Necdin Gene Research Articles

Identification and characterization of Necdin as a target for the Cockayne syndrome B protein in promoting neuronal differentiation and maintenance

Cockayne syndrome (CS) is a devastating autosomal recessive genetic disorder, mainly characterized by photosensitivity, growth failure, neurological abnormalities, and premature aging. Mutations in CSB (ERCC6) are associated with almost all clinical phenotypes resembling classic CS. Using RNA-seq approach in multiple cell types, we identified Necdin (NDN) as a target of the CSB protein. Supportive of the RNA-seq results, CSB directly binds to NDN and manipulates the remodeling of active histone marks and DNA 5mC methylation on the regulatory elements of the NDN gene. Intriguingly, hyperactivation of NDN due to CSB deficiency does not interfere with nucleotide excision repair (1), but greatly affects neuronal cell differentiation. Inhibition of NDN can partially rescue the motor neuron defects in CSB mouse models. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for intervention, these data substantiate a reciprocal communication between CSB and NDN in the context of general transcription regulation.

Necdin, one of the important pathway proteins in the regulation of osteosarcoma progression by microRNA-200c

ABSTRACT MicroRNA-200c (miR-200c) generally acts as a tumor suppressor in multiple cancer types and a promising therapeutic target in tumorigenesis. However, only a few studies have explained the role of miR-200c in the development of osteosarcoma (OS). In this study, we investigated the role of miR-200c in OS progression and identified the regulatory pathway protein NDN involved in inhibiting the occurrence and development of OS. Firstly, we found that miR-200c is downregulated in OS cells and tissues. As well, in vitro and in vivo experiments showed that upregulating miR-200c inhibits the proliferation, invasion, metastasis of Saos-2 cells, promotes the apoptosis of Saos-2 cells and suppresses tumor growth in mice, indicating miR-200c plays a major role in regulating the OS progression. Furthermore, bioinformatics analysis showed that an anti-tumor protein, necdin (NDN), might be a potential target by miR-200c. To verify this hypothesis, we measured the expression level of NDN in OS cells and tissues and found NDN is downregulated, suggesting NDN is functional in OS progression. Moreover, we found that the expression levels of NDN and miR-200c in in vivo and in vitro experiments were positively correlated. However, the results of dual-luciferase reporter gene experiment showed miR-200c does not directly act on the 3ʹ untranslated region (UTR) of NDN gene, indicating that NDN might be an important pathway protein which regulates OS progression in the presence of miR-200c. Therefore, miR-200c/NDN could be potential targets for developing effective treatment against OS.

Necdin: A purposive integrator of molecular interaction networks for mammalian neuron vitality

Necdin was originally found in 1991 as a hypothetical protein encoded by a neural differentiation‐specific gene transcript in murine embryonal carcinoma cells. Virtually all postmitotic neurons and their precursor cells express the necdin gene (Ndn) during neuronal development. Necdin mRNA is expressed only from the paternal allele through genomic imprinting, a placental mammal‐specific epigenetic mechanism. Necdin and its homologous MAGE (melanoma antigen) family, which have evolved presumedly from a subcomplex component of the SMC5/6 complex, are expressed exclusively in placental mammals. Paternal Ndn‐mutated mice totally lack necdin expression and exhibit various types of neuronal abnormalities throughout the nervous system. Ndn‐null neurons are vulnerable to detrimental stresses such as DNA damage. Necdin also suppresses both proliferation and apoptosis of neural stem/progenitor cells. Functional analyses using Ndn‐manipulated cells reveal that necdin consistently exerts antimitotic, anti‐apoptotic and prosurvival effects. Necdin interacts directly with a number of regulatory proteins including E2F1, p53, neurotrophin receptors, Sirt1 and PGC‐1α, which serve as major hubs of protein–protein interaction networks for mitosis, apoptosis, differentiation, neuroprotection and energy homeostasis. This review focuses on necdin as a pleiotropic protein that integrates molecular interaction networks to promote neuronal vitality in modern placental mammals.

Differences in Placental Imprinted Gene Expression across Preeclamptic and Non-Preeclamptic Pregnancies.

