REST-mediated recruitment of polycomb repressor complexes in mammalian cells.

Polycomb Repressive Complex (PRC) 1 and PRC2 regulate genes involved in differentiation and development. However, the mechanism for how PRC1 and PRC2 are recruited to genes in mammalian cells is unclear. Here we present evidence for an interaction between the transcription factor REST, PRC1, and PRC2 and show that RNF2 and REST co-regulate a number of neuronal genes in human teratocarcinoma cells (NT2-D1). Using NT2-D1 cells as a model of neuronal differentiation, we furthermore showed that retinoic-acid stimulation led to displacement of PRC1 at REST binding sites, reduced H3K27Me3, and increased gene expression. Genome-wide analysis of Polycomb binding in Rest⁻/⁻ and Eed⁻/⁻ mouse embryonic stem (mES) cells showed that Rest was required for PRC1 recruitment to a subset of Polycomb regulated neuronal genes. Furthermore, we found that PRC1 can be recruited to Rest binding sites independently of CpG islands and the H3K27Me3 mark. Surprisingly, PRC2 was frequently increased around Rest binding sites located in CpG-rich regions in the Rest⁻/⁻ mES cells, indicating a more complex interplay where Rest also can limit PRC2 recruitment. Therefore, we propose that Rest has context-dependent functions for PRC1- and PRC2- recruitment, which allows this transcription factor to act both as a recruiter of Polycomb as well as a limiting factor for PRC2 recruitment at CpG islands

On molecular mechanisms of neural differentiation and forebrain development

Telencephalic development is an extraordinary complex process where neural structures of highly diverse cell compositions emerge as the result of multiple developmental strategies imposing tight regulation, balance and timing of gene expression. Embryonic telencephalic neural stem cells (NSCs) can form the major cell types found in the telencephalon, and are thus an excellent model for studying transcriptional control of lineage choice mechanisms in CNS development. In paper I, we show that a member of the BTB/POZ family, Zbtb20, is required for proper repression of genes involved in interneuron and oligodendrocyte differentiation as well as proliferation and cell cycle exit in NSCs. Zbtb20 was expressed in the subpallium in a Dlx and Mash1 overlapping fashion and interacted with the co-regulator SMRT. siRNA-mediated knockdown of Zbtb20 in NSCs modulated expression of key regulatory genes in control of differentiation and proliferation, including Dlx1/2/5 and p57kip2, thus affecting the outcome of cell fate acquisition in favor of oligodendrocytic differentiation. Zbtb20 protein was accumulated in regulatory regions of the Dlx1/2 bi-gene cluster along with a T3-dependent accumulation to the MBP M1 promoter. Furthermore, the zebrafish homolog to Zbtb20 appeared to be necessary for proper gastrulation and formation of neuroectoderm in vivo possibly due to precautious expression of p57kip2, analogous to the NSCs phenotype. In paper II, we suggest a novel role for the cyclin-dependent kinase inhibitor p57Kip2 as a context-dependent repressor of neurogenic transcription factors and telencephalic neuronal differentiation. p57Kip2 interacted with pro-neuronal basic helix-loop-helix factors such as Mash1. Increased levels of p57Kip2 inhibited Mash1 transcriptional activity in a cyclin-dependent kinase (CDK) independent manner and acted as a direct repressor in transcriptional assays. Proliferating telencephalic neural progenitors co-expressed basal levels of Mash1 and p57Kip2, and endogenous p57Kip2 accumulated transiently in the nuclei of NSCs during early stages of astrocyte differentiation, independent of cell-cycle exit and at times when Mash1 expression was still prominent. p57Kip2 repressed neuronal differentiation, but exerted little or no effect astroglial differentiation of NSCs. In paper III we propose that Zbtb45 is a novel regulator of glial differentiation. Zbtb45 mRNA was ubiquitously expressed in the developing CNS in mouse embryos but Zbtb45 mRNA knockdown in NSCs resulted in a rapid decrease in the expression of oligodendrocyte characteristic genes and significantly increased numbers of astrocytes. Zbtb45 mRNA knockdown in oligodendrocyte precursors completely inhibited oligodendrocyte differentiation upon mitogen withdrawal as assessed by a down-regulation of the expression of markers for oligodendrocytic differentiation such as CNPase, MBP and Sox10, whereas CD44 expression was found to be increased in all experiments. In paper IV we propose that the previously uncharacterized protein CXXC5 is acting as a BMP4 induced inhibitor of Wnt signaling in neural stem cells. CXXC5 expression overlapped with BMP4 adjacent to Wnt3a expression in the dorsal regions of the telencephalon. CXXC5 showed partial homology with Idax, shown to interact with the Wnt-signaling intermediate Dishevelled (Dvl). Indeed CXXC5 and Dvl colocalized in NSCs and interacted in vitro. CXXC5 over-expression attenuated Wnt signaling. Interestingly, BMP4-induced decrease in Axin2 levels was abolished by siRNA-mediated knockdown of CXXC5

