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Background Frontline healthcare workers responding to coronavirus disease 2019 (COVID-19) inevitably face tremendous psychological burden. Thus, the present study aimed to identify the psychological impact and the factors contributing to the likely increase in emotional distress of healthcare workers. Methods The participants include a total of 99 healthcare workers at Bugok National Hospital. Psychometric scales were used to assess emotional distress (12-item General Health Questionnaire; GHQ-12), depression symptoms (Patient Health Questionnaire-9; PHQ-9), and post-traumatic stress disorder-related symptoms (Impact of Events Scale-Revised; IES-R). A supplementary questionnaire was administered to investigate the experience of healthcare workers exposed to COVID-19-infected patients. Based on the results of GHQ-12 survey, participants were categorized into two groups: distress and non-distress. All the assessed scores were compared between the two groups. A logistic regression model was constructed to identify factors associated with emotional distress. Results Emotional distress was reported by 45.3% (n = 45) of all participants. The emotionally distressed group was more likely to be female, manage close contacts, have higher scores on PHQ-9 and IES-R, feel increased professional risk, and report that proper infection control training was not provided. Female gender, managing close contacts, higher scores on PHQ-9, and a feeling that proper infection control training was not provided were associated with emotional distress in logistic regression. Conclusion Frontline healthcare workers face tremendous psychological burden during the COVID-19 pandemic. Therefore, appropriate psychological interventions should be provided to the HCWs engaged in the management of COVID-19-infected patients., Graphical Abstract

In humans, inactivating mutations in MLL4, which encodes a histone H3-lysine 4-methyltransferase, lead to Kabuki syndrome (KS). While dwarfism is a cardinal feature of KS, the underlying etiology remains unclear. Here we report that Mll4 regulates the development of growth hormone-releasing hormone (GHRH)-producing neurons in the mouse hypothalamus. Our two Mll4 mutant mouse models exhibit dwarfism phenotype and impairment of the developmental programs for GHRH-neurons. Our ChIP-seq analysis reveals that, in the developing mouse hypothalamus, Mll4 interacts with the transcription factor Nrf1 to trigger the expression of GHRH-neuronal genes. Interestingly, the deficiency of Mll4 results in a marked reduction of histone marks of active transcription, while treatment with the histone deacetylase inhibitor AR-42 rescues the histone mark signature and restores GHRH-neuronal production in Mll4 mutant mice. Our results suggest that the developmental dysregulation of Mll4-directed epigenetic control of transcription plays a role in the development of GHRH-neurons and dwarfism phenotype in mice., Mutations in the MLL4 gene can cause Kabuki syndrome, whose underlying molecular mechanisms are unclear. Here, the authors show that Mll4 epigenetically regulates the transcriptional program leading to the formation of GHRH-neurons in the developing mouse hypothalamus.

Despite critical roles of the hypothalamic arcuate neurons in controlling the growth and energy homeostasis, the gene regulatory network directing their development remains unclear. Here we report that the transcription factors Dlx1/2 and Otp coordinate the balanced generation of the two functionally related neurons in the hypothalamic arcuate nucleus, GHRH-neurons promoting the growth and AgRP-neurons controlling the feeding and energy expenditure. Dlx1/2-deficient mice show a loss-of-GHRH-neurons and an increase of AgRP-neurons, and consistently develop dwarfism and consume less energy. These results indicate that Dlx1/2 are crucial for specifying the GHRH-neuronal identity and, simultaneously, for suppressing AgRP-neuronal fate. We further show that Otp is required for the generation of AgRP-neurons and that Dlx1/2 repress the expression of Otp by directly binding the Otp gene. Together, our study demonstrates that the identity of GHRH- and AgRP-neurons is synchronously specified and segregated by the Dlx1/2-Otp gene regulatory axis., In the hypothalamus, the arcuate nucleus (ARC) contains AgRP-neurons that regulate energy balance as well as GHRH-neurons that regulate linear growth. Here, the authors looked at how the transcription factors Dlx1/2 and Otp link development of AgRP- and GHRH-neurons.

Neurons in the hypothalamic arcuate nucleus (ARC) relay and translate important cues from the periphery into the central nervous system to maintain critical homeostatic processes such as energy balance, growth, and reproductive behaviors. Despite extensive studies dedicated to unraveling the physiological functions of various neuronal types in the ARC, the gene regulatory networks that drive their development are still poorly understood. This chapter provides a concise overview of the transcription factors that control the development of ARC neurons, followed by discussions of several outstanding challenges in the field and future directions to address those issues using the recent technological advances in genome-wide studies.

Despite the crucial physiological processes governed by neurons in the hypothalamic arcuate nucleus (ARC), such as growth, reproduction and energy homeostasis, the developmental pathways and regulators for ARC neurons remain understudied. Our single cell RNA-seq analyses of mouse embryonic ARC revealed many cell type-specific markers for developing ARC neurons. These markers include transcription factors whose expression is enriched in specific neuronal types and often depleted in other closely-related neuronal types, raising the possibility that these transcription factors play important roles in the fate commitment or differentiation of specific ARC neuronal types. We validated this idea with the two transcription factors, Foxp2 enriched for Ghrh-neurons and Sox14 enriched for Kisspeptin-neurons, using Foxp2- and Sox14-deficient mouse models. Taken together, our single cell transcriptome analyses for the developing ARC uncovered a panel of transcription factors that are likely to form a gene regulatory network to orchestrate fate specification and differentiation of ARC neurons., Despite the crucial physiological processes governed by neurons in the hypothalamic arcuate nucleus (ARC), the developmental pathways and regulators for ARC neurons remain understudied. In this study the authors use single cell RNA-seq analyses of mouse embryonic ARC to identify cell type-specific markers for developing ARC neurons and give key insight into the underlying developmental pathways and regulators.