Preeclampsia is a multi-systemic syndrome that presents in approximately 5% of pregnancies worldwide and is associated with a range of subsequent postpartum and postnatal outcomes, including fetal growth restriction. As the placenta plays a critical role in the development of preeclampsia, surveying genomic features of the placenta, including expression of imprinted genes, may reveal molecular markers that can further refine subtypes to aid targeted disease management. In this study, we conducted a comprehensive survey of placental imprinted gene expression across early and late onset preeclampsia cases and preterm and term normotensive controls. Placentas were collected at delivery from women recruited at the Magee-Womens Hospital prenatal clinics, and expression levels were profiled across 109 imprinted genes. We observed downregulation of placental Mesoderm-specific transcript (MEST) and Necdin (NDN) gene expression levels (false discovery rate (FDR) < 0.05) among early onset preeclampsia cases compared to preterm controls. No differences in placental imprinted gene expression were observed between late onset preeclampsia cases and term controls. While few studies have linked NDN to pregnancy complications, reductions in MEST expression levels, as observed in our study, are consistently reported in the literature in relation to various pregnancy complications, including fetal growth restriction, suggesting a potential role for placental MEST expression as a biosensor of an adverse in utero environment.

The mouse Necdin gene is expressed from the paternal allele only and lies in the 7C region of the mouse chromosome 7, a region of conserved synteny to the human Prader-Willi syndrome region.

Prader-Willi syndrome (PWS) is a neurogenetic disorder resulting from the loss of paternal expression of gene(s) localized in the 15q11-q12 region. A new human gene encoding a putative protein with high homology to the mouse NECDIN protein has recently been characterized and mapped to chromosome 15q11-q12. It is expressed from the paternal allele only, suggesting its potential involvement in PWS. We now report the localization of the mouse Necdin gene in a region of conserved synteny to the human PWS region. We demonstrate the paternal specific expression of Necdin in the mouse central nervous system, and show that parental alleles display a differential methylation profile in the coding region. Finally, fluorescence in situ hybridization analysis reveals an asynchronous pattern of replication at the Necdin locus. These results clearly demonstrate imprinting of the mouse Necdin gene. Mouse models will be powerful tools in the study of human PWS phenotype and imprinting mechanisms.

Sequence variation of necdin gene in Bovidae

BackgroundNecdin (NDN), a member of the melanoma antigen family showing imprinted pattern of expression, has been implicated as causing Prader-Willi symptoms, and known to participate in cellular growth, cellular migration and differentiation. The region where NDN is located has been associated to QTLs affecting reproduction and early growth in cattle, but location and functional analysis of the molecular mechanisms have not been established.MethodsHere we report the sequence variation of the entire coding sequence from 72 samples of cattle, yak, buffalo, goat and sheep, and discuss its variation in Bovidae. Median-joining network analysis was used to analyze the variation found in the species. Synonymous and non-synonymous substitution rates were determined for the analysis of all the polymorphic sites. Phylogenetic analysis were carried out among the species of Bovidae to reconstruct their relationships.ResultsFrom the phylogenetic analysis with the consensus sequences of the studied Bovidae species, we found that only 11 of the 26 nucleotide changes that differentiate them produced amino acid changes. All the SNPs found in the cattle breeds were novel and showed similar percentages of nucleotides with non-synonymous substitutions at the N-terminal, MHD and C-terminal (12.3, 12.8 and 12.5%, respectively), and were much higher than the percentage of synonymous substitutions (2.5, 2.6 and 4.9%, respectively). Three mutations in cattle and one in sheep, detected in heterozygous individuals were predicted to be deleterious. Additionally, the analysis of the biochemical characteristics in the most common form of the proteins in each species show very little difference in molecular weight, pI, net charge, instability index, aliphatic index and GRAVY (Table 4) in the Bovidae species, except for sheep, which had a higher molecular weight, instability index and GRAVY.ConclusionsThere is sufficient variation in this gene within and among the studied species, and because NDN carry key functions in the organism, it can have effects in economically important traits in the production of these species. NDN sequence is phylogenetically informative in this group, thus we propose this gene as a phylogenetic marker to study the evolution and conservation in Bovidae.

Transgenic mice with a tandem duplication of the Necdin gene overexpress Necdin.

