The transcription factor SRY (sex-determining region)-box 2 (SOX2) is an important functional marker of neural precursor cells (NPCs) and plays a critical role in self-renewal and neuronal differentiation; however, the molecular mechanisms underlying its functions are poorly understood. Using human embryonic stem cellderived NPCs to model neurogenesis, we found that SOX2 is required to maintain optimal levels of LIN28, a well-characterized suppressor of let-7 microRNA biogenesis. Exogenous LIN28 expression rescued the NPC proliferation deficit, as well as the early but not the late stages of the neurogenic deficit associated with the loss of SOX2. We found that SOX2 binds to a proximal site in the LIN28 promoter region and regulates LIN28 promoter acetylation, likely through interactions with the histone acetyltransferase complex. Misexpression of let-7 microRNAs in NPCs reduced proliferation and inhibited neuronal differentiation, phenocopying the loss of SOX2. In particular, we identified let-7i as a novel and potent inhibitor of neuronal differentiation that targets MASH1 and NGN1, two well-characterized proneural genes. In conclusion, we discovered the SOX2-LIN28/let-7 pathway as a unique molecular mechanism governing NPC proliferation and neurogenic potential.neural stem cells | mechanisms of pluripotency S elf-renewing, multipotent neural precursor cells (NPCs) are capable of terminally differentiating into neuronal and glial lineages during development and in the adult nervous system (1, 2). Disruption of the pathways controlling NPC biology has been implicated in various pathologies, including autism (3), Treacher Collins syndrome (4), and neural tube defects (5), emphasizing the importance of gaining a better understanding of the underlying molecular events and how they may be manipulated to treat and prevent such pathologies. The HMG-box transcription factor SOX2 is ubiquitously expressed in NPCs and supports their self-renewal (6). SOX2 is also required for neurogenesis in the central nervous system (7-10). Recently, we used human embryonic stem cells (hESCs) and mouse models to demonstrate a critical requirement for SOX2 for sensory neurogenesis in dorsal root ganglia (11). However, the mechanisms by which SOX2 functions in self-renewal and neuronal differentiation remain poorly understood.Small, noncoding microRNAs (miRNAs) are transcribed as long precursors (pri-miRNAs) that are sequentially processed by the RNases Drosha/Pasha to form pri-miRNAs and by Dicer to form the mature miRNAs of ∼20-25 nucleotides. The miRNAs function through imperfect base-pairing with hundreds of target mRNAs to trigger their degradation (or block their translation) by the RNA-induced silencing complex, RISC (12). In some cases, miRNA maturation is tightly controlled; for example, the RNA-binding protein LIN28 regulates the biogenesis of the let-7 miRNA family by inhibiting their maturation at both the primiRNA (13, 14) and premiRNA (15, 16) processing steps. Intriguingly, LIN28 protein was found to be associated with SO...

A novel family of transcription factors responsible for regulation of various aspects of NAD synthesis in a broad range of bacteria was identified by comparative genomics approach. Regulators of this family (here termed NrtR for Nudix-related transcriptional regulators), currently annotated as ADP-ribose pyrophosphatases from the Nudix family, are composed of an N-terminal Nudix-like effector domain and a C-terminal DNA-binding HTH-like domain. NrtR regulons were reconstructed in diverse bacterial genomes by identification and comparative analysis of NrtR-binding sites upstream of genes involved in NAD biosynthetic pathways. The candidate NrtR-binding DNA motifs showed significant variability between microbial lineages, although the common consensus sequence could be traced for most of them. Bioinformatics predictions were experimentally validated by gel mobility shift assays for two NrtR family representatives. ADP-ribose, the product of glycohydrolytic cleavage of NAD, was found to suppress the in vitro binding of NrtR proteins to their DNA target sites. In addition to a major role in the direct regulation of NAD homeostasis, some members of NrtR family appear to have been recruited for the regulation of other metabolic pathways, including sugar pentoses utilization and biogenesis of phosphoribosyl pyrophosphate. This work and the accompanying study of NiaR regulon demonstrate significant variability of regulatory strategies for control of NAD metabolic pathway in bacteria.

Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts.

