Giovanni Neri, MD
Principal Investigator
Pietro Chiurazzi, MD, PhD
Postdoctoral Fellow
Institute of Medical Genetics
Catholic University, Rome, Italy

by Giovanni Neri, 4/2010

We are now screening dozens of potentially reactivating compounds with a histone hyperacetylating effect, in collaboration with the orphan-drug company Neuropharm (UK). A new collaboration established with Dr. Steve Haggarty at the Broad Institute of Harvard and MIT will also allow us to screen molecules that appear to have an effect comparable to that of 5-azadC, but independent of cell division. The discovery of such reactivating compounds would facilitate subsequent clinical trials.

We also continue studying the epigenetic changes that regulate transcription in FXS patients and in UFM carriers. We observed that in UFM cells histones H3 and H4 are deacetylated, like in FXS cells, while lysine K4 of histone H3 is hypermethylated and lysine K27 is demethylated, like in normally transcribing control cells. In UFM cells we also detected the antisense transcript ASFMR1, that might have a critical role in the epigenetic regulation of FMR1, by generating double stranded RNAs together with the sense transcript. These studies will be extended to a recently described regulatory region, upstream of the FMR1 promoter, and will also include ChIP analysis of CTCF binding to the FMR1 promoter as well as dosage of natural antisense transcripts.

To further clarify the mechanisms of FMR1 inactivation and to establish its timing, we will analyze the epigenetic status of induced Pluripotent Stem (iPS) cells obtained from fibroblasts of FXS males, control individuals and UFM carriers. iPS cells will be generated in collaboration with Dr. Guoping Fan of UCLA.

by Giovanni Neri and Pietro Chiurazzi, 6/2008

Our project is aimed at achieving in vitro reactivation of the inactive FMR1 gene in non-dividing neurons obtained from Fragile X neural progenitor cells (NPC). We hypothesize that silencing of the fully mutated FMR1 gene is a dynamic process, requiring continuous maintenance in somatic cells. If this is the case, interference with the activity of the enzymes involved in the epigenetic inactivation of full mutations may allow FMR1 reactivation in postmitotic neurons like in an adult brain. We will employ both pharmacological and siRNA treatments in order to modulate the activity of DNA methyltransferases as well as histone (de)acetylases and (de)methylases.

Furthermore, we have recently established cell lines from two rare males with normal cognition and completely unmethylated full mutations (UMF). We plan to characterize these cell lines, comparing their epigenetic and transcriptional status to that of "classical" methylated full mutations (MFM) and normal controls. Differences between UMF and MFM cells may hint at other relevant molecules that could be targeted pharmacologically in fragile X neural cells with the goal of reactivating the inactive MFMs of patients and restoring sufficient levels of FMRP protein.

Project grant funded 1999-2000

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.

Update report,1/2000

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.

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.