scholarly journals Concentration Dependent Chromatin States Induced by the Bicoid Morphogen Gradient

2017 ◽  
Author(s):  
Colleen E. Hannon ◽  
Shelby A. Blythe ◽  
Eric F. Wieschaus
Keyword(s):  
Target Genes ◽  
Positional Information ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Genome Wide ◽  
Low Concentrations ◽  
High Concentrations ◽  
Accessible Chromatin ◽  
Anterior Posterior

ABSTRACTIn Drosophila, graded expression of the maternal transcription factor Bicoid (Bcd) provides positional information to activate target genes at different positions along the anterior-posterior axis. We have measured the genome-wide binding profile of Bcd using ChIP-seq in embryos expressing single, uniform levels of Bcd protein, and grouped Bcd-bound targets into four classes based on occupancy at different concentrations. By measuring the biochemical affinity of target enhancers in these classes in vitro and genome-wide chromatin accessibility by ATAC-seq, we found that the occupancy of target sequences by Bcd is not primarily determined by Bcd binding sites, but by chromatin context. Bcd drives an open chromatin state at a subset its targets. Our data support a model where Bcd influences chromatin structure to gain access to concentration-sensitive targets at high concentrations, while concentration-insensitive targets are found in more accessible chromatin and are bound at low concentrations.

scholarly journals Concentration dependent chromatin states induced by the bicoid morphogen gradient

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Colleen E Hannon ◽  
Shelby A Blythe ◽  
Eric F Wieschaus
Keyword(s):  
Large Scale ◽  
Common Property ◽  
Target Genes ◽  
Positional Information ◽  
Chromatin Accessibility ◽  
Chromatin State ◽  
Open Chromatin ◽  
Genome Wide ◽  
High Concentrations

In Drosophila, graded expression of the maternal transcription factor Bicoid (Bcd) provides positional information to activate target genes at different positions along the anterior-posterior axis. We have measured the genome-wide binding profile of Bcd using ChIP-seq in embryos expressing single, uniform levels of Bcd protein, and grouped Bcd-bound targets into four classes based on occupancy at different concentrations. By measuring the biochemical affinity of target enhancers in these classes in vitro and genome-wide chromatin accessibility by ATAC-seq, we found that the occupancy of target sequences by Bcd is not primarily determined by Bcd binding sites, but by chromatin context. Bcd drives an open chromatin state at a subset of its targets. Our data support a model where Bcd influences chromatin structure to gain access to concentration-sensitive targets at high concentrations, while concentration-insensitive targets are found in more accessible chromatin and are bound at low concentrations. This may be a common property of developmental transcription factors that must gain early access to their target enhancers while the chromatin state of the genome is being remodeled during large-scale transitions in the gene regulatory landscape.

scholarly journals Multi-omics analysis for potential inflammation-related genes involved in tumor immune evasion via extended application of epigenetic data

2021 ◽  
Author(s):  
Chenshen Huang ◽  
Ning Wang ◽  
Na Zhang ◽  
Zhizhan Ni ◽  
Xiaohong Liu ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Tumor Immune Evasion ◽  
Omics Analysis ◽  
Extended Application ◽  
Open Chromatin Region ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Accessible Chromatin

Background: Accumulating evidence suggests that inflammation-related genes may play key roles in tumor immune evasion. Programmed cell death ligand 1 (PD-L1) is an important immune checkpoint involved in mediating antitumor immunity. We performed multi-omics analysis to explore key inflammation-related genes affecting the transcriptional regulation of PD-L1 expression. Methods: The open chromatin region of the PD-L1 promoter was mapped using the assay for transposase-accessible chromatin using sequencing (ATAC-seq) profiles. Correlation analysis of epigenetic data (ATAC-seq) and transcriptome data (RNA-seq) were performed to identify inflammation-related transcription factors whose expression levels were correlated with the chromatin accessibility of the PD-L1 promoter. Chromatin immunoprecipitation sequencing (ChIP-seq) profiles were used to confirm the physical binding of the TF STAT2 and the predicted binding regions. We also confirmed the results of the bioinformatics analysis with cell experiments. Results: We identified chr9:5449463-5449962 and chr9:5450250-5450749 as reproducible open chromatin regions in the PD-L1 promoter. Moreover, we observed a correlation between STAT2 expression and the accessibility of the aforementioned regions. Furthermore, we confirmed its physical binding through ChIP-seq profiles and demonstrated the regulation of PD-L1 by STAT2 overexpression in vitro. Multiple databases were also used for the validation of the results. Conclusion: Our study identified STAT2 as a direct upstream TF regulating PD-L1 expression. The interaction of STAT2 and PD-L1 might be associated with tumor immune evasion in cancers, suggesting the potential value for tumor treatment.

