Postsynaptic density radiation signature following space irradiation.

Introduction: The response of the brain to space radiation is an important concern for astronauts during space missions. Therefore, we assessed the response of the brain to 28Si ion irradiation (600 MeV/n), a heavy ion present in the space environment, on cognitive performance and whether the response is associated with altered DNA methylation in the hippocampus, a brain area important for cognitive performance. Methods: We determined the effects of 28Si ion irradiation on object recognition, 6-month-old mice irradiated with 28Si ions (600 MeV/n, 0.3, 0.6, and 0.9 Gy) and cognitively tested two weeks later. In addition, we determined if those effects were associated with alterations in hippocampal networks and/or hippocampal DNA methylation. Results: At 0.3 Gy, but not at 0.6 Gy or 0.9 Gy, 28Si ion irradiation impaired cognition that correlated with altered gene expression and 5 hmC profiles that mapped to specific gene ontology pathways. Comparing hippocampal DNA hydroxymethylation following proton, 56Fe ion, and 28Si ion irradiation revealed a general space radiation synaptic signature with 45 genes that are associated with profound phenotypes. The most significant categories were glutamatergic synapse and postsynaptic density. Discussion: The brain's response to space irradiation involves novel excitatory synapse and postsynaptic remodeling.

H3.3 contributes to chromatin accessibility and transcription factor binding at promoter-proximal regulatory elements in embryonic stem cells.

BACKGROUND: The histone variant H3.3 is enriched at active regulatory elements such as promoters and enhancers in mammalian genomes. These regions are highly accessible, creating an environment that is permissive to transcription factor binding and the recruitment of transcriptional coactivators that establish a unique chromatin post-translational landscape. How H3.3 contributes to the establishment and function of chromatin states at these regions is poorly understood. RESULTS: We perform genomic analyses of features associated with active promoter chromatin in mouse embryonic stem cells (ESCs) and find evidence of subtle yet widespread promoter dysregulation in the absence of H3.3. Loss of H3.3 results in reduced chromatin accessibility and transcription factor (TF) binding at promoters of expressed genes in ESCs. Likewise, enrichment of the transcriptional coactivator p300 and downstream histone H3 acetylation at lysine 27 (H3K27ac) is reduced at promoters in the absence of H3.3, along with reduced enrichment of the acetyl lysine reader BRD4. Despite the observed chromatin dysregulation, H3.3 KO ESCs maintain transcription from ESC-specific genes. However, upon undirected differentiation, H3.3 KO cells retain footprinting of ESC-specific TF motifs and fail to generate footprints of lineage-specific TF motifs, in line with their diminished capacity to differentiate. CONCLUSIONS: H3.3 facilitates DNA accessibility, transcription factor binding, and histone post-translational modification at active promoters. While H3.3 is not required for maintaining transcription in ESCs, it does promote de novo transcription factor binding which may contribute to the dysregulation of cellular differentiation in the absence of H3.3.

Establishment and function of chromatin modification at enhancers.

How a single genome can give rise to distinct cell types remains a fundamental question in biology. Mammals are able to specify and maintain hundreds of cell fates by selectively activating unique subsets of their genome. This is achieved, in part, by enhancers-genetic elements that can increase transcription of both nearby and distal genes. Enhancers can be identified by their unique chromatin signature, including transcription factor binding and the enrichment of specific histone post-translational modifications, histone variants, and chromatin-associated cofactors. How each of these chromatin features contributes to enhancer function remains an area of intense study. In this review, we provide an overview of enhancer-associated chromatin states, and the proteins and enzymes involved in their establishment. We discuss recent insights into the effects of the enhancer chromatin state on ongoing transcription versus their role in the establishment of new transcription programmes, such as those that occur developmentally. Finally, we highlight the role of enhancer chromatin in new conceptual advances in gene regulation such as condensate formation.

Phosphorylation of histone H3.3 at serine 31 promotes p300 activity and enhancer acetylation.

The histone variant H3.3 is enriched at enhancers and active genes, as well as repeat regions such as telomeres and retroelements, in mouse embryonic stem cells (mESCs)1-3. Although recent studies demonstrate a role for H3.3 and its chaperones in establishing heterochromatin at repeat regions4-8, the function of H3.3 in transcription regulation has been less clear9-16. Here, we find that H3.3-specific phosphorylation17-19 stimulates activity of the acetyltransferase p300 in trans, suggesting that H3.3 acts as a nucleosomal cofactor for p300. Depletion of H3.3 from mESCs reduces acetylation on histone H3 at lysine 27 (H3K27ac) at enhancers. Compared with wild-type cells, those lacking H3.3 demonstrate reduced capacity to acetylate enhancers that are activated upon differentiation, along with reduced ability to reprogram cell fate. Our study demonstrates that a single amino acid in a histone variant can integrate signaling information and impact genome regulation globally, which may help to better understand how mutations in these proteins contribute to human cancers20,21.

Bi-directional and shared epigenomic signatures following proton and 56Fe irradiation.

The brain's response to radiation exposure is an important concern for patients undergoing cancer therapy and astronauts on long missions in deep space. We assessed whether this response is specific and prolonged and is linked to epigenetic mechanisms. We focused on the response of the hippocampus at early (2-weeks) and late (20-week) time points following whole body proton irradiation. We examined two forms of DNA methylation, cytosine methylation (5mC) and hydroxymethylation (5hmC). Impairments in object recognition, spatial memory retention, and network stability following proton irradiation were observed at the two-week time point and correlated with altered gene expression and 5hmC profiles that mapped to specific gene ontology pathways. Significant overlap was observed between DNA methylation changes at the 2 and 20-week time points demonstrating specificity and retention of changes in response to radiation. Moreover, a novel class of DNA methylation change was observed following an environmental challenge (i.e. space irradiation), characterized by both increased and decreased 5hmC levels along the entire gene body. These changes were mapped to genes encoding neuronal functions including postsynaptic gene ontology categories. Thus, the brain's response to proton irradiation is both specific and prolonged and involves novel remodeling of non-random regions of the epigenome.