Necdin (Ndn) transgenic (Tg) mice were generated with a bacterial artificial chromosome (BAC) clone. Droplet digital PCR (ddPCR) and inverse PCR methods revealed that the transgene consisted of four fragments with a total length of 171kb. Two of these fragments were tandem tail-to-tail duplicates of 77kb and 37kb that both contained a Ndn gene. The transgene was inserted in chromosome 15qD1. Ndn is a paternally expressed imprinted gene; however, the total expression level of Ndn in hemizygous Tg mice was approximately twofold higher than that in wild-type mice. ddPCR assays with locked nucleic acid (LNA) TaqMan probes revealed that transgenic Ndn expression was almost equal to endogenous Ndn expression, despite there being two copies of the Ndn gene in the transgene, indicating an interaction between the transcriptional regulation of endogenous Ndn and the transgene. ddPCR assays with LNA TaqMan probes were also applied for imprinting analysis to confirm exclusive paternal expression in tissues with low Ndn expression. This is the first report of a Tg mouse with a tandem duplication of a Ndn transgene and Ndn overexpression, which will be useful for the in vivo study of Ndn overexpression and for rescue experiments of the neonatal lethality seen in the Ndn knockout mouse.

Necdin, a p53 target gene, in stem cells

Necdin, a member of the melanoma antigen gene (MAGE) family, originally identified to suppress cell proliferation in post-mitotic neurons, facilitates the entry of the cell into cell cycle, functions like retinoblastoma (Rb) protein, and acts as a transcriptional repressor that recognizes guanosine (G)-rich DNA sequences. Necdin also binds to p53 and inhibits p53-dependent apoptosis in these cells, demonstrating its function as an endogenous anti-mitotic and anti-apoptotic protein in post-mitotic neurons [1]. The necdin gene is located on chromosome 15 in human and its expression is controlled through genomic imprinting; it is one of the genes that have been associated with the human Prader-Willi syndrome, a genetic neurobehavioral disorder [2]. Necdin is expressed in some stem or undifferentiated progenitor cells such as mesoangioblast stem cells [3], vessel-associated stem cells [4], adipocyte progenitor cells [5, 6], and hematopoietic stem cells (HSCs) [7], yet the function of necdin in these cells remains unclear. While highly expressed in long-term HSCs, transcript profiling of hematopoietic stem/progenitor cells (HSPCs) isolated from wild-type and p53 null mice identified necdin as being a p53 target gene [7]. We then demonstrated using chromatin immunoprecipitation assays that the necdin promoter contains a p53 binding site and that necdin is a direct transcriptional activator of p53 in HSPCs. Lentivirus-mediated knock down or overexpression of necdin resulted in decreased or increased HSPC quiescence, respectively [7]. We recently clarified the function of necdin in HSCs and HSPCs using necdin null mice [8]. Necdin null mice show the same phenotype as the patients with Prader-Willi syndrome and because they die perinatally, we transplanted necdin null fetal liver cells into lethally irradiated wild type recipients to reconstitute their bone marrow function and analyzed the HSC intrinsic function of necdin in adult hematopoiesis. Necdin-null HSCs have normal self-renewal capacity after serial transplantation, while we found that necdin-null HSCs are less quiescent and more proliferative than normal, suggesting that necdin has the similar function as p53 in promoting HSC quiescence in the steady state. We then demonstrated that necdin-null HSCs are highly sensitive to genotoxic stress, irradiation or chemotherapy, with increased apoptosis via both cell-cycle-dependent and cell-cycle independent mechanisms. We further found that the function of necdin in hematopoiesis in response to genotoxic stress depends on Gas2L3, a novel member of the Gas2 family, using

Necdin Controls Foxo1 Acetylation in Hypothalamic Arcuate Neurons to Modulate the Thyroid Axis