The novel BTB/POZ and zinc finger factor Zbtb45 is essential for proper glial differentiation of neural and oligodendrocyte progenitor cells

Understanding the regulatory mechanisms controlling the fate decisions of neural stem cells (NSCs) is a crucial issue to shed new light on mammalian central nervous system (CNS) development in health and disease. We have investigated a possible role for the previously uncharacterized BTB/POZ-domain containing zinc finger factor Zbtb45 in the differentiation of NSCs and postnatal oligodendrocyte precursors. In situ hybridization histochemistry and RT-qPCR analysis revealed that Zbtb45 mRNA was ubiquitously expressed in the developing CNS in mouse embryos at embryonic day (E) 12.5 and 14.5. Zbtb45 mRNA knockdown in embryonic forebrain NSCs by siRNA resulted in a rapid decrease in the expression of oligodendrocyte-characteristic genes after mitogen (FGF2) withdrawal, whereas the expression of astrocyte-associated genes such as CD44 and GFAP increased compared to control. Accordingly, the number of astrocytes was significantly increased seven days after Zbtb45 siRNA delivery to NSCs, in contrast to the numbers of neuronal and oligodendrocyte-like cells. Surprisingly, mRNA knockdown of the Zbtb45-associated factor Med31, a subunit of the Mediator complex, did not result in any detectable effect on NSC differentiation. Similar to NSCs, Zbtb45 mRNA knockdown in oligodendrocyte precursors (CG-4) reduced oligodendrocyte maturation upon mitogen withdrawal associated with downregulation of the mRNA expression and protein levels of markers for oligodendrocytic differentiation. Zbtb45 mRNA knockdown did not significantly affect proliferation or cell death in any of the cell types. Based on these observations, we propose that Zbtb45 is a novel regulator of glial differentiation

REST-mediated recruitment of polycomb repressor complexes in mammalian cells.

Polycomb Repressive Complex (PRC) 1 and PRC2 regulate genes involved in differentiation and development. However, the mechanism for how PRC1 and PRC2 are recruited to genes in mammalian cells is unclear. Here we present evidence for an interaction between the transcription factor REST, PRC1, and PRC2 and show that RNF2 and REST co-regulate a number of neuronal genes in human teratocarcinoma cells (NT2-D1). Using NT2-D1 cells as a model of neuronal differentiation, we furthermore showed that retinoic-acid stimulation led to displacement of PRC1 at REST binding sites, reduced H3K27Me3, and increased gene expression. Genome-wide analysis of Polycomb binding in Rest⁻/⁻ and Eed⁻/⁻ mouse embryonic stem (mES) cells showed that Rest was required for PRC1 recruitment to a subset of Polycomb regulated neuronal genes. Furthermore, we found that PRC1 can be recruited to Rest binding sites independently of CpG islands and the H3K27Me3 mark. Surprisingly, PRC2 was frequently increased around Rest binding sites located in CpG-rich regions in the Rest⁻/⁻ mES cells, indicating a more complex interplay where Rest also can limit PRC2 recruitment. Therefore, we propose that Rest has context-dependent functions for PRC1- and PRC2- recruitment, which allows this transcription factor to act both as a recruiter of Polycomb as well as a limiting factor for PRC2 recruitment at CpG islands

A comprehensive map coupling histone modifications with gene regulation in adult dopaminergic and serotonergic neurons

The brain is composed of hundreds of different neuronal subtypes, which largely retain their identity throughout the lifespan of the organism. The mechanisms governing this stability are not fully understood, partly due to the diversity and limited size of clinically relevant neuronal populations, which constitute a technical challenge for analysis. Here, using a strategy that allows for ChIP-seq combined with RNA-seq in small neuronal populations in vivo, we present a comparative analysis of permissive and repressive histone modifications in adult midbrain dopaminergic neurons, raphe nuclei serotonergic neurons, and embryonic neural progenitors. Furthermore, we utilize the map generated by our analysis to show that the transcriptional response of midbrain dopaminergic neurons following 6-OHDA or methamphetamine injection is characterized by increased expression of genes with promoters dually marked by H3K4me3/H3K27me3. Our study provides an in vivo genome-wide analysis of permissive/repressive histone modifications coupled to gene expression in these rare neuronal subtypes.Correction in: NATURE COMMUNICATIONS, Volume: 9, Article Number: 4639, DOI: 10.1038/s41467-018-07154-5</p

Dopamine Signaling Leads to Loss of Polycomb Repression and Aberrant Gene Activation in Experimental Parkinsonism

International audiencePolycomb group (PcG) proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP) experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq) in combination with expression analysis by RNA-sequencing (RNA-seq) showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson's disease