A single-cell atlas of chromatin accessibility in the human genome.

Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with previous single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for ∼1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell-type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems.

Single-cell analysis of chromatin accessibility in the adult mouse brain.

Recent advances in single-cell technologies have led to the discovery of thousands of brain cell types; however, our understanding of the gene regulatory programs in these cell types is far from complete1-4. Here we report a comprehensive atlas of candidate cis-regulatory DNA elements (cCREs) in the adult mouse brain, generated by analysing chromatin accessibility in 2.3 million individual brain cells from 117 anatomical dissections. The atlas includes approximately 1 million cCREs and their chromatin accessibility across 1,482 distinct brain cell populations, adding over 446,000 cCREs to the most recent such annotation in the mouse genome. The mouse brain cCREs are moderately conserved in the human brain. The mouse-specific cCREs-specifically, those identified from a subset of cortical excitatory neurons-are strongly enriched for transposable elements, suggesting a potential role for transposable elements in the emergence of new regulatory programs and neuronal diversity. Finally, we infer the gene regulatory networks in over 260 subclasses of mouse brain cells and develop deep-learning models to predict the activities of gene regulatory elements in different brain cell types from the DNA sequence alone. Our results provide a resource for the analysis of cell-type-specific gene regulation programs in both mouse and human brains.

DNA methylation atlas of the mouse brain at single-cell resolution.

Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing1,2 to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data3 enabled prediction of high-confidence enhancer-gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments4. By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum.

An atlas of gene regulatory elements in adult mouse cerebrum.

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures1. Recent surveys of mouse and human brains with single-cell transcriptomics2-6 and high-throughput imaging technologies7,8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis-regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis-regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans.

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis.

Integrated analysis of single-cell chromatin state and transcriptome identified common vulnerability despite glioblastoma heterogeneity.

In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed in vitro and in vivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.

Single Cell Multiomics Identifies Cells and Genetic Networks Underlying Alveolar Capillary Dysplasia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV (n = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.

Chemoproteomics reveals baicalin activates hepatic CPT1 to ameliorate diet-induced obesity and hepatic steatosis.

Obesity and related metabolic diseases are becoming worldwide epidemics that lead to increased death rates and heavy health care costs. Effective treatment options have not been found yet. Here, based on the observation that baicalin, a flavonoid from the herbal medicine Scutellaria baicalensis, has unique antisteatosis activity, we performed quantitative chemoproteomic profiling and identified carnitine palmitoyltransferase 1 (CPT1), the controlling enzyme for fatty acid oxidation, as the key target of baicalin. The flavonoid directly activated hepatic CPT1 with isoform selectivity to accelerate the lipid influx into mitochondria for oxidation. Chronic treatment of baicalin ameliorated diet-induced obesity (DIO) and hepatic steatosis and led to systemic improvement of other metabolic disorders. Disruption of the predicted binding site of baicalin on CPT1 completely abolished the beneficial effect of the flavonoid. Our discovery of baicalin as an allosteric CPT1 activator opens new opportunities for pharmacological treatment of DIO and associated sequelae.

Study on Active Components of Cuscuta chinensis Promoting Neural Stem Cells Proliferation: Bioassay-Guided Fractionation.

Neural stem cells (NSCs) exist in the central nervous system of adult animals and capable of self-replication. NSCs have two basic functions, namely the proliferation ability and the potential for multi-directional differentiation. In this study, based on the bioassay-guided fractionation, we aim to screen active components in Cuscuta chinensis to promote the proliferation of NSCs. CCK-8 assays were used as an active detection method to track the active components. On the basis of isolating active fraction and monomer compounds, the structures of these were identified by LC-MS and (1H, 13C) NMR. Moreover, active components were verified by pharmacodynamics and network pharmacology. The system solvent extraction method combined with the traditional isolation method were used to ensure that the fraction TSZE-EA-G6 of Cuscuta chinensis exhibited the highest activity. Seven chemical components were identified from the TSZE-EA-G6 fraction by UPLC-QE-Orbitrap-MS technology, which were 4-O-p-coumarinic acid, chlorogenic acid, 5-O-p-coumarinic acid, hyperoside, astragalin, isochlorogenic acid C, and quercetin-3-O-galactose-7-O-glucoside. Using different chromatographic techniques, five compounds were isolated in TSZE-EA-G6 and identified as kaempferol, kaempferol-3-O-glucoside (astragalin), quercetin-3-O-galactoside (hyperoside), chlorogenic acid, and sucrose. The activity study of these five compounds showed that the proliferation rate of kaempferol had the highest effects; at a certain concentration (25 µg/mL, 3.12 µg/mL), the proliferation rate could reach 87.44% and 59.59%, respectively. Furthermore, research results using network pharmacology techniques verified that kaempferol had an activity of promoting NSCs proliferation and the activity of flavonoid aglycones might be greater than that of flavonoid glycosides. In conclusion, this research shows that kaempferol is the active component in Cuscuta chinensis to promote the proliferation of NSCs.