SUMMARY PRC2 creates the repressive mark histone H3 Lys27 trimethylation. Although PRC2 is involved in various biological processes, its role in glial development remains ambiguous. Here, we show that PRC2 is required for oligodendrocyte (OL) differentiation and myelination, but not for OL precursor formation. PRC2-deficient OL lineage cells differentiate into OL precursors, but they fail to trigger the molecular program for myelination, highlighting that PRC2 is essential for directing the differentiation timing of OL precursors. PRC2 null OL lineage cells aberrantly induce Notch pathway genes and acquire astrocytic features. The repression of the Notch pathway restores the myelination program and inhibits abnormal astrocytic differentiation in the PRC2-deficient OL lineage, indicating that Notch is a major target of PRC2. Altogether, our studies propose a specific action of PRC2 as a novel gatekeeper that determines the glial fate choice and the timing of OL lineage progression and myelination by impinging on the Notch pathway., In Brief Wang et al. show that the polycomb repressive complex PRC2 is required for the differentiation of oligodendrocyte precursors to myelinating oligodendrocytes. They further show that PRC2 promotes oligodendrocyte differentiation and inhibits erroneous astrocytic fate by repressing the Notch pathway., Graphical Abstract

Regulation of the protein stability of epigenetic regulators remains ill-defined despite its potential applicability in epigenetic therapies. The histone H3-lysine 4-methyltransferase MLL4 is an epigenetic transcriptional coactivator that directs overnutrition-induced obesity and fatty liver formation, and Mll4(+/−) mice are resistant to both. Here we show that the E3 ubiquitin ligase UBE3A targets MLL4 for degradation thereby suppressing high fat diet (HFD)-induced expression of the hepatic steatosis target genes of MLL4. In contrast to Mll4(+/−) mice, Ube3a(+/−) mice are hypersensitive to HFD-induced obesity and fatty liver development. Ube3a(+/−);Mll4(+/−) mice lose this hypersensitivity, supporting roles of increased MLL4 levels in both phenotypes of Ube3a(+/−) mice. Correspondingly, our comparative studies with wild-type, Ube3a(+/−) and Ube3a(−/−) and UBE3A-overexpressing transgenic mouse livers demonstrate inverse-correlation of UBE3A protein levels with MLL4 protein levels, expression of the steatosis target genes of MLL4, and their decoration by H3-lysine 4-monomethylation, a surrogate marker for the epigenetic action of MLL4. Therefore, UBE3A indirectly exerts an epigenetic regulation of obesity and steatosis by degrading MLL4. This UBE3A-MLL4 regulatory axis provides a potential therapeutic venue for treating various MLL4-directed pathogeneses, including obesity and hepatic steatosis.

The hallmarks of FOXG1 syndrome, which results from mutations in a single FOXG1 allele, include cortical atrophy and corpus callosum agenesis. However, the etiology for these structural deficits and the role of FOXG1 in cortical projection neurons remain unclear. Here we demonstrate that Foxg1 in pyramidal neurons plays essential roles in establishing cortical layers and the identity and axon trajectory of callosal projection neurons. The neuron-specific actions of Foxg1 are achieved by forming a transcription complex with Rp58. The Foxg1-Rp58 complex directly binds and represses Robo1, Slit3, and Reelin genes, the key regulators of callosal axon guidance and neuronal migration. We also found that inactivation of one Foxg1 allele specifically in cortical neurons was sufficient to cause cerebral cortical hypoplasia and corpus callosum agenesis. Together, this study reveals a novel gene regulatory pathway that specifies neuronal characteristics during cerebral cortex development and sheds light on the etiology of FOXG1 syndrome. VIDEO ABSTRACT.

PURPOSE The purpose of this study was to construct an acceptance-commitment therapy (ACT)-based stress management program for inpatients with schizophrenia and to examine its effects on hospitalization stress, self-efficacy, and psychological well-being. METHODS A non-equivalent control group pretest-posttest design was used. Participants were 44 inpatients with a diagnosis of schizophrenia. The experimental group (n=22) received the ACT-based stress management program twice a week for a total of four weeks. The control group (n=22) received the usual care from their primary health care providers. The study was carried out from August 7 to September 1, 2017, and data were analyzed using IBM SPSS/WIN 22.0 with a Chi-square test, Fisher's exact test, and an independent t-test. RESULTS The experimental group showed a significant decrease in hospitalization stress (t=5.09, p

Dilated cardiomyopathy (DCM) is a progressive heart disease characterized by left ventricular dilation and contractile dysfunction. Although many candidate genes have been identified using mouse models, few of them have been shown to be associated with DCM in humans. Germline depletion of Ncoa6, a nuclear hormone receptor coactivator, leads to embryonic lethality including heart defects. However, it is unclear whether Ncoa6 mutations cause heart diseases in adults. Here, we report that two independent mouse models of NCOA6 dysfunction develop severe DCM with decreased mitochondrial number and impaired mitochondrial function and reduced levels of both activity and target gene expression of peroxisome proliferator-activated receptor-δ (PPARδ), an NCOA6 target critical for normal heart function. Sequencing of NCOA6 coding regions revealed three independent non-synonymous mutations present in 5 of 50 (10%) idiopathic DCM (iDCM) patients. These data suggest that malfunction of NCOA6 can cause DCM in humans.

