A genome-wide long noncoding RNA CRISPRi screen identifies PRANCR as a novel regulator of epidermal homeostasis.

Genome-wide association studies indicate that many disease susceptibility regions reside in non-protein-coding regions of the genome. Long noncoding RNAs (lncRNAs) are a major component of the noncoding genome, but their biological impacts are not fully understood. Here, we performed a CRISPR interference (CRISPRi) screen on 2263 epidermis-expressed lncRNAs and identified nine novel candidate lncRNAs regulating keratinocyte proliferation. We further characterized a top hit from the screen, progenitor renewal associated non-coding RNA (PRANCR), using RNA interference-mediated knockdown and phenotypic analysis in organotypic human tissue. PRANCR regulates keratinocyte proliferation, cell cycle progression, and clonogenicity. PRANCR-deficient epidermis displayed impaired stratification with reduced expression of differentiation genes that are altered in human skin diseases, including keratins 1 and 10, filaggrin, and loricrin. Transcriptome analysis showed that PRANCR controls the expression of 1136 genes, with strong enrichment for late cell cycle genes containing a CHR promoter element. In addition, PRANCR depletion led to increased levels of both total and nuclear CDKN1A (also known as p21), which is known to govern both keratinocyte proliferation and differentiation. Collectively, these data show that PRANCR is a novel lncRNA regulating epidermal homeostasis and identify other lncRNA candidates that may have roles in this process as well.

Mutation-specific effects in germline transmission of pathogenic mtDNA variants.

STUDY QUESTION: Does germline selection (besides random genetic drift) play a role during the transmission of heteroplasmic pathogenic mitochondrial DNA (mtDNA) mutations in humans? SUMMARY ANSWER: We conclude that inheritance of mtDNA is mutation-specific and governed by a combination of random genetic drift and negative and/or positive selection. WHAT IS KNOWN ALREADY: mtDNA inherits maternally through a genetic bottleneck, but the underlying mechanisms are largely unknown. Although random genetic drift is recognized as an important mechanism, selection mechanisms are thought to play a role as well. STUDY DESIGN, SIZE, DURATION: We determined the mtDNA mutation loads in 160 available oocytes, zygotes, and blastomeres of five carriers of the m.3243A>G mutation, one carrier of the m.8993T>G mutation, and one carrier of the m.14487T>C mutation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mutation loads were determined in PGD samples using PCR assays and analysed mathematically to test for random sampling effects. In addition, a meta-analysis has been performed on mutation load transmission data in the literature to confirm the results of the PGD samples. MAIN RESULTS AND THE ROLE OF CHANCE: By applying the Kimura distribution, which assumes random mechanisms, we found that mtDNA segregations patterns could be explained by variable bottleneck sizes among all our carriers (moment estimates ranging from 10 to 145). Marked differences in the bottleneck size would determine the probability that a carrier produces offspring with mutations markedly different than her own. We investigated whether bottleneck sizes might also be influenced by non-random mechanisms. We noted a consistent absence of high mutation loads in all our m.3243A>G carriers, indicating non-random events. To test this, we fitted a standard and a truncated Kimura distribution to the m.3243A>G segregation data. A Kimura distribution truncated at 76.5% heteroplasmy has a significantly better fit (P-value = 0.005) than the standard Kimura distribution. For the m.8993T>G mutation, we suspect a skewed mutation load distribution in the offspring. To test this hypothesis, we performed a meta-analysis on published blood mutation levels of offspring-mother (O-M) transmission for the m.3243A>G and m.8993T>G mutations. This analysis revealed some evidence that the O-M ratios for the m.8993T>G mutation are different from zero (P-value <0.001), while for the m.3243A>G mutation there was little evidence that the O-M ratios are non-zero. Lastly, for the m.14487T>G mutation, where the whole range of mutation loads was represented, we found no indications for selective events during its transmission. LARGE SCALE DATA: All data are included in the Results section of this article. LIMITATIONS, REASON FOR CAUTION: The availability of human material for the mutations is scarce, requiring additional samples to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Our data show that non-random mechanisms are involved during mtDNA segregation. We aimed to provide the mechanisms underlying these selection events. One explanation for selection against high m.3243A>G mutation loads could be, as previously reported, a pronounced oxidative phosphorylation (OXPHOS) deficiency at high mutation loads, which prohibits oogenesis (e.g. progression through meiosis). No maximum mutation loads of the m.8993T>G mutation seem to exist, as the OXPHOS deficiency is less severe, even at levels close to 100%. In contrast, high mutation loads seem to be favoured, probably because they lead to an increased mitochondrial membrane potential (MMP), a hallmark on which healthy mitochondria are being selected. This hypothesis could provide a possible explanation for the skewed segregation pattern observed. Our findings are corroborated by the segregation pattern of the m.14487T>C mutation, which does not affect OXPHOS and MMP significantly, and its transmission is therefore predominantly determined by random genetic drift. Our conclusion is that mutation-specific selection mechanisms occur during mtDNA inheritance, which has implications for PGD and mitochondrial replacement therapy. STUDY FUNDING/COMPETING INTEREST(S): This work has been funded by GROW-School of Oncology and Developmental Biology. The authors declare no competing interests.

