Insulin and IGF-1 elicit robust transcriptional regulation to modulate autophagy in astrocytes.

OBJECTIVE: Insulin is a principal metabolic hormone. It regulates a plethora of metabolic pathways in peripheral tissues. The highly homologous insulin-like growth factor 1 (IGF-1), on the other hand, is important for development and growth. Recent studies have shown that insulin and IGF-1 signaling plays fundamental roles in the brain. Loss of insulin or IGF-1 receptors in astrocytes leads to altered glucose handling, mitochondrial metabolism, neurovascular coupling, and behavioral abnormalities in mice. Here, we aim to investigate molecular mechanisms by which insulin and IGF-1 signaling regulates astrocyte functions. METHODS: IR-flox and IRKO primary astrocytes were treated with 100 nM insulin or IGF-1 for 6 h, and their transcriptomes were analyzed. Astrocytes with either IR deletion, IGF1R deletion or both were used to examine receptor-dependent transcriptional regulations using qPCR. Additional immunoblotting and confocal imaging studies were performed to functionally validate pathways involved in protein homeostasis. RESULTS: Using next-generation RNA sequencing, we show that insulin significantly regulates the expression of over 1,200 genes involved in multiple functional processes in primary astrocytes. Insulin-like growth factor 1 (IGF-1) triggers a similar robust transcriptional regulation in astrocytes. Thus, over 50% of the differentially expressed genes are regulated by both ligands. As expected, these commonly regulated genes are highly enriched in pathways involved in lipid and cholesterol biosynthesis. Additionally, insulin and IGF-1 induce the expression of genes involved in ribosomal biogenesis, while suppressing the expression of genes involved in autophagy, indicating a common role of insulin and IGF-1 on protein homeostasis in astrocytes. Insulin-dependent suppression of autophagy genes, including p62, Ulk1/2, and several Atg genes, is blunted only when both IR and IGF1R are deleted. CONCLUSIONS: In summary, insulin and IGF-1 potently suppress autophagy in astrocytes through transcriptional regulation. Both IR and IGF1R can elicit ligand-dependent transcriptional suppression of autophagy. These results demonstrate an important role of astrocytic insulin/IGF-1 signaling on proteostasis. Impairment of this regulation in insulin resistance and diabetes may contribute to neurological complications related to diabetes.

Convergent biological pathways underlying the Kallmann syndrome-linked genes Hs6st1 and Fgfr1.

Kallmann syndrome (KS) is a congenital disorder characterized by idiopathic hypogonadotropic hypogonadism and olfactory dysfunction. KS is linked to variants in >34 genes, which are scattered across the human genome and show disparate biological functions. Although the genetic basis of KS is well studied, the mechanisms by which disruptions of these diverse genes cause the same outcome of KS are not fully understood. Here we show that disruptions of KS-linked genes affect the same biological processes, indicating convergent molecular mechanisms underlying KS. We carried out machine learning-based predictions and found that KS-linked mutations in heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) are likely loss-of-function mutations. We next disrupted Hs6st1 and another KS-linked gene, fibroblast growth factor receptor 1 (Fgfr1), in mouse neuronal cells and measured transcriptome changes using RNA sequencing. We found that disruptions of Hs6st1 and Fgfr1 altered genes in the same biological processes, including the upregulation of genes in extracellular pathways and the downregulation of genes in chromatin pathways. Moreover, we performed genomics and bioinformatics analyses and found that Hs6st1 and Fgfr1 regulate gene transcription likely via the transcription factor Sox9/Sox10 and the chromatin regulator Chd7, which are also associated with KS. Together, our results demonstrate how different KS-linked genes work coordinately in a convergent signaling pathway to regulate the same biological processes, thus providing new insights into KS.

Disruption of GMNC-MCIDAS multiciliogenesis program is critical in choroid plexus carcinoma development.

