The UNC-83/UNC-84 LINC members are required for body wall muscle nuclei positioning in C. elegans

From a mutagenesis screen in the nematode C. elegans we isolated the mutant bar18, showing an accumulation of muscle cell nuclei around the posterior pharyngeal bulb of the worm. Quantification of the overall amount of body wall muscle nuclei, based on the muscle-specific reporter myo-3p::gfp::NLS, revealed that the number of nuclei in bar18 mutants is unchanged compared to WT worms. The accumulation of muscle nuclei around the posterior pharyngeal bulb is due to a positioning defect, which can be precisely quantified by subdividing the worm into head, neck, and posterior body segments. Whole-genome sequencing revealed that bar18 animals carry a mutation in the KASH-domain gene unc-83 causing a premature STOP. An additional unc-83 mutant allele recapitulates the phenotype, as does a mutant allele of UNC-84, a SUN-domain containing protein that interacts with UNC-83. UNC-83 and UNC-84 belong to a Linker of Nucleoskeleton and Cytoskeletonnuclear (LINC) complex that bridges the nuclear lamina with the cytoskeleton. SUN and KASH domain proteins are conserved in mammals and mutations in the corresponding genes have been linked to cancer, autism, muscular dystrophy and other diseases. Additionally, LINC complexes that function in nuclear migration have also been identified in mammals. We were able to rescue the unc-83 mutant phenotype by expressing the WT gene under a muscle-specific (myo-3p) promoter, demonstrating that the effect is cell autonomous. Mutations in either unc-83 or unc-84 have previously been linked to nuclear migration defects in P cells, intestinal cells and hyp7 hypodermal precursors but not in muscles. Whether the mis-positioning of muscle nuclei is due to migration or anchoring defects still needs to be determined

Transdifferentiation: do transition states lie on the path of development?

The direct conversion of one differentiated cell fate into another identity is a process known as Transdifferentiation. During Transdifferentiation, cells pass through intermediate states that are not well understood. Given the potential application of transdifferentiation in regenerative medicine and disease modeling, a better understanding of intermediate states is crucial to avoid uncontrolled conversion or proliferation, which pose a risk for patients. Researchers have begun to analyze the transcriptomes of donor, converting and target cells of Transdifferentiation with single cell resolution to compare transitional states to those found along the path of development. Here, we review examples of Transdifferentiation in a range of model systems and organisms. We propose that cells pass either through a mixed, unspecific intermediate or progenitor-like state during Transdifferentiation, which, to varying degrees, resemble states seen during development

Strategies for in vivo reprogramming

Reprogramming has the potential to provide specific cell types for regenerative medicine applications aiming at replacing tissues that have been lost or damaged due to degenerative diseases and injury. In this review we discuss the latest strategies and advances of in vivo reprogramming to convert cell identities in living organisms, including reprogramming induced by transcription factors (TFs) and CRISPR/dCas9 synthetic TFs, as well as by cell fusion and small molecules. We also provide a brief recap of reprogramming barriers, the effect of senescence on reprogramming efficiency, and strategies to deliver reprogramming factors in vivo. Because of the limited space, we omit dwelling on naturally occurring reprogramming phenomena such as developmentally programmed transdifferentiation found in the nematode Caenorhabditis elegans

Induced neurons from germ cells in Caenorhabditis elegans

Cell fate conversion by the forced overexpression of transcription factors (TFs) is a process known as reprogramming. It leads to de-differentiation or trans-differentiation of mature cells, which could then be used for regenerative medicine applications to replenish patients suffering from, e.g., neurodegenerative diseases, with healthy neurons. However, TF-induced reprogramming is often restricted due to cell fate safeguarding mechanisms, which require a better understanding to increase reprogramming efficiency and achieve higher fidelity. The germline of the nematode Caenorhabditis elegans has been a powerful model to investigate the impediments of generating neurons from germ cells by reprogramming. A number of conserved factors have been identified that act as a barrier for TF-induced direct reprogramming of germ cells to neurons. In this review, we will first summarize our current knowledge regarding cell fate safeguarding mechanisms in the germline. Then, we will focus on the molecular mechanisms underlying neuronal induction from germ cells upon TF-mediated reprogramming. We will shortly discuss the specific characteristics that might make germ cells especially fit to change cellular fate and become neurons. For future perspectives, we will look at the potential of C. elegans research in advancing our knowledge of the mechanisms that regulate cellular identity, and what implications this has for therapeutic approaches such as regenerative medicine

HOT or not: examining the basis of high-occupancy target regions

High-occupancy target (HOT) regions are segments of the genome with unusually high number of transcription factor binding sites. These regions are observed in multiple species and thought to have biological importance due to high transcription factor occupancy. Furthermore, they coincide with house-keeping gene promoters and consequently associated genes are stably expressed across multiple cell types. Despite these features, HOT regions are solemnly defined using ChIP-seq experiments and shown to lack canonical motifs for transcription factors that are thought to be bound there. Although, ChIP-seq experiments are the golden standard for finding genome-wide binding sites of a protein, they are not noise free. Here, we show that HOT regions are likely to be ChIP-seq artifacts and they are similar to previously proposed 'hyper-ChIPable' regions. Using ChIP-seq data sets for knocked-out transcription factors, we demonstrate presence of false positive signals on HOT regions. We observe sequence characteristics and genomic features that are discriminatory of HOT regions, such as GC/CpG-rich k-mers, enrichment of RNA-DNA hybrids (R-loops) and DNA tertiary structures (G-quadruplex DNA). The artificial ChIP-seq enrichment on HOT regions could be associated to these discriminatory features. Furthermore, we propose strategies to deal with such artifacts for the future ChIP-seq studies