scholarly journals Spatially uniform establishment of chromatin accessibility in the early Drosophila embryo

2017 ◽  
Author(s):  
Jenna E. Haines ◽  
Michael B. Eisen
Keyword(s):  
Chromatin Accessibility ◽  
Drosophila Embryo ◽  
Direct Result ◽  
Open Chromatin ◽  
Relative Contribution ◽  
Genome Wide ◽  
Zygotic Transcription ◽  
Early Drosophila Embryo ◽  
Nucleosome Density ◽  
Anterior Posterior

AbstractAs the Drosophila embryo transitions from the use of maternal RNAs to zygotic transcription, domains of open chromatin, with relatively low nucleosome density and specific histone marks, are established at promoters and enhancers involved in patterned embryonic transcription. However, it remains unclear whether open chromatin is a product of activity - transcription at promoters and patterning transcription factor binding at enhancers - or whether it is established by independent mechanisms. Recent work has implicated the ubiquitously expressed, maternal factor Zelda in this process. To assess the relative contribution of activity in the establishment of chromatin accessibility, we have probed chromatin accessibility across the anterior-posterior axis of early Drosophila melanogaster embryos by applying a transposon based assay for chromatin accessibility (ATAC-seq) to anterior and posterior halves of hand-dissected, cellular blastoderm embryos. We find that genome-wide chromatin accessibility is remarkably similar between the two halves. Promoters and enhancers that are active in exclusively one half of the embryo have open chromatin in the other half, demonstrating that chromatin accessibility is not a direct result of activity. However, there is a small skew at enhancers that drive transcription exclusively in either the anterior or posterior half of the embryo, with greater accessibility in the region of activity. Taken together these data support a model in which regions of chromatin accessibility are defined and established by ubiquitous factors, and fine-tuned subsequently by activity.

scholarly journals Dose- and time-dependent effects of hyaluronidase on structural cells and the extracellular matrix of the skin

2020 ◽  
Vol 25 (1) ◽  
Author(s):  
Bettina Alexandra Buhren ◽  
Holger Schrumpf ◽  
Katharina Gorges ◽  
Oliver Reiners ◽  
Edwin Bölke ◽  
...  
Keyword(s):  
Wound Healing ◽  
Ex Vivo ◽  
Time Dependent ◽  
Genome Wide ◽  
Low Concentrations ◽  
High Concentrations ◽  
Dose Dependent ◽  
Genome Wide Expression ◽  
Skin Samples

Abstract Introduction Hyaluronic acid (hyaluronan; HA) is an essential component of the extracellular matrix (ECM) of the skin. The HA-degrading enzyme hyaluronidase (HYAL) is critically involved in the HA-metabolism. Yet, only little information is available regarding the skin’s HA–HYAL interactions on the molecular and cellular levels. Objective To analyze the dose- and time-dependent molecular and cellular effects of HYAL on structural cells and the HA-metabolism in the skin. Materials and methods Chip-based, genome-wide expression analyses (Affymetrix® GeneChip PrimeView™ Human Gene Expression Array), quantitative real-time PCR analyses, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (DAB), and in vitro wound healing assays were performed to assess dose-dependent and time-kinetic effects of HA and HYAL (bovine hyaluronidase, Hylase “Dessau”) on normal human dermal fibroblasts (NHDF), primary human keratinocytes in vitro and human skin samples ex vivo. Results Genome-wide expression analyses revealed an upregulation of HA synthases (HAS) up to 1.8-fold change in HA- and HYAL-treated NHDF. HA and HYAL significantly accelerated wound closure in an in vitro model for cutaneous wound healing. HYAL induced HAS1 and HAS2 mRNA gene expression in NHDF. Interestingly, low concentrations of HYAL (0.015 U/ml) resulted in a significantly higher induction of HAS compared to moderate (0.15 and 1.5 U/ml) and high concentrations (15 U/ml) of HYAL. This observation corresponded to increased concentrations of HA measured by ELISA in conditioned supernatants of HYAL-treated NHDF with the highest concentrations observed for 0.015 U/ml of HYAL. Finally, immunohistochemical analysis of human skin samples incubated with HYAL for up to 48 h ex vivo demonstrated that low concentrations of HYAL (0.015 U/ml) led to a pronounced accumulation of HA, whereas high concentrations of HYAL (15 U/ml) reduced dermal HA-levels. Conclusion HYAL is a bioactive enzyme that exerts multiple effects on the HA-metabolism as well as on the structural cells of the skin. Our results indicate that HYAL promotes wound healing and exerts a dose-dependent induction of HA-synthesis in structural cells of the skin. Herein, interestingly the most significant induction of HAS and HA were observed for the lowest concentration of HYAL.

