Folding makes an imprint.

Imprinted gene clusters are confined genomic regions containing genes with parent-of-origin-dependent transcriptional activity. In this issue of Genes & Development, Loftus and colleagues (pp. 829-843) made use of an insightful combination of descriptive approaches, genetic manipulations, and epigenome-editing approaches to show that differences in nuclear topology precede the onset of imprinted expression at the Peg13-Kcnk9 locus. Furthermore, the investigators provide data in line with a model suggesting that parent-of-origin-specific topological differences could be responsible for parent-of-origin-specific enhancer activity and thus imprinted expression.

Probing cell identity hierarchies by fate titration and collision during direct reprogramming.

Despite the therapeutic promise of direct reprogramming, basic principles concerning fate erasure and the mechanisms to resolve cell identity conflicts remain unclear. To tackle these fundamental questions, we established a single-cell protocol for the simultaneous analysis of multiple cell fate conversion events based on combinatorial and traceable reprogramming factor expression: Collide-seq. Collide-seq revealed the lack of a common mechanism through which fibroblast-specific gene expression loss is initiated. Moreover, we found that the transcriptome of converting cells abruptly changes when a critical level of each reprogramming factor is attained, with higher or lower levels not contributing to major changes. By simultaneously inducing multiple competing reprogramming factors, we also found a deterministic system, in which titration of fates against each other yields dominant or colliding fates. By investigating one collision in detail, we show that reprogramming factors can disturb cell identity programs independent of their ability to bind their target genes. Taken together, Collide-seq has shed light on several fundamental principles of fate conversion that may aid in improving current reprogramming paradigms.

Innate Immune Pathways Promote Oligodendrocyte Progenitor Cell Recruitment to the Injury Site in Adult Zebrafish Brain.

The oligodendrocyte progenitors (OPCs) are at the front of the glial reaction to the traumatic brain injury. However, regulatory pathways steering the OPC reaction as well as the role of reactive OPCs remain largely unknown. Here, we compared a long-lasting, exacerbated reaction of OPCs to the adult zebrafish brain injury with a timely restricted OPC activation to identify the specific molecular mechanisms regulating OPC reactivity and their contribution to regeneration. We demonstrated that the influx of the cerebrospinal fluid into the brain parenchyma after injury simultaneously activates the toll-like receptor 2 (Tlr2) and the chemokine receptor 3 (Cxcr3) innate immunity pathways, leading to increased OPC proliferation and thereby exacerbated glial reactivity. These pathways were critical for long-lasting OPC accumulation even after the ablation of microglia and infiltrating monocytes. Importantly, interference with the Tlr1/2 and Cxcr3 pathways after injury alleviated reactive gliosis, increased new neuron recruitment, and improved tissue restoration.

CRISPR Tools for Physiology and Cell State Changes: Potential of Transcriptional Engineering and Epigenome Editing.

Given the large amount of genome-wide data that have been collected during the last decades, a good understanding of how and why cells change during development, homeostasis, and disease might be expected. Unfortunately, the opposite is true; triggers that cause cellular state changes remain elusive, and the underlying molecular mechanisms are poorly understood. Although genes with the potential to influence cell states are known, the historic dependency on methods that manipulate gene expression outside the endogenous chromatin context has prevented us from understanding how cells organize, interpret, and protect cellular programs. Fortunately, recent methodological innovations are now providing options to answer these outstanding questions, by allowing to target and manipulate individual genomic and epigenomic loci. In particular, three experimental approaches are now feasible due to DNA targeting tools, namely, activation and/or repression of master transcription factors in their endogenous chromatin context; targeting transcription factors to endogenous, alternative, or inaccessible sites; and finally, functional manipulation of the chromatin context. In this article, we discuss the molecular basis of DNA targeting tools and review the potential of these new technologies before we summarize how these have already been used for the manipulation of cellular states and hypothesize about future applications.

Epigenetic regulation of neural lineage elaboration: Implications for therapeutic reprogramming.

The vulnerability of the mammalian brain is mainly due to its limited ability to generate new neurons once fully matured. Direct conversion of non-neuronal cells to neurons opens up a new avenue for therapeutic intervention and has made great strides also for in vivo applications in the injured brain. These great achievements raise the issue of adequate identity and chromatin hallmarks of the induced neurons. This may be particularly important, as aberrant epigenetic settings may reveal their adverse effects only in certain brain activity states. Therefore, we review here the knowledge about epigenetic memory and partially resetting of chromatin hallmarks from other reprogramming fields, before moving to the knowledge in direct neuronal reprogramming, which is still limited. Most importantly, novel tools are available now to manipulate specific epigenetic marks at specific sites of the genome. Applying these will eventually allow erasing aberrant epigenetic memory and paving the way towards new therapeutic approaches for brain repair.

CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion.

Astrocyte-to-neuron conversion is a promising avenue for neuronal replacement therapy. Neurons are particularly dependent on mitochondrial function, but how well mitochondria adapt to the new fate is unknown. Here, we determined the comprehensive mitochondrial proteome of cortical astrocytes and neurons, identifying about 150 significantly enriched mitochondrial proteins for each cell type, including transporters, metabolic enzymes, and cell-type-specific antioxidants. Monitoring their transition during reprogramming revealed late and only partial adaptation to the neuronal identity. Early dCas9-mediated activation of genes encoding mitochondrial proteins significantly improved conversion efficiency, particularly for neuron-enriched but not astrocyte-enriched antioxidant proteins. For example, Sod1 not only improves the survival of the converted neurons but also elicits a faster conversion pace, indicating that mitochondrial proteins act as enablers and drivers in this process. Transcriptional engineering of mitochondrial proteins with other functions improved reprogramming as well, demonstrating a broader role of mitochondrial proteins during fate conversion.

Choroid plexus-derived miR-204 regulates the number of quiescent neural stem cells in the adult brain.

Regulation of adult neural stem cell (NSC) number is critical for lifelong neurogenesis. Here, we identified a post-transcriptional control mechanism, centered around the microRNA 204 (miR-204), to control the maintenance of quiescent (q)NSCs. miR-204 regulates a spectrum of transcripts involved in cell cycle regulation, neuronal migration, and differentiation in qNSCs. Importantly, inhibition of miR-204 function reduced the number of qNSCs in the subependymal zone (SEZ) by inducing pre-mature activation and differentiation of NSCs without changing their neurogenic potential. Strikingly, we identified the choroid plexus of the mouse lateral ventricle as the major source of miR-204 that is released into the cerebrospinal fluid to control number of NSCs within the SEZ. Taken together, our results describe a novel mechanism to maintain adult somatic stem cells by a niche-specific miRNA repressing activation and differentiation of stem cells.

Targeted removal of epigenetic barriers during transcriptional reprogramming.

Master transcription factors have the ability to direct and reverse cellular identities, and consequently their genes must be subject to particular transcriptional control. However, it is unclear which molecular processes are responsible for impeding their activation and safeguarding cellular identities. Here we show that the targeting of dCas9-VP64 to the promoter of the master transcription factor Sox1 results in strong transcript and protein up-regulation in neural progenitor cells (NPCs). This gene activation restores lost neuronal differentiation potential, which substantiates the role of Sox1 as a master transcription factor. However, despite efficient transactivator binding, major proportions of progenitor cells are unresponsive to the transactivating stimulus. By combining the transactivation domain with epigenome editing we find that among a series of euchromatic processes, the removal of DNA methylation (by dCas9-Tet1) has the highest potential to increase the proportion of cells activating foreign master transcription factors and thus breaking down cell identity barriers.

Glucose-Regulated TET2 Activity Links Cancer to Diabetes.

Diabetes has long been associated with an increased risk of cancer. While many molecular connections likely exist between the diseases, a recent publication discovered a clear molecular link, demonstrating that a glucose-dependent destabilisation of the DNA demethylase TET2 can promote malignant transformation via an AMPK-dependent phosphoswitch.

The Aryl Hydrocarbon Receptor Pathway Defines the Time Frame for Restorative Neurogenesis.

Zebrafish have a high capacity to replace lost neurons after brain injury. New neurons involved in repair are generated by a specific set of glial cells, known as ependymoglial cells. We analyze changes in the transcriptome of ependymoglial cells and their progeny after injury to infer the molecular pathways governing restorative neurogenesis. We identify the aryl hydrocarbon receptor (AhR) as a regulator of ependymoglia differentiation toward post-mitotic neurons. In vivo imaging shows that high AhR signaling promotes the direct conversion of a specific subset of ependymoglia into post-mitotic neurons, while low AhR signaling promotes ependymoglial proliferation. Interestingly, we observe the inactivation of AhR signaling shortly after injury followed by a return to the basal levels 7 days post injury. Interference with timely AhR regulation after injury leads to aberrant restorative neurogenesis. Taken together, we identify AhR signaling as a crucial regulator of restorative neurogenesis timing in the zebrafish brain.

Selfish mutations dysregulating RAS-MAPK signaling are pervasive in aged human testes.

