RESUMO
Scene classification in autonomous navigation is a highly complex task due to variations, such as light conditions and dynamic objects, in the inspected scenes; it is also a challenge for small-factor computers to run modern and highly demanding algorithms. In this contribution, we introduce a novel method for classifying scenes in simultaneous localization and mapping (SLAM) using the boundary object function (BOF) descriptor on RGB-D points. Our method aims to reduce complexity with almost no performance cost. All the BOF-based descriptors from each object in a scene are combined to define the scene class. Instead of traditional image classification methods such as ORB or SIFT, we use the BOF descriptor to classify scenes. Through an RGB-D camera, we capture points and adjust them onto layers than are perpendicular to the camera plane. From each plane, we extract the boundaries of objects such as furniture, ceilings, walls, or doors. The extracted features compose a bag of visual words classified by a support vector machine. The proposed method achieves almost the same accuracy in scene classification as a SIFT-based algorithm and is 2.38× faster. The experimental results demonstrate the effectiveness of the proposed method in terms of accuracy and robustness for the 7-Scenes and SUNRGBD datasets.
RESUMO
No single-omic approach completely elucidates the multitude of alterations taking place in Alzheimer's disease (AD). Here, we coupled transcriptomic and phosphoproteomic approaches to determine the temporal sequence of changes in mRNA, protein, and phosphopeptide expression levels from human temporal cortical samples, with varying degree of AD-related pathology. This approach highlighted fluctuation in synaptic and mitochondrial function as the earliest pathological events in brain samples with AD-related pathology. Subsequently, increased expression of inflammation and extracellular matrix-associated gene products was observed. Interaction network assembly for the associated gene products, emphasized the complex interplay between these processes and the role of addressing post-translational modifications in the identification of key regulators. Additionally, we evaluate the use of decision trees and random forests in identifying potential biomarkers differentiating individuals with different degree of AD-related pathology. This multiomic and temporal sequence-based approach provides a better understanding of the sequence of events leading to AD.
Assuntos
Doença de Alzheimer/patologia , Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Humanos , Biologia de Sistemas/métodosRESUMO
The physiological functions of vitamin D are mediated by its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) activating the transcription factor vitamin D receptor (VDR). In THP-1 human monocytes we demonstrated epigenome-wide effects of 1,25(OH)2D3 at 8979 loci with significantly modulated chromatin accessibility. Maximal chromatin opening was observed after 24 h, while after 48 h most sites closed again. The chromatin-organizing protein CTCF bound to 14% of the 1,25(OH)2D3-sensitive chromatin regions. Interestingly, 1,25(OH)2D3 affected the chromatin association of CTCF providing an additional mechanism for the epigenome-wide effects of the VDR ligand. The 1,25(OH)2D3-modulated transcriptome of THP-1 cells comprised 1284 genes, 77.5% of which responded only 24 h after stimulation. During the 1,25(OH)2D3 stimulation time course the proportion of down-regulated genes increased from 0% to 44.9% and the top-ranking physiological function of the respective genes shifted from anti-microbial response to connective tissue disorders. The integration of epigenomic and transcriptomic data identified 165 physiologically important 1,25(OH)2D3 target genes, including HTT and NOD2, whose expression can be predicted primarily from epigenomic data of their genomic loci. Taken together, a large number of 1,25(OH)2D3-triggered epigenome-wide events precede and accompany the transcriptional activation of target genes of the nuclear hormone.