[Study on therapeutic mechanism of Rehmanniae Radix Praeparata- Corni Fructus in sequelae of ischemic stroke based on network pharmacology technology].

In ischemic stroke sequela phase, Rehmanniae Radix Praeparata-Corni Fructus drug pair has the effect in protecting damaged neurons, but its mechanism has not been clear. In this study, network pharmacology was used to predict the mechanism of Rehmanniae Radix Praeparata-Corni Fructus in the treatment of ischemic stroke sequela. Through database search and literature retrie-val, 40 active ingredients of Rehmanniae Radix Praeparata and Corni Fructus were obtained, and their targets were obtained through STITCH and TCMSP databases. The targets of ischemic stroke sequela were obtained through OMIM,GAD,TTD and DrugBank databases. By screening the intersections of active ingredients targets and stroke treatment targets, 21 potential targets were obtained. The DAVID database was used for GO enrichment analysis and KEGG pathway analysis of potential targets. GO enrichment analysis showed that Rehmanniae Radix Praeparata-Corni Fructus were mainly involved in regulation of blood pressure, negative regulation of extrinsic apoptotic signaling and positive regulation of angiogenesis. KEGG pathway analysis showed that Rehmanniae Radix Praeparata-Corni Fructus could inhibit inflammatory response and apoptosis signaling pathway by regulating HIF-VEGFA signaling pathway in neural stem cell proliferation, TNF signaling pathway and NF-kappaB signaling pathway. Molecular docking technique was used to verify that Rehmanniae Radix Praeparata-Corni Fructus component has a good binding activity with potential targets. The results showed that in ischemic stroke sequela phase, Rehmanniae Radix Praeparata-Corni Fructus drug pair could play an important role in recovering neural function, promoting the proliferation of neural stem cells, angiogenesis, preventing neural cells apoptosis and regulating inflammatory factors.

Quantitative and Site-Specific Chemoproteomic Profiling of Targets of Acrolein.

Acrolein exists in common pollutants, such as cigarette smoke and car exhaust, which has been implicated with many pathological processes. It is also one type of endogenous lipid-derived electrophile (LDE) generated from lipid peroxidation when cells are under oxidative stress. Chemically, acrolein is able to covalently modify nucleophilic residues in proteins so as to influence their structures and functions, and identification of targets of acrolein modification in proteomes is critical for understanding its biological roles. Here, we report a quantitative chemoproteomic method to globally profile acrolein modifications using an aldehyde-directed aniline-based probe. Collectively, we identified >2300 proteins and >500 cysteine sites that are targeted by acrolein. Our data provide valuable information for understanding acrolein-mediated toxicity and expanding our knowledge of oxidative stress-mediated damage and signaling.

Pulmonary cryptococcosis characteristics in immunocompetent patients-A 20-year clinical retrospective analysis in China.

