Karine Merienne

Transcriptional Activation of REST by Sp1 in Huntington's Disease Models

In Huntingtons disease (HD), mutant huntingtin (mHtt) disrupts the normal transcriptional program of disease neurons by altering the function of several gene expression regulators such as Sp1. REST (Repressor Element-1 Silencing Transcription Factor), a key regulator of neuronal differentiation, is also aberrantly activated in HD by a mechanism that remains unclear. Here, we show that the level of REST mRNA is increased in HD mice and in NG108 cells differentiated into neuronal-like cells and expressing a toxic mHtt fragment. Using luciferase reporter gene assay, we delimited the REST promoter regions essential for mHtt-mediated REST upregulation and found that they contain Sp factor binding sites. We provide evidence that Sp1 and Sp3 bind REST promoter and interplay to fine-tune REST transcription. In undifferentiated NG108 cells, Sp1 and Sp3 have antagonistic effect, Sp1 acting as an activator and Sp3 as a repressor. Upon neuronal differentiation, we show that the amount and ratio of Sp1/Sp3 proteins decline, as does REST expression, and that the transcriptional role of Sp3 shifts toward a weak activator. Therefore, our results provide new molecular information to the transcriptional regulation of REST during neuronal differentiation. Importantly, specific knockdown of Sp1 abolishes REST upregulation in NG108 neuronal-like cells expressing mHtt. Our data together with earlier reports suggest that mHtt triggers a pathogenic cascade involving Sp1 activation, which leads to REST upregulation and repression of neuronal genes.

Neuronal identity genes regulated by super-enhancers are preferentially down-regulated in the striatum of Huntington's disease mice

Huntingtons disease (HD) is a neurodegenerative disease associated with extensive down-regulation of genes controlling neuronal function, particularly in the striatum. Whether altered epigenetic regulation underlies transcriptional defects in HD is unclear. Integrating RNA-sequencing (RNA-seq) and chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq), we show that down-regulated genes in HD mouse striatum associate with selective decrease in H3K27ac, a mark of active enhancers, and RNA Polymerase II (RNAPII). In addition, we reveal that decreased genes in HD mouse striatum display a specific epigenetic signature, characterized by high levels and broad patterns of H3K27ac and RNAPII. Our results indicate that this signature is that of super-enhancers, a category of broad enhancers regulating genes defining tissue identity and function. Specifically, we reveal that striatal super-enhancers display extensive H3K27 acetylation within gene bodies, drive transcription characterized by low levels of paused RNAPII, regulate neuronal function genes and are enriched in binding motifs for Gata transcription factors, such as Gata2 regulating striatal identity genes. Together, our results provide evidence for preferential down-regulation of genes controlled by super-enhancers in HD striatum and indicate that enhancer topography is a major parameter determining the propensity of a gene to be deregulated in a neurodegenerative disease.

Contribution of Neuroepigenetics to Huntington’s Disease

Unbalanced epigenetic regulation is thought to contribute to the progression of several neurodegenerative diseases, including Huntington’s disease (HD), a genetic disorder considered as a paradigm of epigenetic dysregulation. In this review, we attempt to address open questions regarding the role of epigenetic changes in HD, in the light of recent advances in neuroepigenetics. We particularly discuss studies using genome-wide scale approaches that provide insights into the relationship between epigenetic regulations, gene expression and neuronal activity in normal and diseased neurons, including HD neurons. We propose that cell-type specific techniques and 3D-based methods will advance knowledge of epigenome in the context of brain region vulnerability in neurodegenerative diseases. A better understanding of the mechanisms underlying epigenetic changes and of their consequences in neurodegenerative diseases is required to design therapeutic strategies more effective than current strategies based on histone deacetylase (HDAC) inhibitors. Researches in HD may play a driving role in this process.