Assuntos
Cromatina/metabolismo , Receptores de Calcitriol/metabolismo , Proteínas Repressoras/genética , Ativação Transcricional/efeitos dos fármacos , Vitamina D/análogos & derivados , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Epigênese Genética/genética , Humanos , Proteína Huntingtina/genética , Monócitos/citologia , Monócitos/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Ligação Proteica/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transcrição Gênica/genética , Transcriptoma/genética , Vitamina D/química , Vitamina D/farmacologiaRESUMO
CCCTC-binding factor (CTCF) is a transcription factor being involved in 3D chromatin organization and displays a highly conserved genome-wide binding pattern. In this study, we report the cistrome of CTCF in THP-1 human monocytes and confirm that from the 40,078 CTCF binding sites nearly 85% are identical with those found in K562 monocytes. Quadruplicate chromatin immunoprecipitation sequencing (ChIP-seq) demonstrated that at 2130 loci the association strenght of CTCF with genomic DNA was significantly (p<0.05) modulated by stimulation with the natural vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Some 55% of these CTCF sites contribute to DNA looping and mark the anchors of 587 putative topologically associating domains (TADs) containing at least one VDR binding site and one 1,25(OH)2D3 target gene. These TADs can explain the regulatory scenarios of up to 70% of all 1,25(OH)2D3 target genes. A self-organizing map approach subdivided the vitamin D-sensitive CTCF sites into seven classes that can be distinguished by participation in DNA loop formation, binding to open chromatin, carrying binding motifs for CTCF or its relative BORIS, overlap with transcription start site (TSS) regions and binding of VDR. These variant molecular profiles suggest different mechanisms of the 1,25(OH)2D3-dependent action of CTCF. The co-location of VDR and 1,25(OH)2D3-dependent CTCF sites increases in the context of accessible chromatin and TSS regions but does not show any significant correlation with classical DNA binding mechanisms of CTCF. In conclusion, vitamin D-sensitive CTCF sites provide further mechanistic details to the epigenome-wide understanding of 1,25(OH)2D3-mediated gene regulation.
Assuntos
Cromatina/metabolismo , Monócitos/metabolismo , Proteínas Repressoras/metabolismo , Vitamina D/farmacologia , Fator de Ligação a CCCTC , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Receptores de Calcitriol/metabolismo , Proteínas Repressoras/genéticaRESUMO
The vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the high affinity ligand of the transcription factor vitamin D receptor (VDR) and therefore a direct regulator of transcription. Transcriptome-wide analysis of THP-1 human monocytes had indicated more than 600 genes to be significantly (p<0.05) stimulated after 4h incubation with 1,25(OH)2D3, but only 67 of them where more than 1.5-fold up-regulated. These include the genes encoding for the transcription factors BCL6, NFE2, POU4F2 and ELF4, which are controlled by one or two VDR binding sites within their chromosomal domains. The latter are defined via DNA loop formation mediated by the transcription factor CTCF that is highly conserved in its genome-wide loci. We found BCL6 being most responsive to 1,25(OH)2D3 and selected it for further analysis. An incubation of THP-1 cells with 1,25(OH)2D3 for 24 h resulted in a significant (p<0.001) change in the mRNA expression of more than 1600 genes, of which 132 were at least 2-fold up-regulated. About half of the latter genes are secondary 1,25(OH)2D3 targets, since they do not carry any VDR binding site within their chromosomal domain. Chromatin immunoprecipitation sequencing datasets indicated that the majority of these domains contain a BCL6 binding site. We followed the secondary transcriptional response to 1,25(OH)2D3 for eight representative gene examples and confirmed the binding of CTCF and BCL6 to their respective chromosomal domains. In conclusion, our study indicated that in monocytes most of the physiological responses to 1,25(OH)2D3 involve the action of the transcription factor BCL6.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Subunidade p45 do Fator de Transcrição NF-E2/biossíntese , Fator de Transcrição Brn-3B/biossíntese , Fatores de Transcrição/biossíntese , Vitamina D/administração & dosagem , Sítios de Ligação , Fator de Ligação a CCCTC , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade p45 do Fator de Transcrição NF-E2/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6 , RNA Mensageiro/biossíntese , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição Brn-3B/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Vitamina D/análogos & derivadosRESUMO
Property prediction is a key interest in chemistry. For several decades there has been a continued and incremental development of mathematical models to predict properties. As more data is generated and accumulated, there seems to be more areas of opportunity to develop models with increased accuracy. The same is true if one considers the large developments in machine and deep learning models. However, along with the same areas of opportunity and development, issues and challenges remain and, with more data, new challenges emerge such as the quality and quantity and reliability of the data, and model reproducibility. Herein, we discuss the status of the accuracy of predictive models and present the authors' perspective of the direction of the field, emphasizing on good practices. We focus on predictive models of bioactive properties of small molecules relevant for drug discovery, agrochemical, food chemistry, natural product research, and related fields.
