Differential recruitment of coregulators to the RORA promoter adds another layer of complexity to gene (dys)regulation by sex hormones in autism

Background Our independent cohort studies have consistently shown the reduction of the nuclear receptor RORA (retinoic acid-related orphan receptor-alpha) in lymphoblasts as well as in brain tissues from individuals with autism spectrum disorder (ASD). Moreover, we have found that RORA regulates the gene for aromatase, which converts androgen to estrogen, and that male and female hormones regulate RORA in opposite directions, with androgen suppressing RORA, suggesting that the sexually dimorphic regulation of RORA may contribute to the male bias in ASD. However, the molecular mechanisms through which androgen and estrogen differentially regulate RORA are still unknown. Methods Here we use functional knockdown of hormone receptors and coregulators with small interfering RNA (siRNA) to investigate their involvement in sex hormone regulation of RORA in human neuronal cells. Luciferase assays using a vector containing various RORA promoter constructs were first performed to identify the promoter regions required for inverse regulation of RORA by male and female hormones. Sequential chromatin immunoprecipitation methods followed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses of RORA expression in hormone-treated SH-SY5Y cells were then utilized to identify coregulators that associate with hormone receptors on the RORA promoter. siRNA-mediated knockdown of interacting coregulators was performed followed by qRT-PCR analyses to confirm the functional requirement of each coregulator in hormone-regulated RORA expression. Results Our studies demonstrate the direct involvement of androgen receptor (AR) and estrogen receptor (ER) in the regulation of RORA by male and female hormones, respectively, and that the promoter region between ?10055 bp and ?2344 bp from the transcription start site of RORA is required for the inverse hormonal regulation. We further show that AR interacts with SUMO1, a reported suppressor of AR transcriptional activity, whereas ER? interacts with the coactivator NCOA5 on the RORA promoter. siRNA-mediated knockdown of SUMO1 and NCOA5 attenuate the sex hormone effects on RORA expression. Conclusions AR and SUMO1 are involved in the suppression RORA expression by androgen, while ER? and NCOA5 collaborate in the up-regulation of RORA by estrogen. While this study offers a better understanding of molecular mechanisms involved in sex hormone regulation of RORA, it also reveals another layer of complexity with regard to gene regulation in ASD. Inasmuch as coregulators are capable of interacting with a multitude of transcription factors, aberrant expression of coregulator proteins, as we have seen previously in lymphoblasts from individuals with ASD, may contribute to the polygenic nature of gene dysregulation in ASD

Polygonumins A, a newly isolated compound from the stem of Polygonum minus Huds with potential medicinal activities

Polygonumins A, a new compound, was isolated from the stem of Polygonum minus. Based on NMR results, the compound’s structure is identical to that of vanicoside A, comprising four phenylpropanoid ester units and a sucrose unit. The structure diferences were located at C-3″″′. The cytotoxic activity of polygonumins A was evaluated on several cancer cell lines by a cell viability assay using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The compound showed the highest antiproliferative (p<0.05) activities against K562 (Human Leukaemia Cell Line), MCF7 (Human breast adenocarcinoma cell line), and HCT116 (Colorectal cancer cells) cells. Cytotoxic studies against V79–4 cells were carried out and showed that polygonumins A was toxic at 50µg/ml, suggesting that this compound may be used as an anticancer drug without afecting normal cells. Polygonumins A also showed promising activity as an HIV-1 protease inhibitor with 56% relative inhibition. Molecular docking results indicated that the compound possesses high binding afnity towards the HIV protease over the low binding free energy range of -10.5 to -11.3kcal/mol. P. minus is used in Malaysian traditional medicine for the treatment of tumour cells. This is the frst report on the use of P. minus as an HIV-1 protease inhibitor

MIR137 is the key gene mediator of the syndromic obesity phenotype of patients with 1p21.3 microdeletions.

BACKGROUND: Deletions in the long arm of chromosome 1 have been described in patients with a phenotype consisting primarily of obesity, intellectual disability and autism-spectrum disorder. The minimal region of overlap comprises two genes: DPYD and MIR137. CASE PRESENTATION: We describe a 10-year-old boy with syndromic obesity who carries a novel 1p21.3 deletion overlapping the critical region with the MIR137 gene only. CONCLUSIONS: This study suggests that MIR137 is the mediator of the obesity phenotype of patients carrying 1p21.3 microdeletions

Investigation of post-transcriptional gene regulatory networks associated with autism spectrum disorders (ASD) by miRNA expression profiling of lymphoblastoid cell lines

