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RETT SYNDROME RESEARCH TRUST

Kelly Rae Chi

[links to podcasts are below]

That mutations in the MECP2 gene cause Rett Syndrome has been known for over a decade. But what exactly the protein does is not yet clear.

In the early 90s, Adrian Bird’s group purified MeCP2—which stands for methyl-CpG binding protein 2—and named the protein for its ability to bind parts of the DNA with a chemical tag called a methyl. Methyls tend to dampen the expression of genes, suggesting that MeCP2’s function is to silence genes.

Studies published since then suggest MeCP2 activates or represses the expression of many genes. Other results suggest that the protein binds throughout the genome, influencing the way DNA packs into a cell.

New evidence, published today (December 21) in Cell, shows that MeCP2 binds to spots throughout the genome that are tagged with the chemical, 5-hydroxymethylcytosine (5hmC) in mice, and that this interaction may be important for understanding Rett Syndrome.

“[The study is] a very interesting new development in studying the functional significance of MeCP2, which we have to understand if we’re going to understand Rett Syndrome,” says Bird, professor of genetics at the University of Edinburgh in Scotland who was not involved with work.

Abundant in the DNA of certain brain cells, 5hmC seems to be a signpost of sorts for active spots within the genome — that is, the regions that are churning out new protein — the study found. MeCP2’s latching onto these sites supports its potential role as a gene activator, though it’s clear that the case of what MeCP2 does is far from closed.

“Whether or not [MeCP2 is] directly involved in activation is still a matter of further investigation. But we know that it can localize to a region that contains 5hmC and active genes,” says Skirmantas Kriaucionis, group head in the University of Oxford Nuffield Department of Medicine, who co-led the study with Nathaniel Heintz of the Rockefeller University in New York.

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heintz-skiri

Podcast with Skirmantas Kriaucionis

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Figuring out 5hmC

As a postdoctoral researcher in Heintz’s lab, Kriaucionis found 5hmC in 2009 by accident when he was looking at how a closely related chemical, 5-methylcytosine (5mC), influences genome structure in brain cells. Anjana Rao’s group, then at Harvard Medical School, working independently from Heintz’s lab, confirmed the existence of 5hmC in the same issue of the journal.

Researchers consider 5mC a fifth base, and 5hmC a sixth base, of DNA, which is traditionally thought of as a string of four different chemicals called nucleotides. 5mC and 5hmC resemble the traditional base cytosine, but with a methyl group added on, making 5mC, and a hydroxy group added to the methyl, creating 5hmC. The new study confirmed that patterns of these chemical modifications to the genome are different in each cell and influence which genes are turned on or off and when.

The discovery of 5hmC opened up a new area of work—and hundreds of new papers—focused on where the nucleotide is in the genomes of different cell types, and what it’s doing.

In the new study, using an explorative molecular assay to fish for a binding partner for 5hmC, the group identified the molecule as MeCP2, and nothing else. “It was really a surprise,” Kriaucionis says.

“This paper is the second paper to suggest a candidate binding protein for 5hmC,” says Rao, now a professor of signaling and gene expression research at the La Jolla Institute for Allergy and Immunology, who was not involved with the new study. Other findings have proposed a different molecule, MBD3, as a candidate, but those and the new results need further investigation, she adds.

“So far, both candidates — MBD3 and MeCP2—also bind 5mC, so an exclusive binding protein for 5hmC has not yet emerged,” Rao says.

Indeed, contradicting the new evidence that MeCP2 binds 5hmC and 5mC equally, some previous studies show that MeCP2 much prefers binding to 5mC over 5hmC. For example, a study published earlier this year shows that says MeCP2 is nearly 20 times more likely to bind 5mC than 5hmC.

Relating to Rett

In the new study, Kriaucionis and his colleagues observed that a certain Rett-causing mutation, called R133C—which is responsible for a relatively milder form of the disorder—disrupts MeCP2’s binding to 5hmC.