Nuclear receptors (NRs) regulate diverse physiological processes, including the central nervous system control of energy balance. However, the molecular mechanisms for the central actions of NRs in energy balance remain relatively poorly defined. Here we report a hypothalamic gene network involving two NRs, neuron-derived orphan receptor 1 (NOR1) and glucocorticoid receptor (GR), which directs the regulated expression of orexigenic neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) in response to peripheral signals. Our results suggest that the anorexigenic signal leptin induces NOR1 expression likely via the transcription factor cyclic AMP response element-binding protein (CREB), while the orexigenic signal glucocorticoid mobilizes GR to inhibit NOR1 expression by antagonizing the action of CREB. Also, NOR1 suppresses glucocorticoid-dependent expression of AgRP and NPY. Consistently, relative to wild-type mice, NOR1-null mice showed significantly higher levels of AgRP and NPY and were less responsive to leptin in decreasing the expression of AgRP and NPY. These results identify mutual antagonism between NOR1 and GR to be a key rheostat for peripheral metabolic signals to centrally control energy balance.

The molecular basis underlying the physiologically well-defined orexigenic function of glucocorticoid (Gc) is unclear. Brain-specific homeobox factor (Bsx) is a positive regulator of the orexigenic neuropeptide, agouti-related peptide (AgRP), in AgRP neurons of the hypothalamic arcuate nucleus. Here, we show that in response to fasting-elevated Gc levels, Gc receptor (GR) and Bsx synergize to direct activation of AgRP transcription. This synergy is dictated by unique sequence features in a novel Gc response element in AgRP (AgRP-GRE). In contrast to AgRP-GRE, Bsx suppresses transactivation directed by many conventional GREs, functioning as a gene context-dependent modulator of GR actions or a target selector for GR. Consistent with this finding, AgRP-GRE drives fasting-dependent activation of a target gene specifically in GR(+) Bsx(+) AgRP neurons. These results define AgRP as a common orexigenic target gene of GR and Bsx and provide an opportunity to identify their additional common targets, facilitating our understanding of the molecular basis underlying the orexigenic activity of Gc and Bsx.

Neurons in the hypothalamic arcuate nucleus relay and translate important cues from the periphery into the central nervous system. However, the gene regulatory program directing their development remains poorly understood. Here we report that the LIM-homeodomain transcription factor Isl1 is expressed in several subpopulations of developing arcuate neurons and plays crucial roles in their fate specification. Mice with conditional deletion of the Isl1 gene in developing hypothalamus display severe deficits in both feeding and linear growth. Consistent with these results, their arcuate nucleus fails to express key fate markers of Isl1-expressing neurons that regulate feeding and growth. These include the orexigenic neuropeptides AgRP and NPY for specifying AgRP-neurons, the anorexigenic neuropeptide αMSH for POMC-neurons, and the two growth-stimulatory peptides, growth hormone-releasing hormone (GHRH) for GHRH-neurons and somatostatin (Sst) for Sst-neurons. Finally, we show that Isl1 directly enhances the expression of AgRP by cooperating with the key orexigenic transcription factors glucocorticoid receptor and brain-specific homeobox factor. Our results identify Isl1 as a critical transcription factor that plays critical roles in the gene regulatory program directing development of multiple arcuate neuronal subpopulations.

Combinatorial transcription codes generate the myriad of cell types during development and thus likely provide crucial insights into directed differentiation of stem cells to a specific cell type. The LIM complex composed of Isl1 and Lhx3 directs the specification of spinal motor neurons (MNs) in embryos. Here, we report that Isl1–Lhx3, a LIM-complex mimicking fusion, induces a signature of MN transcriptome and concomitantly suppresses interneuron differentiation programs, thereby serving as a potent and specific inducer of MNs in stem cells. We show that an equimolar ratio of Isl1 and Lhx3 and the LIM domain of Lhx3 are crucial for generating MNs without up-regulating interneuron genes. These led us to design Isl1–Lhx3, which maintains the desirable 1:1 ratio of Isl1 and Lhx3 and the LIM domain of Lhx3. Isl1–Lhx3 drives MN differentiation with high specificity and efficiency in the spinal cord and embryonic stem cells, bypassing the need for sonic hedgehog (Shh). RNA-seq analysis revealed that Isl1–Lhx3 induces the expression of a battery of MN genes that control various functional aspects of MNs, while suppressing key interneuron genes. Our studies uncover a highly efficient method for directed MN generation and MN gene networks. Our results also demonstrate a general strategy of using embryonic transcription complexes for producing specific cell types from stem cells.

This study analyzed the characteristics of stormwater runoff in the orchard areas and quantitatively estimated effluence of nonpoint source pollutants for the volume of runoff. Two target areas under vine cultivation were each and , located in Gyeongju City. Since grape was the only crop on the target area, the characteristics of stormwater runoff at vineyard could be evaluated independently. A total of 51 rainfall events in the vineyard area during two years(2008-2009) was surveyed, and 19 of them became stormwater runoff, with rainfall ranging 16.5 - 79.7 mm and antecedent dry period of 1-13 days. The pollutant runoff loads by volume of stormwater runoff showed BOD ranging 19.5 - 45.3% in 30% of runoff volume. The average pollution discharge rate was 32.4%, indicating small first flush effect of BOD. The range of SS concentrations was 5 - 52.0% in 10% of runoff volume, showing the average 28.7% of discharge rate, about 3 times more than rainfall effluent. TOC and TN appeared to be similar to the results of BOD, the average discharge rate of 30.9% and 30.6% for TOC and TN, respectively, for 30% of stormwater runoff volume. Average discharge rate of COD and TP in the same runoff volume was 35.1% and 36%, respectively, showing comparatively high discharge ratio. As the targeted vineyard area was permeable land, the pollution load ratio against rainfall-runoff volume appeared to be 1:1, implying no strong first flush effect for all the survey items.

