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CpG, 5'-C-phosphate-G-3', islands (CGIs) have long been known for their association with enhancers, silencers, and promoters, and for their epigenetic signatures. They are maintained in embryonic stem cells (ESCs) in a poised but inactive state via the formation of bivalent chromatin containing both active and repressive marks. CGIs also occur within coding sequences, where their functional role has remained obscure. Intragenic CGIs (iCGIs) are largely absent from housekeeping genes, but they are found in all genes associated with organ development and cell lineage control. In this paper, we investigated the epigenetic status of iCGIs and found that they too reside in bivalent chromatin in ESCs. Cell type-specific DNA methylation of iCGIs in differentiated cells was linked to the loss of both the H3K4me3 and H3K27me3 marks, and disruption of physical interaction with promoter regions, resulting in transcriptional activation of key regulators of differentiation such as PAXs, HOXs, and WNTs. The differential epigenetic modification of iCGIs appears to be mediated by cell type-specific transcription factors distinct from those bound by promoter, and these transcription factors may be involved in the hypermethylation of iCGIs upon cell differentiation. iCGIs thus play a key role in the cell type-specific regulation of transcription.

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of "repressive" histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX's ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this "methyl/phos" switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

Mature oocyte cytoplasm can reprogram somatic cell nuclei to the pluripotent state through a series of sequential events including protein exchange between the donor nucleus and ooplasm, chromatin remodeling, and pluripotency gene reactivation. Maternal factors that are responsible for this reprogramming process remain largely unidentified. Here, we demonstrate that knockdown of histone variant H3.3 in mouse oocytes results in compromised reprogramming and down-regulation of key pluripotency genes; and this compromised reprogramming for developmental potentials and transcription of pluripotency genes can be rescued by injecting exogenous H3.3 mRNA, but not H3.2 mRNA, into oocytes in somatic cell nuclear transfer embryos. We show that maternal H3.3, and not H3.3 in the donor nucleus, is essential for successful reprogramming of somatic cell nucleus into the pluripotent state. Furthermore, H3.3 is involved in this reprogramming process by remodeling the donor nuclear chromatin through replacement of donor nucleus-derived H3 with de novo synthesized maternal H3.3 protein. Our study shows that H3.3 is a crucial maternal factor for oocyte reprogramming and provides a practical model to directly dissect the oocyte for its reprogramming capacity.

Dysregulation of the transcriptional repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor is important in a broad range of diseases, including cancer, diabetes, and heart disease. The role of REST-dependent epigenetic modifications in neurodegeneration is less clear. Here, we show that neuronal insults trigger activation of REST and CoREST in a clinically relevant model of ischemic stroke and that REST binds a subset of "transcriptionally responsive" genes (gria2, grin1, chrnb2, nefh, nfκb2, trpv1, chrm4, and syt6), of which the AMPA receptor subunit GluA2 is a top hit. Genes with enriched REST exhibited decreased mRNA and protein. We further show that REST assembles with CoREST, mSin3A, histone deacetylases 1 and 2, histone methyl-transferase G9a, and methyl CpG binding protein 2 at the promoters of target genes, where it orchestrates epigenetic remodeling and gene silencing. RNAi-mediated depletion of REST or administration of dominant-negative REST delivered directly into the hippocampus in vivo prevents epigenetic modifications, restores gene expression, and rescues hippocampal neurons. These findings document a causal role for REST-dependent epigenetic remodeling in the neurodegeneration associated with ischemic stroke and identify unique therapeutic targets for the amelioration of hippocampal injury and cognitive deficits.

The histone variant H3.3 is implicated in the formation and maintenance of specialized chromatin structure in metazoan cells. H3.3-containing nucleosomes are assembled in a replication-independent manner by means of dedicated chaperone proteins. We previously identified the death domain associated protein (Daxx) and the alpha-thalassemia X-linked mental retardation protein (ATRX) as H3.3-associated proteins. Here, we report that the highly conserved N terminus of Daxx interacts directly with variant-specific residues in the H3.3 core. Recombinant Daxx assembles H3.3/H4 tetramers on DNA templates, and the ATRX-Daxx complex catalyzes the deposition and remodeling of H3.3-containing nucleosomes. We find that the ATRX-Daxx complex is bound to telomeric chromatin, and that both components of this complex are required for H3.3 deposition at telomeres in murine embryonic stem cells (ESCs). These data demonstrate that Daxx functions as an H3.3-specific chaperone and facilitates the deposition of H3.3 at heterochromatin loci in the context of the ATRX-Daxx complex.