RESUMO
The transcription factor vitamin D receptor (VDR) is the high affinity nuclear target of the biologically active form of vitamin D3 (1,25(OH)2D3). In order to identify pure genomic transcriptional effects of 1,25(OH)2D3, we used VDR cistrome, transcriptome and open chromatin data, obtained from the human monocytic cell line THP-1, for a novel hierarchical analysis applying three bioinformatics approaches. We predicted 75.6% of all early 1,25(OH)2D3-responding (2.5 or 4 h) and 57.4% of the late differentially expressed genes (24 h) to be primary VDR target genes. VDR knockout led to a complete loss of 1,25(OH)2D3-induced genome-wide gene regulation. Thus, there was no indication of any VDR-independent non-genomic actions of 1,25(OH)2D3 modulating its transcriptional response. Among the predicted primary VDR target genes, 47 were coding for transcription factors and thus may mediate secondary 1,25(OH)2D3 responses. CEBPA and ETS1 ChIP-seq data and RNA-seq following CEBPA knockdown were used to validate the predicted regulation of secondary vitamin D target genes by both transcription factors. In conclusion, a directional network containing 47 partly novel primary VDR target transcription factors describes secondary responses in a highly complex vitamin D signaling cascade. The central transcription factor VDR is indispensable for all transcriptome-wide effects of the nuclear hormone.
Assuntos
Colecalciferol/farmacologia , Receptores de Calcitriol/genética , Transcriptoma/genética , Vitamina D/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Sistemas CRISPR-Cas/genética , Colecalciferol/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Redes Reguladoras de Genes/efeitos dos fármacos , Genoma Humano/genética , Humanos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , RNA-Seq , Transcriptoma/efeitos dos fármacos , Vitamina D/metabolismoRESUMO
Vitamin D is essential for the function of the immune system. In this study, we treated peripheral blood mononuclear cells (PBMCs) of healthy adults with the biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) using two different approaches: single repeats with PBMCs obtained from a cohort of 12 individuals and personalized analysis based on triplicates of five study participants. This identified 877 (cohort approach) and 3951 (personalized approach) genes that significantly (p < 0.05) changed their expression 24 h after 1,25(OH)2D3 stimulation. From these, 333 and 1232 were classified as supertargets, a third of which were identified as novel. Individuals differed largely in their vitamin D response not only by the magnitude of expression change but also by their personal selection of (super)target genes. Functional analysis of the target genes suggested the overarching role of vitamin D in the regulation of metabolism, proliferation and differentiation, but in particular in the control of functions mediated by the innate and adaptive immune system, such as responses to infectious diseases and chronic inflammatory disorders. In conclusion, immune cells are an important target of vitamin D and common genes may serve as biomarkers for personal responses to the micronutrient.
Assuntos
Leucócitos Mononucleares/metabolismo , Micronutrientes/farmacologia , Vitamina D/farmacologia , Adulto , Células Cultivadas , Estudos de Coortes , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma Humano , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The vitamin D-modulated transcriptome of highly responsive human cells, such as THP-1 monocytes, comprises more than 500 genes, half of which are primary targets. Recently, we proposed a chromatin model of vitamin D signaling demonstrating that nearly all vitamin D target genes are located within vitamin D-modulated topologically associated domains (TADs). This model is based on genome-wide binding patterns of the vitamin D receptor (VDR), the pioneer transcription factor PU.1, the chromatin organizer CTCF and histone markers of active promoter regions (H3K4me3) and active chromatin (H3K27ac). In addition, time-dependent data on accessible chromatin and mRNA expression are implemented. For the interrogation and in deep inspection of these high-dimensional datasets unsupervised and supervised machine learning algorithms were applied. Unsupervised methods, such as the vector quantization tool K-means and the dimensionality reduction algorithm self-organizing map, generated descriptions of how attributes, such as VDR binding and chromatin accessibility, affect each other as a function of time and/or co-localization within the same genomic region. Supervised algorithms, such as random forests, allowed the data to be classified into pre-existing categories like persistent (i.e. constant) and time-dependent (i.e. transient) VDR binding sites. The relative amounts of these VDR categories in TADs showed to be the main discriminator for sorting the latter into five classes carrying vitamin D target genes involved in distinct biological processes. In conclusion, via the application of machine learning methods we identified the spatio-temporal VDR binding pattern in TADs as the most critical attribute for specific regulation of vitamin D target genes and the segregation of vitamin D's physiologic function.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Aprendizado de Máquina , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo , Algoritmos , Linhagem Celular , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica , RNA Mensageiro/genética , Transdução de Sinais , Células THP-1RESUMO
