Failure of diet-induced transcriptional adaptations in alpha-synuclein transgenic mice.

Nutritional influences have been discussed as potential modulators of Parkinson's disease (PD) pathology through various epidemiological and physiological studies. In animal models, a high-fat diet (HFD) with greater intake of lipid-derived calories leads to accelerated disease onset and progression. The underlying molecular mechanisms of HFD-induced aggravated pathology, however, remain largely unclear. In this study, we aimed to further illuminate the effects of a fat-enriched diet in PD by examining the brainstem and hippocampal transcriptome of alpha-synuclein transgenic mice exposed to a life-long HFD. Investigating individual transcript isoforms, differential gene expression and co-expression clusters, we observed that transcriptional differences between wild-type (WT) and transgenic animals intensified in both regions under HFD. Both brainstem and hippocampus displayed strikingly similar transcriptomic perturbation patterns. Interestingly, expression differences resulted mainly from responses in WT animals to HFD, while these genes remained largely unchanged or were even slightly oppositely regulated by diet in transgenic animals. Genes and co-expressed gene groups exhibiting this dysregulation were linked to metabolic and mitochondrial pathways. Our findings propose the failure of metabolic adaptions as the potential explanation for accelerated disease unfolding under exposure to HFD. From the identified clusters of co-expressed genes, several candidates lend themselves to further functional investigations.

Alpha-synuclein overexpression induces epigenomic dysregulation of glutamate signaling and locomotor pathways.

Parkinson's disease (PD) is a neurological disorder with complex interindividual etiology that is becoming increasingly prevalent worldwide. Elevated alpha-synuclein levels can increase risk of PD and may influence epigenetic regulation of PD pathways. Here, we report genome-wide DNA methylation and hydroxymethylation alterations associated with overexpression of two PD-linked alpha-synuclein variants (wild-type and A30P) in LUHMES cells differentiated to dopaminergic neurons. Alpha-synuclein altered DNA methylation at thousands of CpGs and DNA hydroxymethylation at hundreds of CpGs in both genotypes, primarily in locomotor behavior and glutamate signaling pathway genes. In some cases, epigenetic changes were associated with transcription. SMITE network analysis incorporating H3K4me1 ChIP-seq to score DNA methylation and hydroxymethylation changes across promoters, enhancers, and gene bodies confirmed epigenetic and transcriptional deregulation of glutamate signaling modules in both genotypes. Our results identify distinct and shared impacts of alpha-synuclein variants on the epigenome, and associate alpha-synuclein with the epigenetic etiology of PD.

Distinct impacts of alpha-synuclein overexpression on the hippocampal epigenome of mice in standard and enriched environments.

Elevated alpha-synuclein (SNCA) gene expression is associated with transcriptional deregulation and increased risk of Parkinson's disease, which may be partially ameliorated by environmental enrichment. At the molecular level, there is emerging evidence that excess alpha-synuclein protein (aSyn) impacts the epigenome through direct and/or indirect mechanisms. However, the extents to which the effects of both aSyn and the environment converge at the epigenome and whether epigenetic alterations underpin the preventive effects of environmental factors on transcription remain to be elucidated. Here, we profiled five DNA and histone modifications in the hippocampus of wild-type and transgenic mice overexpressing human SNCA. Mice of each genotype were housed under either standard conditions or in an enriched environment (EE) for 12 months. SNCA overexpression induced hippocampal CpG hydroxymethylation and histone H3K27 acetylation changes that associated with genotype more than environment. Excess aSyn was also associated with genotype- and environment-dependent changes in non-CpG (CpH) DNA methylation and H3K4 methylation. These H3K4 methylation changes included loci where the EE ameliorated the impacts of the transgene as well as loci resistant to the effects of environmental enrichment in transgenic mice. In addition, select H3K4 monomethylation alterations were associated with changes in mRNA expression. Our results suggested an environment-dependent impact of excess aSyn on some functionally relevant parts of the epigenome, and will ultimately enhance our understanding of the molecular etiology of Parkinson's disease and other synucleinopathies.

Dot1 and histone H3K79 methylation in natural telomeric and HM silencing.

