scholarly journals Tn5 transposase-based epigenomic profiling methods are prone to open chromatin bias

2021 ◽  
Author(s):  
Meng Wang ◽  
Yi Zhang
Keyword(s):  
Transcription Factor ◽  
Comparative Analysis ◽  
Histone Modifications ◽  
Biological Samples ◽  
False Positive ◽  
Protein A ◽  
Preimplantation Embryos ◽  
Open Chromatin ◽  
Tn5 Transposase ◽  
Number Of Cells

Epigenetic studies of rare biological samples like mammalian oocytes and preimplantation embryos require low input or even single cell epigenomic profiling methods. To reduce sample loss and avoid inefficient immunoprecipitation, several chromatin immuno-cleavage-based methods using Tn5 transposase fused with Protein A/G have been developed to profile histone modifications and transcription factor bindings using small number of cells. The Tn5 transposase-based epigenomic profiling methods are featured with simple library construction steps in the same tube, by taking advantage of Tn5 transposase's capability of simultaneous DNA fragmentation and adaptor ligation. However, the Tn5 transposase prefers to cut open chromatin regions. Our comparative analysis shows that Tn5 transposase-based profiling methods are prone to open chromatin bias. The high false positive signals due to biased cleavage in open chromatin could cause misinterpretation of signal distributions and dynamics. Rigorous validation is needed when employing and interpreting results from Tn5 transposase-based epigenomic profiling methods.

scholarly journals Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag

2021 ◽  
Vol 12 ◽  
Author(s):  
Weizhi Ouyang ◽  
Xiwen Zhang ◽  
Yong Peng ◽  
Qing Zhang ◽  
Zhilin Cao ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Protein A ◽  
Transcriptional Factor ◽  
Experimental Procedure ◽  
Histone Marks ◽  
Genome Wide ◽  
Efficient Alternative ◽  
Tn5 Transposase

Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

scholarly journals Evaluation of ChIC-based data requires normalization that properly retains signal-to-noise ratios

2021 ◽  
Author(s):  
Bofeng Liu ◽  
Fengling Chen ◽  
Wei Xie
Keyword(s):  
Transcription Factors ◽  
Histone Modifications ◽  
Chromatin Immunoprecipitation ◽  
Signal To Noise Ratio ◽  
Complete Loss ◽  
Open Chromatin ◽  
Dramatic Reduction ◽  
Signal To Noise ◽  
Noise Ratio ◽  
Tn5 Transposase

Several chromatin immunocleavage-based (ChIC) methods using Tn5 transposase have been developed to profile histone modifications and transcription factors bindings. A recent preprint by Wang et al. raised potential concerns that these methods are prone to open chromatin bias. While the authors are appreciated for alerting the community for this issue, it has been previously described and discussed by Henikoff and colleagues in the original CUT&Tag paper. However, as described for CUT&Tag, the signal-to-noise ratio is essential for Tn5-based profiling methods and all antibody-based enrichment assays. Based on this notion, we would like to point out a major analysis issue in Wang et al. that caused a complete loss or dramatic reduction of enrichment at true targets for datasets generated by Tn5-based methods, which in turn artificially enhanced the relative enrichment of potential open chromatin bias. Such analysis issue is caused by distinct background normalizations used towards ChIP-based (chromatin immunoprecipitation) data and Tn5-based data in Wang et al. Only the normalization for Tn5-based data, but not ChIP-seq based data, yielded such effects. Distortion of such signal-to-noise ratio would consequently lead to misleading results.

scholarly journals Profiling of chromatin accessibility across Aspergillus species and identification of transcription factor binding sites in the Aspergillus genome using filamentous fungi ATAC-seq

2019 ◽  
Author(s):  
Lianggang Huang ◽  
Xuejie Li ◽  
Liangbo Dong ◽  
Bin Wang ◽  
Li Pan
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Filamentous Fungi ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Transcription Factor Binding ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Factor Binding ◽  
Tn5 Transposase

AbstractTo identify cis-regulatory elements (CREs) and motifs of TF binding is an important step in understanding the regulatory functions of TF binding and gene expression. The lack of experimentally determined and computationally inferred data means that the genome-wide CREs and TF binding sites (TFBs) in filamentous fungi remain unknown. ATAC-seq is a technique that provides a high-resolution measurement of chromatin accessibility to Tn5 transposase integration. In filamentous fungi, the existence of cell walls and the difficulty in purifying nuclei have prevented the routine application of this technique. Herein, we modified the ATAC-seq protocol in filamentous fungi to identify and map open chromatin and TF-binding sites on a genome-scale. We applied the assay for ATAC-seq among different Aspergillus species, during different culture conditions, and among TF-deficient strains to delineate open chromatin regions and TFBs across each genome. The syntenic orthologues regions and differential changes regions of chromatin accessibility were responsible for functional conservative regulatory elements and differential gene expression in the Aspergillus genome respectively. Importantly, 17 and 15 novel transcription factor binding motifs that were enriched in the genomic footprints identified from ATAC-seq data of A. niger, were verified in vivo by our artificial synthetic minimal promoter system, respectively. Furthermore, we first confirmed the strand-specific patterns of Tn5 transposase around the binding sites of known TFs by comparing ATAC-seq data of TF-deficient strains with the data from a wild-type strain.

