Leveraging cross-species transcription factor binding site patterns: from diabetes risk loci to disease mechanisms.

Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms.

Conserved reduction of m6A RNA modifications during aging and neurodegeneration is linked to changes in synaptic transcripts.

N6-methyladenosine (m6A) regulates mRNA metabolism. While it has been implicated in the development of the mammalian brain and in cognition, the role of m6A in synaptic plasticity, especially during cognitive decline, is not fully understood. In this study, we employed methylated RNA immunoprecipitation sequencing to obtain the m6A epitranscriptome of the hippocampal subregions CA1, CA3, and the dentate gyrus and the anterior cingulate cortex (ACC) in young and aged mice. We observed a decrease in m6A levels in aged animals. Comparative analysis of cingulate cortex (CC) brain tissue from cognitively intact human subjects and Alzheimer's disease (AD) patients showed decreased m6A RNA methylation in AD patients. m6A changes common to brains of aged mice and AD patients were found in transcripts linked to synaptic function including calcium/calmodulin-dependent protein kinase 2 (CAMKII) and AMPA-selective glutamate receptor 1 (Glua1). We used proximity ligation assays to show that reduced m6A levels result in decreased synaptic protein synthesis as exemplified by CAMKII and GLUA1. Moreover, reduced m6A levels impaired synaptic function. Our results suggest that m6A RNA methylation controls synaptic protein synthesis and may play a role in cognitive decline associated with aging and AD.

Post-transcriptional regulation by the exosome complex is required for cell survival and forebrain development via repression of P53 signaling.

Fine-tuned gene expression is crucial for neurodevelopment. The gene expression program is tightly controlled at different levels, including RNA decay. N6-methyladenosine (m6A) methylation-mediated degradation of RNA is essential for brain development. However, m6A methylation impacts not only RNA stability, but also other RNA metabolism processes. How RNA decay contributes to brain development is largely unknown. Here, we show that Exosc10, a RNA exonuclease subunit of the RNA exosome complex, is indispensable for forebrain development. We report that cortical cells undergo overt apoptosis, culminating in cortical agenesis upon conditional deletion of Exosc10 in mouse cortex. Mechanistically, Exosc10 directly binds and degrades transcripts of the P53 signaling-related genes, such as Aen and Bbc3. Overall, our findings suggest a crucial role for Exosc10 in suppressing the P53 pathway, in which the rapid turnover of the apoptosis effectors Aen and Bbc3 mRNAs is essential for cell survival and normal cortical histogenesis.

Compound heterozygosity of low-frequency promoter deletions and rare loss-of-function mutations in TXNL4A causes Burn-McKeown syndrome.

Mutations in components of the major spliceosome have been described in disorders with craniofacial anomalies, e.g., Nager syndrome and mandibulofacial dysostosis type Guion-Almeida. The U5 spliceosomal complex of eight highly conserved proteins is critical for pre-mRNA splicing. We identified biallelic mutations in TXNL4A, a member of this complex, in individuals with Burn-McKeown syndrome (BMKS). This rare condition is characterized by bilateral choanal atresia, hearing loss, cleft lip and/or palate, and other craniofacial dysmorphisms. Mutations were found in 9 of 11 affected families. In 8 families, affected individuals carried a rare loss-of-function mutation (nonsense, frameshift, or microdeletion) on one allele and a low-frequency 34 bp deletion (allele frequency 0.76%) in the core promoter region on the other allele. In a single highly consanguineous family, formerly diagnosed as oculo-oto-facial dysplasia, the four affected individuals were homozygous for a 34 bp promoter deletion, which differed from the promoter deletion in the other families. Reporter gene and in vivo assays showed that the promoter deletions led to reduced expression of TXNL4A. Depletion of TXNL4A (Dib1) in yeast demonstrated reduced assembly of the tri-snRNP complex. Our results indicate that BMKS is an autosomal-recessive condition, which is frequently caused by compound heterozygosity of low-frequency promoter deletions in combination with very rare loss-of-function mutations.

Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD, MIM #272200) results from pathogenic variants in the SUMF1 gene that impair proper function of the formylglycine-generating enzyme (FGE). FGE is essential for the posttranslational activation of cellular sulfatases. MSD patients display reduced or absent sulfatase activities and, as a result, clinical signs of single sulfatase disorders in a unique combination. Up to date therapeutic options for MSD are limited and mostly palliative. We performed a screen of FDA-approved drugs using immortalized MSD patient fibroblasts. Recovery of arylsulfatase A activity served as the primary readout. Subsequent analysis confirmed that treatment of primary MSD fibroblasts with tazarotene and bexarotene, two retinoids, led to a correction of MSD pathophysiology. Upon treatment, sulfatase activities increased in a dose- and time-dependent manner, reduced glycosaminoglycan content decreased and lysosomal position and size normalized. Treatment of MSD patient derived induced pluripotent stem cells (iPSC) differentiated into neuronal progenitor cells (NPC) resulted in a positive treatment response. Tazarotene and bexarotene act to ultimately increase the stability of FGE variants. The results lay the basis for future research on the development of a first therapeutic option for MSD patients.