Altered enhancer transcription underlies Huntington’s disease striatal transcriptional signature

Epigenetic and transcriptional alterations are both implicated in Huntington’s disease (HD), a progressive neurodegenerative disease resulting in degeneration of striatal neurons in the brain. However, how impaired epigenetic regulation leads to transcriptional dysregulation in HD is unclear. Here, we investigated enhancer RNAs (eRNAs), a class of long non-coding RNAs transcribed from active enhancers. We found that eRNAs are expressed from many enhancers of mouse striatum and showed that a subset of those eRNAs are deregulated in HD vs control mouse striatum. Enhancer regions producing eRNAs decreased in HD mouse striatum were associated with genes involved in striatal neuron identity. Consistently, they were enriched in striatal super-enhancers. Moreover, decreased eRNA expression in HD mouse striatum correlated with down-regulation of associated genes. Additionally, a significant number of RNA Polymerase II (RNAPII) binding sites were lost within enhancers associated with decreased eRNAs in HD vs control mouse striatum. Together, this indicates that loss of RNAPII at HD mouse enhancers contributes to reduced transcription of eRNAs, resulting in down-regulation of target genes. Thus, our data support the view that eRNA dysregulation in HD striatum is a key mechanism leading to altered transcription of striatal neuron identity genes, through reduced recruitment of RNAPII at super-enhancers.

Neuroepigenetics and addictive behaviors: Where do we stand?

Substance use disorders involve long-term changes in the brain that lead to compulsive drug seeking, craving, and a high probability of relapse. Recent findings have highlighted the role of epigenetic regulations in controlling chromatin access and regulation of gene expression following exposure to drugs of abuse. In the present review, we focus on data investigating genome-wide epigenetic modifications in the brain of addicted patients or in rodent models exposed to drugs of abuse, with a particular focus on DNA methylation and histone modifications associated with transcriptional studies. We highlight critical factors for epigenomic studies in addiction. We discuss new findings related to psychostimulants, alcohol, opiate, nicotine and cannabinoids. We examine the possible transmission of these changes across generations. We highlight developing tools, specifically those that allow investigation of structural reorganization of the chromatin. These have the potential to increase our understanding of alteration of chromatin architecture at gene regulatory regions. Neuroepigenetic mechanisms involved in addictive behaviors could explain persistent phenotypic effects of drugs and, in particular, vulnerability to relapse.

A06 Huntington’s disease striatal super-enhancer signature

Huntington’s disease (HD) is a progressive neurodegenerative disease, affecting primarily the striatum. Transcriptional dysregulation is believed to contribute to HD. However, the underlying mechanism is unclear. Using ChIPseq and RNAseq on the striatum of HD R6/1 transgenic mice, we found that down-regulated genes are enriched in striatal identity genes, controlled by a super-enhancer. H3K27ac, enhancer transcription and recruitment of RNA polymerase II (RNAPII) were selectively reduced at R6/1 striatal super-enhancers, indicating that altered super-enhancer activity underlies down-regulation of striatal identity genes in HD. Our 4Cseq data using R6/1 striatum further suggest that disruption of chromatin 3D architecture contributes to altered expression of striatal identity genes regulated by a super-enhancer. To investigate functional consequences of epigenetic alterations in HD, R6/1 mice were trained to learn striatum-dependent cognitive task. In contrast to wild-type (WT) animals, R6/1 mice were impaired in this task. ChIPseq data generated using the striatum of ‘trained’ and ‘home cage’ mice showed an increase of H3K27ac and RNAPII at genes implicated in synaptic plasticity and regulated by a super-enhancer, in trained vs home cage WT animals. However, this ‘plasticity’ signature was absent in trained R6/1 mice, suggesting that aberrant RNAPII dynamics and inadequate histone acetylation at these genes preclude synaptic plasticity and contribute to R6/1 behavioural deficits. Finally, we generated ChIPseq data using the striatum of HD patients and knockin mice. HD striatal ‘super-enhancer’ signature was conserved across models and our analyses further revealed that it establishes early, at presymptomatic stage.

Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator

Chromatin acetylation, a critical regulator of synaptic plasticity and memory processes, is thought to be altered in neurodegenerative diseases. Here, we demonstrate that spatial memory and plasticity (LTD, dendritic spine formation) deficits can be restored in a mouse model of tauopathy following treatment with CSP‐TTK21, a small‐molecule activator of CBP/p300 histone acetyltransferases (HAT). At the transcriptional level, CSP‐TTK21 re‐established half of the hippocampal transcriptome in learning mice, likely through increased expression of neuronal activity genes and memory enhancers. At the epigenomic level, the hippocampus of tauopathic mice showed a significant decrease in H2B but not H3K27 acetylation levels, both marks co‐localizing at TSS and CBP enhancers. Importantly, CSP‐TTK21 treatment increased H2B acetylation levels at decreased peaks, CBP enhancers, and TSS, including genes associated with plasticity and neuronal functions, overall providing a 95% rescue of the H2B acetylome in tauopathic mice. This study is the first to provide in vivo proof‐of‐concept evidence that CBP/p300 HAT activation efficiently reverses epigenetic, transcriptional, synaptic plasticity, and behavioral deficits associated with Alzheimers disease lesions in mice.

The striatal kinase DCLK3 produces neuroprotection against mutant huntingtin

Expression of the neuronal kinase DCLK3 is reduced in Huntington’s disease. Galvan et al. report that DCLK3 silencing in the mouse striatum exacerbates the toxicity of mutant huntingtin, whereas DCLK3 overexpression is neuroprotective, and show that DCLK3 regulates the expression of many genes involved in transcription regulation and chromatin remodelling.

B11 Altered epigenetic signature in the striatum of HD mice and patients

Epigenetic alterations are documented in several models of Huntington’s disease (HD). However, it remains unclear whether similar alterations also occur in HD patients. Using the striatum of HD R6/1 mice and genome-wide approaches, we found that down-regulated genes display a particular epigenetic signature and this signature is altered in HD mouse striatum. We showed that H3K27ac signal is selectively decreased at super-enhancers, a category of enhancers regulating cell-type specific genes. Our results suggest that the mechanism linking epigenetic and transcriptional defects in HD striatum involves altered expression of non-coding RNA expressed from super-enhancers (seRNAs). To assess whether the mechanism is conserved in HD patients, we generated H3K27ac ChIP-seq data from the striatum of HD patients and control individuals. Our results indicate that striatal super-enhancer signature is also altered in HD patients. Together, our data support a model where altered epigenetic regulation of super-enhancers would be responsible for gene down-regulation in HD brain tissues. We suggest that targeting enhancer activity may be of therapeutic interest.

A07 Transcriptional activation of Neuron Restrictive Silencer Factor by sp1 in Huntington's disease

Background In Huntingtons disease (HD), mutant huntingtin (mHtt) disrupts the normal transcriptional programme of disease neurons by altering the function of several gene expression regulators such as Sp1. The Neuron Restrictive Silencer Factor (NRSF), a key regulator of neuronal differentiation, is also aberrantly activated in HD by a mechanism that remains unclear. Aim We studied the mechanism underlying NRSF activation in HD mouse and cellular models. Results We show that the level of NRSF mRNA is increased in HD mouse and in neuroblastoma/glioma NG108 cells differentiated into neurons and expressing a toxic mHtt fragment. We further provide evidence that Sp1 and Sp3 bind NRSF promoter and interplay to finely regulate NRSF transcription. In undifferentiated NG108 cells, Sp1 and Sp3 have an antagonistic effect, Sp1 being an activator and Sp3 a repressor. On neuronal differentiation, the amount and ratio of Sp1/Sp3 proteins decline, correlating with reduced expression of NRSF, and the transcriptional role of Sp3 shifts towards a faint activator. Importantly, in NG108 neuronal cells expressing mHtt, specific knockdown of Sp1 abolishes the NRSF upregulation. Conclusions Thus our data provide new molecular information to the transcriptional regulation of NRSF during neuronal differentiation and indicate that mHtt triggers an unexpected pathogenic cascade involving activation of Sp1, which in turn upregulates NRSF to repress neuronal genes.