Multiciliated cells (MCCs) in the brain reside in the ependyma and the choroid plexus (CP) epithelia. The CP secretes cerebrospinal fluid that circulates within the ventricular system, driven by ependymal cilia movement. Tumors of the CP are rare primary brain neoplasms mostly found in children. CP tumors exist in three forms: CP papilloma (CPP), atypical CPP, and CP carcinoma (CPC). Though CPP and atypical CPP are generally benign and can be resolved by surgery, CPC is a particularly aggressive and little understood cancer with a poor survival rate and a tendency for recurrence and metastasis. In contrast to MCCs in the CP epithelia, CPCs in humans are characterized by solitary cilia, frequent TP53 mutations, and disturbances to multiciliogenesis program directed by the GMNC-MCIDAS transcriptional network. GMNC and MCIDAS are early transcriptional regulators of MCC fate differentiation in diverse tissues. Consistently, components of the GMNC-MCIDAS transcriptional program are expressed during CP development and required for multiciliation in the CP, while CPC driven by deletion of Trp53 and Rb1 in mice exhibits multiciliation defects consequent to deficiencies in the GMNC-MCIDAS program. Previous studies revealed that abnormal NOTCH pathway activation leads to CPP. Here we show that combined defects in NOTCH and Sonic Hedgehog signaling in mice generates tumors that are similar to CPC in humans. NOTCH-driven CP tumors are monociliated, and disruption of the NOTCH complex restores multiciliation and decreases tumor growth. NOTCH suppresses multiciliation in tumor cells by inhibiting the expression of GMNC and MCIDAS, while Gmnc-Mcidas overexpression rescues multiciliation defects and suppresses tumor cell proliferation. Taken together, these findings indicate that reactivation of the GMNC-MCIDAS multiciliogenesis program is critical for inhibiting tumorigenesis in the CP, and it may have therapeutic implications for the treatment of CPC.

Neuronal Yin Yang1 in the prefrontal cortex regulates transcriptional and behavioral responses to chronic stress in mice.

Although the synaptic alterations associated with the stress-related mood disorder major depression has been well-documented, the underlying transcriptional mechanisms remain poorly understood. Here, we perform complementary bulk nuclei- and single-nucleus transcriptome profiling and map locus-specific chromatin interactions in mouse neocortex to identify the cell type-specific transcriptional changes associated with stress-induced behavioral maladaptation. We find that cortical excitatory neurons, layer 2/3 neurons in particular, are vulnerable to chronic stress and acquire signatures of gene transcription and chromatin structure associated with reduced neuronal activity and expression of Yin Yang 1 (YY1). Selective ablation of YY1 in cortical excitatory neurons enhances stress sensitivity in both male and female mice and alters the expression of stress-associated genes following an abbreviated stress exposure. These findings demonstrate how chronic stress impacts transcription in cortical excitatory neurons and identify YY1 as a regulator of stress-induced maladaptive behavior in mice.

Recursive splicing is a rare event in the mouse brain.

Recursive splicing (RS) is a splicing mechanism to remove long introns from messenger RNA precursors of long genes. Compared to the hundreds of RS events identified in humans and drosophila, only ten RS events have been reported in mice. To further investigate RS in mice, we analyzed RS in the mouse brain, a tissue that is enriched in the expression of long genes. We found that nuclear total RNA sequencing is an efficient approach to investigate RS events. We analyzed 1.15 billion uniquely mapped reads from the nuclear total RNA sequencing data in the mouse cerebral cortex. Unexpectedly, we only identified 20 RS sites, suggesting that RS is a rare event in the mouse brain. We also identified that RS is constitutive between excitatory and inhibitory neurons and between sexes in the mouse cerebral cortex. In addition, we found that the primary sequence context is associated with RS splicing intermediates and distinguishes RS AGGT site from non-RS AGGT sites, indicating the importance of the primary sequence context in RS sites. Moreover, we discovered that cryptic exons may use an RS-like mechanism for splicing. Overall, we provide novel findings about RS in long genes in the mouse brain.

Spatial, temporal and cell-type-specific expression profiles of genes encoding heparan sulfate biosynthesis enzymes and proteoglycan core proteins.