The forkhead transcription factor Foxo1 regulates energy homeostasis by modulating gene expression in the hypothalamus. Foxo1 undergoes post-translational modifications such as phosphorylation and acetylation, which modulate its functional activities. Sirtuin1 (Sirt1), a nicotinamide adenine dinucleotide-dependent protein deacetylase, regulates the acetylation status of Foxo1 in mammalian cells. Necdin, a pleiotropic protein required for neuronal development and survival, interacts with both Sirt1 and p53 to facilitate p53 deacetylation. The necdin gene (Ndn), an imprinted gene transcribed only from the paternal allele, is strongly expressed in hypothalamic neurons. Here, we demonstrate that necdin controls the acetylation status of Foxo1 in vivo in hypothalamic arcuate neurons to modulate the thyroid function. Necdin forms a stable ternary complex with Sirt1 and Foxo1, diminishes Foxo1 acetylation, and suppresses the transcriptional activity of Foxo1 in vitro. Paternal Ndn mutant mice express high levels of acetylated Foxo1 and mRNAs encoding agouti-related protein and neuropeptide Y in the hypothalamus in vivo during the juvenile period. The mutant mice exhibit endocrine dysfunction characteristic of hypothalamic hypothyroidism. Chemically induced hyperthyroidism and hypothyroidism lead to hypothalamic responses similar to those under necdin-deficient and excessive conditions, respectively, suggesting that thyroid hormone serves as a negative regulator of this system. These results suggest that necdin regulates Foxo1 acetylation and neuropeptide gene expression in the arcuate neurons to modulate the hypothalamic-pituitary-thyroid axis during development.

Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism

Necdin activates GNRH gene expression and is fundamental for the development, migration, and axonal extension of murine GNRH neurons. In humans, necdin plays a potential role in the hypogonadotropic hypogonadism phenotype in patients with Prader-Willi syndrome. To investigate necdin gene (NDN) variants in patients with isolated hypogonadotropic hypogonadism (IHH). We studied 160 Brazilian patients with IHH, which includes 92 with Kallmann syndrome and 68 with normosmic IHH. Genomic DNA was extracted and the single NDN exon was amplified and sequenced. To measure GNRH transcriptional activity, luciferase reporter plasmids containing GNRH regulatory regions were transiently transfected into GT1-7 cells in the presence and absence of overexpressed wild-type or mutant necdin. A heterozygous variant of necdin, p.V318A, was identified in a 23-year-old male with Kallmann syndrome. The p.V318A was also present in affected aunt and his father and was absent in 100 Brazilian control subjects. Previous FGFR1 gene analysis revealed a missense mutation (p.P366L) in this family. Functional studies revealed a minor difference in the activation of GNRH transcription by mutant protein compared with wild type in that a significant impairment of the necdin protein activity threshold was observed. A rare variant of necdin (p.V318A) was described in a family with Kallmann syndrome associated with a FGFR1 mutation. Familial segregation and in vitro analysis suggested that this non-synonymous variant did not have a direct causative role in the hypogonadism phenotype. NDN mutations are not a frequent cause of congenital IHH.

The Necdin-Wnt Pathway Causes Epigenetic Peroxisome Proliferator-activated Receptor γ Repression in Hepatic Stellate Cells

Hepatic stellate cells (HSCs), vitamin A-storing liver pericytes, undergo myofibroblastic trans-differentiation or "activation" to participate in liver wound healing. This cellular process involves loss of regulation by adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ). Necdin, a melanoma antigen family protein, promotes neuronal and myogenic differentiation while inhibiting adipogenesis. The present study demonstrates that necdin is selectively expressed in HSCs among different liver cell types and induced during their activation in vitro and in vivo. Silencing of necdin with adenovirally expressed shRNA, reverses activated HSCs to quiescent cells in a manner dependent on PPARγ and suppressed canonical Wnt signaling. Promoter analysis, site-directed mutagenesis, and chromatin immunoprecipitation demonstrate that Wnt10b, a canonical Wnt induced in activated HSCs, is a direct target of necdin. Necdin silencing abrogates three epigenetic signatures implicated in repression of PPARγ: increased MeCP2 (methyl CpG binding protein 2) and HP-1α co-repressor recruitments to Pparγ promoter and enhanced H3K27 dimethylation at the exon 5 locus, again in a manner dependent on suppressed canonical Wnt. These epigenetic effects are reproduced by antagonism of canonical Wnt signaling with Dikkopf-1. Our results demonstrate a novel necdin-Wnt pathway, which serves to mediate antiadipogenic HSC trans-differentiation via epigenetic repression of PPARγ.