The myeloid master regulator CCAAT enhancer-binding protein alpha (CEBPA) is known as a pioneer factor. In this study, we report the CEBPA cistrome of THP-1 human monocytes after stimulation with the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) for 2, 8 and 24â¯h. About a third of the genomic VDR binding sites co-located with those of CEBPA. In parallel, the binding strength of 5% of the CEBPA cistrome, i.e. some 1500 sites, is significantly (pâ¯<â¯0.001) affected by 1,25(OH)2D3. Transcriptome-wide analysis after CEBPA silencing indicated that the pioneer factor enhances both the basal expression and ligand inducibility of 70 vitamin D target genes largely involved in lipid signaling and metabolism. In contrast, CEBPA suppresses 82 vitamin D target genes many of which are related to the modulation of T cell activity by monocytes. The inducibility of the promoter-specific histone marker H3K4me3 distinguishes the former class of genes from the latter. Moreover, prominent occupancy of the myeloid pioneer factor PU.1 on 1,25(OH)2D3-sensitive CEBPA enhancers mechanistically explains the dichotomy of vitamin D target genes. In conclusion, CEBPA supports vitamin D signaling concerning actions of the innate immune system, but uses the antagonism with PU.1 for suppressing possible overreactions of adaptive immunity.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Receptores de Calcitriol/metabolismo , Vitamina D/análogos & derivados , Imunidade Adaptativa , Humanos , Imunidade Inata , Monócitos/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais , Células THP-1 , Transativadores/fisiologia , Vitamina D/metabolismoRESUMO
In the vitamin D intervention study VitDbol (NCT02063334) blood samples were drawn directly before an oral bolus (2000 µg vitamin D3) and 24 h later. The focus of phase II of VitDbol was the transcriptome-wide analysis of the effects of vitamin D gene expression in human peripheral blood mononuclear cells (PBMCs). All five participants responded in an individual fashion to the bolus by increases in serum levels of the vitamin D metabolites 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). RNA sequencing identified 15.040 commonly expressed genes in PBMCs, 702 (4,7%) of which were significantly (p < 0,05) affected by the vitamin D3 bolus. KEGG pathway analysis suggested that these genes are involved in general protein translation, monocyte differentiation and cellular growth control. Previously published transcriptome-wide studies in comparable cell systems confirmed 234 of the 702 vitamin D target genes, leaving many genes, such as HLA-A and HLA-C, as novel discoveries. Interestingly, in vivo stimulated PBMCs of this study showed a larger number of common vitamin D target genes with the monocytic cell line THP-1 than with in vitro stimulated PBMCs. The expression pattern of vitamin D target genes differed significantly between individuals and the average expression change can serve as a marker for vitamin D responsiveness. In conclusion, this study demonstrates that under in vivo conditions changes in 25(OH)D3 and 1,25(OH)2D3 serum concentrations alter the expression of more than 700 vitamin D target genes in human leukocytes.
Assuntos
Leucócitos Mononucleares/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Vitamina D/farmacologia , Vitaminas/farmacologia , Adulto , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
We tested whether lifelong modification of vitamin D signaling can alter the progression of early prostate carcinogenesis in studies using mice that develop high-grade prostatic intraepithelial neoplasia that is similar to humans. Two tissue-limited models showed that prostate vitamin D receptor (VDR) loss increased prostate carcinogenesis. In another study, we fed diets with three vitamin D3 levels (inadequate = 25 IU/kg diet, adequate for bone health = 150 IU/kg, or high = 1,000 IU/kg) and two calcium levels (adequate for bone health = 0.5% and high = 1.5%). Dietary vitamin D caused a dose-dependent increase in serum 25-hydroxyvitamin D levels and a reduction in the percentage of mice with adenocarcinoma but did not improve bone mass. In contrast, high calcium suppressed serum 1,25-dihydroxyvitamin D levels and improved bone mass but increased the incidence of adenocarcinoma. Analysis of the VDR cistrome in RWPE1 prostate epithelial cells revealed vitamin D-mediated regulation of multiple cancer-relevant pathways. Our data support the hypothesis that the loss of vitamin D signaling accelerates the early stages of prostate carcinogenesis, and our results suggest that different dietary requirements may be needed to support prostate health or maximize bone mass. SIGNIFICANCE: This work shows that disrupting vitamin D signaling through diet or genetic deletion increases early prostate carcinogenesis through multiple pathways. Higher-diet vitamin D levels are needed for cancer than bone.
