’s reproductive endocrinology and infertility program. “It’s also possible that we could one day use other, correlating features to harmlessly identify healthy sperm for use in IVF. In the end, the DNA is the raw material that ultimately defines a sperm’s potential. If we can learn more about this process, we can better understand human fertility.”

The research was supported by the National Institute of Health, the Chinese Scholarship Council and the Siebel Foundation.

 

Link:

http://med.stanford.edu/ism/2012/july/sperm.html

Journal Reference:

1. Jianbin Wang, H. Christina Fan, Barry Behr, Stephen R. Quake. Genome-wide Single-Cell Analysis of Recombination Activity and De Novo Mutation Rates in Human Sperm. Cell, 20 July 2012 DOI: 10.1016/j.cell.2012.06.030

Citation:

Stanford University Medical Center (2012, July 19). Entire genetic sequence of individual human sperm determined. ScienceDaily. Retrieved July 21, 2012, from http://www.sciencedaily.com­