ALS Researchers Tackle Epigenetics of C9ORF72 and the Entire Genome

This is part 3 of a 3-part series from the 27th International Symposium on ALS/MND. See also part 1 and part 2 here.

Likable ME? Histone methylation (red) and hypermethylation of CpG islands (white) near the expanded C9orf72 repeat may help reduce levels of toxic RNAs and dipeptide repeat proteins in C9orf72 disease by reducing C9orf72 expression. [Perry et al., 2010. Courtesy of Nature Publishing Group.]

How do epigenetic marks—covalent changes to DNA or histones—affect ALS risk, onset, or progression? Do disease models replicate these changes? And, could altering epigenetic markers be therapeutic? These questions in the young field of ALS epigenomics were the focus of a group of talks at the 27th International Symposium on ALS/MND, held in Dublin in December 2016.

Chemical changes to the genome help control which genes are switched on and off. These changes, known as epigenetic marks, include methylation of cytosines and multiple modifications of histones, including lysine acetylation. Such changes affect accessibility of DNA to the RNA polymerase II machinery, leading to transcriptional silencing of some genes.

Rewriting these epigenetic marks enzymatically helps people adapt to key changes including in the environment. But these changes according to a number of studies may also make them more vulnerable to complex genetic disease.

Recent methodological advances have made it feasible to conduct epigenome-wide association studies (EWAS) to study these changes in hopes to understand their role in complex genetic disease, said Jonathan Mill of the University of Exeter in the United Kingdom. His lab is part of the multinational PsychENCODE consortium, which is building a publicly accessible database of epigenetic changes in schizophrenia, autism, and bipolar disorder. “While ALS is not a core disease for this effort, a lot of the information coming out from it will be directly relevant to ALS,” Mill said. Mill is also studying epigenomic changes in neurodegenerative disorders including Alzheimer’s disease.

“DNA sequence is very stable, but epigenetic regulation is highly dynamic over the course of the lifespan,” Mill said. Development, the immune response, and the environment all influence the epigenome. Environmental exposures may be especially influential during certain vulnerable times, such as the prenatal and perinatal periods, and other times of rapid cell division.

In other diseases, PsychENCODE is already offering insights into the epigenomic landscape. Work from Mill’s lab and others has revealed that an unexpected proportion of methylation sites in the fetal brain were also implicated in adult schizophrenia, suggesting that epigenetic changes during early development may increase the risk for neuropsychiatric disease later in life (Pidsley et al., 2014).

What’s more, these epigenetics changes may also play a role in neurodegenerative diseases including Alzheimer’s disease. Mill’s team in 2014 identified specific methylation changes in genes encoding ankyrin1, ribosomal protein L13 (RPL13), rhomboid 5 homolog 2 (RHBDF2) and cadherin-related protein 23 (CDH23) in the brains of people who died of complications due to Alzheimer’s disease. The genes were previously not associated with the disease (deJager et al., 2014).

Could epigenomics do the same for ALS? Mill and his collaborators hope so. They will compare epigenomes of ALS cases and controls matched for age, smoking status, and other variables. “Smoking produces really robust changes in the epigenome,” he noted, and is associated with a higher risk for ALS. Aging is the strongest known ALS risk factor, and also produces important epigenetic changes.

The ALS neuroprotector?

Some researchers however suspect that these epigenetics changes might also be neuroprotective – at least in C9orf72 ALS. The expansion is associated with hypermethylation of CpG islands near the repeat in the promoter region as well as repressive histone 3 and histone 4 modifications (See March 2015 news; Belzil et al., 2013; Xi et al., 2013). This leads to partial epigenetic repression of the C9orf72 locus (Belzil et al., 2014).

Protect and silence? Researchers found in 2016 that hypermethylation in patient-derived stem cells appears to reduce levels of toxic C9 repeat-containing RNAs (Cohen-Hadad et al., 2016). [Image: Stem Cell Reports, CC BY-NC-ND 4.0.]

At the meeting, Marka Van Blitterswijk of the Mayo Clinic in Jacksonville, Florida, reported that promoter hypermethylation can be detected in carriers of the C9orf72 expansion, with methylation levels that may be as much as 40%. The promoter is typically unmethylated in control subjects. Within a family, she said, methylation is generally lower in those who do not carry the expansion than in those who do, but the levels are variable. Levels of C9orf72 promoter methylation are much higher in the blood than in the brain, she has found, though at the moment, she noted, little is known about the significance of these differences.

Reduced C9orf72 expression, potentially due to hypermethylation, or other mechanisms related to the expansion, may also lead to expression changes of other genes in patients with C9orf72 disease. Van Blitterswijk reported that expansion carriers have highly elevated expression of multiple homeobox genes, master transcriptional regulators of development most commonly associated with patterning during embryogenesis. She compared the expression profile of the brains post mortem of 32 patients with either ALS or FTD with the expansion, to 30 ALS or FTD patients without expansions, and 20 controls without neurologic disease. Among the 40 genes most upregulated, 23 were homeobox genes she said. Upregulation of homeobox genes in expansion carriers was greatest in the cerebellum, in particular the gene for homeobox A5. Expression of transthyretin, which transports thyroid hormones and vitamin A in the blood and CSF, was also elevated.

While it’s too soon to know whether the epigenetic modification of C9orf72 is directly responsible for the elevation, Van Blitterswijk did find that knocking down C9orf72 expression in culture led to an increase in expression of these homeobox genes and transthyretin. Since reduced C9orf72 expression is one consequence of the expansion, she said, “our findings may point to the presence of compensatory mechanisms to cope with the pathogenic effects of the repeat expansion.”

