trichostatin-a and Fragile-X-Syndrome

Since it is extremely difficult to establish an animal model for human chromosomal abnormalities, induced pluripotent stem cells (iPSCs) provide a powerful alternative to study underlying mechanisms of these disorders and identify potential therapeutic interventions. In this study we established iPSCs from a young girl with a hemizygous deletion of Xq27.3-q28 who exhibited global developmental delay and intellectual disability from early in infancy. The deletion site on the X chromosome includes Fragile X Mental Retardation 1 (FMR1), the gene responsible for fragile X syndrome, which likely contributes to the patient's neurodevelopmental abnormalities. The FMR1 gene was expressed in approximately half of the iPSC clones we generated while it was absent in the other half due to the random inactivation of normal and abnormal X chromosomes. The normal or absent expression pattern of the FMR1 gene was not altered when the iPSCs were differentiated into neural progenitor cells (NPCs). Moreover, chromosome reactivating reagents such as 5-aza-2-deoxycytidine, trichostatin A, and UNC0638, were tested in an attempt to reactivate the suppressed FMR1 gene in affected iPSC-NPCs. The affected and control isogenic iPSCs developed in this study are ideal models with which to identify downstream consequences caused by the Xq27.3-q28 deletion and also to provide tools for high-throughput screening to identify compounds potentially improving the well-being of this patient population.

Topics: Cell Differentiation; Cells, Cultured; Child, Preschool; Chromosome Deletion; Chromosomes, Human, X; Decitabine; Developmental Disabilities; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Induced Pluripotent Stem Cells; Intellectual Disability; Quinazolines

Topics: Animals; Antimetabolites, Antineoplastic; Azacitidine; Cell Differentiation; Cells, Cultured; Chromatin Assembly and Disassembly; Epigenesis, Genetic; Fragile X Mental Retardation Protein; Fragile X Syndrome; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Induced Pluripotent Stem Cells; Mice; Neurons

Expansion of the CGG.CCG-repeat tract in the 5' UTR of the FMR1 gene to >200 repeats leads to heterochromatinization of the promoter and gene silencing. This results in Fragile X syndrome (FXS), the most common heritable form of mental retardation. The mechanism of gene silencing is unknown. We report here that a Class III histone deacetylase, SIRT1, plays an important role in this silencing process and show that the inhibition of this enzyme produces significant gene reactivation. This contrasts with the much smaller effect of inhibitors like trichostatin A (TSA) that inhibit Class I, II and IV histone deacetylases. Reactivation of silenced FMR1 alleles was accompanied by an increase in histone H3 lysine 9 acetylation as well as an increase in the amount of histone H4 that is acetylated at lysine 16 (H4K16) by the histone acetyltransferase, hMOF. DNA methylation, on the other hand, is unaffected. We also demonstrate that deacetylation of H4K16 is a key downstream consequence of DNA methylation. However, since DNA methylation inhibitors require DNA replication in order to be effective, SIRT1 inhibitors may be more useful for FMR1 gene reactivation in post-mitotic cells like neurons where the effect of the gene silencing is most obvious.

Topics: Alleles; Azacitidine; Base Sequence; Cell Line; Decitabine; DNA Methylation; DNA Primers; Enzyme Inhibitors; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; Gene Silencing; Histones; Humans; Hydroxamic Acids; Models, Biological; Mutation; Naphthalenes; Niacinamide; Promoter Regions, Genetic; Pyrones; Sirtuin 1; Sirtuins; Trinucleotide Repeat Expansion

Although expansion of trinucleotide repeats accounts for over 30 human diseases, mechanisms of repeat instability remain poorly understood. We show that a Drosophila model for the CAG/polyglutamine (polyQ) disease spinocerebellar ataxia type 3 recapitulates key features of human CAG-repeat instability, including large repeat changes and strong expansion bias. Instability is dramatically enhanced by transcription and modulated by nuclear excision repair and a regulator of DNA repair adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB)-binding protein-a histone acetyltransferase (HAT) whose decreased activity contributes to polyQ disease. Pharmacological treatment to normalize acetylation suppressed instability. Thus, toxic consequences of pathogenic polyQ protein may include enhancing repeat instability.

