|Reactivation of the FMR1 Gene in Fragile X Patients Cells in Culture
FRAXA funded this project grant during 1999-2000.
In individuals with fragile X syndrome a chemical switch called methylation "turns off" the FMR1 gene. Simple molecules known as methyl groups attach to the DNA sequence constituting the "promoter" region of the gene, effectively blocking the transcription of its genetic code. The result is gene inactivation and lack of its specific protein product FMRP.
In addition, proteins known as histones make up a spool around which the DNA thread is wound. If the histones are loaded with acetyl groups (again, simple modifying molecules like methyl groups), the DNA is loosely packed and the FMR1 gene can be freely transcribed. On the other hand, loss of acetyl groups (deacetylation) results in tight packing of the DNA, which becomes inaccessible to the molecular machinery responsible for the transcription of the gene and, ultimately, for the production of FMRP. Thus, DNA hypermethylation and histone deacetylation need to be reversed to enable the FMR1 gene to express FMRP.
by Giovanni Neri, 1/30/1999
Our project is based on the fact that in 99% of the fragile X syndrome patients, the coding region of the FMR1 gene is intact and its loss of function is due to the CGG expansion and methylation of the regulatory region at one of its extremities. One can say, in a simplified manner, that the gene is switched off and that we are trying to find a way to switch it back on.
Since methylation is a key factor in the silencing of the gene, our first approach was to treat in vitro cell lines from fragile X patients with the demethylating drug 5-azadeoxycytidine. This treatment reactivated FMR1 gene transcription and restored production of its protein product FMRP (Chiurazzi et al., Hum Mol Genet 7, 109-113, 1998). We plan on extending our original observations to a larger number of cell lines and at the same time on trying other compounds that have the potential to reactivate the FMR1 gene.
One promising compound is butyrate, which acts on DNA-associated proteins called histones. Histones play an important role in the regulation of gene activity and in the past we were able to show that treating fragile X lymphocytes (blood cells) with butyrate resulted in a reduced manifestation of the fragile site FRAXA (Pomponi and Neri, Am J Med Genet 51, 447-450, 1994). Butyrate is a drug of limited toxicity and the results obtained with the in vitro experiments could lead to eventual in vivo trials (in live animals or human patients.) International collaboration with other distinguished groups (Ben A. Oostra, Rotterdam; Steve Warren, Atlanta) will allow the sharing of expertise and will help in developing new ideas and new approaches.
After showing that demethylating drugs such as 5-azadeoxycytidine (5-azadC) can reverse hypermethylation and reactivate the FMR1 gene, Dr. Neri and his team have run two sets of experiments in this project aimed at:
1) reversing the silencing of the FMR1 gene by treating fragile X lymphoblastoid cell lines with histone-acetylating drugs such as 4-phenylbutyrate (4-PBA), butyrate (BA) and trichostatin A (TSA).
2) Verifying the possible synergistic action of the histone-acetylating drugs with the DNA- demethylating drug 5-azadC, when used in a combined treatment.
The team has found that 4-PBA and BA are able to reactivate the expression of the FMR1 gene, although at low level (only 1-2% of its normal activity). On the other hand, when 4-PBA or BA were employed together with 5-azadC, a marked synergistic effect was observed. The FMR1 reactivation obtained with 5-azadC alone was enhanced up to fivefold when 4-PBA or BA were added, thus confirming the hypothesis that DNA hypermethylation and histone deacetylation are sequential steps in a single pathway that leads to the silencing of the fully mutated FMR1 gene. The team also found that the extent of the FMR1 reactivation seems to correlate with the size of the full mutation: cells with 600-700 CGG repeats responded less to to the treatments than cells with 270-350 repeats.