The expression of genes residing near telomeres is attenuated through telomere position-effect variegation (TPEV). By using a URA3 reporter located at TEL-VII-L of Saccharomyces cerevisiae, it was proposed that the disruptor of telomeric silencing-1 (Dot1) regulates TPEV by catalyzing H3K79 methylation. URA3 reporter assays also indicated that H3K79 methylation is required for HM silencing. Surprisingly, a genome-wide expression analysis of H3K79 methylation-defective mutants identified only a few telomeric genes, such as COS12 at TEL-VII-L, to be subject to H3K79 methylation-dependent natural silencing. Consistently, loss of Dot1 did not globally alter Sir2 or Sir3 occupancy in subtelomeric regions, but only led to some telomere-specific changes. Furthermore, H3K79 methylation by Dot1 did not play a role in the maintenance of natural HML silencing. Therefore, commonly used URA3 reporter assays may not report on natural PEV, and therefore, studies concerning the epigenetic mechanism of silencing in yeast should also employ assays reporting on natural gene expression patterns.

The specificity and topology of chromatin interaction pathways in yeast.

Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin.

Splitting the task: Ubp8 and Ubp10 deubiquitinate different cellular pools of H2BK123.

Monoubiquitination of H2BK123 (H2BK123ub), catalyzed by Rad6/Bre1, is a transient histone modification with roles in transcription and is essential for establishing H3K4 and H3K79 trimethylations (H3K4me3 and H3K79me3). Here, we investigated the chromatin network around H2BK123ub by examining its localization and co-occurrence with its dependent marks as well as the transcription elongation mark H3K36me3 across the genome of Saccharomyces cerevisiae. In yeast, H2BK123ub is removed by the deubiquitinases Ubp8 and Ubp10, but their genomic target regions remain to be determined. Genome-wide maps of H2BK123ub in the absence of Ubp8 and Ubp10 revealed their distinct target loci, which were genomic sites enriched for H3K4me3 and H3K79me3, respectively. We propose an extended model of the H2BK123ub cross-talk by integrating existing relationships with the substrate specificities of Ubp8 and Ubp10 reported here.

Alpha-synuclein deregulates the expression of COL4A2 and impairs ER-Golgi function.

Alpha-synuclein (aSyn) is the major protein component of Lewy bodies and Lewy neurites, the typical pathological hallmarks in Parkinson's disease (PD) and Dementia with Lewy bodies. aSyn is capable of inducing transcriptional deregulation, but the precise effect of specific aSyn mutants associated with familial forms of PD, remains unclear. Here, we used transgenic mice overexpressing human wild-type (WT) or A30P aSyn to compare the transcriptional profiles of the two animal models. We found that A30P aSyn promotes strong transcriptional deregulation and increases DNA binding. Interestingly, COL4A2, a major component of basement membranes, was found to be upregulated in both A30P aSyn transgenic mice and in dopaminergic neurons expressing A30P aSyn, suggesting a crucial role for collagen related genes in aSyn-induced toxicity. Finally, we observed that A30P aSyn alters Golgi morphology and increases the susceptibility to endoplasmic reticulum (ER) stress in dopaminergic cells. In total, our findings provide novel insight into the putative role of aSyn on transcription and on the molecular mechanisms involved, thereby opening novel avenues for future therapeutic interventions in PD and other synucleinopathies.

Linking cell cycle to histone modifications: SBF and H2B monoubiquitination machinery and cell-cycle regulation of H3K79 dimethylation.

To identify regulators involved in determining the differential pattern of H3K79 methylation by Dot1, we screened the entire yeast gene deletion collection by GPS for genes required for normal levels of H3K79 di- but not trimethylation. We identified the cell cycle-regulated SBF protein complex required for H3K79 dimethylation. We also found that H3K79 di- and trimethylation are mutually exclusive, with M/G1 cell cycle-regulated genes significantly enriched for H3K79 dimethylation. Since H3K79 trimethylation requires prior monoubiquitination of H2B, we performed genome-wide profiling of H2BK123 monoubiquitination and showed that H2BK123 monoubiquitination is not detected on cell cycle-regulated genes and sites containing H3K79me2, but is found on H3K79me3-containing regions. A screen for genes responsible for the establishment/removal of H3K79 dimethylation resulted in identification of NRM1 and WHI3, both of which impact the transcription by the SBF and MBF protein complexes, further linking the regulation of methylation status of H3K79 to the cell cycle.