Assuntos
Adenocarcinoma/prevenção & controle , Carcinogênese/metabolismo , Neoplasias da Próstata/prevenção & controle , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo , Vitaminas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose , Carcinogênese/patologia , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Binding motifs of the ETS-domain transcription factor GABPA are found with high significance below the summits of the vitamin D receptor (VDR) cistrome. VDR is the nuclear receptor for the biologically most active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In this study, we determined the GABPA cistrome in THP-1 human monocytes and found that it is comprised of 3822 genomic loci, some 20% of which were modulated by 1,25(OH)2D3. The GABPA cistrome showed a high overlap rate with accessible chromatin and the pioneer transcription factor PU.1. Interestingly, 23 and 12% of persistent and transient VDR binding sites, respectively, co-localized with GABPA, which is clearly higher than the rate of secondary VDR loci (4%). Some 40% of GABPA binding sites were found at transcription start sites, nearly 100 of which are of 1,25(OH)2D3 target genes. On 593 genomic loci VDR and GABPA co-localized with PU.1, while only 175 VDR sites bound GABPA in the absence of PU.1. In total, VDR sites with GABPA co-localization may control some 450 vitamin D target genes. Those genes that are co-controlled by PU.1 preferentially participate in cellular and immune signaling processes, while the remaining genes are involved in cellular metabolism pathways. In conclusion, GABPA may contribute to differential VDR target gene regulation.
Assuntos
Fator de Transcrição de Proteínas de Ligação GA/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Calcitriol/fisiologia , Transativadores/fisiologia , Sítios de Ligação , Regulação da Expressão Gênica , Humanos , Células K562 , Proteínas Proto-Oncogênicas c-ets , Células THP-1RESUMO
The micronutrient vitamin D significantly modulates the human epigenome via enhancing genome-wide the rate of accessible chromatin and vitamin D receptor (VDR) binding. This study focuses on histone marks of active chromatin at promoter and enhancer regions and investigates, whether these genomic loci are sensitive to vitamin D. The epigenome of THP-1 human monocytes contains nearly 23,000 sites with H3K4me3 histone modifications, 550 of which sites are significantly (pâ¯<â¯0.05) modulated by stimulation with the VDR ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). H3K27ac histone modifications mark active chromatin and 2473 of 45,500 sites are vitamin D sensitive. The two types of ligand-dependent histone marks allow to distinguish promoter and enhancer regulation by vitamin D, respectively. Transcription start site overlap is the prime attribute of ligand-dependent H3K4me3 marks, while VDR co-location is the top ranking parameter describing 1,25(OH)2D3-sensitive H3K27ac marks at enhancers. A categorization of 1,25(OH)2D3-sensitive histone marks by machine learning algorithms - using the attributes overall peak strength and ligand inducibility - highlights 260 and 287 regions with H3K4me3 and H3K27ac modifications, respectively. These loci are found at the promoter regions of 59 vitamin D target genes and their associated enhancers. In this way, ligand-dependent histone marks provide a link of the effects of 1,25(OH)2D3 on the epigenome with previously reported mRNA expression changes of vitamin D target genes. In conclusion, the human epigenome responds also on the level of histone modifications to 1,25(OH)2D3 stimulation. This allows a more detailed understanding of vitamin D target gene regulation.
Assuntos
Calcitriol/fisiologia , Epigênese Genética , Código das Histonas , Receptores de Calcitriol/metabolismo , Cromatina , Genoma , Humanos , Ligantes , Células THP-1 , Sítio de Iniciação de TranscriçãoRESUMO
In vitro cell culture studies showed that the hormonal form of vitamin D3, 1α,25-dihydroxyvitamin D3, significantly (pâ¯<â¯0.05) affects the human epigenome at thousands of genomic loci. Phase II of the VitDbol vitamin D intervention trial (NCT02063334) involved a proof-of-principle study of one individual, who was exposed three times every 28 days to an oral bolus (2000⯵g) of vitamin D3. Blood samples were taken directly before each supplementation as well as one and two days after, chromatin was isolated from peripheral blood mononuclear cells without any further in vitro culture and at all nine time points epigenome-wide chromatin accessibility was assessed by applying FAIRE-seq (formaldehyde-assisted isolation of regulatory elements sequencing). The vitamin D3 bolus resulted in an average raise in 25-hydroxyvitamin D3 (25(OH)D3) serum concentration of 11.9 and 19.4â¯nM within one and two days, respectively. Consistently accessible chromatin was detected at 5205 genomic loci, the 853 most prominent of which a self-organizing map algorithm classified into early, delayed and non-responding genomic regions: 70 loci showed already after one day and 361 sites after two days significant (pâ¯<â¯0.0001) chromatin opening or closing. Interestingly, more than half of these genomic regions overlap with transcription start sites, but the change of chromatin accessibility at these sites has no direct effect on the transcriptome. Some of the vitamin D responsive chromatin sites cluster at specific loci within the human genome, the most prominent of which is the human leukocyte antigen region in chromosome 6. In conclusion, this study demonstrates that under in vivo conditions a rather minor rise in 25(OH)D3 serum levels is sufficient to result in significant changes at hundreds of sites within the epigenome of human leukocytes.