scholarly journals Dose- and time-dependent effects of hyaluronidase on structural cells and the extracellular matrix of the skin

2020 ◽  
Author(s):  
Bettina Buhren ◽  
Holger Schrumpf ◽  
Katharina Gorges ◽  
Oliver Reiners ◽  
Edwin Boelke ◽  
...  
Keyword(s):  
Wound Healing ◽  
Ex Vivo ◽  
Time Dependent ◽  
Genome Wide ◽  
Low Concentrations ◽  
High Concentrations ◽  
Dose Dependent ◽  
Genome Wide Expression ◽  
Skin Samples

Abstract Hyaluronic acid (hyaluronan; HA) is an essential component of the extracellular matrix (ECM) of the skin. The HA-degrading enzyme hyaluronidase (HYAL) is critically involved in the HA-metabolism. Yet, only little information is available regarding the skin´s HA-HYAL interactions on the molecular and cellular levels.OBJECTIVE To analyze the dose- and time-dependent molecular and cellular effects of HYAL on structural cells and the HA-metabolism in the skin.MATERIALS AND METHODS Chip-based, genome-wide expression analyses (Affymetrix® GeneChip PrimeView™ Human Gene Expression Array), quantitative real-time PCR analyses, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (DAB), and in vitro wound healing assays were performed to assess dose-dependent and time-kinetic effects of HA and HYAL (bovine hyaluronidase, Hylase “Dessau”) on normal human dermal fibroblasts (NHDF), primary human keratinocytes in vitro and human skin samples ex vivo.RESULTS Genome-wide expression analyses revealed an upregulation of HA synthases (HAS) up to 1.8-fold change in HA- and HYAL-treated NHDF. HA and HYAL significantly accelerated wound closure in an in vitro model for cutaneous wound healing. HYAL induced HAS1 and HAS2 mRNA gene expression in NHDF. Interestingly, low concentrations of HYAL (0.015 U/ml) resulted in a significantly higher induction of HAS compared to moderate (0.15 and 1.5 U/ml) and high concentrations (15 U/ml) of HYAL. This observation corresponded to increased concentrations of HA measured by ELISA in conditioned supernatants of HYAL-treated NHDF with the highest concentrations observed for 0.015 U/ml of HYAL. Finally, immunohistochemical analysis of human skin samples incubated with HYAL for up to 48 h ex vivo demonstrated that low concentrations of HYAL (0.015 U/ml) led to a pronounced accumulation of HA, whereas high concentrations of HYAL (15 U/ml) reduced dermal HA-levels.CONCLUSION HYAL is a bioactive enzyme that exerts multiple effects on the HA-metabolism as well as on the structural cells of the skin. Our results indicate that HYAL promotes wound healing and exerts a dose-dependent induction of HA-synthesis in structural cells of the skin. Herein, interestingly the most significant induction of HAS and HA were observed for the lowest concentration of HYAL.

scholarly journals Effects of Hyaluronidase on Structural Cells and the Extracellular Matrix of the Skin

2020 ◽  
Author(s):  
Bettina Alexandra Buhren ◽  
Holger Schrumpf ◽  
Katharina Gorges ◽  
Oliver Reiners ◽  
Edwin Boelke ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wound Healing ◽  
Ex Vivo ◽  
Genome Wide ◽  
Low Concentrations ◽  
High Concentrations ◽  
Dose Dependent ◽  
Genome Wide Expression ◽  
Skin Samples