Histone H3K4 demethylation is negatively regulated by histone H3 acetylation in Saccharomyces cerevisiae.

Histone H3 lysine 4 trimethylation (H3K4me3) is a hallmark of transcription initiation, but how H3K4me3 is demethylated during gene repression is poorly understood. Jhd2, a JmjC domain protein, was recently identified as the major H3K4me3 histone demethylase (HDM) in Saccharomyces cerevisiae. Although JHD2 is required for removal of methylation upon gene repression, deletion of JHD2 does not result in increased levels of H3K4me3 in bulk histones, indicating that this HDM is unable to demethylate histones during steady-state conditions. In this study, we showed that this was due to the negative regulation of Jhd2 activity by histone H3 lysine 14 acetylation (H3K14ac), which colocalizes with H3K4me3 across the yeast genome. We demonstrated that loss of the histone H3-specific acetyltransferases (HATs) resulted in genome-wide depletion of H3K4me3, and this was not due to a transcription defect. Moreover, H3K4me3 levels were reestablished in HAT mutants following loss of JHD2, which suggested that H3-specific HATs and Jhd2 serve opposing functions in regulating H3K4me3 levels. We revealed the molecular basis for this suppression by demonstrating that H3K14ac negatively regulated Jhd2 demethylase activity on an acetylated peptide in vitro. These results revealed the existence of a general mechanism for removal of H3K4me3 following gene repression.

LRRK2 guides the actin cytoskeleton at growth cones together with ARHGEF7 and Tropomyosin 4.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent the most common genetic cause of Parkinson's disease (PD). However, LRRK2 function and molecular mechanisms causing the parkinsonian phenotype remain widely unknown. Most of LRRK2 knockdown and overexpression models strengthen the relevance of LRRK2 in regulating neurite outgrowth. We have recently identified ARHGEF7 as the first guanine nucleotide exchange factor (GEF) of LRRK2. This GEF is influencing neurite outgrowth through regulation of actin polymerization. Here, we examined the expression profile of neuroblastoma cells with reduced LRRK2 and ARHGEF7 levels to identify additional partners of LRRK2 in this process. Tropomyosins (TPMs), and in particular TPM4, were the most interesting candidates next to other actin cytoskeleton regulating transcripts in this dataset. Subsequently, enhanced neurite branching was shown using primary hippocampal neurons of LRRK2 knockdown animals. Furthermore, we observed an enhanced number of growth cones per neuron and a mislocalization and dysregulation of ARHGEF7 and TPM4 in these neuronal compartments. Our results reveal a fascinating connection between the neurite outgrowth phenotype of LRRK2 models and the regulation of actin polymerization directing further investigations of LRRK2-related pathogenesis.

The RORÉ£/SREBP2 pathway is a master regulator of cholesterol metabolism and serves as potential therapeutic target in t(4;11) leukemia.

Dysregulated cholesterol homeostasis promotes tumorigenesis and progression. Therefore, metabolic reprogramming constitutes a new hallmark of cancer. However, until today, only few therapeutic approaches exist to target this pathway due to the often-observed negative feedback induced by agents like statins leading to controversially increased cholesterol synthesis upon inhibition. Sterol regulatory element-binding proteins (SREBPs) are key transcription factors regulating the synthesis of cholesterol and fatty acids. Since SREBP2 is difficult to target, we performed pharmacological inhibition of retinoic acid receptor (RAR)-related orphan receptor gamma (RORγ), which acts upstream of SREBP2 and serves as master regulator of the cholesterol metabolism. This resulted in an inactivated cholesterol-related gene program with significant downregulation of cholesterol biosynthesis. Strikingly, these effects were more pronounced than the effects of fatostatin, a direct SREBP2 inhibitor. Upon RORγ inhibition, RNA sequencing showed strongly increased cholesterol efflux genes leading to leukemic cell death and cell cycle changes in a dose- and time-dependent manner. Combinatorial treatment of t(4;11) cells with the RORγ inhibitor showed additive effects with cytarabine and even strong anti-leukemia synergism with atorvastatin by circumventing the statin-induced feedback. Our results suggest a novel therapeutic strategy to inhibit tumor-specific cholesterol metabolism for the treatment of t(4;11) leukemia.