Assuntos
Cromatina/genética , Epigenômica , Genoma Humano , Leucócitos Mononucleares/metabolismo , Transcriptoma , Vitamina D/farmacologia , Vitaminas/farmacologia , Cromatina/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-IdadeRESUMO
The transcription factor vitamin D receptor (VDR) is the exclusive nuclear target of the biologically active form of vitamin D (1,25(OH)2D3). In THP-1 human monocytes we obtained a highly accurate VDR cistrome after 2 and 24h ligand stimulation comprising >11,600 genomic loci, 78% of which were detected exclusively after 24h. In contrast, a group of 510 persistent VDR sites occurred at all conditions and some 2100 VDR loci were only transiently occupied. Machine learning and statistical analysis as well as a comparison with the re-analyzed B cell VDR cistrome indicated a subgroup of 339 highly conserved persistent VDR sites that were suited best for describing vitamin D-triggered gene regulatory scenarios. The 1,25(OH)2D3-dependent transcriptome of THP-1 cells comprised 587 genes, 311 of which were primary targets with main functions in the immune system. More than 97% of the latter genes were located within 1,25(OH)2D3-modulated topologically associated domains (TADs). The number of persistent and transient VDR sites was found to be the main discriminator for sorting these TADs into five classes carrying vitamin D target genes involved in distinct biological processes. In conclusion, specific regulation of biological processes by vitamin D depends on differences in time-dependent VDR binding.
Assuntos
Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo , Linfócitos B/metabolismo , Fenômenos Biológicos , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Humanos , Monócitos/metabolismo , Ligação Proteica/fisiologia , Fatores de Transcrição/metabolismo , Transcriptoma/fisiologiaRESUMO
The ETS-domain transcription factor PU.1 acts as a pioneer factor for other transcription factors including nuclear receptors. In this study, we report that in THP-1 human monocytes the PU.1 cistrome comprises 122,319 genomic sites. Interestingly, at 6498 (5.3%) of these loci PU.1 binding was significantly modulated by the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In most cases 1,25(OH)2D3 increased PU.1 association, which correlated strongly with VDR co-location and overlap ratios for canonical DR3-type VDR binding sites. Genome-wide 6488 sites associating both with PU.1 and VDR as well as 5649 non-VDR overlapping, 1,25(OH)2D3-sensitive PU.1 loci represent the PU.1-VDR crosstalk and can be described by four gene regulatory scenarios, each. Chromatin accessibility was the major discriminator between these models. The location of the PU.1 binding loci in open chromatin coincided with a significantly smaller mean distance to the closest 1,25(OH)2D3 target gene. PU.1 knockdown indicated that the pioneer factor is relevant for the transcriptional activation of 1,25(OH)2D3 target genes but its impact differed in magnitude and orientation. In conclusion, PU.1 is an important modulator of VDR signaling in monocytes, including but also exceeding its role as a pioneer factor, but we found no evidence for a direct interaction of both proteins.