Short- and long-term effects of 56Fe irradiation on cognition and hippocampal DNA methylation and gene expression.

BACKGROUND: Astronauts are exposed to 56Fe ions that may pose a significant health hazard during and following prolonged missions in deep space. We showed previously that object recognition requiring the hippocampus, a structure critical for cognitive function, is affected in 2-month-old mice irradiated with 56Fe ions. Here we examined object recognition in 6-month-old mice irradiated with 56Fe ions, a biological age more relevant to the typical ages of astronauts. Moreover, because the mechanisms mediating the detrimental effects of 56Fe ions on hippocampal function are unclear, we examined changes in hippocampal networks involved in synaptic plasticity and memory, gene expression, and epigenetic changes in cytosine methylation (5mC) and hydroxymethylation (5hmC) that could accompany changes in gene expression. We assessed the effects of whole body 56Fe ion irradiation at early (2 weeks) and late (20 weeks) time points on hippocampus-dependent memory and hippocampal network stability, and whether these effects are associated with epigenetic changes in hippocampal DNA methylation (both 5mC and 5hmC) and gene expression. RESULTS: At the two-week time point, object recognition and network stability were impaired following irradiation at the 0.1 and 0.4 Gy dose, but not following irradiation at the 0.2 Gy dose. No impairments in object recognition or network stability were seen at the 20-week time point at any irradiation dose used. Consistent with this pattern, the significance of pathways for gene categories for 5hmC was lower, though not eliminated, at the 20-week time point compared to the 2-week time point. Similarly, significant changes were observed for 5mC gene pathways at the 2-week time point, but no significant gene categories were observed at the 20-week time point. Only the 5hmC changes tracked with gene expression changes. CONCLUSIONS: Dose- and time-dependent epigenomic remodeling in the hippocampus following 56Fe ion exposure correlates with behavioral changes.

Proton irradiation induces persistent and tissue-specific DNA methylation changes in the left ventricle and hippocampus.

BACKGROUND: Proton irradiation poses a potential hazard to astronauts during and following a mission, with post-mitotic cells at most risk because they cannot dilute resultant epigenetic changes via cell division. Persistent epigenetic changes that result from environmental exposures include gains or losses of DNA methylation of cytosine, which can impact gene expression. In the present study, we compared the long-term epigenetic effects of whole body proton irradiation in the mouse hippocampus and left ventricle. We used an unbiased genome-wide DNA methylation study, involving ChIP-seq with antibodies to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) to identify DNA regions in which methylation levels have changed 22 weeks after a single exposure to proton irradiation. We used DIP-Seq to profile changes in genome-wide DNA methylation and hydroxymethylation following proton irradiation. In addition, we used published RNAseq data to assess whether differentially methylated regions were linked to changes in gene expression. RESULTS: The DNA methylation data showed tissue-dependent effects of proton irradiation and revealed significant major pathway changes in response to irradiation that are related to known pathophysiologic processes. Many regions affected in the ventricle mapped to genes involved in cardiovascular function pathways, whereas many regions affected in the hippocampus mapped to genes involved in neuronal functions. In the ventricle, increases in 5hmC were associated with decreases in 5mC. We also observed spatial overlap for regions where both epigenetic marks decreased in the ventricle. In hippocampus, increases in 5hmC were most significantly correlated (spatially) with regions that had increased 5mC, suggesting that deposition of hippocampal 5mC and 5hmC may be mechanistically coupled. CONCLUSIONS: The results demonstrate long-term changes in DNA methylation patterns following a single proton irradiation, that these changes are tissue specific, and that they map to pathways consistent with tissue specific responses to proton irradiation. Further, the results suggest novel relationships between changes in 5mC and 5hmC.

Antagonism between Gdf6a and retinoic acid pathways controls timing of retinal neurogenesis and growth of the eye in zebrafish.

Maintaining neurogenesis in growing tissues requires a tight balance between progenitor cell proliferation and differentiation. In the zebrafish retina, neuronal differentiation proceeds in two stages with embryonic retinal progenitor cells (RPCs) of the central retina accounting for the first rounds of differentiation, and stem cells from the ciliary marginal zone (CMZ) being responsible for late neurogenesis and growth of the eye. In this study, we analyse two mutants with small eyes that display defects during both early and late phases of retinal neurogenesis. These mutants carry lesions in gdf6a, a gene encoding a BMP family member previously implicated in dorsoventral patterning of the eye. We show that gdf6a mutant eyes exhibit expanded retinoic acid (RA) signalling and demonstrate that exogenous activation of this pathway in wild-type eyes inhibits retinal growth, generating small eyes with a reduced CMZ and fewer proliferating progenitors, similar to gdf6a mutants. We provide evidence that RA regulates the timing of RPC differentiation by promoting cell cycle exit. Furthermore, reducing RA signalling in gdf6a mutants re-establishes appropriate timing of embryonic retinal neurogenesis and restores putative stem and progenitor cell populations in the CMZ. Together, our results support a model in which dorsally expressed gdf6a limits RA pathway activity to control the transition from proliferation to differentiation in the growing eye.