Necdin Promotes Tangential Migration of Neocortical Interneurons from Basal Forebrain

Necdin is a pleiotropic protein that promotes neuronal differentiation and survival. In mammals, the necdin gene on the maternal chromosome is silenced by genomic imprinting, and only the paternal necdin gene is expressed in virtually all postmitotic neurons. Necdin forms a complex with the homeodomain protein Dlx2 to enhance its transcriptional activity. Dlx2 plays a major role in controlling tangential migration of GABAergic interneurons from the basal forebrain to the neocortex. Here, we examined whether Dlx2-expressing interneurons migrate properly in vivo in mutant mice lacking the paternal necdin gene. In necdin-deficient mice at birth, the population of Dlx2-expressing cells significantly decreased in the neocortex but increased in the preoptic area. DiI-labeled cell migration assay using organotypic forebrain slice cultures revealed that the number of cells migrating from the medial ganglionic eminence into the neocortex was significantly reduced in necdin-deficient embryos. Furthermore, necdin-deficient mice had a decreased population of neocortical GABA-containing neurons and were highly susceptible to pentylenetetrazole-induced seizures. These results suggest that necdin promotes tangential migration of neocortical GABAergic interneurons during mammalian forebrain development.

Epigenetic regulation of necdin gene expression during neural development

Prader-Willi syndrome (PWS) is a rare, multifaceted genetic disorder resulting from the absence of normally active paternally expressed genes from the 15q11-q13 chromosome region. Due to a lack of anthropometric and intellectual data in Taiwan, we attempted these evaluations. Twenty patients (14 males/6 females) aged 7–23 years with molecularly confirmed PWS were enrolled with parental consent. Their mean height standard deviation score (SDS) was −1.26 ± 1.89 (from −4.3 to +2.16); mean weight SDS was +1.77 ± 2.00 (from −0.44 to +5.89); mean body mass index SDS was +3.84 ± 10.54 (from −0.08 to +10.48); and mean body fat tissue SDS was 39.4 ± 10.54% (14.7-57.8%) by an InBody 3.0 analyzer. All were hypogonadal. Nine of them had once been given growth hormone therapy, and were taller and slimmer than the rest. Their in telligence tests showed full intelligence quotient = 52.0 ± 7.6; verbal intelligence quotient = 55.9 ± 8.77; performance intelligence quotient = 53.2 ± 9.0. Chronic health status revealed that diabetes was prevalent among the older population. Their IQ was in the range of those with moderate retardation.

Necdin Regulates p53 Acetylation via Sirtuin1 to Modulate DNA Damage Response in Cortical Neurons

Sirtuin1 (Sirt1), a mammalian homolog of yeast Sir2, deacetylates the tumor suppressor protein p53 and attenuates p53-mediated cell death. Necdin, a p53-interacting protein expressed predominantly in postmitotic neurons, is a melanoma antigen family protein that promotes neuronal differentiation and survival. In mammals, the necdin gene (Ndn) is maternally imprinted, and mutant mice carrying mutated paternal Ndn show abnormalities of neuronal development. Here we report that necdin regulates the acetylation status of p53 via Sirt1 to suppress p53-dependent apoptosis in postmitotic neurons. Double-immunostaining analysis demonstrated that necdin colocalizes with Sirt1 in postmitotic neurons of mouse embryonic forebrain in vivo. Coimmunoprecipitation and in vitro binding analyses revealed that necdin interacts with both p53 and Sirt1 to potentiate Sirt1-mediated p53 deacetylation by facilitating their association. Primary cortical neurons prepared from paternal Ndn-deficient mice have high p53 acetylation levels and are sensitive to the DNA-damaging compounds camptothecin and hydrogen peroxide. Moreover, DNA transfection per se increases p53 acetylation and apoptosis in paternal Ndn-deficient neurons, whereas small interfering RNA-mediated p53 knockdown completely blocks these changes. However, Sirt1 knockdown increases both acetylated p53 level and apoptosis in wild-type neurons but fails to affect them in paternal Ndn-deficient neurons. In organotypic forebrain slice cultures treated with hydrogen peroxide, p53 is accumulated and colocalized with necdin and Sirt1 in cortical neurons. These results suggest that necdin downregulates p53 acetylation levels by forming a stable complex with p53 and Sirt1 to protect neurons from DNA damage-induced apoptosis.