BACKGROUND: Pulmonary cryptococcosis (PC) is not considered an rare, opportunistic infection anymore. The immunocompetent population accounts for an increasing proportion of the morbidity. OBJECTIVE: This study investigated the clinical characteristics of PC patients spanning 20 years, in a referral centre of China. PATIENTS/METHODS: We retrospectively investigated the clinical data of 99 patients with PC who were diagnosed at Peking Union Medical College Hospital (PUMCH) from January 1998 to December 2017. RESULTS: Pulmonary cryptococcosis incidence in PUMCH has seen sharp increase in two decades. There were 40.4% (40/99), 17.2% (17/99) and 42.4% (42/99) immunocompetent, mildly immunocompromised and severe immunocompromised patients, respectively. Significantly higher (P = .035) male predominance in immunocompetent and mildly immunocompromised groups (68.4%, 39/57) compared with severe immunocompromised group (45.2%, 19/42) was found. Overall, 27.5% (11/40) immunocompetent patients reported a significant difference (P = .02) in history of more than weekly drinking, higher than mildly or severe immunocompromised. No significant difference occurred in symptoms and radiographic characteristics among the groups. In pulmonary computerised tomography findings, the non-air pathway feature was the dominant distribution characteristics in all patients with PC (P = .002). The gap in body dissemination frequency between immunocompetent combined with mildly immunocompromised (5.26%, 3/57) and severe immunocompromised (19.0%, 8/42) was marginally significant (P = .05). CONCLUSIONS: Gender and alcohol drinking could be PC risk factors of concern in patients without severe immunodeficiency. No significant difference occurred in symptoms or radiographic characteristics between patients with different levels of immune status. The unique radiographic non-air pathway distribution in the lung may be the feature of Cryptococcus invasion that may enhance accurate diagnosis.

IgG4-related disease can present as recurrent spontaneous hemothorax: a case report.

BACKGROUND: Immunoglobulin G4-related disease (IgG4-RD) encompasses a group of immune-mediated disorders that are gaining increasing recognition. Pulmonary presentations are common, with four types of patterns been described on radiography, including solid nodular, bronchovascular, ground glass opacities, and alveolar interstitial. Pleural thickening and pleural effusion have also been reported. However, there have been no reports of IgG4-RD that presents as spontaneous hemothorax. CASE PRESENTATION: A 61-year-old Chinese woman experienced recurrent right-sided chest pain and transient syncope. A significant decrease in her hemoglobin level and thick bloody pleural fluid demonstrated spontaneous hemothorax. The elevated serum IgG4 and histopathological analysis of the right pleura and intercostal lymph node specimens all supported the diagnosis of IgG4-RD in this patient. Further diagnostic evaluation did not reveal other causes for spontaneous hemothorax. She received steroids and no recurrent bleeding event occurred during a follow-up period of more than 1 year. CONCLUSION: Recurrent spontaneous hemothorax can be a rare manifestation of IgG4-RD, with pleural involvement as the most probable mechanism.

Quantitative Profiling of Protein Carbonylations in Ferroptosis by an Aniline-Derived Probe.

Ferroptosis is a regulated form of necrotic cell death implicated in carcinogenesis and neurodegeneration that is driven by phospholipid peroxidation. Lipid-derived electrophiles (LDEs) generated during this process can covalently modify proteins ("carbonylation") and affect their functions. Here we report the development of a quantitative chemoproteomic method to profile carbonylations in ferroptosis by an aniline-derived probe. Using the method, we established a global portrait of protein carbonylations in ferroptosis with >400 endogenously modified proteins and for the first time, identified >20 residue sites with endogenous LDE modifications in ferroptotic cells. Specifically, we discovered and validated a novel cysteine site of modification on voltage-dependent anion-selective channel protein 2 (VDAC2) that might play an important role in sensitizing LDE signals and mediating ferroptosis. Our results will contribute to the understanding of ferroptotic signaling and pathogenesis and provide potential biomarkers for ferroptosis detection.

Sialyltransferase-Based Chemoenzymatic Histology for the Detection of N- and O-Glycans.

Profiling specific glycans in histopathological samples is hampered by the lack of selective and sensitive tools for their detection. Here, we report on the development of chemoenzymatic histology of membrane polysaccharide (CHoMP)-based methods for the detection of O- and N-linked glycans on tissue sections via the use of sialyltransferases ST3Gal1 and ST6Gal1, respectively. Combining these two methods, we developed tandem labeling and double labeling strategies that permit the detection of unsialylated and sialylated glycans or the detection of O- and N-linked glycans on the same tissue section, respectively. We applied these methods to screen murine tissue specimens, human multiple-organ cancer arrays, and lymphoma and prostate cancer arrays. Using tandem labeling with ST6Gal1 to analyze N-glycans in a prostate cancer array, we found striking differences in expression patterns of both sialylated and unsialylated N-glycans between cancerous and healthy samples. Such differences were also observed between normal tissue from healthy donors and healthy tissue adjacent to tumors. Our double labeling technique identified significant differences in unsialylated O-glycans between B-cell and T-cell lymphomas and between B-cell lymphomas and normal adjacent lymph nodes. Remarkable differences were also detected between adjacent lymph nodes and spleen tissue samples. These new chemoenzymatic histology methods therefore provide valuable tools for the analysis of glycans in clinically relevant tissue samples.

