REST and CoREST Modulate Neuronal Subtype Specification, Maturation and Maintenance

BACKGROUND: The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST functions as a modular scaffold for dynamic recruitment of epigenetic regulatory factors including its primary cofactor, the corepressor for element-1-silencing transcription factor (CoREST), to genomic loci that contain the repressor element-1 (RE1) binding motif. While REST was initially believed to silence RE1 containing neuronal genes in neural stem cells (NSCs) and non-neuronal cells, emerging evidence shows an increasingly complex cell type- and developmental stage-specific repertoire of REST target genes and functions that include regulation of neuronal lineage maturation and plasticity. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we utilized chromatin immunoprecipitation on chip (ChIP-chip) analysis to examine REST and CoREST functions during NSC-mediated specification of cholinergic neurons (CHOLNs), GABAergic neurons (GABANs), glutamatergic neurons (GLUTNs), and medium spiny projection neurons (MSNs). We identified largely distinct but overlapping profiles of REST and CoREST target genes during neuronal subtype specification including a disproportionately high percentage that are exclusive to each neuronal subtype. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that the differential deployment of REST and CoREST is an important regulatory mechanism that mediates neuronal subtype specification by modulating specific gene networks responsible for inducing and maintaining neuronal subtype identity. Our observations also implicate a broad array of factors in the generation of neuronal diversity including but not limited to those that mediate homeostasis, cell cycle dynamics, cell viability, stress responses and epigenetic regulation

Long noncoding RNAs in neuronal-glial fate specification and oligodendrocyte lineage maturation

Background: Long non-protein-coding RNAs (ncRNAs) are emerging as important regulators of cellular differentiation and are widely expressed in the brain.Results: Here we show that many long ncRNAs exhibit dynamic expression patterns during neuronal and oligodendrocyte (OL) lineage specification, neuronal-glial fate transitions, and progressive stages of OL lineage elaboration including myelination. Consideration of the genomic context of these dynamically regulated ncRNAs showed they were part of complex transcriptional loci that encompass key neural developmental protein-coding genes, with which they exhibit concordant expression profiles as indicated by both microarray and in situ hybridization analyses. These included ncRNAs associated with differentiation-specific nuclear subdomains such as Gomafu and Neat1, and ncRNAs associated with developmental enhancers and genes encoding important transcription factors and homeotic proteins. We also observed changes in ncRNA expression profiles in response to treatment with trichostatin A, a histone deacetylase inhibitor that prevents the progression of OL progenitors into post-mitotic OLs by altering lineage-specific gene expression programs.Conclusion: This is the first report of long ncRNA expression in neuronal and glial cell differentiation and of the modulation of ncRNA expression by modification of chromatin architecture. These observations explicitly link ncRNA dynamics to neural stem cell fate decisions, specification and epigenetic reprogramming and may have important implications for understanding and treating neuropsychiatric diseases

Differential Deployment of REST and CoREST Promotes Glial Subtype Specification and Oligodendrocyte Lineage Maturation

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a master transcriptional regulator that binds to numerous genomic RE1 sites where it acts as a molecular scaffold for dynamic recruitment of modulatory and epigenetic cofactors, including corepressor for element-1-silencing transcription factor (CoREST). CoREST also acts as a hub for various cofactors that play important roles in epigenetic remodeling and transcriptional regulation. While REST can recruit CoREST to its macromolecular complex, CoREST complexes also function at genomic sites independently of REST. REST and CoREST perform a broad array of context-specific functions, which include repression of neuronal differentiation genes in neural stem cells (NSCs) and other non-neuronal cells as well as promotion of neurogenesis. Despite their involvement in multiple aspects of neuronal development, REST and CoREST are not believed to have any direct modulatory roles in glial cell maturation.We challenged this view by performing the first study of REST and CoREST in NSC-mediated glial lineage specification and differentiation. Utilizing ChIP on chip (ChIP-chip) assays, we identified distinct but overlapping developmental stage-specific profiles for REST and CoREST target genes during astrocyte (AS) and oligodendrocyte (OL) lineage specification and OL lineage maturation and myelination, including many genes not previously implicated in glial cell biology or linked to REST and CoREST regulation. Amongst these factors are those implicated in macroglial (AS and OL) cell identity, maturation, and maintenance, such as members of key developmental signaling pathways and combinatorial transcription factor codes.Our results imply that REST and CoREST modulate not only neuronal but also glial lineage elaboration. These factors may therefore mediate critical developmental processes including the coupling of neurogenesis and gliogenesis and neuronal-glial interactions that underlie synaptic and neural network plasticity and homeostasis in health and in specific neurological disease states