Sensory defects in Necdin deficient mice result from a loss of sensory neurons correlated within an increase of developmental programmed cell death

BackgroundThe human NECDIN gene is involved in a neurodevelopmental disorder, Prader-Willi syndrome (PWS). Previously we reported a mouse Necdin knock-out model with similar defects to PWS patients. Despite the putative roles attributed to Necdin, mainly from in vitro studies, its in vivo function remains unclear. In this study, we investigate sensory-motor behaviour in Necdin deficient mice. We reveal cellular defects and analyse their cause.ResultsWe report sensory differences in Necdin deficient mice compared to wild type animals. These differences led us to investigate sensory neuron development in Necdin deficient mouse embryos. First, we describe the expression pattern of Necdin in developing DRGs and report a reduction of one-third in specified sensory neurons in dorsal roots ganglia and show that this neuronal loss is achieved by E13.5, when DRGs sensory neurons are specified. In parallel, we observed an increase of 41% in neuronal apoptosis during the wave of naturally occurring cell death at E12.5. Since it is assumed that Necdin is a P75NTR interactor, we looked at the P75NTR-expressing cell population in Necdin knock-out embryos. Unexpectedly, Necdin loss of function has no effect on p75NTR expressing neurons suggesting no direct genetic interaction between Necdin and P75NTR in this context.Although we exclude a role of Necdin in axonal outgrowth from spinal sensory neurons in early developmental stages; such a role could occur later in neuronal differentiation. Finally we also exclude an anti-proliferative role of Necdin in developing sensory neurons.ConclusionOverall, our data show clearly that, in early development of the nervous system, Necdin is an anti-apoptotic or survival factor.

Differences in the Pattern of Histone H3 (K4) and (K27) Methylation throughout the β-Globin Gene Locus in Baboon Fetal Liver and Adult Bone Marrow Erythroblasts Pre- and Post-Decitabine Treatment.

Understanding the role of chromatin structure in specifying the pattern of β-like globin gene expression during development would be important in the design of future pharmacologic therapies to increase fetal hemoglobin in patients with sickle cell disease and β-thalassemia. The baboon is an important experimental animal model to study the regulation of globin gene expression because the structure of the β-globin gene complex and developmental pattern of globin gene expression are similar to man, and HbF levels are greatly increased in baboons treated with the DNA methyltransferase inhibitor decitabine (5-aza-2′-deoxycytidine). To investigate the relationship between chromatin structure, DNA methylation, and globin gene regulation, the distribution of acetyl histone H3 (ac-H3), acetyl histone H4 (ac-H4), histone H3 (K4) dimethyl and trimethyl, and histone H3 (K27) dimethyl throughout the β-globin gene locus was determined in purified primary erythroblasts from baboon fetal liver (FL), and adult bone marrow (BM) pre- and post-decitabine treatment. Analysis was performed by chromatin immunoprecipitation (ChIP) of formaldehyde-fixed chromatin followed by real time PCR using 18 primer sets spanning the baboon β-globin gene locus from the 5′ region of the ε-globin gene to the β-globin gene. Comparison of the pattern of ac-H3 and ac-H4 suggested the presence of three subdomains of chromatin within the β-globin locus characterized by different levels of histone acetylation that exhibited a differential response to decitabine treatment. Histone H3 (K4) dimethyl was relatively enriched in the region containing the ε- and γ-globin genes and in the γ-β intergenic region 5′ to the duplicated Alu sequence in FL. Levels associated with the ε-, γ-, and γ-globin genes in adult BM were similar and relatively unaffected by decitabine treatment. In contrast, high levels of histone H3 (K4) trimethylation and pol II distribution were associated with the promoters and transcribed regions of active genes. Differences in the levels of H3 (K4) trimethylation and pol II associated with individual genes were well correlated with differences in their relative levels of expression in FL and adult BM pre- and post-decitabine treatment. The level of histone H3 (K4) trimethyl associated with the promoter of the developmentally inactive ε-globin gene was very low and not enriched compared to inactive necdin gene or the γ-β intergenic regon in adult BM suggesting that the ε-globin gene is not maintained in a “poised” transcriptional state by the presence of the histone H3 (K4) trimethyl mark near the ε-globin promoter. The pattern of histone H3 (K27) dimethyl differed in FL and adult BM. Levels of H3 (K27) dimethyl associated with the ε- and γ-globin genes in FL were 2–4 fold less than near the duplicated Alu sequence in the γ-β intergenic region, while levels were 4–10 fold higher near the ε- and γ-globin genes and γ-β intergenic region compared to the promoter and transcribed region of the β-globin gene in adult BM. Reactivation of γ-globin expression following decitabine treatment was associated with a relative decrease in the level of H3 (K27) dimethyl near the γ-globin gene. Increased H3 (K27) methylation in regions surrounding the silenced ε- and γ-globin genes suggests that the polycomb group (PcG) protein EZH2, a histone H3 (K27) methyltransferase, may be involved in globin gene silencing.