Heparan sulfate (HS) is a linear polysaccharide found in almost all animal cells and plays an important role in various biological processes. HS functions mainly via covalently binding to core proteins to form HS proteoglycans (HSPGs), which are heterogeneous in the lengths of the HS chain, the modifications on HS and the core proteins. The molecular mechanisms underlying HSPG heterogeneity, although widely studied, are not yet fully defined. The expression profiles of HS biosynthesis enzymes and HSPG core proteins likely contribute to the HSPG heterogeneity, but these expression profiles remain poorly characterized. To investigate the expression profiles of genes encoding HS biosynthesis enzymes and HSPG core proteins, we systematically integrated the publicly available RNA sequencing data in mice. To reveal the spatial expression of these genes, we analyzed their expression in 21 mouse tissues. To reveal the temporal expression of these genes, we analyzed their expression at 17 time points during the mouse forebrain development. To determine the cell-type-specific expression of these genes, we obtained their expression profiles in 23 cell types in the mouse cerebral cortex by integrating single nucleus RNA sequencing data. Our findings demonstrate the spatial, temporal and cell-type-specific expression of genes encoding HS biosynthesis enzymes and HSPG core proteins and represent a valuable resource to the HS research community.

Self-powered portable melt electrospinning for in situ wound dressing.

BACKGROUND: Electrospun (e-spun) nanofibers for wound dressing have attracted wide attention due to its large specific surface area, large porosity and breathability. Compared with solution electrospinning (e-spinning), melt e-spinning is more bio-friendly without toxic solvent participation, which provides the possibility of in situ e-spinning on wounds directly. However, previously reported melt e-spinning devices were usually bulky and cumbersome due to their necessary heating unit, and different components were separated to avoid electrostatic interference. RESULTS: In this article, we report on a self-powered hand-held melt e-spinning gun which can work without any external power supply (outdoors). The problem of electrostatic interference for this integrated device was solved by using a special high heat transfer insulation unit. The apparatus is easy and safe to operate by a single hand due to its small volume (24 × 6 × 13 cm3) and light weight (about 450 g). Some biodegradable polymers, for example, polycaprolactone (PCL) fibers were successful e-spun onto wounds directly by using this dressing gun. CONCLUSIONS: PCL fibrous membrane has good biocompatibility and can be in situ electrospun to wound surface as a wound dressing by the portable melt e-spinning gun. Besides wound dressing, this hand-held melt e-spinning gun may be used in 3D printing and experimental teaching demonstration aids.

In situ melt electrospun polycaprolactone/Fe3O4 nanofibers for magnetic hyperthermia.

Magnetic fibrous membrane used to generate heat under the alternating magnetic field (AMF) has attracted wide attention due to their application in magnetic hyperthermia. However, there is not magnetic fibrous membrane prepared by melt electrospinning (e-spinning) which is a solvent-free, bio-friendly technology. In this work, polycaprolactone (PCL)/Fe3O4 fiber membrane was prepared by melt e-spinning and using homemade self-powered portable melt e-spinning apparatus. The hand-held melt e-spinning apparatus has a weight of about 450 g and a precise size of 24 cm in length, 6 cm in thickness and 13 cm in height, which is more portable for widely using in the medical field. The PCL/Fe3O4 composite fibers with diameters of 4-17 µm, are very uniform. In addition, the magnetic composite fiber membrane has excellent heating efficiency and thermal cycling characteristics. The results indicated that self-powered portable melt e-spinning apparatus and PCL/Fe3O4 fiber membrane may provide an attractive way for hyperthermia therapy.

Genomic architecture of Shh-dependent cochlear morphogenesis.

The mammalian cochlea develops from a ventral outgrowth of the otic vesicle in response to Shh signaling. Mouse embryos lacking Shh or its essential signal transduction components display cochlear agenesis; however, a detailed understanding of the transcriptional network mediating this process is unclear. Here, we describe an integrated genomic approach to identify Shh-dependent genes and associated regulatory sequences that promote cochlear duct morphogenesis. A comparative transcriptome analysis of otic vesicles from mouse mutants exhibiting loss (Smoecko ) and gain (Shh-P1) of Shh signaling reveal a set of Shh-responsive genes partitioned into four expression categories in the ventral half of the otic vesicle. This target gene classification scheme provides novel insight into several unanticipated roles for Shh, including priming the cochlear epithelium for subsequent sensory development. We also mapped regions of open chromatin in the inner ear by ATAC-seq that, in combination with Gli2 ChIP-seq, identified inner ear enhancers in the vicinity of Shh-responsive genes. These datasets are useful entry points for deciphering Shh-dependent regulatory mechanisms involved in cochlear duct morphogenesis and establishment of its constituent cell types.

