Programmed genomic instability regulates neural transdifferentiation of human brain microvascular pericytes

Background Transdifferentiation describes transformation in vivo of specialized cells from one lineage into another. While there is extensive literature on forced induction of lineage reprogramming in vitro, endogenous mechanisms that govern transdifferentiation remain largely unknown. The observation that human microvascular pericytes transdifferentiate into neurons provided an opportunity to explore the endogenous molecular basis for lineage reprogramming. Results We show that abrupt destabilization of the higher-order chromatin topology that chaperones lineage memory of pericytes is driven by transient global transcriptional arrest. This leads within minutes to localized decompression of the repressed competing higher-order chromatin topology and expression of pro-neural genes. Transition to neural lineage is completed by probabilistic induction of R-loops in key myogenic loci upon re-initiation of RNA polymerase activity, leading to depletion of the myogenic transcriptome and emergence of the neurogenic transcriptome. Conclusions These findings suggest that the global transcriptional landscape not only shapes the functional cellular identity of pericytes, but also stabilizes lineage memory by silencing the competing neural program within a repressed chromatin state.

Genome-wide mapping implicates R-loops in lineage-specific processes and formation of DNA break hotspots in neural stem/progenitor cells

ABSTRACTRecent work has revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes. However, the mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of DNA:RNA hybrid structures called “R-loops” in primary neural stem/progenitor cells in order to assess their contribution to the different classes of DNA break “hotspots”. We report that R-loops in neural stem/progenitor cells are associated primarily with transcribed regions that replicate early and genes that show GC skew in their promoter region. Surprisingly, the majority of genes with recurrent DSB clusters in long, neural genes does not show substantial R-loop accumulation. We implicate R-loops in promoter-proximal DNA break formation in highly transcribed, early replicating regions and conclude that R-loops are not a driver of recurrent double-strand break cluster formation in most long, neural genes. Together, our study provides an understanding of how R-loops may contribute to DNA break hotspots and affect lineage-specific processes in neural stem/progenitor cells.

Newly-Discovered Neural Features Expand the Pathobiological Knowledge of Blastic Plasmacytoid Dendritic Cell Neoplasm

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.

Coordination of EZH2 and SOX2 specifies human neural fate decision

Polycomb repressive complexes (PRCs) are essential in mouse gastrulation and specify neural ectoderm in human embryonic stem cells (hESCs), but the underlying molecular basis remains unclear. Here in this study, by employing an array of different approaches, such as gene knock-out, RNA-seq, ChIP-seq, et al., we uncover that EZH2, an important PRC factor, specifies the normal neural fate decision through repressing the competing meso/endoderm program. EZH2−/− hESCs show an aberrant re-activation of meso/endoderm genes during neural induction. At the molecular level, EZH2 represses meso/endoderm genes while SOX2 activates the neural genes to coordinately specify the normal neural fate. Moreover, EZH2 also supports the proliferation of human neural progenitor cells (NPCs) through repressing the aberrant expression of meso/endoderm program during culture. Together, our findings uncover the coordination of epigenetic regulators such as EZH2 and lineage factors like SOX2 in normal neural fate decision.

Comparative Gene Analysis in Chordate Embryonic Development Reveals Co-existence of Von Baer’s and Haeckel’s Postulates

The relationship between embryonic development and evolution historically investigated based on embryo morphology could now be reassessed using mRNA expression endophenotype. Here, we analyzed the conservation of divergence of the developmental mRNA expression profiles in nine evolutionarily distinct species, from oyster to mouse, and compared them to the original concepts formulated by von Baer and Haeckel. We find nearly linear conservation of species’ developmental programs among these species, compatible with models rooted on von Baer’s postulates, for approximately a third of expressed orthologous genes. By contrast, 5-15% of developmental expression profiles, enriched in neural genes, displayed an alignment pattern compatible with the terminal edition paradigm proposed by Haeckel. Thus, the development-evolution relationship based on mRNA expression agrees with early concepts based on embryo morphology and demonstrates that the corresponding patterns coexist in chordate development.

