Role for Chromatin Remodeling Factor Chd1 in Learning and Memory.

Precise temporal and spatial regulation of gene expression in the brain is a prerequisite for cognitive processes such as learning and memory. Epigenetic mechanisms that modulate the chromatin structure have emerged as important regulators in this context. While posttranslational modification of histones or the modification of DNA bases have been examined in detail in many studies, the role of ATP-dependent chromatin remodeling factors (ChRFs) in learning- and memory-associated gene regulation has largely remained obscure. Here we present data that implicate the highly conserved chromatin assembly and remodeling factor Chd1 in memory formation and the control of immediate early gene (IEG) response in the hippocampus. We used various paradigms to assess short-and long-term memory in mice bearing a mutated Chd1 gene that gives rise to an N-terminally truncated protein. Our data demonstrate that the Chd1 mutation negatively affects long-term object recognition and short- and long-term spatial memory. We found that Chd1 regulates hippocampal expression of the IEG early growth response 1 (Egr1) and activity-regulated cytoskeleton-associated (Arc) but not cFos and brain derived neurotrophic factor (Bdnf), because the Chd1-mutation led to dysregulation of Egr1 and Arc expression in naive mice and in mice analyzed at different stages of object location memory (OLM) testing. Of note, Chd1 likely regulates Egr1 in a direct manner, because chromatin immunoprecipitation (ChIP) assays revealed enrichment of Chd1 upon stimulation at the Egr1 genomic locus in the hippocampus and in cultured cells. Together these data support a role for Chd1 as a critical regulator of molecular mechanisms governing memory-related processes, and they show that this function involves the N-terminal serine-rich region of the protein.

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity.

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.

Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain.

BACKGROUND: Recent work has identified and mapped a range of posttranscriptional modifications in mRNA, including methylation of the N6 and N1 positions in adenine, pseudouridylation, and methylation of carbon 5 in cytosine (m5C). However, knowledge about the prevalence and transcriptome-wide distribution of m5C is still extremely limited; thus, studies in different cell types, tissues, and organisms are needed to gain insight into possible functions of this modification and implications for other regulatory processes. RESULTS: We have carried out an unbiased global analysis of m5C in total and nuclear poly(A) RNA of mouse embryonic stem cells and murine brain. We show that there are intriguing differences in these samples and cell compartments with respect to the degree of methylation, functional classification of methylated transcripts, and position bias within the transcript. Specifically, we observe a pronounced accumulation of m5C sites in the vicinity of the translational start codon, depletion in coding sequences, and mixed patterns of enrichment in the 3' UTR. Degree and pattern of methylation distinguish transcripts modified in both embryonic stem cells and brain from those methylated in either one of the samples. We also analyze potential correlations between m5C and micro RNA target sites, binding sites of RNA binding proteins, and N6-methyladenosine. CONCLUSION: Our study presents the first comprehensive picture of cytosine methylation in the epitranscriptome of pluripotent and differentiated stages in the mouse. These data provide an invaluable resource for future studies of function and biological significance of m5C in mRNA in mammals.

Dysregulation of select ATP-dependent chromatin remodeling factors in high trait anxiety.

Enhanced anxiety is a salient feature of a number of psychiatric disorders including anxiety disorders, trauma-related disorders and depression. Although aberrant expression of various genes has been detected in patients suffering from persistent high anxiety as well as in high anxiety rodent models, the molecular mechanisms responsible for altered transcription regulation have been poorly addressed. Transcription regulation intimately involves the contribution of chromatin modifying processes, such as histone modification and ATP-dependent chromatin remodeling, yet their role in pathological anxiety is not known. Here, we investigated for the first time if altered levels of several ATP-dependent chromatin remodeling factors (ChRFs) and histone deacetylases (HDACs) may be linked to high trait anxiety in mice. While we found protein levels of the ChRFs SNF2H, ATRX, CHD1, CHD3 and CHD5 and of HDACs 1-3 and 6 to be similar in most of the tested brain areas of mice with high (HAB) versus normal (NAB) anxiety-related behavior, we observed distinctly altered regulation of SNF2H in the amygdala, and of CHD3 and CHD5 in the ventral hippocampus. In particular, CHD3 and CHD5 exhibited altered expression of protein but not of mRNA in HAB mice. Since both proteins are components of NuRD-like complexes, these results may indicate an impaired equilibrium between different NuRD-like complexes in the ventral hippocampus. Overall, our data provide novel evidence for localized differences of specific ATP-dependent chromatin remodeling factors in mice with high trait anxiety that may ultimately contribute to altered transcriptional programs resulting in the manifestation of pathological anxiety.