Specificity Protein 1 (Sp1) Consensus Binding Sequences Flank Many Functional GATA-1 Binding Sites and Are Sufficient To Allow GATA-1 Binding to a WGATAR Sequence in Nuclear Chromatin.

GATA-1 is an hematopoietic transcription factor known for its roles in erythropoiesis and erythroid gene expression. In vitro, GATA-1 readily binds to WGATAR and closely related sequences. However, in vivo, GATA-1 appears to bind only a small subset of potential sites - those associated with gene regulatory elements. It is currently unclear what allows stable binding of GATA-1 at these sites in nuclear chromatin. We hypothesized that the binding of factors adjacent to WGATAR sites may “open” the local chromatin region to allow GATA-1 access to binding sites, while other factors may stabilize GATA-1 binding. To begin to test this model, we examined 11 well-characterized regulatory elements which are known to bind GATA-1 to determine whether there were any common associated binding sites. To do this we used the Transcription Element Search Software (TESS). These sites were from the human β-globin LCR HSs 2–4, human and mouse β-globin promoters, human γ-globin, NF-E2, EKLF and SCl/TALI (proximal and distal) promoters and the chicken 3′ β-globin enhancer. This analysis revealed that in each case, Sp1 consensus binding sites were found flanking the WGATAR elements. While this association between Sp1 and GATA binding motifs has been noted before, the reason for this association has not been determined. On average, the 5′ Sp1 motifs were located 18 +/− 8 bp upstream of the WGATAR elements, and the 3′ Sp1 motifs were located 30 +/− 17 bp downstream. To determine whether these flanking Sp1 elements were sufficient to allow GATA-1 binding in chromatin, we developed a new approach based on the Bouhassira lab's recombinase mediated cassette exchange (RMCE) system. This system allows us to design oligonucleotides up to ~120 bp in length containing clusters of test binding sites and then insert them as single copies into a defined genomic location in MEL cells. ChIP experiments, analyzed by quantitative RT-PCR, can then be used to characterize factor binding and local histone modification. The positive internal control for GATA-1 binding and histone H3 acetylation was the −2.8G region of the mGATA-1 promoter. The negative control was the murine necdin gene promoter. As an additional positive control, we inserted the 100 bp LCR HS4 core element and found, as expected, that it was able to mediate GATA-1 binding. We then inserted 3 different GATA-1 binding elements and two derivative sites which have been shown to bind GATA-1 with high affinity in vitro. None of these sites alone was able to bind GATA-1 in our test system. We next placed one of the high affinity sites, a GATA-PAL motif from the murine gata-1 gene promoter, between two exact Sp1 consensus sequences (GGGGCGGGG) located 18 bp upstream and 30 bp downstream of the WGATAR element. When tested in our system, the flanking Sp1 elements were sufficient to allow GATA-1 binding to the test site at levels equivalent to those seen for the positive control site. As expected, no GATA-1 bound when the Sp1 sites were used alone, but H3 histones in the region did become hyperacetylated, indicating that factors interacting with the Sp1 direct localized histone acetylation and mediate GATA-1 binding. These results begin to provide a mechanism for GATA-1 targeting to sites in nuclear chromatin and provide an explanation for the observed association of WGATAR and Sp1 binding elements within erythroid gene regulatory elements.