CHROMATRA: a Galaxy tool for visualizing genome-wide chromatin signatures.

UNLABELLED: CHROMATRA (CHROmatin Mapping Across TRAnscripts) is a visualization tool available as plug-in for the Galaxy platform. It allows detailed yet concise presentations of data derived from ChIP-chip or ChIP-seq experiments by visualizing enrichment scores across genes or other genomic features while accounting for their length and additional characteristics such as gene expression. It integrates into typical analysis workflows and enables rapid graphical assessment and comparison of genome-wide data at a glance. AVAILABILITY: https://github.com/cmmt/chromatra.

Blood transcriptome analysis suggests an indirect molecular association of early life adversities and adult social anxiety disorder by immune-related signal transduction.

Social anxiety disorder (SAD) is a psychiatric disorder characterized by severe fear in social situations and avoidance of these. Multiple genetic as well as environmental factors contribute to the etiopathology of SAD. One of the main risk factors for SAD is stress, especially during early periods of life (early life adversity; ELA). ELA leads to structural and regulatory alterations contributing to disease vulnerability. This includes the dysregulation of the immune response. However, the molecular link between ELA and the risk for SAD in adulthood remains largely unclear. Evidence is emerging that long-lasting changes of gene expression patterns play an important role in the biological mechanisms linking ELA and SAD. Therefore, we conducted a transcriptome study of SAD and ELA performing RNA sequencing in peripheral blood samples. Analyzing differential gene expression between individuals suffering from SAD with high or low levels of ELA and healthy individuals with high or low levels of ELA, 13 significantly differentially expressed genes (DEGs) were identified with respect to SAD while no significant differences in expression were identified with respect to ELA. The most significantly expressed gene was MAPK3 (p = 0.003) being upregulated in the SAD group compared to control individuals. In contrary, weighted gene co-expression network analysis (WGCNA) identified only modules significantly associated with ELA (p ≤ 0.05), not with SAD. Furthermore, analyzing interaction networks of the genes from the ELA-associated modules and the SAD-related MAPK3 revealed complex interactions of those genes. Gene functional enrichment analyses indicate a role of signal transduction pathways as well as inflammatory responses supporting an involvement of the immune system in the association of ELA and SAD. In conclusion, we did not identify a direct molecular link between ELA and adult SAD by transcriptional changes. However, our data indicate an indirect association of ELA and SAD mediated by the interaction of genes involved in immune-related signal transduction.

Targeting MYC in combination with epigenetic regulators induces synergistic anti-leukemic effects in MLLr leukemia and simultaneously improves immunity.

MLL rearranged (MLLr) leukemias are associated with a poor prognosis and a limited response to conventional therapies. Moreover, chemotherapies result in severe side effects with significant impairment of the immune system. Therefore, the identification of novel treatment strategies is mandatory. Recently, we developed a human MLLr leukemia model by inducing chromosomal rearrangements in CD34+ cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This MLLr model authentically mimics patient leukemic cells and can be used as a platform for novel treatment strategies. RNA sequencing of our model revealed MYC as one of the most important key drivers to promote oncogenesis. However, in clinical trials the BRD4 inhibitor JQ-1 leading to indirect blocking of the MYC pathway shows only modest activity. We and others previously reported that epigenetic drugs targeting MAT2A or PRMT5 promote cell death in MLLr cells. Therefore, we use these drugs in combination with JQ-1 leading to augmented anti-leukemic effects. Moreover, we found activation of T, NK and iNKT cells, release of immunomodulatory cytokines and downregulation of the PD-1/PD-L1 axis upon inhibitor treatment leading to improved cytotoxicity. In summary, the inhibition of MYC and MAT2A or PRMT5 drives robust synergistic anti-leukemic activity in MLLr leukemia. Moreover, the immune system is concomitantly activated upon combinatorial inhibitor treatment, hereby further augmenting the therapeutic efficiency.