Transient forebrain or global ischemia induces delayed neuronal death in vulnerable CA1 pyramidal cells with many features of apoptosis. A brief period of ischemia, i.e., ischemic preconditioning, affords robust protection of CA1 neurons against a subsequent more prolonged ischemic challenge. Here we show that preconditioning acts via PI3K/Akt signaling to block the ischemia-induced cascade involving mitochondrial translocation of Bad, assembly of Bad with Bcl-x(L), cleavage of Bcl-x(L) to form its prodeath fragment, DeltaN-Bcl-x(L), activation of large-conductance channels in the mitochondrial outer membrane, mitochondrial release of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI), caspase activation, and neuronal death. These findings show how preconditioning acts to prevent the release of cytochrome c and Smac/DIABLO from mitochondria and to preserve the integrity of the mitochondrial membrane. The specific PI3K inhibitor LY294002 administered in vivo 1 h before or immediately after ischemia or up to 120 h later significantly reverses preconditioning-induced protection, indicating a requirement for sustained PI3K signaling in ischemic tolerance. These findings implicate PI3K/Akt signaling in maintenance of the integrity of the mitochondrial outer membrane.

Transient global ischemia is a neuronal insult that induces delayed, selective death of hippocampal CA1 pyramidal neurons. A mechanism underlying ischemia-induced cell death is activation of the gene silencing transcription factor REST (repressor element-1 silencing transcription factor)/NRSF (neuron-restrictive silencing factor) and REST-dependent suppression of the AMPA receptor subunit GluR2 in CA1 neurons destined to die. Here we show that REST regulates an additional gene target, OPRM1 (mu opioid receptor 1 or MOR-1). MORs are abundantly expressed by basket cells and other inhibitory interneurons of CA1. Global ischemia induces a marked decrease in MOR-1 mRNA and protein expression that is specific to the selectively vulnerable area CA1, as assessed by quantitative real-time RT-PCR, Western blotting, and ChIP. We further show that OPRM1 gene silencing is REST-dependent and occurs via epigenetic modifications. Ischemia promotes deacetylation of core histone proteins H3 and H4 and dimethylation of histone H3 at lysine-9 (H3-K9) over the MOR-1 promoter, an signature of epigenetic gene silencing. Acute knockdown of MOR-1 gene expression by administration of antisense oligodeoxynucleotides to hippocampal slices in vitro or injection of the MOR antagonist naloxone to rats in vivo affords protection against ischemia-induced death of CA1 pyramidal neurons. These findings implicate MORs in ischemia-induced death of CA1 pyramidal neurons and document epigenetic remodeling of expression of OPRM1 in CA1 inhibitory interneurons.

Transient global or forebrain ischemia induced experimentally in animals can cause selective, delayed neuronal death of hippocampal CA1 pyramidal neurons. A striking feature is a delayed rise in intracellular free Zn(2+) in CA1 neurons just before the onset of histologically detectable cell death. Here we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) at Schaffer collateral to CA1 synapses in postischemic hippocampus exhibit properties of Ca(2+)/Zn(2+)-permeable, Glu receptor 2 (GluR2)-lacking AMPARs before the rise in Zn(2+) and cell death. At 42 h after ischemia, AMPA excitatory postsynaptic currents exhibited pronounced inward rectification and marked sensitivity to 1-naphthyl acetyl spermine (Naspm), a selective channel blocker of GluR2-lacking AMPARs. In control hippocampus, AMPA excitatory postsynaptic currents were electrically linear and relatively insensitive to Naspm. Naspm injected intrahippocampally at 9-40 h after insult greatly reduced the late rise in intracellular free Zn(2+) in postischemic CA1 neurons and afforded partial protection against ischemia-induced cell death. These results implicate GluR2-lacking AMPA receptors in the ischemia-induced rise in free Zn(2+) and death of CA1 neurons, although a direct action at the time of the rise in Zn(2+) is unproven. This receptor subtype appears to be an important therapeutic target for intervention in ischemia-induced neuronal death in humans.