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.
“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.
Note: Materials may be edited for content and length. For further information, please contact the source cited above.
- 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
Jan. 20, 2013 — In 1953, Cambridge researchers Watson and Crick published a paper describing the interweaving ‘double helix’ DNA structure — the chemical code for all life.
Now, in the year of that scientific landmark’s 60th Anniversary, Cambridge researchers have published a paper proving that four-stranded ‘quadruple helix’ DNA structures — known as G-quadruplexes — also exist within the human genome. They form in regions of DNA that are rich in the building block guanine, usually abbreviated to ‘G’.
The findings mark the culmination of over 10 years investigation by scientists to show these complex structures in vivo — in living human cells — working from the hypothetical, through computational modelling to synthetic lab experiments and finally the identification in human cancer cells using fluorescent biomarkers.
The research, published January 20 in Nature Chemistry and funded by Cancer Research UK, goes on to show clear links between concentrations of four-stranded quadruplexes and the process of DNA replication, which is pivotal to cell division and production.
By targeting quadruplexes with synthetic molecules that trap and contain these DNA structures — preventing cells from replicating their DNA and consequently blocking cell division — scientists believe it may be possible to halt the runaway cell proliferation at the root of cancer.
“We are seeing links between trapping the quadruplexes with molecules and the ability to stop cells dividing, which is hugely exciting,” said Professor Shankar Balasubramanian from the University of Cambridge’s Department of Chemistry and Cambridge Research Institute, whose group produced the research.
“The research indicates that quadruplexes are more likely to occur in genes of cells that are rapidly dividing, such as cancer cells. For us, it strongly supports a new paradigm to be investigated — using these four-stranded structures as targets for personalised treatments in the future.”
Physical studies over the last couple of decades had shown that quadruplex DNA can form in vitro — in the ‘test tube’, but the structure was considered to be a curiosity rather than a feature found in nature. The researchers now know for the first time that they actually form in the DNA of human cells.
“This research further highlights the potential for exploiting these unusual DNA structures to beat cancer — the next part of this pipeline is to figure out how to target them in tumour cells,” said Dr Julie Sharp, senior science information manager at Cancer Research UK.
“It’s been sixty years since its structure was solved but work like this shows us that the story of DNA continues to twist and turn.”
The study published January 20 was led by Giulia Biffi, a researcher in Balasubramaninan’s lab at the Cambridge Research Institute.
By building on previous research, Biffi was able to generate antibody proteins that detect and bind to areas in a human genome rich in quadruplex-structured DNA, proving their existence in living human cells.
Using fluorescence to mark the antibodies, the researchers could then identify ‘hot spots’ for the occurrence of four-stranded DNA — both where in the genome and, critically, at what stage of cell division.
While quadruplex DNA is found fairly consistently throughout the genome of human cells and their division cycles, a marked increase was shown when the fluorescent staining grew more intense during the ‘s-phase’ — the point in a cell cycle where DNA replicates before the cell divides.
Cancers are usually driven by genes called oncogenes that have mutated to increase DNA replication — causing cell proliferation to spiral out of control, and leading to tumour growth.
The increased DNA replication rate in oncogenes leads to an intensity in the quadruplex structures. This means that potentially damaging cellular activity can be targeted with synthetic molecules or other forms of treatments.
“We have found that by trapping the quadruplex DNA with synthetic molecules we can sequester and stabilise them, providing important insights into how we might grind cell division to a halt,” said Balasubramanian.
“There is a lot we don’t know yet. One thought is that these quadruplex structures might be a bit of a nuisance during DNA replication — like knots or tangles that form.
“Did they evolve for a function? It’s a philosophical question as to whether they are there by design or not — but they exist and nature has to deal with them. Maybe by targeting them we are contributing to the disruption they cause.”
The study showed that if an inhibitor is used to block DNA replication, quadruplex levels go down — proving the idea that DNA is dynamic, with structures constantly being formed and unformed.
The researchers also previously found that an overactive gene with higher levels of Quadruplex DNA is more vulnerable to external interference.