Topics: Alleles; Animals; Animals, Genetically Modified; Anticipation, Genetic; CREB-Binding Protein; DNA Repair; Drosophila melanogaster; Drosophila Proteins; Female; Fragile X Syndrome; Genomic Instability; Histone Deacetylase Inhibitors; Humans; Huntington Disease; Hydroxamic Acids; Machado-Joseph Disease; Male; Models, Animal; Peptides; Transcription, Genetic; Transgenes; Trinucleotide Repeat Expansion; Trinucleotide Repeats

Expansion of an unstable (CGG)n repeat to over 200 triplets within the promoter region of the human FMR1 gene leads to extensive local methylation and transcription silencing, resulting in the loss of FMRP protein and the development of the clinical features of fragile X syndrome. The causative link between (CGG)n expansion, methylation and gene silencing is unknown, although gene silencing is associated with extensive changes to local chromatin architecture.. In order to determine the direct effects of increased repeat length on gene transcription in a chromatin context, we have examined the influence of FMR1 (CGG)n repeats upon transcription from the HSV thymidine kinase promoter in the Xenopus laevis oocyte. We observe a reduction in mRNA production directly associated with increasing repeat length, with a 90% reduction in mRNA production from arrays over 100 repeats in length. Using a kinetic approach, we show that this transcriptional repression is concomitant with chromatin maturation and, using in vitro transcription, we show that chromatin formation is a fundamental part of the repressive pathway mediated by (CGG)n repeats. Using Trichostatin A, a histone deacetylase inhibitor, we show reactivation of the silenced promoter.. Thus, isolated fragile X associated (CGG)n repeat arrays can exert a modifying and transcriptionally repressive influence over adjacent promoters and this repressive phenomenon is, in part, mediated by histone deacetylation.

Topics: Animals; Chromatin; Cytosine; Disease Models, Animal; Enzyme Inhibitors; Fragile X Syndrome; Gene Expression Regulation; Gene Silencing; Genes, Reporter; Genes, Viral; Genetic Linkage; Guanine; Herpesvirus 1, Human; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Oocytes; Plasmids; Promoter Regions, Genetic; RNA-Binding Proteins; Thymidine Kinase; Transcription, Genetic; Trinucleotide Repeat Expansion; Viral Structural Proteins; Xenopus laevis

Mutation of FMR1 results in fragile X mental retardation. The most common FMR1 mutation is expansion of a CGG repeat tract at the 5' end of FMR1, which leads to cytosine methylation and transcriptional silencing. Both DNA methylation and histone deacetylation have been associated with transcriptional inactivity. The finding that the methyl cytosine-binding protein MeCP2 binds to histone deacetylases and represses transcription in vivo supports a model in which MeCP2 recruits histone deacetylases to methylated DNA, resulting in histone deacetylation, chromatin condensation and transcriptional silencing. Here we demonstrate that the 5' end of FMR1 is associated with acetylated histones H3 and H4 in cells from normal individuals, but acetylation is reduced in cells from fragile X patients. Treatment of fragile X cells with 5-aza-2'-deoxycytidine (5-aza-dC) resulted in reassociation of acetylated histones H3 and H4 with FMR1 and transcriptional reactivation, whereas treatment with trichostatin A (TSA) led to almost complete acetylated histone H4 and little acetylated histone H3 reassociation with FMR1, as well as no detectable transcription. Our results represent the first description of loss of histone acetylation at a specific locus in human disease, and advance understanding of the mechanism of FMR1 transcriptional silencing.

Topics: Acetylation; Acetyltransferases; Azacitidine; Cells, Cultured; Chromatin; Decitabine; DNA; Enzyme Inhibitors; Fragile X Mental Retardation Protein; Fragile X Syndrome; Histone Acetyltransferases; Histones; Humans; Hydroxamic Acids; Male; Nerve Tissue Proteins; Protein Binding; RNA-Binding Proteins; Saccharomyces cerevisiae Proteins; Transcription, Genetic