ASC-2, a multifunctional coactivator, forms a steady-state complex, named ASCOM (for AS C-2 COM plex), that contains the histone H3-lysine-4 (H3K4)-methyltransferase MLL3 or its paralogue MLL4. Somewhat surprisingly, given prior indications of redundancy between MLL3 and MLL4, targeted inactivation of the MLL3 H3K4-methylation activity in mice is found to result in ureter epithelial tumors. Interestingly, this phenotype is exacerbated in a p53 +/− background and the tumorigenic cells are heavily immunostained for γH2AX, indicating a contribution of MLL3 to the DNA damage response pathway through p53. Consistent with the in vivo observations, and the demonstration of a direct interaction between p53 and ASCOM, cell-based assays have revealed that ASCOM, through ASC-2 and MLL3/4, acts as a p53 coactivator and is required for H3K4-trimethyation and expression of endogenous p53-target genes in response to the DNA damaging agent doxorubicin. In support of redundant functions for MLL3 and MLL4 for some events, siRNA-mediated down-regulation of both MLL3 and MLL4 is required to suppress doxorubicin-inducible expression of several p53-target genes. Importantly, this study identifies a specific H3K4 methytransferase complex, ASCOM, as a physiologically relevant coactivator for p53 and implicates ASCOM in the p53 tumor suppression pathway in vivo.

SummaryDuring development, two cell types born from closely related progenitor pools often express identical transcriptional regulators despite their completely distinct characteristics. This phenomenon implies the need for a mechanism that operates to segregate the identities of the two cell types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). We demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing the MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate the V2a and MN pathways. Our study uncovers a widely applicable gene regulatory principle for segregating related cell fates.

LIM homeodomain factors regulate the development of many cell types. However, transcriptional coactivators that mediate their developmental function remain poorly defined. To address these, we examined how two related NLI-dependent LIM complexes, which govern the development of spinal motor neurons and V2a interneurons, activate the transcription in the embryonic spinal cord. We found that single-stranded DNA-binding proteins are recruited to these LIM complexes via NLI, and enhance their transcriptional activation potential. Ssdp1 and Ssdp2 (Ssdp1/2) are highly expressed in the neural tube and promote motor neuron differentiation in the embryonic spinal cord and P19 stem cells. Inhibition of Ssdp1/2 activity in mouse and chick embryos suppresses the generation of motor neurons and V2a interneurons. Furthermore, Ssdp1/2 recruit histone-modifying enzymes to the motor neuron-specifying LIM complex and trigger acetylation and lysine 4 trimethylation of histone H3, which are well-established chromatin marks for active transcription. Our results suggest that Ssdp1/2 function as crucial transcriptional coactivators for LIM complexes to specify spinal neuronal identities during development.

Activating signal cointegrator 2 (ASC-2) is a transcriptional coactivator of many nuclear receptors (NRs) and other transcription factors and contains two NR-interacting LXXLL motifs (NR boxes). In the pancreas, ASC-2 is expressed only in the endocrine cells of the islets of Langerhans, but not in the exocrine cells. Thus, we examined the potential role of ASC-2 in insulin secretion from pancreatic beta-cells. Overexpressed ASC-2 increased glucose-elicited insulin secretion, whereas insulin secretion was decreased in islets from ASC-2+/- mice. DN1 and DN2 are two dominant-negative fragments of ASC-2 that contain NR boxes 1 and 2, respectively, and block the interactions of cognate NRs with the endogenous ASC-2. Primary rat islets ectopically expressing DN1 or DN2 exhibited decreased insulin secretion. Furthermore, relative to the wild type, ASC-2+/- mice showed reduced islet mass and number, which correlated with increased apoptosis and decreased proliferation of ASC-2+/- islets. These results suggest that ASC-2 regulates insulin secretion and beta-cell survival and that the regulatory role of ASC-2 in insulin secretion appears to involve, at least in part, its interaction with NRs via its two NR boxes.

The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones.

The expression of Mullerian inhibiting substance (MIS), a key molecule in sex differentiation and reproduction, is tightly regulated. It has been suggested that meiotic germ cells repress MIS expression in testicular Sertoli cells, although the substance responsible for this cell-cell communication remains unknown. Here, we present the cytokine tumor necrosis factor alpha (TNF-alpha) as a strong candidate for such a substance and its downstream molecular events. TNF-alpha inhibited MIS expression in testis organ cultures, and TNF-alpha(-/-) testes showed high and prolonged MIS expression. Furthermore, in transient-transfection assays TNF-alpha suppressed the MIS promoter that was activated by steroidogenic factor 1 (SF-1), one of the major transcription factors that regulate MIS expression. The modulation of SF-1 transactivation by TNF-alpha is through the activation of NF-kappa B, which subsequently interacts with SF-1 and represses its transactivation. The physical association of NF-kappa B with SF-1 was shown by yeast two-hybrid protein interaction, glutathione S-transferase pull-down, and coimmunoprecipitation (ChIP) analyses. ChIP assays also revealed that endogenous NF-kappa B, as well as SF-1, is recruited to the MIS promoter upon TNF-alpha signaling. SF-1-bound NF-kappa B subsequently recruits histone deacetylases to inhibit the SF-1-activated gene expression. These results may identify, for the first time, the responsible substance and its action mechanism underlying the repression of MIS expression by meiotic germ cells in the testis.