“[The R133C mutation] is really interesting because it allows us to speculate that MeCP2 binding to 5hmC is important as a part of the function which causes Rett Syndrome,” Kriaucionis says.

The evidence now “strongly suggests” the potential involvement of 5hmC in Rett, says Peng Jin, an associate professor of human genetics who was not involved with the new study. A study his group published last year in Nature Neuroscience found that patterns of 5hmC are altered in mouse models of Rett.

Interestingly, the R133C mutation only slightly dampens MeCP2’s interaction with 5mC, suggesting that MeCP2’s binding with 5mC serves a different purpose than that of MeCP2 and 5hmC.

Other Rett-causing mutations in MeCP2 examined by the group don’t seem to affect binding to 5hmC, meaning 5hmC binding does not fully explain the symptoms of Rett.  “It will be important to test further the contribution of 5hmC to Rett Syndrome,” notes Jin, adding that there are mouse models available to do so. Those mutant mice lack the enzymes needed to convert 5mC to 5hmC.

In the new study, researchers studied only a few types of neurons, but there are hundreds of cell types in the brain. Kriaucionis thinks that MeCP2 binds to 5hmC in other cells.

The 5hmC patterns themselves are cell-specific, however, perhaps further complicating the story of MeCP2.

“We really need to get more data to understand whether or not, how different 5hmC and MeCP2 localization would be in different cell types,” Kriaucionis says. “It’s an important component to understand MeCP2 function,” and how scientists might think about future treatment.

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Cell podcast with Nat Heintz    (click on Paperclick on right)

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HEINTZ LAB
Isolated nuclei from cerebellum. Blue color represents DNA. Green spot is the nucleolus of a Purkinje cell. (photo from Heintz lab)

Isolated nuclei from cerebellum. Blue color represents DNA. Green spot is the nucleolus of a Purkinje cell. (photo from Heintz lab)

The readers of this blog will have noted the frequent mention of epigenetics – a young but hot area of research which holds promise for novel therapeutic interventions for a myriad of diseases.  The term epigenetic means over and above the genome. It refers to changes in gene expression caused by mechanisms other than changes in the underlying DNA sequence.  It helps to think of epigenetics in terms of the charm bracelet analogy – the DNA is the charm bracelet and epigenetic modifications are the charms that can be added to or removed from the bracelet.

Every cell has about 30,000 genes. What distinguishes a heart cell from a liver cell is the pattern of gene expression (what genes are on and what genes are off). Considering the vast number of cell types in the human body it’s easy to appreciate how the instructions for turning genes on and off must be finely tuned and exquisitely calibrated.  When missteps occur – disease often follows.

Epigenetic modifications catalyze gene expression changes. The best known epigenetic modification is methylation.  Adding methyl “charms” to DNA leads to gene silencing. MeCP2, the protein that is mutated in Rett syndrome as well as a variety of other disorders, binds to DNA that has already been methylated and plays a role in regulating downstream genes.

The excitement surrounding epigenetics is that it’s dynamic. “Charms” can be added and they can be removed. The ability to catalyze these modifications through drugs has already led to some treatments for cancers. The hope is that these treatments are the mere tip of the iceberg.

5-6-box1

The latest breakthrough in epigenetics comes to us from the laboratory of RSRT advisor Nat Heintz of Rockefeller University. The connection to Rett Syndrome, however, does not end there. The post-doc in the Heintz lab who made this discovery is Skirmantas Kriaucionis. As a graduate student, he spent five years in the lab of RSRT trustee Professor Adrian Bird who in 2007 announced the dramatic Rett reversal experiments. The Heintz and Kriaucionis paper was published online, April 16th, in the high profile journal Science.

Nat Heintz, Ph.D.

Nat Heintz, Ph.D.

We all remember the four nucleotide bases (A-adenine, T-thymine, G-guanine, C-cystosine) from high school biology. Together these four bases make up DNA. If we were taking high school biology today we would also learn about a fifth nucleotide, methylcytosine (mC) that replaces cytosine depending on whether a gene needs to be turned on or off. Kriaucionis has now added a sixth, hydroxymethylcytosine (hmC), a nucleotide base previously observed only in the simplest of organisms, bacterial viruses.