Impaired Contextual Fear Extinction Learning is Associated with Aberrant Regulation of CHD-Type Chromatin Remodeling Factors.

Successful attenuation of fearful memories is a cognitive process requiring initiation of highly coordinated transcription programs. Chromatin-modulating mechanisms such as DNA methylation and histone modifications, including acetylation, are key regulators of these processes. However, knowledge concerning the role of ATP-dependent chromatin remodeling factors (ChRFs) being required for successful fear extinction is lacking. Underscoring the potential importance of these factors that alter histone-DNA contacts within nucleosomes are recent genome-wide association studies linking several ChRFs to various human cognitive and psychiatric disorders. To better understand the role of ChRFs in the brain, and since to date little is known about ChRF expression in the brain, we performed a comprehensive survey of expression levels of 24 ATP-dependent remodelers across different brain areas, and we identified several distinct high molecular weight complexes by chromatographic methods. We next aimed to gain novel insight into the potential regulation of ChRFs in different brain regions in association with normal and impaired fear extinction learning. To this end, we established the 129S1/SvImJ (S1) laboratory mouse strain as a model for compromised contextual fear extinction learning that can be rescued by dietary zinc restriction (ZnR). Using this model along with genetically related but fear extinction-competent 129S6/SvEv (S6) mice as controls, we found that impaired fear extinction in S1 was associated with enhanced ventral hippocampal expression of CHD1 and reduced expression of CHD5 that was normalized following successful rescue of impaired fear extinction. Moreover, a select reduction in CHD3 expression was observed in the ventral hippocampus (vHC) following successful rescue of fear extinction in S1 mice. Taken together, these data provide novel insight into the regulation of specific ChRFs following an impaired cognitive process and its rescue, and they suggest that imbalance of CHD-type remodeler levels, which consequently may lead to changes of transcriptional programs, may be an underlying mechanism involved in impaired fear extinction learning and its therapeutic rescue.

Differential neuronal targeting of a new and two known calcium channel ß4 subunit splice variants correlates with their regulation of gene expression.

The ß subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain ß subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of ß isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual ß variants in specific neuronal functions. In the present study, an alternatively spliced ß4 subunit lacking the variable N terminus (ß4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length ß4 variants (ß4a and ß4b), ß4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of ß4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (ß4-null) mice individually reconstituted with ß4a, ß4b, and ß4e. Notably, the number of genes regulated by each ß4 splice variant correlated with the rank order of their nuclear-targeting properties (ß4b > ß4a > ß4e). Together, these findings support isoform-specific functions of ß4 splice variants in neurons, with ß4b playing a dual role in channel modulation and gene regulation, whereas the newly detected ß4e variant serves exclusively in calcium-channel-dependent functions.

Long non-coding RNAs as targets for cytosine methylation.

Post-synthetic modifications of nucleic acids have long been known to affect their functional and structural properties. For instance, numerous different chemical modifications modulate the structural organization, stability or translation efficiency of tRNAs and rRNAs. In contrast, little is known about modifications of poly(A)RNAs. Here, we demonstrate for the first time that the two well-studied regulatory long non-coding RNAs HOTAIR and XIST are targets of site-specific cytosine methylation. In both XIST and HOTAIR, we found methylated cytosines located within or near functionally important regions that are known to mediate interaction with chromatin-associated protein complexes. We show that cytosine methylation in the XIST A structure strongly affects binding to the chromatin-modifying complex PRC2 in vitro. These results suggest that cytosine methylation may serve as a general strategy to regulate the function of long non-coding RNAs.