While microRNAs have emerged as an important component of gene regulatory networks, it remains unclear how microRNAs collaborate with transcription factors in the gene networks that determines neuronal cell fate. Here we show that in the developing spinal cord, the expression of miR-218 is directly upregulated by the Isl1–Lhx3 complex, which drives motor neuron fate. Inhibition of miR-218 suppresses the generation of motor neurons in both chick neural tube and mouse embryonic stem cells, suggesting that miR-218 plays a crucial role in motor neuron differentiation. Results from unbiased RISC-trap screens, in vivo reporter assays and overexpression studies indicated that miR-218 directly represses transcripts that promote developmental programs for interneurons. In addition, we found that miR-218 activity is required for Isl1–Lhx3 to effectively induce motor neurons and suppress interneuron fates. Together our results reveal an essential role of miR-218 as a downstream effector of the Isl1–Lhx3 complex in establishing motor neuron identity. microRNAs have emerged as important components of numerous gene regulatory networks. Here the authors demonstrate that miR-218 is an essential component of the gene regulatory network that controls motor neuron fate specification in the developing spinal cord.

Activating signal cointegrator 1 (ASC-1) has been recently reported as a coactivator of some nuclear receptors. In the present study, we have analyzed the expression of ASC-1 in the mouse testis and investigated its capacity to modulate the transcriptional activity of androgen receptor (AR). We found that although ASC-1 mRNA was ubiquitously expressed at a low level in mouse tissues, a couple of testis-specific mRNAs were expressed in the adult testis. Cloning of one testis-specific variant revealed that the ubiquitous and testis-specific transcripts of ASC-1 share at least the same open reading frame. The expression of the testis-specific ASC-1 mRNAs was developmentally regulated, and the onset of their expression coincided with the initiation of spermatogenesis. In situ hybridization of mouse testis with ASC-1 antisense probe demonstrated predominant expression of ASC-1 in the interstitial Leydig cells that express AR. Moreover, yeast two-hybrid tests and glutathione S-transferase pull-down assays revealed that ASC-1 associates directly with AR and that the hinge domain of AR and a putative zinc-finger motif of ASC-1 are major determinants for their interaction. Transient transfection assays performed by expressing ASC-1 in combination with AR and an androgen-responsive reporter gene showed that ASC-1 moderately alters the induction of the reporter gene. Taken together, these results suggest that ASC-1 may function as an AR coregulator and have a role in testicular functions.

The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development., Author Summary Neurons utilize various chemicals to transmit signals to a target cell. Distinct types of neurons in the spinal cord and forebrain, collectively termed cholinergic neurons, utilize the same chemical, acetylcholine, for signal transmission. These neurons play critical roles in controlling locomotion and cognition. In this study, we have found that the Isl1 gene orchestrates the process to generate cholinergic neurons in the spinal cord and forebrain. Isl1 forms two different types of multi-protein complexes in the spinal cord and forebrain. Both complexes bind the same genomic regions in a group of genes critical for cholinergic signal transmission, and promote their simultaneous expression. These cholinergic genes include enzymes that synthesize acetylcholine and proteins required to package acetylcholine into vesicles. The Isl1-containing multi-protein complexes were able to trigger the generation of cholinergic neurons in embryonic stem cells and neural stem cells. Our study reveals crucial mechanisms to coordinate the expression of genes in the same biological pathway in different cell types. Furthermore, it suggests a new strategy to produce cholinergic neurons from stem cells.

For the orphan nuclear receptor subfamily that includes Nur77 (NGFI-B), Nurr1, and NOR-1, no transcriptional coregulators have been identified thus far. In this report, we found that Ca(2+)/calmodulin-dependent protein kinase IV enhances Nur77 transactivation in cotransfections either alone or in synergy with AF2dependent coactivator ASC-2, whereas corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) is repressive. Interestingly, Nur77 interacted with SMRT but did not directly bind ASC-2, and accordingly, the putative AF2 core domain of Nur77 did not affect the Nur77 transactivation. SMRT harbors transferable repression domains that associate with various histone deacetylases. Surprisingly, histone deacetylase inhibitor trichostatin A was unable to block the repressive effect of SMRT while dramatically stimulating the Nur77 transactivation. These results suggest that SMRT and ASC-2 are specific coregulators of Nur77 and that SMRT may dynamically compete with a putative adaptor molecule, which links ASC-2 to Nur77, for the identical binding sites within Nur77 in vivo.

Transcriptional coactivators SRC-1 and p300 specifically interact with liganded-nuclear receptors and also modulate other transcription factors, including serum response factor (SRF). Here, we report that retinoids repress transactivation by SRF and specific interactions exist between the DNA binding domains of SRF and retinoic acid and retinoid X receptors. We further demonstrate that the repression may involve retinoid-dependent competition for a limiting amount of SRC-1 and p300 between SRF and retinoid receptors. We propose that the well-defined anti-proliferative action of retinoids could, at least in part, result from this novel transrepressive action on the mitogenic transcription factor SRF.

Silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is known to interact with Sin3 and recruit the histone deacetylases (HDACs) that lead to hypoacetylation of histones and transrepression of target transcription factors. Herein, we found that coexpression of SMRT significantly repressed transactivations by activating protein-1 (AP-1), nuclear factor-kappaB (NFkappaB), and serum response factor (SRF) in a dose-dependent manner, but not in the presence of trichostatin A, a specific inhibitor of HDAC. Similarly, coexpression of HDAC1 and mSin3A also showed repressive effects. Consistent with these results, the C-terminal region of SMRT directly interacted with SRF, the AP-1 components c-Jun and c-Fos, and the NFkappaB components p50 and p65, as demonstrated by the yeast and mammalian two hybrid tests as well as the glutathione S-transferase pull down assays. Thus, we concluded that SMRT serves to recruit Sin3/HDACs to SRF, NFkappaB, and AP-1 in vivo and modulate their transactivation.

Small guanosine triphosphatases, typified by the mammalian Ras proteins, play major roles in the regulation of numerous cellular pathways. A subclass of evolutionarily conserved Ras-like proteins was identified, members of which differ from other Ras proteins in containing amino acids at positions 12 and 61 that are similar to those present in the oncogenic forms of Ras. These proteins, kappaB-Ras1 and kappaB-Ras2, interact with the PEST domains of IkappaBalpha and IkappaBbeta [inhibitors of the transcription factor nuclear factor kappa B (NF-kappaB)] and decrease their rate of degradation. In cells, kappaB-Ras proteins are associated only with NF-kappaB:IkappaBbeta complexes and therefore may provide an explanation for the slower rate of degradation of IkappaBbeta compared with IkappaBalpha.

Control of oestradiol-responsive gene regulation by oestrogen receptors (ERs) may involve complex cross-talk with retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Recently, we have shown that ERalpha directly interacts with RARalpha and RXRalpha through their ligand binding domains (LBDs). In the present work, we extend these results by showing that ERbeta binds similarly to RARalpha and RXRalpha but not to the glucocorticoid receptor, as demonstrated by the yeast two-hybrid tests and glutathione S-transferase pull-down assays. These direct interactions were also demonstrated in gel-shift assays, in which the oestrogen response element (ERE) binding by ERalpha was enhanced by the RXRalpha LBD but was abolished by the RARalpha LBD. In addition, we showed that RARalpha and RXRalpha bound the ERE as efficiently as ERalpha, suggesting that competition for DNA binding may affect the transactivation function of the ER. In transient transfection experiments, co-expression of RARalpha or RXRalpha, along with ERalpha or ERbeta, revealed differential modulation of the ERE-dependent transactivation, which was distinct from the results when each receptor alone was co-transfected. Importantly, when the LBD of RARalpha was co-expressed with ERalpha, transactivation of ERalpha on the ERE was repressed as efficiently as when wild-type RARalpha was co-expressed. Furthermore, liganded RARalpha or unliganded RXRalpha enhanced the ERalpha transactivation, suggesting the formation of transcriptionally active heterodimer complexes between the ER and retinoid receptors. Taken together, these results suggest that direct protein-protein interactions may play major roles in the determination of the biological consequences of cross-talk between ERs and RARalpha or RXRalpha.

Steroid receptor coactivator-1 (SRC-1) specifically bound to serum response factor (SRF), as demonstrated by glutathione S-transferase pull down assays, and the yeast and mammalian two-hybrid tests. In mammalian cells, SRC-1 potentiated serum response element (SRE)-mediated transactivations in a dose-dependent manner. Coexpression of p300 synergistically enhanced this SRC-1-potentiated level of transactivations, consistent with the recent finding (Ramirez, S., Ali, S. A. S., Robin, P., Trouche, D., and Harel-Bellan, A. (1997) J. Biol. Chem. 272, 31016-31021) in which the p300 homologue CREB-binding protein was shown to be a transcription coactivator of SRF. Thus, we concluded that at least two distinct classes of coactivator molecules may cooperate to regulate SRF-dependent transactivations in vivo.

The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord.

Metabolic changes in cancer have been observed for almost a century. The mechanisms underlying these changes have begun to emerge from the recent studies implicating the tumor suppressor p53 in multiple metabolic pathways. The ability of p53 to regulate metabolism may also play important roles in the physiology of normal cells and organs. Here we demonstrate that p53 lowers bile acid (BA) levels under both normal and stressed conditions primarily through up-regulating expression of small heterodimer partner, a critical inhibitor of BA synthesis. Our results uncover a unique metabolic regulatory axis that unexpectedly couples p53 to BA homeostasis. Our results also warrant future studies to investigate a possible role of this axis in the tumor suppression by p53, because excessive quantities of BAs are cytotoxic and can cause liver damage and promote gastrointestinal cancers.

The steroid hormone-inducible promoter from mouse mammary tumor virus is associated with a distal negative regulatory element that represses its inherent basal activity. Deletion analysis localized the sequences required for repression to 64 base pairs of DNA between -427 and -364 with respect to the transcription initiation site. Transient transfection experiments with a series of linker scanning and small internal deletion mutations revealed two mutation-sensitive domains separated by a region of relative resistance to sequence alterations. DNase I footprinting and gel electrophoresis mobility shift experiments with crude nuclear extracts identified at least one protein-binding site within each of the two mutation-sensitive regions. An oligonucleotide corresponding to one of these sites is able to repress transcription, but only when linked to the promoter in multiple copies. This negative regulatory element functions synergistically with a promoter proximal negative element to mediate efficient promoter repression, selectively affecting basal relative to steroid hormone-induced transcription and thus increasing the ratio of promoter activity observed in the presence and absence of hormone.