The human retinoblastoma gene is imprinted.

Genomic imprinting is an epigenetic process leading to parent-of-origin-specific DNA methylation and gene expression. To date, approximately 60 imprinted human genes are known. Based on genome-wide methylation analysis of a patient with multiple imprinting defects, we have identified a differentially methylated CpG island in intron 2 of the retinoblastoma (RB1) gene on chromosome 13. The CpG island is part of a 5'-truncated, processed pseudogene derived from the KIAA0649 gene on chromosome 9 and corresponds to two small CpG islands in the open reading frame of the ancestral gene. It is methylated on the maternal chromosome 13 and acts as a weak promoter for an alternative RB1 transcript on the paternal chromosome 13. In four other KIAA0649 pseudogene copies, which are located on chromosome 22, the two CpG islands have deteriorated and the CpG dinucleotides are fully methylated. By analysing allelic RB1 transcript levels in blood cells, as well as in hypermethylated and 5-aza-2'-deoxycytidine-treated lymphoblastoid cells, we have found that differential methylation of the CpG island skews RB1 gene expression in favor of the maternal allele. Thus, RB1 is imprinted in the same direction as CDKN1C, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation.

The Coding and Small Non-coding Hippocampal Synaptic RNAome.

Neurons are highly compartmentalized cells that depend on local protein synthesis. Messenger RNAs (mRNAs) have thus been detected in neuronal dendrites, and more recently in the pre- and postsynaptic compartments as well. Other RNA species such as microRNAs have also been described at synapses where they are believed to control mRNA availability for local translation. A combined dataset analyzing the synaptic coding and non-coding RNAome via next-generation sequencing approaches is, however, still lacking. Here, we isolate synaptosomes from the hippocampus of young wild-type mice and provide the coding and non-coding synaptic RNAome. These data are complemented by a novel approach for analyzing the synaptic RNAome from primary hippocampal neurons grown in microfluidic chambers. Our data show that synaptic microRNAs control almost the entire synaptic mRNAome, and we identified several hub microRNAs. By combining the in vivo synaptosomal data with our novel microfluidic chamber system, our findings also support the hypothesis that part of the synaptic microRNAome may be supplied to neurons via astrocytes. Moreover, the microfluidic system is suitable for studying the dynamics of the synaptic RNAome in response to stimulation. In conclusion, our data provide a valuable resource and point to several important targets for further research.

Epigenetic gene expression links heart failure to memory impairment.

In current clinical practice, care of diseased patients is often restricted to separated disciplines. However, such an organ-centered approach is not always suitable. For example, cognitive dysfunction is a severe burden in heart failure patients. Moreover, these patients have an increased risk for age-associated dementias. The underlying molecular mechanisms are presently unknown, and thus, corresponding therapeutic strategies to improve cognition in heart failure patients are missing. Using mice as model organisms, we show that heart failure leads to specific changes in hippocampal gene expression, a brain region intimately linked to cognition. These changes reflect increased cellular stress pathways which eventually lead to loss of neuronal euchromatin and reduced expression of a hippocampal gene cluster essential for cognition. Consequently, mice suffering from heart failure exhibit impaired memory function. These pathological changes are ameliorated via the administration of a drug that promotes neuronal euchromatin formation. Our study provides first insight to the molecular processes by which heart failure contributes to neuronal dysfunction and point to novel therapeutic avenues to treat cognitive defects in heart failure patients.

A microRNA signature that correlates with cognition and is a target against cognitive decline.

While some individuals age without pathological memory impairments, others develop age-associated cognitive diseases. Since changes in cognitive function develop slowly over time in these patients, they are often diagnosed at an advanced stage of molecular pathology, a time point when causative treatments fail. Thus, there is great need for the identification of inexpensive and minimal invasive approaches that could be used for screening with the aim to identify individuals at risk for cognitive decline that can then undergo further diagnostics and eventually stratified therapies. In this study, we use an integrative approach combining the analysis of human data and mechanistic studies in model systems to identify a circulating 3-microRNA signature that reflects key processes linked to neural homeostasis and inform about cognitive status. We furthermore provide evidence that expression changes in this signature represent multiple mechanisms deregulated in the aging and diseased brain and are a suitable target for RNA therapeutics.

Changes in m6A RNA methylation contribute to heart failure progression by modulating translation.