Selective Roles of Normal and Mutant Huntingtin in Neural Induction and Early Neurogenesis

<div><p>Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal polyglutamine expansion in the amino-terminal end of the huntingtin protein (Htt) and characterized by progressive striatal and cortical pathology. Previous reports have shown that Htt is essential for embryogenesis, and a recent study by our group revealed that the pathogenic form of Htt (mHtt) causes impairments in multiple stages of striatal development. In this study, we have examined whether HD-associated striatal developmental deficits are reflective of earlier maturational alterations occurring at the time of neurulation by assessing differential roles of Htt and mHtt during neural induction and early neurogenesis using an <i>in vitro</i> mouse embryonic stem cell (ESC) clonal assay system. We demonstrated that the loss of Htt in ESCs (KO ESCs) severely disrupts the specification of primitive and definitive neural stem cells (pNSCs, dNSCs, respectively) during the process of neural induction. In addition, clonally derived KO pNSCs and dNSCs displayed impaired proliferative potential, enhanced cell death and altered multi-lineage potential. Conversely, as observed in HD knock-in ESCs (Q111 ESCs), mHtt enhanced the number and size of pNSC clones, which exhibited enhanced proliferative potential and precocious neuronal differentiation. The transition from Q111 pNSCs to fibroblast growth factor 2 (FGF2)-responsive dNSCs was marked by potentiation in the number of dNSCs and altered proliferative potential. The multi-lineage potential of Q111 dNSCs was also enhanced with precocious neurogenesis and oligodendrocyte progenitor elaboration. The generation of Q111 epidermal growth factor (EGF)-responsive dNSCs was also compromised, whereas their multi-lineage potential was unaltered. These abnormalities in neural induction were associated with differential alterations in the expression profiles of <i>Notch</i>, <i>Hes1</i> and <i>Hes5</i>. These cumulative observations indicate that Htt is required for multiple stages of neural induction, whereas mHtt enhances this process and promotes precocious neurogenesis and oligodendrocyte progenitor cell elaboration.</p></div

Functions of Huntingtin in Germ Layer Specification and Organogenesis

<div><p>Huntington’s disease (HD) is a neurodegenerative disease caused by abnormal polyglutamine expansion in the huntingtin protein (Htt). Although both Htt and the HD pathogenic mutation (mHtt) are implicated in early developmental events, their individual involvement has not been adequately explored. In order to better define the developmental functions and pathological consequences of the normal and mutant proteins, respectively, we employed embryonic stem cell (ESC) expansion, differentiation and induction experiments using huntingtin knock-out (KO) and mutant huntingtin knock-in (Q111) mouse ESC lines. In KO ESCs, we observed impairments in the spontaneous specification and survival of ectodermal and mesodermal lineages during embryoid body formation and under inductive conditions using retinoic acid and Wnt3A, respectively. Ablation of BAX improves cell survival, but failed to correct defects in germ layer specification. In addition, we observed ensuing impairments in the specification and maturation of neural, hepatic, pancreatic and cardiomyocyte lineages. These developmental deficits occurred in concert with alterations in Notch, Hes1 and STAT3 signaling pathways. Moreover, in Q111 ESCs, we observed differential developmental stage-specific alterations in lineage specification and maturation. We also observed changes in Notch/STAT3 expression and activation. Our observations underscore essential roles of Htt in the specification of ectoderm, endoderm and mesoderm, in the specification of neural and non-neural organ-specific lineages, as well as cell survival during early embryogenesis. Remarkably, these developmental events are differentially deregulated by mHtt, raising the possibility that HD-associated early developmental impairments may contribute not only to region-specific neurodegeneration, but also to non-neural co-morbidities.</p> </div

Specification and survival of three germ layers requires Htt whereas mHtt differentially impairs these processes.