Summary Spinal motor neurons (MNs) and V2 interneurons (V2-INs) are specified by two related LIM-complexes, MN-hexamer and V2-tetramer, respectively. Here we show how multiple parallel and complementary feedback loops are integrated to assign these two cell fates accurately. While MN-hexamer response elements (REs) are specific to MN-hexamer, V2-tetramer-REs can bind both LIM-complexes. In embryonic MNs, however, two factors cooperatively suppress the aberrant activation of V2-tetramer-REs. First, LMO4 blocks V2-tetramer assembly. Second, MN-hexamer induces a repressor, Hb9, which binds V2-tetramer-REs and suppresses their activation. V2-INs use a similar approach; V2-tetramer induces a repressor, Chx10, which binds MN-hexamer-REs and blocks their activation. Thus, our study uncovers a regulatory network to segregate related cell fates, which involves reciprocal feedforward gene regulatory loops.

Activating signal cointegrator-2 (ASC-2), a cancer-amplified transcription coactivator of nuclear receptors and numerous other transcription factors, was previously shown to contain two LXXLL motifs, each of which interacts with a distinct set of nuclear receptors. In this work, we showed that ASC-2 has an indirect, separate binding site for androgen receptor (AR). Interestingly, this region overlapped with the direct interaction interfaces with the tumor suppressor retinoblastoma (Rb). Although ASC-2 alone stimulated AR transactivation in cotransfections of HeLa cells, ectopic expression of Rb effected ASC-2 to act as a transcription coactivator of AR in Rb-null Saos2 cells. These results, along with the previous report in which AR was shown to directly interact with Rb (Yeh, S., Miyamoto, H., Nishimura, K., Kang, H., Ludlow, J., Hsiao, P., Wang, C., Su, C., and Chang C. (1998) Biochem. Biophys. Res. Commun. 248, 361-367), suggest that the AR-ASC-2 interactions in vivo may involve Rb. Thus, ASC-2 appears to contain at least three distinct nuclear receptor interaction domains.

Mucoceles are relatively common cystic lesions of the paranasal sinuses. However, mucocele of the nasal septum is extremely rare. We report a case of a mucocele present in this unusual location. Mucocele of the nasal septum should be considered in the differential diagnosis of a mass of the nasal septum and/or median canthal region. Nasal septal mucocele can be effectively treated with endoscopic marsupialization or complete excision.

BLK mouse fibroblasts (H-2b) were genetically engineered to express costimulatory B7.1 and interleukin-2 (BLK/IL2/B7.1). The BLK/IL2/B7.1 cells were then pulsed with an ovalbumin (OVA) epitope as a model antigen (Ag) (BLK/IL2/B7.1/OVA), and tested for the induction of OVA-specific cytotoxic T lymphocytes (CTLs) in C57BL/6 mice (H-2b). The genetically engineered fibroblasts lacking one or two of three factors (interleukin-2, B7.1, and OVA) were constructed and used as controls. Immunization with the BLK/IL2/B7.1/OVA cells induced strong cytotoxic activities against OVA-expressing EL4 (EG7) tumor cells, but not against other H-2b tumor cells, such as EL4, C1498 and B16F1 cells. The magnitude of the cytotoxic response in mice with the BLK/IL2/B7.1/OVA cells was significantly higher than the response in mice immunized with any other cell constructs. CD8+ T cells with OVA-specific cytotoxic activities were predominant in mice immunized with the BLK/IL2/B7.1/OVA cells. Furthermore, immunization with the BLK/IL2/B7.1/OVA cells significantly prolonged the survival of mice, compared with any other cell constructs, when the mice were challenged with EG7 tumor cells at 2 weeks postimmunization. Induction of antitumoral CTL immunity by the BLK/IL2/B7.1/OVA cells was independent of host Ag-presenting cells and of CD4+ T-cell and natural killer 1.1+ cell help. These results suggest that fibroblasts can be genetically modified to efficient Ag-presenting cells for the induction of an Ag-specific CTL response.

Bcl3, an IkappaB protein, was originally isolated as a putative proto-oncogene in a subset of B cell chronic lymphocytic leukemias. Bcl3 was subsequently shown to associate tightly with and transactivate the NFkappaB p50 or p52 homodimer. Herein, we show that Bcl3 stimulates the activating protein-1 (AP-1) transactivation, either alone or in conjunction with transcription integrators steroid receptor coactivator-1 and CREB-binding protein/p300. The C-terminal 158 residues of Bcl3 exhibited an autonomous transactivation function and interacted with specific subregions of the AP-1 components c-Jun and c-Fos, CREB-binding protein/p300, and steroid receptor coactivator-1, as demonstrated by the yeast and mammalian two-hybrid tests as well as glutathione S-transferase pull-down assays. In addition, anti-HA antibody co-precipitated c-Jun from HeLa cells co-expressing c-Jun and HA-tagged Bcl3, consistent with the idea that Bcl3 directly associates with AP-1 in vivo. Furthermore, microinjection of Bcl3 expression vector into Rat-1 fibroblast cells significantly enhanced DNA synthesis and expression of c-jun, one of the cellular target genes of AP-1. These results suggest that Bcl3 may directly participate in the tumorigenesis processes as a novel transcription coactivator of the mitogenic transcription factor AP-1 in vivo.