“The data supports the idea that certain cancer genes can be usefully interfered with by small molecules designed to bind specific DNA sequences,” said Balasubramanian.
“Many current cancer treatments attack DNA, but it’s not clear what the rules are. We don’t even know where in the genome some of them react — it can be a scattergun approach.
“The possibility that particular cancer cells harbouring genes with these motifs can now be targeted, and appear to be more vulnerable to interference than normal cells, is a thrilling prospect.
“The ‘quadruple helix’ DNA structure may well be the key to new ways of selectively inhibiting the proliferation of cancer cells. The confirmation of its existence in human cells is a real landmark.”
Note: Materials may be edited for content and length. For further information, please contact the source cited above.
- Giulia Biffi, David Tannahill, John McCafferty, Shankar Balasubramanian. Quantitative visualization of DNA G-quadruplex structures in human cells. Nature Chemistry, 2013; DOI: 10.1038/nchem.1548
Jan. 17, 2013 — In a collaborative effort published online in the January 18, 2013 issue of the journalScience, researchers at the University of North Carolina and Columbia University show for the first time how two key proteins in messenger RNA communicate via a molecular twist to help maintain the balance of histones to DNA.
Histone proteins are the proteins that package DNA into chromosomes. Every time the cell replicates its DNA it must make large amounts of newly made histones to organize DNA within the nucleus.
An imbalance in the production of DNA and histones is usually lethal for the cell, which is why the levels of the messenger RNA (mRNA) encoding the histone proteins must be tightly controlled to ensure the proper amounts of histones (not too many and not too few) are made.
In a collaborative effort published online in the January 18, 2013 issue of the journal Science, researchers at the University of North Carolina and Columbia University show for the first time how two key proteins in messenger RNA communicate via a molecular twist to help maintain the balance of histones to DNA.
“This is one of the safeguards that our cells have evolved and it is part of the normal progression through cell division — all growing cells have to use this all of the time,” said study co-author William F. Marzluff, PhD, Kenan Distinguished Professor of biochemistry and biophysics at UNC’s School of Medicine.
Every time a cell divides, Marzluff adds, it has to replicate both DNA and histone proteins and then package them together into chromosomes. “That way, each of the two cells resulting from division has one complete set of genes.”
In humans, the 23 chromosomes that house roughly 35,000 genes are made up of both DNA and histone proteins. The DNA for a histone protein is first transcribed into RNA, which then acts as a guide for building a histone protein. Because the RNA relays a message — in this case a blueprint for a histone protein, it is referred to as messenger RNA, or mRNA.
Histone mRNAs differ from all other mRNAs and end in a stem-loop [or hairpin] sequence that is required for proper regulation of histone mRNAs. In this study, the Columbia team of Liang Tong, PhD, Professor of biological sciences and the corresponding author on this project, and graduate student Dazhi Tan used crystallography to reveal the structure of two important proteins near the end of the histone mRNA stem-loop. This molecular complex is required for regulating the levels of the histone mRNA.
One of these proteins, stem-loop binding protein (SLBP) is required for translation of histone mRNA into protein, and the other is an exonuclease, which is required to destroy the mRNA. Both were initially identified at UNC by Marzluff and colleague Zbigniew Dominski, PhD, Professor of biochemistry and biophysics, also a study co-author.
“We knew there was some interaction between SLBP and the exonuclease, so we asked Liang to explain how they bind and communicate,” Dominski said. “And the surprising thing was that the proteins do it not by binding to each other but by changing the RNA structure at the site.”
“From the science point of view, that was the most dramatic thing,” Marzluff said. “The way these proteins help each other is either one can twist the RNA so the other can recognize it easier, and they don’t have to touch each other to do that.”
This protein complex is a critical regulator of histone synthesis, and is an important component of cell growth, he adds. “Interfering with it could provide a new method for interfering with cancer cell growth.”
Funding for the research came from the National Institute of General Medical Sciences (NIGMS), a component of the National Institutes of Health.