AIMS: Deregulation of epigenetic processes and aberrant gene expression are important mechanisms in heart failure. Here we studied the potential relevance of m6A RNA methylation in heart failure development. METHODS AND RESULTS: We analysed m6A RNA methylation via next-generation sequencing. We found that approximately one quarter of the transcripts in the healthy mouse and human heart exhibit m6A RNA methylation. During progression to heart failure we observed that changes in m6A RNA methylation exceed changes in gene expression both in mouse and human. RNAs with altered m6A RNA methylation were mainly linked to metabolic and regulatory pathways, while changes in RNA expression level mainly represented changes in structural plasticity. Mechanistically, we could link m6A RNA methylation to altered RNA translation and protein production. Interestingly, differentially methylated but not differentially expressed RNAs showed differential polysomal occupancy, indicating transcription-independent modulation of translation. Furthermore, mice with a cardiomyocyte restricted knockout of the RNA demethylase Fto exhibited an impaired cardiac function compared to control mice. CONCLUSIONS: We could show that m6A landscape is altered in heart hypertrophy and heart failure. m6A RNA methylation changes lead to changes in protein abundance, unconnected to mRNA levels. This uncovers a new transcription-independent mechanisms of translation regulation. Therefore, our data suggest that modulation of epitranscriptomic processes such as m6A methylation might be an interesting target for therapeutic interventions.

A combined miRNA-piRNA signature to detect Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder causing huge emotional and economic burden to our societies. An effective therapy has not been implicated yet, which is in part also due to the fact that pathological changes occur years before clinical symptoms manifest. Thus, there is a great need for the development of a translatable biomarker. Recent evidence highlights microRNAs as candidate biomarkers. In this study, we use next-generation sequencing to study the small noncoding RNAome (sncRNAome) in exosomes derived from human cerebrospinal fluid (CSF). We show that the sncRNAome from CSF-derived exosomes is dominated not only by microRNAs (miRNAs) but also by PIWI-interacting RNAs (piRNAs). We define a combined signature consisting of three miRNAs and three piRNAs that are suitable to detect AD with an AUC of 0.83 in a replication cohort and furthermore predict the conversion of mild-cognitive impaired (MCI) patients to AD dementia with an AUC of 0.86 for the piRNA signature. When combining the smallRNA signature with pTau and Aß 42/40 ratio the AUC reaches 0.98. Our study reports a novel exosomal small noncoding RNA signature to detect AD pathology and provides the first evidence that in addition to miRNAs, piRNAs should also be considered as a candidate biomarker for AD.

Epigenetic dynamics of monocyte-to-macrophage differentiation.

BACKGROUND: Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium. RESULTS: Numerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response. CONCLUSIONS: In summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation.

N6-adenosine methylation in MiRNAs.

Methylation of N6-adenosine (m6A) has been observed in many different classes of RNA, but its prevalence in microRNAs (miRNAs) has not yet been studied. Here we show that a knockdown of the m6A demethylase FTO affects the steady-state levels of several miRNAs. Moreover, RNA immunoprecipitation with an anti-m6A-antibody followed by RNA-seq revealed that a significant fraction of miRNAs contains m6A. By motif searches we have discovered consensus sequences discriminating between methylated and unmethylated miRNAs. The epigenetic modification of an epigenetic modifier as described here adds a new layer to the complexity of the posttranscriptional regulation of gene expression.

FTO levels affect RNA modification and the transcriptome.

A block of single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity associated) gene is associated with variation in body weight. Previous works suggest that increased expression of FTO, which encodes a 2-oxoglutarate-dependent nucleic acid demethylase, leads to increased body weight, although the underlying mechanism has remained unclear. To elucidate the function of FTO, we examined the consequences of altered FTO levels in cultured cells and murine brain. Here we show that a knockdown of FTO in HEK293 cells affects the transcripts levels of genes involved in the response to starvation, whereas overexpression of FTO affects the transcript levels of genes related to RNA processing and metabolism. Subcellular localization of FTO further strengthens the latter notion. Using immunocytochemistry and confocal laser scanning microscopy, we detected FTO in nuclear speckles and--to a lesser and varying extent--in the nucleoplasm and nucleoli of HEK293, HeLa and MCF-7 cells. Moreover, RNA modification analyses revealed that loss of Fto affects the 3-methyluridine/uridine and pseudouridine/uridine ratios in total brain RNA. We conclude that altered levels of FTO have multiple and diverse consequences on RNA modifications and the transcriptome.

The obesity-associated SNPs in intron 1 of the FTO gene affect primary transcript levels.

As shown by genome-wide association studies single-nucleotide polymorphisms (SNPs) within intron 1 of the FTO gene are associated with the body mass index and type II diabetes, although the functional significance of these SNPs has remained unclear. Using primer extension assays, we have determined the ratio of allelic FTO transcript levels in unspliced heterogeneous nuclear RNA preparations from blood of individuals heterozygous for SNP rs9939609. Allelic expression ratios of the neighboring RPGRIP1L gene were investigated in individuals who were heterozygous for SNP rs4784319 and heterozygous or homozygous for rs9939609. In each of five individuals, the FTO transcripts containing the A (risk) allele of rs9939609 were more abundant than those with T allele (mean 1.38; 95% confidence interval 1.31-1.44). Similar results were obtained in a fibroblast sample. We also observed skewed allelic expression of the RPGRIP1L gene in blood, but skewing was independent of the FTO genotype. Our data suggest that increased expression of FTO is associated with increased body mass.