To elucidate the molecular action of the NFkappaB inhibitor IkappaBbeta, we isolated a number of IkappaBbeta interactors using the yeast two-hybrid system. These include the retinoid X receptor (RXR), whose interaction with IkappaBbeta is significantly stimulated by the RXR ligand 9-cis-retinoic acid, as shown in the yeast system as well as the glutathione S-transferase pull down assays. RXR is a nuclear protein, whereas IkappaBbeta accumulates in the nucleus only in cells stimulated with lipopolysaccharide or other inducers that result in prolonged activation of NFkappaB. Consistent with this, cotransfection with IkappaBbeta specifically repressed the 9-cis-RA-induced transcriptional activities of RXR in an lipopolysaccharide-dependent manner. These results suggest a novel IkappaBbeta-mediated antagonism between the signaling pathways of NFkappaB and RXR.

Guillain-Barre syndrome (GBS) presenting as bilateral vocal cord paralysis is extremely rare. We report an unusual case of GBS in which the patient manifested hoarseness resulting from bilateral vocal cord paralysis. In conclusion, GBS needs to be considered as possible causes of new onset bilateral vocal cord paralysis. We emphasize that early recognition of atypical presentations of GBS warrants further evaluation and appropriate management. � Korean J Otorhinolaryngol-Head Neck Surg 2013;56:169-71 Key WordsZZGuillain-Barre syndrome ㆍIntravenous immunoglobulin ㆍVocal cord paralysis.

Amyloidosis is a condition of abnormal deposition of extracellular insoluble fibrillar proteins, termed amyloid, in tissue and organs throughout the body. Systemic amyloidosis, consisting of light amyloid protein (AL protein), the spectrum of multiple myeloma and plasmacytoma, is categorized as a primary amyloidosis; secondary amyloidosis consists of an acute phase reactant serum amyloid A protein (AA protein), which is associated with neoplasm. Amyloid involvement of the tongue is almost always secondary to systemic AL amyloidosis. Macroglossia due to amyloid depositions can lead to serious airway obstruction. We report a case of a 68-year-old woman suffering from dyspnea due to macroglossia. She was diagnosed with amyloidosis associated with multiple myeloma. � Korean J Otorhinolaryngol-Head Neck Surg 2013;56:37-40 Key WordsZZAmyloidosis ㆍMacroglossia ㆍMultiple Myeloma.

Thyroid hormone receptors (TRs) bind as dimers to specific DNA response elements. We have used a genetic approach to identify amino acid sequences required for dimerization of the TR beta isoform. Bacteria expressing a chimeric repressor composed of the DNA binding domain of the bacteriophage lambda cl repressor fused to the TR beta ligand binding domain are immune to lambda infection as a consequence of homodimerization activity provided by the receptor sequences. The phenotypes of deletions and point mutations of the TR beta sequences map dimerization activity to a subregion of the ligand binding domain that is highly conserved among all members of the nuclear hormone receptor superfamily. These results confirm and extend previous findings indicating that this subregion plays an important role in the dimerization of TR beta and other superfamily members.

The pathophysiologic continuum of non-alcoholic fatty liver disease begins with steatosis. Despite recent advances in our understanding of the gene regulatory program directing steatosis, how it is orchestrated at the chromatin level is unclear. PPAR gamma 2 is a hepatic steatotic transcription factor induced by overnutrition. Here, we report that the histone H3 lysine 4 methyltransferase MLL4/KMT2D directs overnutrition-induced murine steatosis via its coactivator function for PPAR gamma 2. We demonstrate that overnutrition facilitates the recruitment of MLL4 to steatotic target genes of PPAR gamma 2 and their transactivation via H3 lysine 4 methylation because PPAR gamma 2 phosphorylated by overnutrition-activated ABL1 kinase shows enhanced interaction with MLL4. We further show that Ppar gamma 2 (encoding PPAR gamma 2) is also a hepatic target gene of ABL1-PPAR gamma 2-MLL4. Consistently, inhibition of ABL1 improves the fatty liver condition of mice with overnutrition by suppressing the pro-steatotic action of MLL4. Our results uncover a murine hepatic steatosis regulatory axis consisting of ABL1-PPAR gamma 2-MLL4, which may serve as a target of anti-steatosis drug development.

Summary Multiple excitatory and inhibitory interneurons form the motor circuit with motor neurons in the ventral spinal cord. Notch signaling initiates the diversification of immature V2-interneurons into excitatory V2a-interneurons and inhibitory V2b-interneurons. Here, we provide a transcriptional regulatory mechanism underlying their balanced production. LIM-only protein LMO4 controls this binary cell fate choice by regulating the activity of V2a- and V2b-specific LIM complexes inversely. In the spinal cord, LMO4 induces GABAergic V2b-interneurons in collaboration with SCL and inhibits Lhx3 from generating glutamatergic V2a-interneuons. In LMO4;SCL compound mutant embryos, V2a-interneurons increase markedly at the expense of V2b-interneurons. We further demonstrate that LMO4 nucleates the assembly of a novel LIM-complex containing SCL, Gata2, and NLI. This complex activates specific enhancers in V2b-genes consisting of binding sites for SCL and Gata2, thereby promoting V2b-interneuron fate. Thus, LMO4 plays essential roles in directing a balanced generation of inhibitory and excitatory neurons in the ventral spinal cord.