Abstract Hyaluronic acid (hyaluronan; HA) is an essential component of the extracellular matrix (ECM) of the skin. The HA-degrading enzyme hyaluronidase (HYAL) is critically involved in the HA-metabolism. Yet, only little information is available regarding the skin´s HA-HYAL interactions on the molecular and cellular levels.OBJECTIVETo analyze the dose- and time-dependent molecular and cellular effects of HYAL on structural cells and the HA-metabolism in the skin.MATERIALS AND METHODSChip-based, genome-wide expression analyses (Affymetrix® GeneChip PrimeView™ Human Gene Expression Array), quantitative real-time PCR analyses, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (DAB), and in vitro wound healing assays were performed to assess dose-dependent and time-kinetic effects of HA and HYAL (bovine hyaluronidase, Hylase “Dessau”) on normal human dermal fibroblasts (NHDF), primary human keratinocytes in vitro and human skin samples ex vivo.RESULTSGenome-wide expression analyses revealed an upregulation of HA synthases (HAS) up to 1.8-fold change in HA- and HYAL-treated NHDF. HA and HYAL significantly accelerated wound closure in an in vitro model for cutaneous wound healing. HYAL induced HAS1 and HAS2 mRNA gene expression in NHDF. Interestingly, low concentrations of HYAL (0.015 U/ml) resulted in a significantly higher induction of HAS compared to moderate (0.15 and 1.5 U/ml) and high concentrations (15 U/ml) of HYAL. This observation corresponded to increased concentrations of HA measured by ELISA in conditioned supernatants of HYAL-treated NHDF with the highest concentrations observed for 0.015 U/ml of HYAL. Finally, immunohistochemical analysis of human skin samples incubated with HYAL for up to 48 h ex vivo demonstrated that low concentrations of HYAL (0.015 U/ml) led to a pronounced accumulation of HA, whereas high concentrations of HYAL (15 U/ml) reduced dermal HA-levels.CONCLUSIONHYAL is a bioactive enzyme that exerts multiple effects on the HA-metabolism as well as on the structural cells of the skin. Our results indicate that HYAL promotes wound healing and exerts a dose-dependent induction of HA-synthesis in structural cells of the skin. Herein, interestingly the most significant induction of HAS and HA were observed for the lowest concentration of HYAL.

scholarly journals The LEAFY floral regulator displays pioneer transcription factor properties

2021 ◽  
Author(s):  
Xuelei Lai ◽  
Romain Blanc-Mathieu ◽  
Loic GrandVuillemin ◽  
Ying Huang ◽  
Arnaud Stigliani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Chromatin Accessibility ◽  
Nucleosomal Dna ◽  
Genome Wide ◽  
Pioneer Transcription Factor ◽  
Floral Gene ◽  
Expression Program

Pioneer transcription factors (TFs) are a special category of TFs with the capacity to bind to closed chromatin regions in which DNA is wrapped around histones and often highly methylated. Subsequently, they are able to modify the chromatin state to initiate gene expression. In plants, LEAFY (LFY) is a master floral regulator and has been suggested to act as a pioneer TF in Arabidopsis. Here, we demonstrate that LFY is able to bind both methylated and non-methylated DNA using a combination of in vitro genome-wide binding experiments and structural modeling. Comparisons between regions bound by LFY in vivo and chromatin accessibility data suggest that LFY binds a subset of regions occupied by nucleosomes. We confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes. Finally, we show that constitutive LFY expression in seedling tissues is sufficient to induce chromatin accessibility in the LFY direct target genes, APETALA1 and AGAMOUS. Taken together, our study suggests that LFY possesses key pioneer TF features that contribute to launch the floral gene expression program.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Aggregation of Cat Platelets in Vitro

1970 ◽  
Vol 23 (03) ◽  
pp. 601-620 ◽  
Author(s):  
Th. B Tschopp
Keyword(s):  
Adenosine Monophosphate ◽  
Blood Collection ◽  
Base Line ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Irreversible Aggregation ◽  
High Concentrations ◽  
Two Phases ◽  
Improved Technique

SummaryAggregation of cat platelets in the citrated plasma is examined by means of Born’s absorptiometer. A marked tendency of the platelets of this species to spontaneous aggregation necessitated first of all the development of an improved technique of blood collection.A hypothesis according to which 5-HT is released from the platelets, explains the absence of oscillations on the base line of the absorptiometer, the absence of platelet swelling, when ADP is added, and the effect of stirring on the aggregation curves in cat PRP. The average volume of cat platelets amounts to 10.46 μ3 when directly fixed in the blood, when fixed from PRP to 12.17 μ3, when fixed from stirred PRP to 13.51 μ3.In low concentrations (0.3-2 μM) ADP produce reversible aggregation; in narrowly restricted, individually dissimilar mean concentrations irreversible aggregation in two phases and in high concentrations, irreversible aggregation in one phase. Like ADP serotonin produces 2 phase irreversible aggregation in concentrations of 3-10 μM, but unlike ADP, the aggregation velocity decreases again with high 5-HT concentrations (>100 μM). Adrenaline does not produce aggregation and it is likely that adenosine and adenosine monophosphate inhibit the aggregation by serotonin but not by ADP. Species differences in the aggregation of human, rabbit and cat platelets are discussed.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

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