Summary The transcription factor SOX2 is widely known to play a critical role in the central nervous system; however, its role in peripheral neurogenesis remains poorly understood. We recently developed an hESC-based model in which migratory cells undergo epithelial to mesenchymal transition (EMT) to acquire properties of neural crest (NC) cells. In this model, we found that migratory NC progenitors downregulate SOX2, but then start re-expressing SOX2 as they differentiate to form neurogenic dorsal root ganglion (DRG)- like clusters. SOX2 downregulation was sufficient to induce EMT and resulted in massive apoptosis when neuronal differentiation was induced. In vivo, downregulation of SOX2 in chick and mouse NC cells significantly reduced the numbers of neurons within DRG. We found that SOX2 binds directly to NGN1 and MASH1 promoters and is required for their expression. Our data suggest that SOX2 plays a key role for NGN1-dependent acquisition of neuronal fates in sensory ganglia.

Newborn granule neurons generated from neural progenitor cells (NPCs) in the adult hippocampus play a key role in spatial learning and pattern separation. However, the molecular mechanisms that control activation of their neurogenic program remain poorly understood. Here, we report a novel function for the pluripotency factor sex-determining region Y (SRY)-related HMG box 2 (SOX2) in regulating the epigenetic landscape of poised genes activated at the onset of neuronal differentiation. We found that SOX2 binds to bivalently marked promoters of poised proneural genes [neurogenin 2 (Ngn2) and neurogenic differentiation 1 (NeuroD1)] and a subset of neurogenic genes [e.g., SRY-box 21 (Sox21), brain-derived neurotrophic factor (Bdnf), and growth arrest and DNA-damage-inducible, beta (Gadd45b)] where it functions to maintain the bivalent chromatin state by preventing excessive polycomb repressive complex 2 activity. Conditional ablation of SOX2 in adult hippocampal NPCs impaired the activation of proneural and neurogenic genes, resulting in increased neuroblast death and functionally aberrant newborn neurons. We propose that SOX2 sets a permissive epigenetic state in NPCs, thus enabling proper activation of the neuronal differentiation program under neurogenic cue.SOX2 | neurogenesis | epigenetics C ell fate and differentiation decisions of adult neural progenitor cells (NPCs) are controlled by intrinsic and extrinsic signals from the neurogenic niche (1-3). Recent genomewide analyses of epigenetic regulators in the brain have provided considerable insight into the mechanisms that regulate neural development, neurological disease, and aging (4, 5). The chromatin states of NPCs change dynamically during cell-fate determination and cell differentiation, and chromatin marks such as histone H3 trimethylated Lys 27 (H3K27me3), histone H3 trimethylated Lys 4 (H3K4me3), and histone H3 acetylated Lys 9 (H3K9ac) are essential for regulating the expression of key genes involved in these processes (6). Sex-determining region Y (SRY)-related HMG box 2 (SOX2) is a member of the SOXB1 family of transcription factors, which play important roles in maintaining neural stem/progenitor cell properties, including their capacity to proliferate and self-renew (7,8). In humans, SOX2 mutations are associated with anophthalmia, defective hippocampal development, and seizures (9-11). Most patients with this syndrome experience intellectual disabilities (11), suggesting that loss of SOX2 function affects areas of the brain involved in cognition (e.g., hippocampus). SOX2 deficiency also causes neurodegeneration and impaired neurogenesis in the adult mouse brain (12, 13). However, the molecular mechanisms underlying the function of SOX2 in adult neurogenesis and its role in the human SOX2 anophthalmia syndrome are largely unknown.The subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus is a germinal zone of active neurogenesis during adulthood (14). Indeed, approximately one-third of DG neurons lost during this period are replaced ...