scholarly journals Subtype-associated epigenomic landscape and 3D genome structure in bladder cancer

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Tejaswi Iyyanki ◽  
Baozhen Zhang ◽  
Qixuan Wang ◽  
Ye Hou ◽  
Qiushi Jin ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Transcription Factor ◽  
Cancer Cell ◽  
Genome Structure ◽  
Transcription Factor Binding ◽  
Specific Gene ◽  
Open Chromatin ◽  
Factor Binding ◽  
3D Genome ◽  
Genome Wide

Abstract Muscle-invasive bladder cancers are characterized by their distinct expression of luminal and basal genes, which could be used to predict key clinical features such as disease progression and overall survival. Transcriptionally, FOXA1, GATA3, and PPARG are shown to be essential for luminal subtype-specific gene regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are critical for regulation of basal subtype-specific genes. Despite these advances, the underlying epigenetic mechanisms and 3D chromatin architecture responsible for subtype-specific regulation in bladder cancer remain unknown. Result We determine the genome-wide transcriptome, enhancer landscape, and transcription factor binding profiles of FOXA1 and GATA3 in luminal and basal subtypes of bladder cancer. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct transcription factor binding at distal enhancers of luminal and basal bladder cancers. Finally, we identify a novel clinically relevant transcription factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration. Conclusion In summary, our work identifies unique epigenomic signatures and 3D genome structures in luminal and basal urinary bladder cancers and suggests a novel link between the circadian transcription factor NPAS2 and a clinical bladder cancer subtype.

scholarly journals Compromised Function of the Pancreatic Transcription Factor PDX1 in a Lineage of Desert Rodents

2021 ◽  
Author(s):  
Yichen Dai ◽  
Sonia Trigueros ◽  
Peter W. H. Holland
Keyword(s):  
Transcription Factor ◽  
Gene Conversion ◽  
Cell Function ◽  
Protein A ◽  
Gene Promoter ◽  
Homeodomain Protein ◽  
Β Cell ◽  
Desert Rodents ◽  
Biased Gene Conversion ◽  
Β Cell Function

AbstractGerbils are a subfamily of rodents living in arid regions of Asia and Africa. Recent studies have shown that several gerbil species have unusual amino acid changes in the PDX1 protein, a homeodomain transcription factor essential for pancreatic development and β-cell function. These changes were linked to strong GC-bias in the genome that may be caused by GC-biased gene conversion, and it has been hypothesized that this caused accumulation of deleterious changes. Here we use two approaches to examine if the unusual changes are adaptive or deleterious. First, we compare PDX1 protein sequences between 38 rodents to test for association with habitat. We show the PDX1 homeodomain is almost totally conserved in rodents, apart from gerbils, regardless of habitat. Second, we use ectopic gene overexpression and gene editing in cell culture to compare functional properties of PDX1 proteins. We show that the divergent gerbil PDX1 protein inefficiently binds an insulin gene promoter and ineffectively regulates insulin expression in response to high glucose in rat cells. The protein has, however, retained the ability to regulate some other β-cell genes. We suggest that during the evolution of gerbils, the selection-blind process of biased gene conversion pushed fixation of mutations adversely affecting function of a normally conserved homeodomain protein. We argue these changes were not entirely adaptive and may be associated with metabolic disorders in gerbil species on high carbohydrate diets. This unusual pattern of molecular evolution could have had a constraining effect on habitat and diet choice in the gerbil lineage.

scholarly journals Inferring time series chromatin states for promoter-enhancer pairs based on Hi-C data

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Henriette Miko ◽  
Yunjiang Qiu ◽  
Bjoern Gaertner ◽  
Maike Sander ◽  
Uwe Ohler
Keyword(s):  
Time Series ◽  
Histone Modifications ◽  
Gene Activation ◽  
Expectation Maximization Algorithm ◽  
Chromatin State ◽  
Open Chromatin ◽  
Multiple Time ◽  
Enhancer Activity ◽  
Chromatin States ◽  
Multiple Time Points