- D. Tan, W. F. Marzluff, Z. Dominski, L. Tong. Structure of Histone mRNA Stem-Loop, Human Stem-Loop Binding Protein, and 3’hExo Ternary Complex. Science, 2013; 339 (6117): 318 DOI: 10.1126/science.1228705
Jan. 17, 2013 — In each cell, thousands of regulatory regions control which genes are active at any time. Scientists at the Research Institute of Molecular Pathology (IMP) in Vienna have developed a method that reliably detects these regions and measures their activity.
Information on the new technology was just published online in the journal Science.
Genome sequences store the information about an organism’s development in the DNA’s four-letter alphabet. Genes carry the instruction for proteins, which are the building blocks of our bodies. However, genes make up only a minority of the entire genome sequence — roughly two percent in humans. The remainder was once dismissed as “junk,” mostly because its function remained elusive. “Dark matter” might be more appropriate, but gradually light is being shed on this part of the genome, too.
Far from being useless, the non-coding part of DNA contains so-called regulatory regions or enhancers that determine when and where each gene is expressed. This regulation ensures that each gene is only active in appropriate cell-types and tissues, e.g. hemoglobin in red blood cell precursors, digestive enzymes in the stomach, or ion channels in neurons. If gene regulation fails, cells express the wrong genes and acquire inappropriate functions such as the ability to divide and proliferate, leading to diseases such as cancer.
Despite the importance of gene regulatory regions, scientists have been limited in their ability to study them on a genome-wide scale. Their identification relied on indirect means, which were error prone and required tedious experiments for validating and quantifying enhancer activities..
Alexander Stark and his team at the IMP in Vienna now closed this gap with the development of a new technology called STARR-seq (self-transcribing active regulatory region sequencing), published online by Science this week. STARR-seq allows the direct identification of DNA sequences that function as enhancers and simultaneously measures their activity quantitatively in entire genomes.
Applying their technology to Drosophila cells, the IMP-scientists surprisingly find that the strongest enhancers reside in both regulatory genes that determine the respective cell-types as well as in broadly active “housekeeping” genes that are required for basic cell survival in most or all cells. In addition, they find several enhancers for each active gene, which might provide redundancy to ensure robustness of gene regulation.
The new method combines advanced sequencing technology and highly specialized know-how in bio-computing. It is a powerful tool which, according to Alexander Stark, will prove immensely valuable in the future. “STARR-seq is like a magic microscope that lets us zoom in on the regulatory regions of DNA. It will be crucial to study gene regulation and how it is encoded in the genome — both during normal development and when it goes wrong in disease.”
- Arnold Cosmas D. Arnold, Daniel Gerlach, Christoph Stelzer, Łukasz M. Boryń, Martina Rath, Alexander Stark.Genome-Wide Quantitative Enhancer Activity Maps Revealing Complex cis-Regulation of Transcription.Science, January 17, 2013 DOI: 10.1126/science.1232542
Jan. 17, 2013 — The enormously diverse complexity seen amongst individual species within the animal kingdom evolved from a surprisingly small gene pool. For example, mice effectively serve as medical research models because humans and mice share 80-percent of the same protein-coding genes. The key to morphological and behavioral complexity, a growing body of scientific evidence suggests, is the regulation of gene expression by a family of DNA-binding proteins called “transcription factors.” Now, a team of researchers with the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley has discovered the secret behind how one these critical transcription factors is able to perform — a split personality.
Using a technique called single-particle cryo-electron microscopy, the team, which was led by biophysicist Eva Nogales, showed that the transcription factor known as “TFIID” can co-exist in two distinct structural states. These two states — the canonical and the rearranged — differ only in the translocation of a single substructural element — known as lobe A — by 100 angstroms (an atom of hydrogen is about one angstrom in diameter). This structural shift enables initiation of the transcription process by which the genetic message of DNA is copied to RNA for the eventual production of proteins.
“TFIID by itself fluctuates between the canonical and rearranged states,” Nogales says. “When TFIID becomes bound to another transcription factor, TFIIA, it shifts mostly to the canonical state, but in the presence of both TFIIA and DNA, the TFIID shifts to the rearranged state, which enables recognition and binding to key DNA sequences and marks the start of the transcriptional process.”