A Chemoenzymatic Histology Method for O-GlcNAc Detection.

Modification of nuclear and cytoplasmic proteins by the addition or removal of O-GlcNAc dynamically impacts multiple biological processes. Here, we present the development of a chemoenzymatic histology method for the detection of O-GlcNAc in tissue specimens. We applied this method to screen murine organs, uncovering specific O-GlcNAc distribution patterns in different tissue structures. We then utilized our histology method for O-GlcNAc detection in human brain specimens from healthy donors and donors with Alzheimer's disease and found higher levels of O-GlcNAc in specimens from healthy donors. We also performed an analysis using a multiple cancer tissue array, uncovering different O-GlcNAc levels between healthy and cancerous tissues, as well as different O-GlcNAc cellular distributions within certain tissue specimens. This chemoenzymatic histology method therefore holds great potential for revealing the biology of O-GlcNAc in physiopathological processes.

Biopsy-proven IgG4-related lung disease.

BACKGROUND: Immunoglobulin G4-related disease (IgG4-RD) is a fibroinflammatory disorder that may involve single or multiple organs. Biopsy-proven lung involvement of this disease is occasionally reported, but not well understood. METHODS: Patients with the diagnosis of biopsy-proven IgG4-related lung disease (IgG4-RLD) from Peking Union Medical College Hospital between January 2011 and July 2015 were retrospectively analyzed. Age, sex, clinical symptoms, laboratory findings, pulmonary function test results, chest CT tests, positron emission tomography (PET) examinations, treatments and prognoses were retrieved from medical records and analyzed. RESULTS: Seventeen patients were included in this study (mean age: 44.8 ± 15.0 years). Ten patients were diagnosed via surgery, and 7 patients were diagnosed via percutaneous transthoracic core-needle lung biopsy. Extrapulmonary involvement was observed in only one patient. The clinical symptoms included cough, fever, dyspnea, chest pain and hemoptysis. The serum IgG4 concentration was elevated in 7/13 patients (mean: 1955 ± 1968 mg/L). The chest CT findings included mainly nodules and masses with spiculated borders, alveolar consolidations with air bronchograms, and ground glass opacities with or without reticular opacities. PET scans indicated increased standardized uptake values, and 7/8 patients were correctly diagnosed with benign inflammation. Corticosteroids and immunosuppressants were administered to 14/17 patients and effectively alleviated the disease. CONCLUSIONS: In biopsy-proven IgG4-RLD, a normal serum IgG4 concentration is commonly seen, while extrapulmonary involvement is infrequent. Alveolar consolidation with air bronchograms is an important imaging finding of IgG4-RLD, which has not been emphasized before.

Drosophila miR-932 modulates hedgehog signaling by targeting its co-receptor Brother of ihog.

Hedgehog (Hh) proteins act as morphogens in a variety of developmental contexts to control cell fates and growth in a concentration-dependent manner. Therefore, secretion, distribution, and reception of Hh proteins must be tightly regulated and deregulation of these processes contributes to numerous human diseases. Brother of ihog (Boi) and its close relative Ihog (Interference hedgehog) are cell surface proteins that act as Hh co-receptors required for Hh signaling response and cell-surface maintenance of Hh protein. MicroRNAs (miRNAs) are a group of widely expressed 21-23 nucleotides non-coding RNAs that repress gene function through interactions with target mRNAs. Here, we have identified a novel miRNA, miR-932, as an important regulator for Boi. We show that overexpression of miR-932 in the wing disc can enhance Hh signaling strength, but reduce its signaling range, a phenotype similar to that of boi knockdown. In both in vivo sensor assay and in vitro luciferase assay, miR-932 can suppress Boi by directly binding to its 3'UTR. Meanwhile, down-regulation of miR-932 by sponge elevates the protein level of Boi, confirming that miR-932 is an in vivo regulator of Boi expression. Further, we demonstrate that miR-932 can block Hh signaling when co-expressed with ihog-RNAi. Moreover, we find that other predicted miRNAs of Boi fail to suppress it as strong as miR-932. Taken together, our data demonstrate that miR-932 can modulate Hh activity by specifically targeting Boi in Drosophila, illustrating the important roles of miRNAs in fine regulation of the Hh signaling pathway.