The results add to growing studies that suggest that hypermethylation of CpG islands near the C9orf72 expanded repeat may be neuroprotective – by reducing production of dipeptide repeat proteins and toxic RNAs (see April 2015 news; McMillan et al., 2015; Liu et al., 2014).

Meanwhile, Zane Zeier of the University of Miami is hoping to understand the mechanistic basis of these epigenetics changes in ALS in hopes to develop a treatment for the disease. He reasoned that if epigenetics contributes to ALS, it will be important to know how faithfully cell models of C9orf72 ALS being studied replicate epigenetic changes seen in the human disease. To investigate this, Zeier examined the epigenetic state of the C9orf72 gene in patient lymphocytes and in motor neurons derived from them. Zeier found that methylation levels dropped during reprogramming of lymphocytes to iPS cells, and increased again during differentiation to motor neurons. “Our data show that DNA hypermethylation of the C9orf72 promoter is recapitulated in reprogrammed motor neurons,” Zeier said, “indicating they are a relevant epigenetic model system.”

He also found, in the C9Orf72 expansions in both reprogrammed motor neurons and patient tissues, the presence of a common type of modification, 5-hydroxymethylcytosine (Esanov et al., 2016). The epigenetic mark is hypothesized to counteract methylated-based repression by displacing methylated CpG island repeat binding proteins (Tahiliani et al., 2009). Thus, these changes might facilitate the production of toxic dipeptide repeat proteins and RNAs, Zeier said. Reducing 5hmC and increasing 5-methylcytosine may therefore be beneficial. Zeier is investigating small molecules targeting epigenetic proteins as a means to reduce this underlying neurotoxicity. Using iPS neurons, he hopes to “pursue an epigenetic strategy to rescue C9orf72 cellular phenotypes, including nucleocytoplasmic transport defects.” Stay tuned.

Read more:

Methyl Groups Put the Brakes on FTD Progression

Expansion and Methylation Go Hand in Hand in C9ORF72


Cohen-Hadad Y, Altarescu G, Eldar-Geva T, Levi-Lahad E, Zhang M, Rogaeva E, Gotkine M, Bartok O, Ashwal-Fluss R, Kadener S, Epsztejn-Litman S, Eiges R. Marked Differences in C9orf72 Methylation Status and Isoform Expression between C9/ALS Human Embryonic and Induced Pluripotent Stem Cells. Stem Cell Reports. 2016 Nov 8;7(5):927-940. [PubMed]

Esanov R, Belle KC, van Blitterswijk M, Belzil VV, Rademakers R, Dickson DW, Petrucelli L, Boylan KB, Dykxhoorn DM, Wuu J, Benatar M, Wahlestedt C, Zeier Z. C9orf72 promoter hypermethylation is reduced while hydroxymethylation is acquired during reprogramming of ALS patient cells. Exp Neurol. 2016 Mar;277:171-7. [PubMed]

McMillan CT, Russ J, Wood EM, Irwin DJ, Grossman M, McCluskey L, Elman L, Van Deerlin V, Lee EB.  C9orf72 promoter hypermethylation is neuroprotective: Neuroimaging and neuropathologic evidence. Neurology. 2015 Apr 21;84(16):1622-30. [PubMed]

Liu EY, Russ J, Wu K, Neal D, Suh E, McNally AG, Irwin DJ, Van Deerlin VM, Lee EB. C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD. Acta Neuropathol. 2014 Oct;128(4):525-41. [PubMed]

Belzil VV, Bauer PO, Gendron TF, Murray ME, Dickson D, Petrucelli L. Characterization of DNA hypermethylation in the cerebellum of c9FTD/ALS patients. Brain Res. 2014 Oct 10;1584:15-21. [PubMed]

Xi Z, Zinman L, Moreno D, Schymick J, Liang Y, Sato C, Zheng Y, Ghani M, Dib S, Keith J, Robertson J, Rogaeva E. Hypermethylation of the CpG island near the G4C2 repeat in ALS with a C9orf72 expansion. Am J Hum Genet. 2013 Jun 6;92(6):981-9. [PubMed]

Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009 May 15;324(5929):930-5. doi: 10.1126/science.1170116. [PubMed]

 Further Reading:

Paez-Colasante X, Figueroa-Romero C, Sakowski SA, Goutman SA, Feldman EL. Amyotrophic lateral sclerosis: mechanisms and therapeutics in the epigenomic era. Nat Rev Neurol. 2015 May;11(5):266-79. [PubMed]

De Jager PL, Srivastava G, Lunnon K, Burgess J, Schalkwyk LC, Yu L, Eaton ML, Keenan BT, Ernst J, McCabe C, Tang A, Raj T, Replogle J, Brodeur W, Gabriel S, Chai HS, Younkin C, Younkin SG, Zou F, Szyf M, Epstein CB, Schneider JA, Bernstein BE, Meissner A, Ertekin-Taner N, Chibnik LB, Kellis M, Mill J, Bennett DA. Alzheimer’s disease: early alterations in brain DNA methylation at ANK1, BIN1, RHBDF2 and other loci. Nat Neurosci. 2014 Sep;17(9):1156-63. [PubMed]

Pidsley R, Viana J, Hannon E, Spiers H, Troakes C, Al-Saraj S, Mechawar N, Turecki G, Schalkwyk LC, Bray NJ, Mill J. Methylomic profiling of human brain tissue supports a neurodevelopmental origin for  schizophrenia. Genome Biol. 2014;15(10):483. [PubMed]

conf-international symposium on ALS/MND disease-ad disease-als disease-schizophrenia epigenetics topic-preclinical
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