Endometrial organoids derived from Mayer-Rokitansky-Küster-Hauser syndrome patients provide insights into disease-causing pathways.

The uterus is responsible for the nourishment and mechanical protection of the developing embryo and fetus and is an essential part in mammalian reproduction. Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by agenesis of the uterus and upper part of the vagina in females with normal ovarian function. Although heavily studied, the cause of the disease is still enigmatic. Current research in the field of MRKH mainly focuses on DNA-sequencing efforts and, so far, has been unable to decipher the nature and heterogeneity of the disease, thereby holding back scientific and clinical progress. Here, we developed long-term expandable organoid cultures from endometrium found in uterine rudiment horns of MRKH patients. Phenotypically, they share great similarity with healthy control organoids and are surprisingly fully hormone responsive. Transcriptome analyses, however, identified an array of dysregulated genes that point to potentially disease-causing pathways altered during the development of the female reproductive tract. We consider the endometrial organoid cultures to be a powerful research tool that promise to enable an array of studies into the pathogenic origins of MRKH syndrome and possible treatment opportunities to improve patient quality of life.

Genome Sequencing and Transcriptome Profiling in Twins Discordant for Mayer-Rokitansky-Küster-Hauser Syndrome.

To identify potential genetic causes for Mayer-Rokitansky-Küster-Hauser syndrome (MRKH), we analyzed blood and rudimentary uterine tissue of 5 MRKH discordant monozygotic twin pairs. Assuming that a variant solely identified in the affected twin or affected tissue could cause the phenotype, we identified a mosaic variant in ACTR3B with high allele frequency in the affected tissue, low allele frequency in the blood of the affected twin, and almost absent in blood of the unaffected twin. Focusing on MRKH candidate genes, we detected a pathogenic variant in GREB1L in one twin pair and their unaffected mother showing a reduced phenotypic penetrance. Furthermore, two variants of unknown clinical significance in PAX8 and WNT9B were identified. In addition, we conducted transcriptome analysis of affected tissue and observed perturbations largely similar to those in sporadic cases. These shared transcriptional changes were enriched for terms associated with estrogen and its receptors pointing at a role of estrogen in MRKH pathology. Our genome sequencing approach of blood and uterine tissue of discordant twins is the most extensive study performed on twins discordant for MRKH so far. As no clear pathogenic differences were detected, research to evaluate other regulatory layers are required to better understand the complex etiology of MRKH.

A Novel SCA3 Knock-in Mouse Model Mimics the Human SCA3 Disease Phenotype Including Neuropathological, Behavioral, and Transcriptional Abnormalities Especially in Oligodendrocytes.

Spinocerebellar ataxia type 3 is the most common autosomal dominant inherited ataxia worldwide, caused by a CAG repeat expansion in the Ataxin-3 gene resulting in a polyglutamine (polyQ)-expansion in the corresponding protein. The disease is characterized by neuropathological, phenotypical, and specific transcriptional changes in affected brain regions. So far, there is no mouse model available representing all the different aspects of the disease, yet highly needed for a better understanding of the disease pathomechanisms. Here, we characterized a novel Ataxin-3 knock-in mouse model, expressing a heterozygous or homozygous expansion of 304 CAACAGs in the murine Ataxin-3 locus using biochemical, behavioral, and transcriptomic approaches. We compared neuropathological, and behavioral features of the new knock-in model with the in SCA3 research mostly used YAC84Q mouse model. Further, we compared transcriptional changes found in cerebellar samples of the SCA3 knock-in mice and post-mortem human SCA3 patients. The novel knock-in mouse is characterized by the expression of a polyQ-expansion in the murine Ataxin-3 protein, leading to aggregate formation, especially in brain regions known to be vulnerable in SCA3 patients, and impairment of Purkinje cells. Along these neuropathological changes, the mice showed a reduction in body weight accompanied by gait and balance instability. Transcriptomic analysis of cerebellar tissue revealed age-dependent differential expression, enriched for genes attributed to myelinating oligodendrocytes. Comparing these changes with those found in cerebellar tissue of SCA3 patients, we discovered an overlap of differentially expressed genes pointing towards similar gene expression perturbances in several genes linked to myelin sheaths and myelinating oligodendrocytes.