Abstract Background Co-localized combinations of histone modifications (“chromatin states”) have been shown to correlate with promoter and enhancer activity. Changes in chromatin states over multiple time points (“chromatin state trajectories”) have previously been analyzed at promoter and enhancers separately. With the advent of time series Hi-C data it is now possible to connect promoters and enhancers and to analyze chromatin state trajectories at promoter-enhancer pairs. Results We present TimelessFlex, a framework for investigating chromatin state trajectories at promoters and enhancers and at promoter-enhancer pairs based on Hi-C information. TimelessFlex extends our previous approach Timeless, a Bayesian network for clustering multiple histone modification data sets at promoter and enhancer feature regions. We utilize time series ATAC-seq data measuring open chromatin to define promoters and enhancer candidates. We developed an expectation-maximization algorithm to assign promoters and enhancers to each other based on Hi-C interactions and jointly cluster their feature regions into paired chromatin state trajectories. We find jointly clustered promoter-enhancer pairs showing the same activation patterns on both sides but with a stronger trend at the enhancer side. While the promoter side remains accessible across the time series, the enhancer side becomes dynamically more open towards the gene activation time point. Promoter cluster patterns show strong correlations with gene expression signals, whereas Hi-C signals get only slightly stronger towards activation. The code of the framework is available at https://github.com/henriettemiko/TimelessFlex. Conclusions TimelessFlex clusters time series histone modifications at promoter-enhancer pairs based on Hi-C and it can identify distinct chromatin states at promoter and enhancer feature regions and their changes over time.

scholarly journals EPEN-30. C11ORF95-RELA FUSION PROTEIN ENGAGES NOVEL GENOMIC LOCI TO DRIVE MURINE EPENDYMOMA GROWTH

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii314-iii314
Author(s):  
Amir Arabzade ◽  
Yanhua Zhao ◽  
Srinidhi Varadharajan ◽  
Hsiao-Chi Chen ◽  
Austin Stuckert ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Molecular Targets ◽  
Lead Target ◽  
Open Chromatin ◽  
Rna Seq ◽  
In Utero Electroporation ◽  
Pathway Activation ◽  
Mouse Cells ◽  
Shed Light

Abstract RATIONALE Over 70% of supratentorial (ST) ependymoma are characterized by an oncogenic fusion between C11ORF95 and RELA. C11ORF95-RELA fusion is frequently the sole genetic driver detected in ST ependymoma, thus ranking this genomic event as a lead target for therapeutic investigation. RELA is a transcription factor (TF) central to mediating NF-kB pathway activation in processes such as inflammation, cellular metabolism, and chemotaxis. HYPOTHESIS: We posited that C11ORF95-RELA acts as an oncogenic TF that aberrantly shapes the tumor epigenome to drive aberrant transcription. Approach: To this end we developed an in utero electroporation (IUE) mouse model of ependymoma to express C11ORF95-RELA during embryonic development. Our IUE approach allowed us to develop C11ORF95-RELA driven tumor models and cell lines. We comprehensively characterized the epigenome and transcriptome of C11ORF95-RELA fusion driven mouse cells by H3K27ac ChIP-seq, ATAC-seq, and RNA-seq. RESULTS This data revealed that: 1) C11ORF95-RELA directly engages ‘open’ chromatin and is enriched at regions with known RELA TF binding sites as well as novel genomic loci/motifs, 2) C11ORF95-RELA preferentially binds to both H3K27ac (active) enhancers and promoters, and 3) Bound C11ORF95-RELA promoter loci are associated with increased transcription of genes shared with human ependymoma. CONCLUSION Our findings shed light on the transcriptional mechanisms of C11ORF95-RELA, and reveal downstream targets that may represent cancer dependency genes and molecular targets.

scholarly journals False Positive Values of Biomarkers of Prenatal Screening on Chromosomopathy as Indicators of a Risky Pregnancy

2011 ◽  
Vol 30 (2) ◽  
pp. 126-130 ◽  
Author(s):  
Jasmina Durković ◽  
Luka Anđelić ◽  
Bojana Mandić ◽  
Denis Lazar
Keyword(s):  
Genetic Screening ◽  
False Positive ◽  
Prenatal Screening ◽  
Protein A ◽  
First Trimester ◽  
Maternal Serum ◽  
Fetal Hypoxia ◽  
Beta Hcg ◽  
First Trimester Of Pregnancy ◽  
Fetal Karyotype

False Positive Values of Biomarkers of Prenatal Screening on Chromosomopathy as Indicators of a Risky PregnancyGenetic screening on chromosomopathy has been performed on 2000 pregnant women in their first trimester of pregnancy by determining Pregnancy associated plasma protein-A and free-beta HCG biomarkers in maternal serum. After obtaining a normal fetal karyotype, the pathological values of the biomarkers have been correlated with other pregnancy disorders, and the possible causes of the positive genetic screening have been tested. 340 false positive biomarkers (17%) have been detected. The increased free-beta HCG (48.24%) had a significant influence. A significant correlation (p > 0.01) between the increased free-beta HCG and bleeding during pregnancy has been established. Complications occurred in 78.52% pregnancies with pathological biomarkers, MISSed in 13.82%, miscarriages in 10.88%, induced pregnancy terminations caused by fetal anomalies in 8.82% and births with disturbed fetal vitality in 45%. The research results have shown a significant correlation (p > 0.01) between the increased value of the free-beta HCG biomarkers and fetal hypoxia. The false positive genetic screening, caused by the increased free-beta HCG, can indicate placental dysfunction and fetal vitality disruption.

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