Drs. Heintz and Kriaucionis discuss their findings with Monica Coenraads.

MC: It’s not every day that a new nucleotide is discovered. Congratulations! Were you surprised by this finding?

NH: Yes, I was. I like to think that in most cases I know where my research is heading. But, in this particular case, the finding was completely unexpected. Broadly speaking, I think these experiments validate that it’s worthwhile spending time analyzing detailed properties of cells as they exist and operate in vivo, rather than studying their properties after they have been adapted for growth in culture.

Ironically, as a graduate student in David Shub’s lab I worked on bacterial viruses carrying modified nucleotides in their genomes. Now, many years later, that research has come full circle.

MC: As is often the case in science, the discovery was rather accidental. Dr. Kriaucionis, can you tell us what kind of research you were undertaking and how you stumbled on hydroxymethylcytosine (hmC)?

Skirmantas Kriaucionis, Ph.D.

Skirmantas Kriaucionis, Ph.D.

SK: The discovery of hmC can be traced back to several key reasons. One, we were interested in investigating chromatin structure (DNA/protein material which makes up the chromosomes) in different brain cell types. A finding which attracted our attention is that certain cell types have distinct looking chromatin. A dramatic example is Purkinje cells which have a very open chromatin configuration, in contrast to cerebellar granule cells which have very condensed looking chromatin (see above picture) We wanted to investigate this further. Second, was the availability of technological tools in the Heintz lab, which not only gave us access to these specific cells but allowed us to isolate very pure material to investigate. Thirdly, was the rather outdated technique that I was using to quantify the absolute levels of the different nucleotides. Scientists rarely use this technique now because it does not tell you where in the genome the nucleotides are. Since location gives insight into biological function this technique is rarely employed. But the decision to use this method turned out to play a large part of how I was able to identify the hmC.

At first I didn’t believe my results, and it took several months to reproduce the results many times using different experimental conditions to give us the necessary confidence that indeed, we were seeing a novel nucleotide and not an artifact of the experiment.

MC: So hmC is an example of a further modification to a nucleotide base, cytosine, that has already been modified by a methyl group. Has your discovery added more complexity to the process by which genes are regulated?

SK: Yes, indeed it does add a layer of complexity to what sort of biological message is being encoded by these modifications. The next important step will be to figure out what outcome is expected when DNA sequences have unmethylated cytosine, methylated cytosine or hydroxymethylated cystosine.

It’s interesting to note that while histone proteins have many epigenetic modifications mammalian DNA had, until now, only one – methylation. This finding is totally unexpected. Although RNA has plenty of modifications, its functional repertoire is complex as well including structural, enzymatic and coding roles.

MC: Do you have any hypothesis about what hmC might be doing?

SK: hmC is very abundant in brain so this gives us confidence that it’s doing something biologically significant. We currently have two hypotheses regarding its function. One hypothesis is that the hmC might be an intermediate step to get methylated cytosine back to its original unmethylated state.

MC: So in essence hmC might transform a silent gene (methylated) to an active gene (unmethylated).

SK: Correct. Another possibility is that hmC is a stable final modification. It could still activate a silent gene without demethylation. We see different amounts of hmC in different cell types so it may be that in some cell types hmC is an intermediate while in other cell types it’s a stable modification. And obviously we are keeping open mind as there may be an unpredicted function of hmC.

MC: There was a second paper in the same issue of Science as your paper that identified the enzyme that catalyzes the conversion from mC to hmC. Can you elaborate on the synergies between these two papers?