Alternative mRNA splicing events and regulators in epidermal differentiation.

Alternative splicing (AS) of messenger RNAs occurs in ∼95% of multi-exon human genes and generates diverse RNA and protein isoforms. We investigated AS events associated with human epidermal differentiation, a process crucial for skin function. We identified 6,413 AS events, primarily involving cassette exons. We also predicted 34 RNA-binding proteins (RBPs) regulating epidermal AS, including 19 previously undescribed candidate regulators. From these results, we identified FUS as an RBP that regulates the balance between keratinocyte proliferation and differentiation. Additionally, we characterized the function of a cassette exon AS event in MAP3K7, which encodes a kinase involved in cell signaling. We found that a switch from the short to long isoform of MAP3K7, triggered during differentiation, enforces the demarcation between proliferating basal progenitors and overlying differentiated strata. Our findings indicate that AS occurs extensively in the human epidermis and has critical roles in skin homeostasis.

Research Techniques Made Simple: CRISPR Genetic Screens.

CRISPR and Cas proteins, often referred to as CRISPR/Cas, are the components of a bacterial genome editing system that can be used to perturb genes in cells and tissues. A classic application is to use CRISPR/Cas to generate genetic loss-of-function. When performed at large scale and combined with deep sequencing techniques, CRISPR-based perturbations can be performed in a high throughput setting to screen many candidate genomic elements for their roles in a phenotype of interest. Here, we discuss major considerations in the design, execution, and analysis of CRISPR screens. We focus on CRISPR knockout screens but also review adaptations to the CRISPR/Cas system that highlight the versatility of the system to make other types of experimental genetic changes as well. We also discuss examples of CRISPR genetic screens in investigative dermatology and how they may be used to answer key scientific questions in the field.

Tfam Knockdown Results in Reduction of mtDNA Copy Number, OXPHOS Deficiency and Abnormalities in Zebrafish Embryos.

High mitochondrial DNA (mtDNA) copy numbers are essential for oogenesis and embryogenesis and correlate with fertility of oocytes and viability of embryos. To understand the pathology and mechanisms associated with low mtDNA copy numbers, we knocked down mitochondrial transcription factor A (tfam), a regulator of mtDNA replication, during early zebrafish development. Reduction of tfam using a splice-modifying morpholino (MO) resulted in a 42 ± 17% decrease in mtDNA copy number in embryos at 4 days post fertilization. Morphant embryos displayed abnormal development of the eye, brain, heart, and muscle, as well as a 50 ± 22% decrease in ATP production. Transcriptome analysis revealed a decrease in protein-encoding transcripts from the heavy strand of the mtDNA, and down-regulation of genes involved in haem production and the metabolism of metabolites, which appear to trigger increased rRNA and tRNA synthesis in the nucleoli. However, this stress or compensatory response appears to fall short as pathology emerges and expression of genes related to eye development are severely down-regulated. Taken together, this study highlights the importance of sufficient mtDNA copies for early zebrafish development. Zebrafish is an excellent model to manipulate the mtDNA bottleneck and study its effect on embryogenesis rapidly and in large numbers of offspring.

Expression of pathogenic SCN9A mutations in the zebrafish: A model to study small-fiber neuropathy.

Small-fiber neuropathy (SFN) patients experience a spectrum of sensory abnormalities, including attenuated responses to non-noxious temperatures in combination with a decreased density of the small-nerve fibers. Gain-of-function mutations in the voltage-gated sodium channels SCN9A, SCN10A and SCN11A have been identified as an underlying genetic cause in a subpopulation of patients with SFN. Based on clinical-diagnostic tests for SFN, we have set up a panel of two read-outs reflecting SFN in zebrafish, being nerve density and behavioral responses. Nerve density was studied using a transgenic line in which the sensory neurons are GFP-labelled. For the behavioral experiments, a temperature-controlled water compartment was developed. This device allowed quantification of the behavioral response to temperature changes. By using these read-outs we demonstrated that zebrafish embryos transiently overexpressing the pathogenic human SCN9A p.(I228M) or p.(G856D) mutations both have a significantly decreased density of the small-nerve fibers. Additionally, larvae overexpressing the p.(I228M) mutation displayed a significant increase in activity induced by temperature change. As these features closely resemble the clinical hallmarks of SFN, our data suggest that transient overexpression of mutant human mRNA provides a model for SFN in zebrafish. This disease model may provide a basis for testing the pathogenicity of novel genetic variants identified in SFN patients. Furthermore, this model could be used for studying SFN pathophysiology in an in vivo model and for testing therapeutic interventions.