Long genes linked to autism spectrum disorders harbor broad enhancer-like chromatin domains.

Genetic variants associated with autism spectrum disorders (ASDs) are enriched in genes encoding synaptic proteins and chromatin regulators. Although the role of synaptic proteins in ASDs is widely studied, the mechanism by which chromatin regulators contribute to ASD risk remains poorly understood. Upon profiling and analyzing the transcriptional and epigenomic features of genes expressed in the cortex, we uncovered a unique set of long genes that contain broad enhancer-like chromatin domains (BELDs) spanning across their entire gene bodies. Analyses of these BELD genes show that they are highly transcribed with frequent RNA polymerase II (Pol II) initiation and low Pol II pausing, and they exhibit frequent chromatin-chromatin interactions within their gene bodies. These BELD features are conserved from rodents to humans, are enriched in genes involved in synaptic function, and appear post-natally concomitant with synapse development. Importantly, we find that BELD genes are highly implicated in neurodevelopmental disorders, particularly ASDs, and that their expression is preferentially down-regulated in individuals with idiopathic autism. Finally, we find that the transcription of BELD genes is particularly sensitive to alternations in ASD-associated chromatin regulators. These findings suggest that the epigenomic regulation of BELD genes is important for post-natal cortical development and lend support to a model by which mutations in chromatin regulators causally contribute to ASDs by preferentially impairing BELD gene transcription.

Bubble Melt Electrospinning for Production of Polymer Microfibers.

In this paper, we report an interesting bubble melt electrospinning (e-spinning) to produce polymer microfibers. Usually, melt e-spinning for fabricating ultrafine fibers needs "Taylor cone", which is formed on the tip of the spinneret. The spinneret is also the bottleneck for mass production in melt e-spinning. In this work, a metal needle-free method was tried in the melt e-spinning process. The "Taylor cone" was formed on the surface of the broken polymer melt bubble, which was produced by an airflow. With the applied voltage ranging from 18 to 25 kV, the heating temperature was about 210⁻250 °C, and polyurethane (TPU) and polylactic acid (PLA) microfibers were successfully fabricated by this new melt e-spinning technique. During the melt e-spinning process, polymer melt jets ejected from the burst bubbles could be observed with a high-speed camera. Then, polymer microfibers could be obtained on the grounded collector. The fiber diameter ranged from 45 down to 5 µm. The results indicate that bubble melt e-spinning may be a promising method for needleless production in melt e-spinning.

Biotin tagging of MeCP2 in mice reveals contextual insights into the Rett syndrome transcriptome.

Mutations in MECP2 cause Rett syndrome (RTT), an X-linked neurological disorder characterized by regressive loss of neurodevelopmental milestones and acquired psychomotor deficits. However, the cellular heterogeneity of the brain impedes an understanding of how MECP2 mutations contribute to RTT. Here we developed a Cre-inducible method for cell-type-specific biotin tagging of MeCP2 in mice. Combining this approach with an allelic series of knock-in mice carrying frequent RTT-associated mutations (encoding T158M and R106W) enabled the selective profiling of RTT-associated nuclear transcriptomes in excitatory and inhibitory cortical neurons. We found that most gene-expression changes were largely specific to each RTT-associated mutation and cell type. Lowly expressed cell-type-enriched genes were preferentially disrupted by MeCP2 mutations, with upregulated and downregulated genes reflecting distinct functional categories. Subcellular RNA analysis in MeCP2-mutant neurons further revealed reductions in the nascent transcription of long genes and uncovered widespread post-transcriptional compensation at the cellular level. Finally, we overcame X-linked cellular mosaicism in female RTT models and identified distinct gene-expression changes between neighboring wild-type and mutant neurons, providing contextual insights into RTT etiology that support personalized therapeutic interventions.

Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC.

Efforts to manipulate locus-specific histone acetylation to assess their causal role in gene expression and cellular and behavioural phenotypes have been impeded by a lack of experimental tools. The Cas9 nuclease has been adapted to target epigenomic modifications, but a detailed description of the parameters of such synthetic epigenome remodellers is still lacking. Here we describe a Cas9-based histone deacetylase (HDAC) and the design principles required to achieve locus-specific histone deacetylation. We assess its range of activity and specificity, and analyse target gene expression in two different cell types to investigate cellular context-dependent effects. Our findings demonstrate that the chromatin environment is an important element to consider when utilizing this synthetic HDAC.

Cis-regulatory architecture of a brain signaling center predates the origin of chordates.

Genomic approaches have predicted hundreds of thousands of tissue-specific cis-regulatory sequences, but the determinants critical to their function and evolutionary history are mostly unknown. Here we systematically decode a set of brain enhancers active in the zona limitans intrathalamica (zli), a signaling center essential for vertebrate forebrain development via the secreted morphogen Sonic hedgehog (Shh). We apply a de novo motif analysis tool to identify six position-independent sequence motifs together with their cognate transcription factors that are essential for zli enhancer activity and Shh expression in the mouse embryo. Using knowledge of this regulatory lexicon, we discover new Shh zli enhancers in mice and a functionally equivalent element in hemichordates, indicating an ancient origin of the Shh zli regulatory network that predates the chordate phylum. These findings support a strategy for delineating functionally conserved enhancers in the absence of overt sequence homologies and over extensive evolutionary distances.

Locus- and cell type-specific epigenetic switching during cellular differentiation in mammals.

BACKGROUND: Epigenomic reconfiguration, including changes in DNA methylation and histone modifications, is crucial for the differentiation of embryonic stem cells (ESCs) into somatic cells. However, the extent to which the epigenome is reconfigured and the interplay between components of the epigenome during cellular differentiation remain poorly defined. METHODS: We systematically analyzed and compared DNA methylation, various histone modification, and transcriptome profiles in ESCs with those of two distinct types of somatic cells from human and mouse. RESULTS: We found that global DNA methylation levels are lower in somatic cells compared to ESCs in both species. We also found that 80% of regions with histone modification occupancy differ between human ESCs and the two human somatic cell types. Approximately 70% of the reconfigurations in DNA methylation and histone modifications are locus- and cell type-specific. Intriguingly, the loss of DNA methylation is accompanied by the gain of different histone modifications in a locus- and cell type-specific manner. Further examination of transcriptional changes associated with epigenetic reconfiguration at promoter regions revealed an epigenetic switching for gene regulation-a transition from stable gene silencing mediated by DNA methylation in ESCs to flexible gene repression facilitated by repressive histone modifications in somatic cells. CONCLUSIONS: Our findings demonstrate that the epigenome is reconfigured in a locus- and cell type-specific manner and epigenetic switching is common during cellular differentiation in both human and mouse.

Dynamic and Coordinated Expression Changes of Rice Small RNAs in Response to Xanthomonas oryzae pv. oryzae.

Endogenous small RNAs are newly identified players in plant immune responses, yet their roles in rice (Oryza sativa) responding to pathogens are still less understood, especially for pathogens that can cause severe yield losses. We examined the small RNA expression profiles of rice leaves at 2, 6, 12, and 24 hours post infection of Xanthomonas oryzae pv. oryzae (Xoo) virulent strain PXO99, the causal agent of rice bacterial blight disease. Dynamic expression changes of some miRNAs and trans-acting siRNAs were identified, together with a few novel miRNA targets, including an RLK gene targeted by osa-miR159a.1. Coordinated expression changes were observed among some small RNAs in response to Xoo infection, with small RNAs exhibiting the same expression pattern tended to regulate genes in the same or related signaling pathways, including auxin and GA signaling pathways, nutrition and defense-related pathways. These findings reveal the dynamic and complex roles of small RNAs in rice-Xoo interactions, and identify new targets for regulating plant responses to Xoo.