Expression of Drosophila MAGE gene encoding a necdin homologous protein in postembryonic neurogenesis

The MAGE (melanoma antigen) family is characterized by a large conserved domain termed MAGE homology domain. Originally identified MAGE genes encoding tumor rejection antigens are expressed only in cancers and male germ cells. Necdin, which contains the MAGE homology domain, is highly expressed in postmitotic cells such as neurons and skeletal muscle cells. The human necdin gene NDN is transcribed only from the paternal allele through genomic imprinting, and its deficiency is implicated in the pathogenesis of the neurodevelopmental disorder Prader–Willi syndrome. Although over 30 MAGE genes have been identified in humans, fruit fly ( Drosophila melanogaster) has only a single MAGE gene that encodes a protein similar to necdin homologous MAGE proteins. In this study, we analyzed the spatiotemporal expression patterns of MAGE mRNA and the encoded protein during fly development. Whole-mount embryo in situ hybridization analysis revealed that MAGE mRNA was highly expressed at the syncytial blastoderm stage and in the ventral and procephalic neurogenic regions of the ectoderm during gastrulation. In contrast, MAGE expression was nearly undetectable in postmitotic neurons of the central nervous system at late embryonic stages. During postembryonic neurogenesis, MAGE was highly expressed in neural stem cells (neuroblasts) and their progeny (ganglion mother cells and postmitotic neurons) at larval and pupal stages. MAGE was also expressed in postmitotic neurons including mushroom body neurons and retinal photoreceptors in adulthood. These results indicate that MAGE expression lasts throughout the postembryonic neurogenesis in Drosophila.

Thyroid hormone-mediated negative transcriptional regulation of Necdin expression

Unliganded thyroid hormone receptors (apoTRs) repress transcription of hormone-activated genes by recruiting corepressors to the promoters. In contrast, on promoters containing so-called negative thyroid hormone response elements (nTREs), apoTRs activate transcription. A number of different molecular mechanisms have been described as to how apoTRs activate transcription varying with the target gene of the study. Here we demonstrate that thyroid hormone regulates the transcription of the Necdin gene, a developmentally regulated candidate gene for the genomic imprinting-associated neurobehavioural disorder, Prader-Willi syndrome. ApoTRs activate Necdin expression through an nTRE in its promoter, downstream of the transcription start site. The nTRE of the Necdin gene resembles the nTREs of the TSHbeta genes of the hypothalamus-pituitary-thyroid axis in the sequence, position in the promoter, and mode of activation. We show that this group of nTRE-driven genes shares the requirements for binding of the retinoic X receptor and nuclear receptor corepressor/silencing mediator of retinoid and thyroid hormone receptors (NCoR/SMRT) for full ligand-independent activation, whereas there is no need for association of the p160 family of coactivators. In accordance with the requirement for corepressors, Necdin expression is influenced by deacetylase activity, suggesting that histone deacetylases and corepressors as well could function as activators of transcription, depending on the promoter context.

Disruption of the Paternal Necdin Gene Diminishes TrkA Signaling for Sensory Neuron Survival

Necdin is a multifunctional signaling protein that stabilizes terminal differentiation of postmitotic neurons. The human necdin gene in chromosome 15q11-q12 is maternally imprinted, paternally transcribed, and not expressed in Prader-Willi syndrome, a human genomic imprinting-associated neurodevelopmental disorder. Although necdin-deficient mice display several abnormal phenotypes reminiscent of this syndrome, little is known about molecular mechanisms that lead to the neurodevelopmental defects. Here, we demonstrate that paternally expressed necdin is required for physiological development of nerve growth factor (NGF)-dependent sensory neurons. Mouse embryos defective in the paternal necdin allele displayed absent necdin expression in the dorsal root ganglia, in which the tropomyosin-related kinase A (TrkA) receptor tyrosine kinase and the p75 neurotrophin receptor were expressed in a normal manner. Necdin interacted with both TrkA and p75 to facilitate the association between these receptors. NGF-induced phosphorylation of TrkA and mitogen-activated protein kinase was significantly diminished in the necdin-null sensory ganglia. Furthermore, the mice lacking the paternal necdin allele displayed augmented apoptosis in the sensory ganglia in vivo and had a reduced population of substance P-containing neurons. These mutant mice showed significantly high tolerance to thermal pain, which is often seen in individuals with Prader-Willi syndrome. These results suggest that paternally expressed necdin facilitates TrkA signaling to promote the survival of NGF-dependent nociceptive neurons.