Two distinct motifs for Zic-r.a drive specific gene expression in two cell lineages

Zic-r.a, a maternal transcription factor, specifies posterior fate in ascidian embryos. However, its direct target, Tbx6-r.b, does not contain typical Zic-r.a binding sites in its regulatory region. Using an in vitro selection assay, we found that Zic-r.a binds to sites dissimilar from the canonical motif, by which it activates Tbx6-r.b in a sub-lineage of muscle cells. These sites with non-canonical motifs have weak affinity for Zic-r.a; therefore, it activates Tbx6-r.b only in cells expressing Zic-r.a abundantly. Meanwhile, we found that Zic-r.a expressed zygotically in late embryos activates neural genes through canonical sites. Because different zinc-finger domains of Zic-r.a are important for driving reporters with canonical and non-canonical sites, it is likely that the non-canonical motif is not a divergent version of the canonical motif. In other words, our data indicate that the non-canonical motif represents a motif distinct from the canonical motif. Thus, Zic-r.a recognizes two distinct motifs to activate two sets of genes at two timepoints in development.

Effect of Long-Term 3D Spheroid Culture on WJ-MSC

The aim of our work was to develop a protocol enabling a derivation of mesenchymal stem/stromal cell (MSC) subpopulation with increased expression of pluripotent and neural genes. For this purpose we used a 3D spheroid culture system optimal for neural stem cells propagation. Although 2D culture conditions are typical and characteristic for MSC, under special treatment these cells can be cultured for a short time in 3D conditions. We examined the effects of prolonged 3D spheroid culture on MSC in hope to select cells with primitive features. Wharton Jelly derived MSC (WJ-MSC) were cultured in 3D neurosphere induction medium for about 20 days in vitro. Then, cells were transported to 2D conditions and confront to the initial population and population constantly cultured in 2D. 3D spheroids culture of WJ-MSC resulted in increased senescence, decreased stemness and proliferation. However long-termed 3D spheroid culture allowed for selection of cells exhibiting increased expression of early neural and SSEA4 markers what might indicate the survival of cell subpopulation with unique features.

Neurotoxicity in Marine Invertebrates: An Update

Invertebrates represent about 95% of existing species, and most of them belong to aquatic ecosystems. Marine invertebrates are found at intermediate levels of the food chain and, therefore, they play a central role in the biodiversity of ecosystems. Furthermore, these organisms have a short life cycle, easy laboratory manipulation, and high sensitivity to marine pollution and, therefore, they are considered to be optimal bioindicators for assessing detrimental chemical agents that are related to the marine environment and with potential toxicity to human health, including neurotoxicity. In general, albeit simple, the nervous system of marine invertebrates is composed of neuronal and glial cells, and it exhibits biochemical and functional similarities with the vertebrate nervous system, including humans. In recent decades, new genetic and transcriptomic technologies have made the identification of many neural genes and transcription factors homologous to those in humans possible. Neuroinflammation, oxidative stress, and altered levels of neurotransmitters are some of the aspects of neurotoxic effects that can also occur in marine invertebrate organisms. The purpose of this review is to provide an overview of major marine pollutants, such as heavy metals, pesticides, and micro and nano-plastics, with a focus on their neurotoxic effects in marine invertebrate organisms. This review could be a stimulus to bio-research towards the use of invertebrate model systems other than traditional, ethically questionable, time-consuming, and highly expensive mammalian models.

PAUPAR and PAX6 sequentially regulate human embryonic stem cell cortical differentiation

Long noncoding RNAs (lncRNAs) play a wide range of roles in the epigenetic regulation of crucial biological processes, but the functions of lncRNAs in cortical development are poorly understood. Using human embryonic stem cell (hESC)-based 2D neural differentiation approach and 3D cerebral organoid system, we identified that the lncRNA PAUPAR, which is adjacent to PAX6, plays essential roles in cortical differentiation by interacting with PAX6 to regulate the expression of a large number of neural genes. Mechanistic studies showed that PAUPAR confers PAX6 proper binding sites on the target neural genes by directly binding the genomic regions of these genes. Moreover, PAX6 recruits the histone methyltransferase NSD1 through its C-terminal PST enrichment domain, then regulate H3K36 methylation and the expression of target genes. Collectively, our data reveal that the PAUPAR/PAX6/NSD1 complex plays a critical role in the epigenetic regulation of hESC cortical differentiation and highlight the importance of PAUPAR as an intrinsic regulator of cortical differentiation.