Differences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells.

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 10(6) mtDNA molecules with high variation between oocytes. During embryogenesis, the mtDNA copy number decreases to ∼170 mtDNA molecules per primordial germ cell (PGC), a number similar to that in mammals, and to ∼50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-)PGCs of the same female, dictated by variation in the mature oocyte. The presence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo) mutations can reach high mutation loads within a single generation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germline and non-germline cells, due to early differentiation of PGCs, may account for different distribution patterns of familial mutations.

Replication Errors Made During Oogenesis Lead to Detectable De Novo mtDNA Mutations in Zebrafish Oocytes with a Low mtDNA Copy Number.

Of all pathogenic mitochondrial DNA (mtDNA) mutations in humans, ∼25% is de novo, although the occurrence in oocytes has never been directly assessed. We used next-generation sequencing to detect point mutations directly in the mtDNA of 3-15 individual mature oocytes and three somatic tissues from eight zebrafish females. Various statistical and biological filters allowed reliable detection of de novo variants with heteroplasmy ≥1.5%. In total, we detected 38 de novo base substitutions, but no insertions or deletions. These 38 de novo mutations were present in 19 of 103 mature oocytes, indicating that ∼20% of the mature oocytes carry at least one de novo mutation with heteroplasmy ≥1.5%. This frequency of de novo mutations is close to that deducted from the reported error rate of polymerase gamma, the mitochondrial replication enzyme, implying that mtDNA replication errors made during oogenesis are a likely explanation. Substantial variation in the mutation prevalence among mature oocytes can be explained by the highly variable mtDNA copy number, since we previously reported that ∼20% of the primordial germ cells have a mtDNA copy number of ≤73 and would lead to detectable mutation loads. In conclusion, replication errors made during oogenesis are an important source of de novo mtDNA base substitutions and their location and heteroplasmy level determine their significance.

Evolutionary defined role of the mitochondrial DNA in fertility, disease and ageing.

BACKGROUND: The endosymbiosis of an alpha-proteobacterium and a eubacterium a billion years ago paved the way for multicellularity and enabled eukaryotes to flourish. The selective advantage for the host was the acquired ability to generate large amounts of intracellular hydrogen-dependent adenosine triphosphate. The price was increased reactive oxygen species (ROS) inside the eukaryotic cell, causing high mutation rates of the mitochondrial DNA (mtDNA). According to the Muller's ratchet theory, this accumulation of mutations in asexually transmitted mtDNA would ultimately lead to reduced reproductive fitness and eventually extinction. However, mitochondria have persisted over the course of evolution, initially due to a rapid, extreme evolutionary reduction of the mtDNA content. After the phylogenetic divergence of eukaryotes into animals, fungi and plants, differences in evolution of the mtDNA occurred with different adaptations for coping with the mutation burden within these clades. As a result, mitochondrial evolutionary mechanisms have had a profound effect on human adaptation, fertility, healthy reproduction, mtDNA disease manifestation and transmission and ageing. An understanding of these mechanisms might elucidate novel approaches for treatment and prevention of mtDNA disease. METHODS: The scientific literature was investigated to determine how mtDNA evolved in animals, plants and fungi. Furthermore, the different mechanisms of mtDNA inheritance and of balancing Muller's ratchet in these species were summarized together with the consequences of these mechanisms for human health and reproduction. RESULTS: Animal, plant and fungal mtDNA have evolved differently. Animals have compact genomes, little recombination, a stable number of genes and a high mtDNA copy number, whereas plants have larger genomes with variable gene counts, a low mtDNA copy number and many recombination events. Fungal mtDNA is somewhere in between. In plants, the mtDNA mutation rate is kept low by effective ROS defence and efficient recombination-mediated mtDNA repair. In animal mtDNA, these mechanisms are not or less well-developed and the detrimental mutagenesis events are controlled by a high mtDNA copy number in combination with a genetic bottleneck and purifying selection during transmission. The mtDNA mutation rates in animals are higher than in plants, which allow mobile animals to adapt more rapidly to various environmental conditions in terms of energy production, whereas static plants do not have this need. Although at the level of the species, these mechanisms have been extremely successful, they can have adverse effects for the individual, resulting, in humans, in severe or unpredictably segregating mtDNA diseases, as well as fertility problems and unhealthy ageing. CONCLUSIONS: Understanding the forces and processes that underlie mtDNA evolution among different species increases our knowledge on the detrimental consequences that individuals can have from these evolutionary end-points. Alternative outcomes in animals, fungi and plants will lead to a better understanding of the inheritance of mtDNA disorders and mtDNA-related fertility problems. These will allow the development of options to ameliorate, cure and/or prevent mtDNA diseases and mtDNA-related fertility problems.