<p>(A) Representative images of different sizes of CTL and KO EBs at 10 DIV. (B, C) Quantification of the size and number of CTL and KO EBs at 4DIV, 6DIV, 8DIV and 10DIV. Error bars represent ±SEM; *p<0.0001. (D) Representative images of TUNEL assay in CTL and KO EB at 4 DIV. (E) Representative images of different sizes of Q18 and Q111 EBs at 10 DIV. (F, G) Quantification of the size and number of Q18 and Q111 EBs at 4DIV, 6DIV, 8DIV and 10DIV. Error bars represent ±SEM; *p<0.0001. (H) Representative images of TUNEL assay in Q18 and Q111 EBs at 4 DIV. (I, J) QPCR analysis of developmental markers representing the ectodermal (FGF5), endodermal (NODAL) and mesodermal (BRACHYURY) germ layers. Error bars represent ±95% CI; *p<0.0001. (K) Quantification of the size of CTL and KO EBs formed after knocking down BAX using lentiviral transfection with a double short hairpin RNA (shRNA). (L) QPCR analysis of germ layer developmental markers in CTL-shSCR, CTL-shBAX, KO-shSCR and KO-shBAX specimens. Error bars represent ±95% CI; *p<0.0001, #p<0.0001 unless otherwise noted. Scale bar = 20 µm (A, E); 200 µm (D, H).</p

mHtt impairs the spontaneous differentiation of ESCs in ways analogous to Htt ablation.

<p>(A, D) Immunofluorescence analysis of the ESC marker (SSEA1) and the pluripotency factors (Nanog, Oct4, Sox2) in undifferentiated ESC maintained with LIF. (B, E) Quantification of Nanog+, Oct4+, Sox2+ (n=1203, 995, 1138 and 608 for CTL, KO, Q18 and Q111, respectively) and Klf4+ (n=1228, 592, 1113 and 1112 for CTL, KO, Q18 and Q111, respectively) cells present in ESCs after 4 DIV following removal of LIF. (C, F) ESC cultures pulsed with BrdU for 4 hrs in media without LIF. The ESCs were then fixed at 1DIV, 2DIV, and 4DIV and quantification of BrdU+ cells was assessed at these three time points (n=1426, 1162, 1571 and 2033 for CTL, KO, Q18 and Q111, respectively). All error bars represent ±95% CI; *p-values < 0.0001 unless otherwise noted. Scale bar = 20 µm.</p

Htt is required for the maintenance of lineage potential of pNSCs, whereas mHtt promotes neurogenesis.

<p>(A, B) QPCR expression analysis of proneural genes in KO pNSs as compared to CTL pNSs, and in Q111 pNSs as compared to Q18 pNSs, respectively. (C, D) QPCR expression analysis of the endodermal gene, <i>GATA4</i>, and the mesodermal genes, <i>Brachyury</i> and <i>Hnf-4A</i>, in KO pNSs as compared to CTL pNSs, and in Q111 pNSs as compared to Q18 pNSs, respectively. (E, F) pNSs were cultured under differentiating conditions for 7DIV and analyzed by immunofluorescence microscopy for SSEA1, Nestin and Doublecortin (DCX), which are markers for ESCs, NSCs and neuronal precursor species respectively. Error bars represent ±95% CI; unless otherwise stated, *p-value<0.05. All scale bars = 25 μm.</p