BackgroundNeural crest stem cells (NCSCs) are a transient multipotent embryonic cell population that represents a defining characteristic of vertebrates. The neural crest (NC) gives rise to many derivatives including the neurons and glia of the sensory and autonomic ganglia of the peripheral nervous system, enteric neurons and glia, melanocytes, and the cartilaginous, bony and connective tissue of the craniofacial skeleton, cephalic neuroendocrine organs, and some heart vessels.Methodology/Principal FindingsWe present evidence that neural crest (NC) competence can be acquired very early when human embryonic stem cells (hESCs) are selectively neuralized towards dorsal neuroepithelium in the absence of feeder cells in fully defined conditions. When hESC-derived neurospheres are plated on fibronectin, some cells emigrate onto the substrate. These early migratory Neural Crest Stem Cells (emNCSCs) uniformly upregulate Sox10 and vimentin, downregulate N-cadherin, and remodel F-actin, consistent with a transition from neuroepithelium to a mesenchymal NC cell. Over 13% of emNCSCs upregulate CD73, a marker of mesenchymal lineage characteristic of cephalic NC and connexin 43, found on early migratory NC cells. We demonstrated that emNCSCs give rise in vitro to all NC lineages, are multipotent on clonal level, and appropriately respond to developmental factors. We suggest that human emNCSC resemble cephalic NC described in model organisms. Ex vivo emNCSCs can differentiate into neurons in Ret.k- mouse embryonic gut tissue cultures and transplanted emNCSCs incorporate into NC-derived structures but not CNS tissues in chick embryos.Conclusions/SignificanceThese findings will provide a framework for further studying early human NC development including the epithelial to mesenchymal transition during NC delamination.

2‐Amino 3‐carboxymuconate 6‐semialdehyde decarboxylase (ACMSD, EC 4.1.1.45) plays a key role in tryptophan catabolism. By diverting 2‐amino 3‐carboxymuconate semialdehyde from quinolinate production, the enzyme regulates NAD biosynthesis from the amino acid, directly affecting quinolinate and picolinate formation. ACMSD is therefore an attractive therapeutic target for treating disorders associated with increased levels of tryptophan metabolites. Through an isoform‐specific real‐time PCR assay, the constitutive expression of two alternatively spliced ACMSD transcripts (ACMSD I and II) has been examined in human brain, liver and kidney. Both transcripts are present in kidney and liver, with highest expression occurring in kidney. In brain, no ACMSD II expression is detected, and ACMSD I is present at very low levels. Cloning of the two cDNAs in yeast expression vectors and production of the recombinant proteins, revealed that only ACMSD I is endowed with enzymatic activity. After purification to homogeneity, this enzyme was found to be a monomer, with a broad pH optimum ranging from 6.5 to 8.0, a Km of 6.5 µm, and a kcat of 1.0 s−1. ACMSD I is inhibited by quinolinic acid, picolinic acid and kynurenic acid, and it is activated slightly by Fe2+ and Co2+. Site‐directed mutagenesis experiments confirmed the catalytic role of residues, conserved in all ACMSDs so far characterized, which in the bacterial enzyme participate directly in the metallocofactor binding. Even so, the properties of the human enzyme differ significantly from those reported for the bacterial counterpart, suggesting that the metallocofactor is buried deep within the protein and not as accessible as it is in bacterial ACMSD.

Dermal Papillae (DP) is a unique population of mesenchymal cells that was shown to regulate hair follicle formation and growth cycle. During development most DP cells are derived from mesoderm, however, functionally equivalent DP cells of cephalic hairs originate from Neural Crest (NC). Here we directed human embryonic stem cells (hESCs) to generate first NC cells and then hair-inducing DP-like cells in culture. We showed that hESC-derived DP-like cells (hESC-DPs) express markers typically found in adult human DP cells (e.g. p-75, nestin, versican, SMA, alkaline phosphatase) and are able to induce hair follicle formation when transplanted under the skin of immunodeficient NUDE mice. Engineered to express GFP, hESC-derived DP-like cells incorporate into DP of newly formed hair follicles and express appropriate markers. We demonstrated that BMP signaling is critical for hESC-DP derivation since BMP inhibitor dorsomorphin completely eliminated hair-inducing activity from hESC-DP cultures. DP cells were proposed as the cell-based treatment for hair loss diseases. Unfortunately human DP cells are not suitable for this purpose because they cannot be obtained in necessary amounts and rapidly loose their ability to induce hair follicle formation when cultured. In this context derivation of functional hESC-DP cells capable of inducing a robust hair growth for the first time shown here can become an important finding for the biomedical science.