Increased expression of myelin-associated genes in frontal cortex of SNCA overexpressing rats and Parkinson's disease patients.

Parkinson's disease (PD) is an age-dependent neurodegenerative disorder. Besides characteristic motor symptoms, patients suffer from cognitive impairments linked to pathology in cortical areas. Due to obvious challenges in tracing the underlying molecular perturbations in human brain over time, we took advantage of a well-characterized PD rat model. Using RNA sequencing, we profiled the frontocortical transcriptome of post-mortem patient samples and aligned expression changes with perturbation patterns obtained in the model at 5 and 12 months of age reflecting a presymptomatic and symptomatic time point. Integrating cell type-specific reference data, we identified a shared expression signature between both species that pointed to oligodendrocyte-specific, myelin-associated genes. Drawing on longitudinal information from the model, their nearly identical upregulation in both species could be traced to two distinctive perturbance modes. While one mode exhibited age-independent alterations that affected genes including proteolipid protein 1 (PLP1), the other mode, impacting on genes like myelin-associated glycoprotein (MAG), was characterized by interferences of disease gene and adequate expression adaptations along aging. Our results highlight that even for a group of functionally linked genes distinct interference mechanisms may underlie disease progression that cannot be distinguished by examining the terminal point alone but instead require longitudinal interrogation of the system.

The Endometrial Transcription Landscape of MRKH Syndrome.

The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome (OMIM 277000) is characterized by agenesis of the uterus and upper part of the vagina in females with normal ovarian function. While genetic causes have been identified for a small subset of patients and epigenetic mechanisms presumably contribute to the pathogenic unfolding, too, the etiology of the syndrome has remained largely enigmatic. A comprehensive understanding of gene activity in the context of the disease is crucial to identify etiological components and their potential interplay. So far, this understanding is lacking, primarily due to the scarcity of samples and suitable tissue. In order to close this gap, we profiled endometrial tissue of uterus rudiments in a large cohort of MRKH patients using RNA-seq and thereby provide a genome-wide view on the altered transcription landscape of the MRKH syndrome. Differential and co-expression analyses of the data identified cellular processes and candidate genes that converge on a core network of interconnected regulators that emerge as pivotal for the perturbed expression space. With these results and browsable access to the rich data through an online tool we seek to accelerate research to unravel the underlying biology of the syndrome.

Only Hematopoietic Stem and Progenitor Cells from Cord Blood Are Susceptible to Malignant Transformation by MLL-AF4 Translocations.

Mixed lineage leukemia (MLL) (KMT2A) rearrangements (KMT2Ar) play a crucial role in leukemogenesis. Dependent on age, major differences exist regarding disease frequency, main fusion partners and prognosis. In infants, up to 80% of acute lymphoid leukemia (ALL) bear a MLL translocation and half of them are t(4;11), resulting in a poor prognosis. In contrast, in adults only 10% of acute myeloid leukemia (AML) bear t(9;11) with an intermediate prognosis. The reasons for these differences are poorly understood. Recently, we established an efficient CRISPR/Cas9-based KMT2Ar model in hematopoietic stem and progenitor cells (HSPCs) derived from human cord blood (huCB) and faithfully mimicked the underlying biology of the disease. Here, we applied this model to HSPCs from adult bone marrow (huBM) to investigate the impact of the cell of origin and fusion partner on disease development. Both genome-edited infant and adult KMT2Ar cells showed monoclonal outgrowth with an immature morphology, myelomonocytic phenotype and elevated KMT2Ar target gene expression comparable to patient cells. Strikingly, all KMT2Ar cells presented with indefinite growth potential except for MLL-AF4 huBM cells ceasing proliferation after 80 days. We uncovered FFAR2, an epigenetic tumor suppressor, as potentially responsible for the inability of MLL-AF4 to immortalize adult cells under myeloid conditions.