Bidirectional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1.

MicroRNAs (miRNAs) originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures, and their accurate processing is required for the production of functional miRNAs. Here, using the pri-miR-166 family in Arabidopsis thaliana as a paradigm, we report the crucial role of pri-miRNA terminal loops in miRNA biogenesis. We found that multibranched terminal loops in pri-miR-166s substantially suppress miR-166 expression in vivo. Unlike canonical processing of pri-miRNAs, terminal loop-branched pri-miRNAs can be processed by Dicer-like 1 (DCL1) complexes bidirectionally from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In both cases, DCL1 complexes canonically cut pri-miRNAs at a distance of 16-17 bp from a reference single-stranded loop region. DCL1 also adjusts processing sites toward an internal loop through its helicase domain. These results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complex secondary structures.

Loss of MeCP2 function is associated with distinct gene expression changes in the striatum.

Rett syndrome (RTT) is a neurodevelopmental disorder characterized by developmental regression beginning 6-18months after birth, followed by a lifetime of intellectual disability, stereotyped behaviors, and motor deficits. RTT is caused by mutations in the gene encoding MeCP2, a methyl-CpG binding protein believed to modulate gene transcription. Gene expression studies of individual brain regions have reported that Mecp2 loss-of-function leads to both activation and repression of its gene targets in mice. Conditional deletion of MeCP2 from different brain regions has revealed unique insights into the role of these structures in mediating particular RTT-like phenotypes. However, the function of MeCP2 in the striatum, a major brain region involved in motor control and executive cognitive functions, has yet to be studied. Here, we characterized the gene expression changes in the striatum of Mecp2 mutant mice. We found a number of differentially expressed genes in the striatum of both constitutive Mecp2-null mice and mice lacking MeCP2 only from forebrain GABAergic neurons. These changes only occurred when MeCP2 expression levels had reached mature levels and RTT-like symptoms were manifest, supporting a role for MeCP2 in maintaining proper brain function. Many of the gene expression changes identified in the striatum have not previously been shown to change in the hypothalamus or cerebellum. Bioinformatic analysis of differentially expressed genes in striatum as well as hypothalamus and cerebellum revealed that loss of MeCP2 does not affect the global landscape of gene expression. Additionally, we uncovered a number of differentially expressed genes in the liver of Mecp2-null mice suggesting an important role for MeCP2 in non-neuronal tissues. Collectively, our data suggest that the differential expression of genes following loss of MeCP2 occurs in a tissue- or cell-type specific manner and thus MeCP2 function should be understood in a cellular context.

Identification and characterization of small RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus.

The term RNA silencing (RNA interference, RNAi) describes a set of mechanisms that regulate gene expression in eukaryotes. Small interfering RNAs (siRNA) and microRNAs (miRNAs) are two major types of RNAi-associated small RNAs (smRNAs) found in most eukaryotic organisms. Despite the presence of a plethora of non-coding RNAs longer than 50-nucleotide (nt) in length in various species of Archaea, little is known about smRNAs in archaea that resemble the 20-24-nt long smRNAs found in eukaryotes, which have been implicated in the post-transcriptional control of gene expression. Here, we report the finding of a large number of smRNAs approximatelly 20-nt in length, including phased smRNAs and potential miRNAs, from the hyperthermophilic archaeon Sulfolobus solfataricus p2 (Ssp2) based on deep sequencing. The expression of some of the miRNA candidates in Ssp2 was confirmed. Consistent with the Ssp2 hyperthermophilic properties, we found that higher temperatures more efficiently induced the production of the miRNA candidates in an in vitro system using the putative foldback precursor transcripts incubated with Ssp2 extract. Although we initially predicted putative target genes of some miRNA candidates, further analysis mapped the cleavage sites downstream of the miRNA candidate complementary regions, similar to those involved in plant miRNA-mediated TAS transcript cleavage. We also identified smRNAs from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, which play important roles in prokaryotic microbial defense systems. Archaea represent a unique life form next to Bacteria and Eukarya, and our results may provide a useful resource for further in-depth study on the regulation and evolution of smRNAs in this special organism.