It is a decades-old mystery: How does a small DNA repeat cause Fragile X syndrome, the most common form of inherited mental retardation? Researchers led by Samie Jaffrey, Cornell University, New York, report in the February 28 Science that an mRNA-DNA interaction may be to blame. They found that in human embryonic stem cells, a trinucleotide repeat expansion in the mRNA for fragile X mental retardation 1 (FMR1) binds its own DNA. The DNA-RNA duplex somehow alters the chromosome packaging around the gene, turning it off. “It is pretty exciting to have a first glimpse of how the mutation shuts down the FMR1 gene,” said David Nelson, Baylor College of Medicine, Houston. Nelson had a hand in discovering the mutation. “The authors provide interesting and convincing data that FMR1 mRNA causes the gene silencing,” he said.  

More than 20 years ago, Nelson and others found that people with Fragile X syndrome shared a long CGG repeat in the promoter region of the FMR1 gene, which codes for an RNA-binding protein involved in RNA metabolism (see Verkerk et al., 1991). While a normal FMR1 gene contains about 30 of these repeats, people with Fragile X have more than 200. That expansion causes the gene to turn off about 11 weeks into gestation, but researchers could not figure out why. Put into mice or human cells, the mutant gene left them unchanged, which only added to the puzzle. More recently, researchers found that human embryonic stem cells (hESCs) differentiating in culture did mimic the disease’s development—cells briefly expressed FMR1, but stopped after several weeks (see Eiges et al., 2007). 

To figure out why, first author Dilek Colak and colleagues examined hESC lines that express the mutant gene. In neurons derived from these cells, FMR1 expression started falling after 48 days and stopped completely at day 51 (see image below). At the same time, the FMR1 promoter took on a pattern of histone methylation consistent with gene repression, suggesting that an epigenetic change was behind the drop. To their surprise, the researchers found that if short hairpin RNAs were injected into the cells to bind FMR1 mRNA, the cells maintained a normal methylation pattern. That suggested that FMR1 mRNA was responsible for silencing its own gene. The group wondered if the mRNA bound directly to the DNA, an interaction that silences genes in plants (see Sun et al., 2013).

Suppressing FMR1.

Neurons derived from hESCs express FMR1 (yellow/green) at 45 days, but stop by day 51. [Image courtesy of Science/AAAS.]

To explore this idea, Colak treated the neurons with a small molecule called 1a, which binds to the G-G bonds that form when the repeats in FMR1 mRNA fold back on themselves. The compound stabilizes the folded mRNA and prevents it from binding other nucleic acids. In the presence of 1a, the FMR1 gene stayed active. However, once the repressive methylation pattern had set in and the gene was turned off, 1a could not reactivate it. These findings suggested that while the mRNA-DNA duplex initiates FMR1 silencing, it is not required to maintain it.

To figure out where in the DNA the FMR1 mRNA bound, Colak isolated the chromatin from the stem cell-derived neurons and chemically cross-linked the mRNA-DNA hybrids. He was then able to chop up the DNA and identify sequences bound to the RNA. He found that the mRNA CGG repeat region latched on to the complementary sequence in the DNA. However, the two only bound in cells that were cultured for at least 45 days, in keeping with the time frame for FMR1 suppression.

Why would this mechanism take that long to kick in? Jaffrey is not sure, but hypothesized that it may be related to repackaging of the DNA in chromatin after transcription. Helicases that rewind DNA are involved in that process, but their levels decline as stem cells take on neuronal characteristics (see Wu et al., 2010). That could give mRNA more time to slip in and bind the DNA before it is repackaged. Why this occurs only when the repeats exceed 200 is unclear, but Jaffrey thinks that as the transcript is being made, the CGG repeat needs to be that long to reach back and bind the DNA, or that at least that many repeats are needed for the RNA-DNA duplex to bind tightly enough to modulate transcription. 

Either way, this mechanism suggests new ways to prevent Fragile X syndrome, Jaffrey said. A 1a-like drug applied early in gestation and maintained throughout life might prevent the initial suppression of FMR1 transcription. While such a drug on its own could not reverse silencing in patients who already have the disorder, it might work if other agents first remove the repressive methylation. Nelson thought the prospect was interesting, but cautioned that if reactivated, the gene’s mutant mRNA might lead to negative effects on its own. People who express the FMR1 transcript with 55 to 200 CGG repeats carry a higher risk of neurodegenerative diseases (see Hagerman, 2006). 

Jaffrey says he next plans to work out how this DNA-RNA duplex alters FMR1 epigenetics. He will also look to see if a similar mechanism plays into other triplet repeat diseases, such as Huntington’s disease and Friedreich’s ataxia. Nelson pointed out that not all gene expansions are characterized by declines in protein production, however. He suggested the mechanism could apply in diseases caused by a hexanucleotide repeat in the C9ORF72 gene. That mutation leads to a drop in C9ORF72 protein expression and is a major cause of familial amyotrophic lateral sclerosis and frontotemporal lobar degeneration (see Jun 2013 news story). 

Cynthia McMurray, Lawrence Berkeley National Laboratory, California, agreed that the proposed mechanism may not apply in all repeat expansion diseases, because other repeats tend to be shorter. However, she thinks it may be at work in a subset of diseases with long trinucleotide repeats, such as myotonic dystrophy. She would have liked to see more details about methylation changes on DNA, as opposed to histones, and is curious to know what happens with different lengths of repeats.

“This is a very interesting and entirely novel mechanism for gene silencing in Fragile X Syndrome that may lead to new prevention tactics,” wrote Jennifer Darnell, The Rockefeller University, New York, to Alzforum in an email. “The better we understand the molecular basis of FMR1’s silencing, the more options will open up for exploring novel therapy.”—Gwyneth Dickey Zakaib


No Available Comments

Make a Comment

To make a comment you must login or register.


News Citations

  1. Methylation a Turn Off for Disease Gene C9ORF72?

Paper Citations

  1. . Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell. 1991 May 31;65(5):905-14. PubMed.
  2. . Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos. Cell Stem Cell. 2007 Nov;1(5):568-77. PubMed.
  3. . R-loop stabilization represses antisense transcription at the Arabidopsis FLC locus. Science. 2013 May 3;340(6132):619-21. PubMed.
  4. . Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing. Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):5254-9. Epub 2010 Mar 1 PubMed.
  5. . Lessons from fragile X regarding neurobiology, autism, and neurodegeneration. J Dev Behav Pediatr. 2006 Feb;27(1):63-74. PubMed.

Further Reading


  1. . The unstable repeats--three evolving faces of neurological disease. Neuron. 2013 Mar 6;77(5):825-43. PubMed.

Primary Papers

  1. . Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome. Science. 2014 Feb 28;343(6174):1002-5. PubMed.