Assuntos
Epigenômica , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Vitamina D/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Modelos Genéticos , Ligação Proteica/genética , Transcrição Gênica , Transcriptoma/genética , Vitamina D/análogos & derivadosRESUMO
Vitamin D3 has via its metabolites 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) direct effects on the transcriptome and the epigenome of most human cells. In the VitDbol study we exposed 35 healthy young adults to an oral vitamin D3 dose (2000µg) or placebo and took blood samples directly before the supplementation as well as at days 1, 2 and 30. Within 24h the vitamin D3 intake raised the average serum levels of both 25(OH)D3 and 1,25(OH)2D3 by approximately 20%. However, we observed large inter-individual differences in these serum levels, reflected by the average ratios between 25(OH)D3 and 1,25(OH)2D3 concentrations ranging from 277 to 1365. Interestingly, average serum parathyroid hormone (PTH) levels increased at day 1 by some 10% but then decreased within the following four weeks to levels 5% below baseline. In peripheral blood mononuclear cells (PBMCs) that were isolated at the same time points we determined vitamin D-modulated chromatin accessibility by FAIRE-qPCR at selected genomic loci. This method is well suited to evaluate both short-term and long-term in vivo effects of vitamin D on the epigenome of human subjects. The differential vitamin D responsiveness of the VitDbol study participants was determined via individual changes in their PTH levels or chromatin accessibility in relation to alterations in 25(OH)D3 concentrations. This led to the segregation of the subjects into 14 high, 11 mid and 10 low responders. In summary, the vitamin D responsiveness classification provides additional information compared to a vitamin D status assessment based on single 25(OH)D3 serum measurements. The study was registered at Clinicaltrials.gov (NCT02063334).
Assuntos
Colecalciferol/farmacologia , Vitaminas/farmacologia , Calcifediol/sangue , Calcitriol/sangue , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucócitos Mononucleares/metabolismo , Masculino , Hormônio Paratireóideo/sangue , Adulto Jovem , Proteínas rap de Ligação ao GTP/genéticaRESUMO
Monocytes are important cells of the innate immune system that can differentiate into macrophages and dendritic cells. The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), serves as a ligand of the nuclear receptor vitamin D receptor (VDR). A key physiological function of 1,25(OH)2D3 is the defense against pathogens, such as those causing tuberculosis, that involves the modulation of the monocyte transcriptome. THP-1 cells are an established model of human monocytes, for which the at present largest set of 1,25(OH)2D3-affected genome-wide data are available. Here we summarize the insight obtained from the recent transcriptome of 1,25(OH)2D3-stimulated THP-1 cells, that was determined by triplicate RNA sequencing (RNA-seq). Primary and secondary vitamin D target genes being up- and down-regulated were related to changes in the epigenome of THP-1 cells, such as 1,25(OH)2D3-dependent chromatin opening and modulation of the genome-wide association of the transcription factors VDR and CCCTC-binding factor (CTCF) with their respective genomic binding sites. The anti-microbial response is the top-ranking early physiological function represented by 1,25(OH)2D3-stimulated genomic regions and genes, but also other immunity-related pathways, such as IL10 signaling, are activated. Taken together, the epigenomic and transcriptomic responses of THP-1 cells to 1,25(OH)2D3 represent a master example of the impact of vitamin D on human physiology.
Assuntos
Calcitriol/metabolismo , Epigênese Genética , Monócitos/metabolismo , Transcriptoma , Fator de Ligação a CCCTC , Humanos , Receptores de Calcitriol/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Vitamin D3 has transcriptome- and genome-wide effects and activates, via the binding of its metabolite 1α,25-dihydroxyvitamin D3 to the transcription factor vitamin D receptor (VDR), several hundred target genes. Using samples from a 5-month vitamin D3 intervention study (VitDmet), we recently reported that the expression of 12 VDR target genes in peripheral blood mononuclear cells (PBMCs) as well as 12 biochemical and clinical parameters of the study participants are significantly triggered by vitamin D3. In this study, we performed a more focused selection of further 12 VDR target genes and demonstrated that changes of their mRNA expression in PBMCs of VitDmet subjects significantly correlate with alterations of 25-hydroxyvitamin D3 serum levels. Network and self-organizing map analysis of these datasets together with that of the other 24 parameters was followed by relevance calculations and identified changes in parathyroid hormone serum levels and the expression of the newly selected genes STS, BCL6, ITGAM, LRRC25, LPGAT1 and TREM1 as well as of the previously reported genes DUSP10 and CD14 as the most relevant parameters for describing vitamin D responsiveness in vivo. Moreover, parameter relevance ranking allowed the segregation of study subjects into high and low responders. Due to the long intervention period the vitamin D response was not too prominent on the level of transcriptional activation. Therefore, we performed in the separate VitDbol trial a short-term but high dose stimulation with a vitamin D3 bolus. In PBMCs of VitDbol subjects we observed direct transcriptional effects on the selected VDR target genes, such as an up to 2.1-fold increase already one day after supplementation onset. In conclusion, both long-term and short-term vitamin D3 supplementation studies allow monitoring the vitamin D responsiveness of human individuals and represent new types of human in vivo vitamin D3 investigations.