NH: The second paper is from the laboratory of Anjana Rao at Harvard. We were unaware of her ongoing research until very recently. Our papers complement and actually help each other quite a bit. The Rao lab identified an enzyme which catalyzes the conversion from mC to hmC. This is a very significant finding. A criticism that our paper was receiving was that hydroxylation of methyl groups could be a spontaneous reaction that was happening due to the oxidation of DNA. We addressed this question in our study, but the accompanying paper completely puts this criticism to rest since the group found the enzyme catalyzing the conversion of mC to hmC. So in that respect their paper was helpful to us. Our data strengthened their paper because we showed that hmC was highly enriched in brain, providing a link to the biology of neurons and, perhaps, neurological disease. Taken together, the two papers establish the importance of hmC in the mammalian genome, and suggest that this new epigenetic mark will provide an entry into a previously unanticipated and important field of biology.

MC: Dr. Kriaucionis, previous to your post-doc position in the Heintz lab you were in Adrian Bird’s lab for five years. Prof Bird’s lab focuses on DNA methylation and discovered MECP2, the “Rett gene” in the early 1990s. You co-authored 5 papers on MECP2 and Rett Syndrome with Prof. Bird. Do you think there is any possible connection between MECP2 and hmC?

SK: I think there is indeed a connection. As your readers know, MECP2 binds to methylated DNA. There is data showing that MECP2 resists binding to DNA that contains hmC. If these findings hold up, then hydroxylating mC could release MECP2 from the chromatin and influence for example nearby gene expression. Finding genomic MeCP2, mC and hmC distribution in neuronal cell types will provide us with valuable insights into the biological function of MECP2 and the role of DNA modifications.

NH: So Skirmantas’ hypothesis implies that hmC can modulate the distribution of where MeCP2 binds in the nucleus. There is an alternative scenario in which a different and, as of yet, unidentified protein binds to hmC. My theory is that this protein is likely not a repressor, like MeCP2, but perhaps is acting as an activator. The beauty of science is that we can test these hypotheses. I suspect we’ll have our answer in a relatively short amount of time.

MC: What are the next steps that the lab is pursuing?

SK: Of utmost importance is to answer the following question: where is hmC and what is it doing? To answer that question we are developing a new set of tools which will allow us to adequately map where hmC is in the genome. I hope that having a clear understanding of location will provide clues as to its role.

MC: Dr. Heintz, you are known in the scientific world as a “big idea man” who identifies and then develops the necessary tools to go after key neurobiological questions. Can you elaborate on the development of the novel tools to which Dr Kriaucionis just alluded?

NH: The fact that as currently applied bisulfate sequencing techniques,which are used to detect sites of methylation in the genome, do not distinguish between a cytosine that is methylated or hydroxymethylated is a major problem. We hope to develop new methodology that will allow us to map the precise sites of genomic hmC and mC separately.

Our strength as a laboratory fits in nicely with the task ahead. We have at our disposal gene expression data that is specific to a large variety of specific cell types. This information will be very useful as we begin to map where in the genome hmC is found. These two separate but very complementary sides of our lab will help elucidate not only the function of hmC but perhaps also the function of the proteins that bind to it.

MC: Dr. Heintz, your lab does not have a history of working on epigenetics and yet you made a remarkable discovery. Given this finding do you envision changing the focus of your lab in any way?

NH: Yes, I think it will change our focus somewhat. We feel this discovery is a critically important finding and the role of hmC in neurons will be of high interest both fundamentally to understand brain function and also for investigation of epigenetic influences on disease states. So we will be focusing a lot of our attention on what the biological role of hmC is in the healthy and diseased brain .

MC: You are not a “Rett researcher” yet you have kept up with the literature, attended a number of Rett scientific meetings that I have organized over the years and have been a highly respected advisor to the field. Do you have any insight on recent progress and where you think the field is heading?

NH: I’m very enthusiastic about the developments of the last few years. Adrian Bird’s reversal experiments are stunning. It has also become very clear, largely as a consequence of Huda Zoghbi’s work, that the impact of MeCP2 in different brain cell types is distinct. This means that strategies aimed at treating particular symptoms of the disease can be devised in the nearer term while approaches to reversing the entire phenotype, a much taller order, are explored.

MC: On behalf of our readers I thank you both for your time and wish you the very best for your ongoing research. I look forward to staying in touch and hearing how this work unravels. Congratulations again on your paper.