Mosaic mutations present in the germline have important implications for reproductive risk and disease transmission. We previously demonstrated a phenomenon occurring in the male germline, whereby specific mutations arising spontaneously in stem cells (spermatogonia) lead to clonal expansion, resulting in elevated mutation levels in sperm over time. This process, termed "selfish spermatogonial selection," explains the high spontaneous birth prevalence and strong paternal age-effect of disorders such as achondroplasia and Apert, Noonan and Costello syndromes, with direct experimental evidence currently available for specific positions of six genes (FGFR2, FGFR3, RET, PTPN11, HRAS, and KRAS). We present a discovery screen to identify novel mutations and genes showing evidence of positive selection in the male germline, by performing massively parallel simplex PCR using RainDance technology to interrogate mutational hotspots in 67 genes (51.5 kb in total) in 276 biopsies of testes from five men (median age, 83 yr). Following ultradeep sequencing (about 16,000×), development of a low-frequency variant prioritization strategy, and targeted validation, we identified 61 distinct variants present at frequencies as low as 0.06%, including 54 variants not previously directly associated with selfish selection. The majority (80%) of variants identified have previously been implicated in developmental disorders and/or oncogenesis and include mutations in six newly associated genes (BRAF, CBL, MAP2K1, MAP2K2, RAF1, and SOS1), all of which encode components of the RAS-MAPK pathway and activate signaling. Our findings extend the link between mutations dysregulating the RAS-MAPK pathway and selfish selection, and show that the aging male germline is a repository for such deleterious mutations.

One step generation of customizable gRNA vectors for multiplex CRISPR approaches through string assembly gRNA cloning (STAgR).

Novel applications based on the bacterial CRISPR system make genetic, genomic, transcriptional and epigenomic engineering widely accessible for the first time. A significant advantage of CRISPR over previous methods is its tremendous adaptability due to its bipartite nature. Cas9 or its engineered variants define the molecular effect, while short gRNAs determine the targeting sites. A majority of CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into single cells, either as an essential precondition, to increase responsive cell populations or to enhance phenotypic outcomes. Despite these requirements, methods allowing the efficient generation and delivery of multiple gRNA expression units into single cells are still sparse. Here we present STAgR (String assembly gRNA cloning), a single step gRNA multiplexing system, that obtains its advantages by employing the N20 targeting sequences as necessary homologies for Gibson assembly. We show that STAgR allows reliable and cost-effective generation of vectors with high numbers of gRNAs enabling multiplexed CRISPR approaches. Moreover, STAgR is easily customizable, as vector backbones as well as gRNA structures, numbers and promoters can be freely chosen and combined. Finally, we demonstrate STAgR's widespread functionality, its efficiency in multi-targeting approaches, using it for both, genome and transcriptome editing, as well as applying it in vitro and in vivo.

Entering the post-epigenomic age: back to epigenetics.

It is undeniably one of the greatest findings in biology that (with some very minor exceptions) every cell in the body possesses the whole genetic information needed to generate a complete individual. Today, this concept has been so thoroughly assimilated that we struggle to still see how surprising this finding actually was: all cellular phenotypes naturally occurring in one person are generated from genetic uniformity, and thus are per definition epigenetic. Transcriptional mechanisms are clearly critical for developing and protecting cell identities, because a mis-expression of few or even single genes can efficiently induce inappropriate cellular programmes. However, how transcriptional activities are molecularly controlled and which of the many known epigenomic features have causal roles remains unclear. Today, clarification of this issue is more pressing than ever because profiling efforts and epigenome-wide association studies (EWAS) continuously provide comprehensive datasets depicting epigenomic differences between tissues and disease states. In this commentary, we propagate the idea of a widespread follow-up use of epigenome editing technology in EWAS studies. This would enable them to address the questions of which features, where in the genome, and which circumstances are essential to shape development and trigger disease states.

DNA-Methylation: Master or Slave of Neural Fate Decisions?

The pristine formation of complex organs depends on sharp temporal and spatial control of gene expression. Therefore, epigenetic mechanisms have been frequently attributed a central role in controlling cell fate determination. A prime example for this is the first discovered and still most studied epigenetic mark, DNA methylation, and the development of the most complex mammalian organ, the brain. Recently, the field of epigenetics has advanced significantly: new DNA modifications were discovered, epigenomic profiling became widely accessible, and methods for targeted epigenomic manipulation have been developed. Thus, it is time to challenge established models of epigenetic gene regulation. Here, we review the current state of knowledge about DNA modifications, their epigenomic distribution, and their regulatory role. We will summarize the evidence suggesting they possess crucial roles in neurogenesis and discuss whether this likely includes lineage choice regulation or rather effects on differentiation. Finally, we will attempt an outlook on how questions, which remain unresolved, could be answered soon.

A Customizable Protocol for String Assembly gRNA Cloning (STAgR).

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.