Understanding the reorganization of TFIID and its role in transcription provides new insight into the regulation of gene expression, Nogales says, a process critical to the growth, development, health and survival of all organisms.
Nogales is a leading authority on electron microscopy and holds joint appointments with Berkeley Lab, the University of California (UC) at Berkeley, and the Howard Hughes Medical Institute (HHMI). She is the corresponding author of a paper describing this research in the journal Cell, titled “Human TFIID Binds to Core Promoter DNA in a Reorganized Structural State.” Co-authors are Michael Cianfrocco, George Kassavetis, Patricia Grob, Jie Fang, Tamar Juven-Gershon and James Kadonaga.
The growing number of organisms whose genomes have been sequenced and made available for comparative analyses shows that the total number of genes in an organism’s genome is no measure of its complexity. The fruit fly, Drosophila, for example, is far more complex than the nematode worm, Caenorhabditis elegans, but has about 6,000 fewer genes than the worm’s 20,000. The total number of human genes is estimated to fall somewhere between 30,000 and 40,000. By comparison, the expression of the genes of both the fruit fly and the nematode are regulated through about 1,000 transcription factors, whereas the human genome boasts approximately 3,000 transcription factors. That multiple transcription factors often act in various combinations with one another creates even more evolutionary roads to organism complexity.
“Although the number of protein coding genes has remained fairly constant throughout metazoan evolution, the number of regulatory DNA elements has increased dramatically,” Nogales says. “Our discovery of the existence of two structurally and functionally distinct forms of TFIID suggests a potential molecular mechanism by which a combination of transcription factors can tune the expression level of genes and thereby give rise to a diversity of outcomes.”
Despite its critical role in transcription, high-resolution structural information of TFIID has been restricted to crystal structures of a handful of protein subunits and domains. Nogales and her colleagues are the first group to obtain three-dimensional visualization of human TFIID that is bound to DNA. The single-particle cryo-electron microscopy technique they employed records a series of two-dimensional images of an individual molecules or macromolecular complexes frozen in random orientations, then computationally combines these images into high-resolution 3D reconstructions.
“Through cryo-EM and extensive image-sorting, we found that TFIID exhibits a surprising degree of flexibility, moving its lobe A, a region that covers approximately one-third of the complex, by 100 angstroms across its central channel,” says Cianfrocco, lead author of the Cell paper. “This movement of the lobe A is absolutely essential for TFIID to bind to DNA.”
Nogales says that while many macromolecular complexes are known to be flexible, this typically involves the limited movement of a small region within the complex, or some tiny motion of the entire complex. The movement of TFIID’s lobe A represents an entire restructuring that dramatically alters what the molecule can do. In the canonical state, TFIID’s lobe A is bound to its lobe C, which appears to be the preferred form of free TFIID. In the rearranged state, TFIID’s lobe A is bound to its lobe B, which is the state in which it can then strongly bind to DNA promoters.
“The TFIIA molecule serves as the mediator for this transition, maintaining TFIID in the canonical state in the absence of DNA and initiating the formation of the rearranged state in the presence of promoter DNA,” Cianfrocco says. “Without the presence of TFIIA, the binding of TFIID to DNA is very weak.”
Nogales and her colleagues are now studying how TFIID, once it is bound to DNA, recruits the rest of the machinery required to transcribe the genetic message into RNA.
“Our new work will involve constructing a macromolecular complex that is well over two million Daltons in size, which is about the size of a bacterial ribosome,” Nogales says. “The size and relative instability of our complex will represent a major experimental challenge.”
This work was supported by grants from the National Institutes of Health and the Human Frontier in Science Program in Strasbourg, Germany, and the Howard Hughes Medical Institute.
- Michael A. Cianfrocco, George A. Kassavetis, Patricia Grob, Jie Fang, Tamar Juven-Gershon, James T. Kadonaga, Eva Nogales. Human TFIID Binds to Core Promoter DNA in a Reorganized Structural State. Cell, 2013; 152 (1-2): 120 DOI: 10.1016/j.cell.2012.12.005