Autism spectrum disorders (ASD) are neurodevelopmental disorders with a pattern of qualitative abnormalities in three behavioral areas: reciprocal social interactions, communication, and restricted interests and repetitive behaviors. Gene expression profiling of monozygotic twins discordant in diagnosis or severity of autism revealed differentially expressed, neurologically relevant genes, suggesting that epigenetic factors, such as DNA methylation or miRNA, may be involved in ASD. In this study, global miRNA expression profiling using lymphoblastoid cell lines derived from 5 autistic individuals and their monozygotic co-twins and/or their siblings were performed using high-throughput miRNA microarray analysis. The majority of the significantly differentially expressed miRNAs were found to target genes highly involved in neurological functions and disorders. The potential targets, moreover, were also linked to gastrointestinal diseases and estrogen receptor signaling, including androgen and estrogen metabolism. A number of the predicted potential target genes were also found to be significantly differentially expressed in cDNA microarray analyses. Novel network prediction analyses of the potential target genes whose transcript levels show an inverse relationship to that of the respective putative regulatory miRNAs reveal association with autism and other co-morbid disorders, i.e. muscular dystrophy and gastrointestinal diseases. Many interesting biological functions implicated in autism, such as memory and synaptic plasticity, were also highlighted. Findings from this study strongly suggest that dysregulation of miRNA expression is a contributing factor to the observed alterations in gene expression and, in turn, may lead to the pathophysiological conditions underpinning autism

Genome-wide identification of transcriptional targets of RORA reveals direct regulation of multiple genes associated with autism spectrum disorder

BACKGROUND: We have recently identified the nuclear hormone receptor RORA (retinoic acid-related orphan receptor-alpha) as a novel candidate gene for autism spectrum disorder (ASD). Our independent cohort studies have consistently demonstrated the reduction of RORA transcript and/or protein levels in blood-derived lymphoblasts as well as in the postmortem prefrontal cortex and cerebellum of individuals with ASD. Moreover, we have also shown that RORA has the potential to be under negative and positive regulation by androgen and estrogen, respectively, suggesting the possibility that RORA may contribute to the male bias of ASD. However, little is known about transcriptional targets of this nuclear receptor, particularly in humans. METHODS: Here we identify transcriptional targets of RORA in human neuronal cells on a genome-wide level using chromatin immunoprecipitation (ChIP) with an anti-RORA antibody followed by whole-genome promoter array (chip) analysis. Selected potential targets of RORA were then validated by an independent ChIP followed by quantitative PCR analysis. To further demonstrate that reduced RORA expression results in reduced transcription of RORA targets, we determined the expression levels of the selected transcriptional targets in RORA-deficient human neuronal cells, as well as in postmortem brain tissues from individuals with ASD who exhibit reduced RORA expression. RESULTS: The ChIP-on-chip analysis reveals that RORA1, a major isoform of RORA protein in human brain, can be recruited to as many as 2,764 genomic locations corresponding to promoter regions of 2,544 genes across the human genome. Gene ontology analysis of this dataset of genes that are potentially directly regulated by RORA1 reveals statistically significant enrichment in biological functions negatively impacted in individuals with ASD, including neuronal differentiation, adhesion and survival, synaptogenesis, synaptic transmission and plasticity, and axonogenesis, as well as higher level functions such as development of the cortex and cerebellum, cognition, memory, and spatial learning. Independent ChIP-quantitative PCR analyses confirm binding of RORA1 to promoter regions of selected ASD-associated genes, including A2BP1, CYP19A1, ITPR1, NLGN1, and NTRK2, whose expression levels (in addition to HSD17B10) are also decreased in RORA1-repressed human neuronal cells and in prefrontal cortex tissues from individuals with ASD. CONCLUSIONS: Findings from this study indicate that RORA transcriptionally regulates A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2, and strongly suggest that reduction of this sex hormone-sensitive nuclear receptor in the brain causes dysregulated expression of these ASD-relevant genes as well as their associated pathways and functions which, in turn, may contribute to the underlying pathobiology of ASD

Sex hormones in autism: Androgens and estrogens differentially and reciprocally regulate RORA, a novel candidate gene for autism

Autism, a pervasive neurodevelopmental disorder manifested by deficits in social behavior and interpersonal communication, and by stereotyped, repetitive behaviors, is inexplicably biased towards males by a ratio of ∼4∶1, with no clear understanding of whether or how the sex hormones may play a role in autism susceptibility. Here, we show that male and female hormones differentially regulate the expression of a novel autism candidate gene, retinoic acid-related orphan receptor-alpha (RORA) in a neuronal cell line, SH-SY5Y. In addition, we demonstrate that RORA transcriptionally regulates aromatase, an enzyme that converts testosterone to estrogen. We further show that aromatase protein is significantly reduced in the frontal cortex of autistic subjects relative to sex- and age-matched controls, and is strongly correlated with RORA protein levels in the brain. These results indicate that RORA has the potential to be under both negative and positive feedback regulation by male and female hormones, respectively, through one of its transcriptional targets, aromatase, and further suggest a mechanism for introducing sex bias in autism