Conversion of germ cells to somatic cell types in C. elegans

The potential of a cell to produce all types of differentiated cells in an organism is termed totipotency. Totipotency is an essential property of germ cells, which constitute the germline and pass on the parental genetic material to the progeny. The potential of germ cells to give rise to a whole organism has been the subject of intense research for decades and remains important in order to better understand the molecular mechanisms underlying totipotency. A better understanding of the principles of totipotency in germ cells could also help to generate this potential in somatic cell lineages. Strategies such as transcription factor-mediated reprogramming of differentiated cells to stem cell-like states could benefit from this knowledge. Ensuring pluripotency or even totipotency of reprogrammed stem cells are critical improvements for future regenerative medicine applications. The C. elegans germline provides a unique possibility to study molecular mechanisms that maintain totipotency and the germ cell fate with its unique property of giving rise to meiotic cells Studies that focused on these aspects led to the identification of prominent chromatin-repressing factors such as the C. elegans members of the Polycomb Repressive Complex 2 (PRC2). In this review, we summarize different factors that were recently identified, which use molecular mechanisms such as control of protein translation or chromatin repression to ensure maintenance of totipotency and the germline fate. Additionally, we focus on recently identified factors involved in preventing transcription-factor-mediated conversion of germ cells to somatic lineages. These so-called reprogramming barriers have been shown in some instances to be conserved with regard to their function as a cell fate safeguarding factor in mammals. Overall, continued studies assessing the different aspects of molecular pathways involved in maintaining the germ cell fate in C. elegans may provide more insight into cell fate safeguarding mechanisms also in other species

Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprograming of germ cells into neurons

Cell-fate reprograming is at the heart of development, yet very little is known about the molecular mechanisms promoting or inhibiting reprograming in intact organisms. In the C. elegans germline, reprograming germ cells into somatic cells requires chromatin perturbation. Here, we describe that such reprograming is facilitated by GLP-1/Notch signaling pathway. This is surprising, since this pathway is best known for maintaining undifferentiated germline stem cells/progenitors. Through a combination of genetics, tissue-specific transcriptome analysis, and functional studies of candidate genes, we uncovered a possible explanation for this unexpected role of GLP-1/Notch. We propose that GLP-1/Notch promotes reprograming by activating specific genes, silenced by the Polycomb repressive complex 2 (PRC2), and identify the conserved histone demethylase UTX-1 as a crucial GLP-1/Notch target facilitating reprograming. These findings have wide implications, ranging from development to diseases associated with abnormal Notch signaling

The C. elegans pseudogene sspt-16 (F55A3.7) is required to safeguard germ cells against reprogramming

We recently identified FAcilitates Chromatin Transcription (FACT) as a reprogramming barrier of transcription factor (TF) mediated conversion of germ cells into neurons in C. elegans. FACT is a conserved heterodimer consisting of SPT16 and SSRP1 in mammals. Duplication events during evolution in C. elegans generated two SSRP1 homologs named HMG-3 and HMG-4, while SPT-16 is the only homolog of SPT16. Yet, the pseudogene F55A3.7 has nearly complete nucleotide sequence homology to the spt-16 gene. However, F55A3.7 lacks some spt-16 exons and DNA pieces so we named it sspt-16 (short spt-16). Surprisingly, the deletion mutant ok1829, which affects only the sspt-16 pseudogene, shows similar germ cell reprogramming effects as described previously for FACT-depleted animals. We examined whether lack of sspt-16 affects other genes or chromatin accessibility, which may explain the permissiveness for germ cell reprogramming

Two distinct types of neuronal asymmetries are controlled by the Caenorhabditis elegans zinc finger transcription factor die-1

Left/right asymmetric features of animals are either randomly distributed on either the left or right side within a population ("antisymmetries") or found stereotypically on one particular side of an animal ("directional asymmetries"). Both types of asymmetries can be found in nervous systems, but whether the regulatory programs that establish these asymmetries share any mechanistic features is not known. We describe here an unprecedented molecular link between these two types of asymmetries in Caenorhabditis elegans. The zinc finger transcription factor die-1 is expressed in a directionally asymmetric manner in the gustatory neuron pair ASE left (ASEL) and ASE right (ASER), while it is expressed in an antisymmetric manner in the olfactory neuron pair AWC left (AWCL) and AWC right (AWCR). Asymmetric die-1 expression is controlled in a fundamentally distinct manner in these two neuron pairs. Importantly, asymmetric die-1 expression controls the directionally asymmetric expression of gustatory receptor proteins in the ASE neurons and the antisymmetric expression of olfactory receptor proteins in the AWC neurons. These asymmetries serve to increase the ability of the animal to discriminate distinct chemosensory inputs