Contemporary research methods in business

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Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated.

BACKGROUND: DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. RESULTS: Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. CONCLUSIONS: Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

A novel RNA-mediated mechanism causing down-regulation of insulating promoter interactions in human embryonic stem cells

From Springer Nature via Jisc Publications RouterHistory: received 2021-02-10, accepted 2021-11-15, registration 2021-11-16, collection 2021-12, pub-electronic 2021-12-01, online 2021-12-01Publication status: PublishedFunder: British Heart Foundation; doi: http://dx.doi.org/10.13039/501100000274; Grant(s): RG/15/12/31616Funder: University of Manchester; doi: http://dx.doi.org/10.13039/501100000770Abstract: The genome-wide promoter interactome is primarily maintained and regulated by architectural proteins such as CTCF and cohesin. However, some studies suggest a role for non-coding RNAs (ncRNAs) in this process. We aimed to characterise the regulatory role of RNA-mediated promoter interactions in the control of gene expression. We integrated genome-wide datasets of RNA-chromatin and promoter-genome interactions in human embryonic stem cells (hESCs) to identify putative RNA-mediated promoter interactions. We discovered that CTCF sites were enriched in RNA-PIRs (promoter interacting regions co-localising with RNA-chromatin interaction sites) and genes interacting with RNA-PIRs containing CTCF sites showed higher expression levels. One of the long noncoding RNAs (lncRNAs) expressed in hESCs, Syntaxin 18-Antisense 1 (STX18-AS1), appeared to be involved in an insulating promoter interaction with the neighbouring gene, MSX1. By knocking down STX18-AS1, the MSX1 promoter-PIR interaction was intensified and the target gene (MSX1) expression was down-regulated. Conversely, reduced MSX1 promoter-PIR interactions, resulting from CRISPR-Cas9 deletion of the PIR, increased the expression of MSX1. We conclude that STX18-AS1 RNA antagonised local CTCF-mediated insulating promoter interactions to augment gene expression. Such down-regulation of the insulating promoter interactions by this novel mechanism may explain the higher expression of genes interacting with RNA-PIRs linked to CTCF sites

The landscape of DNA repeat elements in human heart failure.

BACKGROUND: The epigenomes of healthy and diseased human hearts were recently examined by genome-wide DNA methylation profiling. Repetitive elements, heavily methylated in post-natal tissue, have variable methylation profiles in cancer but methylation of repetitive elements in the heart has never been examined. RESULTS: We analyzed repetitive elements from all repeat families in human myocardial samples, and found that satellite repeat elements were significantly hypomethylated in end-stage cardiomyopathic hearts relative to healthy normal controls. Satellite repeat elements are almost always centromeric or juxtacentromeric, and their overexpression correlates with disease aggressiveness in cancer. Similarly, we found that hypomethylation of satellite repeat elements correlated with up to 27-fold upregulation of the corresponding transcripts in end-stage cardiomyopathic hearts. No other repeat family exhibited differential methylation between healthy and cardiomyopathic hearts, with the exception of the Alu element SINE1/7SL, for which a modestly consistent trend of increased methylation was observed. CONCLUSIONS: Satellite repeat element transcripts, a form of non-coding RNA, have putative functions in maintaining genomic stability and chromosomal integrity. Further studies will be needed to establish the functional significance of these non-coding RNAs in the context of heart failure

Data on cardiac lncRNA STX18-AS1 expression in developing human hearts and function during in vitro hESC-cardiomyocyte differentiation

This article presents data concerning STX18-AS1, a long noncoding RNA gene identified from a Genome-wide association study of Atrial Septal Defect (ASD). The data describes its expression patterns in human tissues and functions in regulating cardiomyocyte differentiation in vitro. STX18-AS1 is a lncRNA with a higher abundance in developing tissues, including hearts. Its transcription distribution within the embryonic hearts during key heart septation stages supports STX18-AS1’s association with risk SNPs for ASD. The CRISPR stem cell pool in which STX18-AS1 was knocked down, showed reduced CM differentiation efficiency and lower expression of key cardiac transcriptional factors. This indicated its regulative role in supporting the lineage specification from cardiac mesoderm into cardiac progenitors and cardiomyocytes. These data can benefit the understanding of human embryonic heart developmental biology, and the time-course changes of cardiac transcriptional factors during in vitro cardiomyocyte differentiation from human embryonic stem cells

High-throughput sequencing identifies STAT3 as the DNA-associated factor for p53-NF-kappaB-complex-dependent gene expression in human heart failure.

BACKGROUND: Genome-wide maps of DNA regulatory elements and their interaction with transcription factors may form a framework for understanding regulatory circuits and gene expression control in human disease, but how these networks, comprising transcription factors and DNA-binding proteins, form complexes, interact with DNA and modulate gene expression remains largely unknown. METHODS: Using microRNA-21 (mir-21), which is an example of genes that are regulated in heart failure, we performed chromatin immunoprecipitation (ChIP) assays to determine the occupancy of transcription factors at this genetic locus. Tissue ChIP was further performed using human hearts and genome-wide occupancies of these transcription factors were analyzed by high-throughput sequencing. RESULTS: We show that the transcription factor p53 piggy-backs onto NF-kappaB/RELA and utilizes the kappaB-motif at a cis-regulatory region to control mir-21 expression. p53 behaves as a co-factor in this complex because despite a mutation in its DNA binding domain, mutant p53 was still capable of binding RELA and the cis-element, and inducing mir-21 expression. In dilated human hearts where mir-21 upregulation was previously demonstrated, the p53-RELA complex was also associated with this cis-element. Using high-throughput sequencing, we analyzed genome-wide binding sites for the p53-RELA complex in diseased and control human hearts and found a significant overrepresentation of the STAT3 motif. We further determined that STAT3 was necessary for the p53-RELA complex to associate with this cis-element and for mir-21 expression. CONCLUSIONS: Our results uncover a mechanism by which transcription factors cooperate in a multi-molecular complex at a cis-regulatory element to control gene expression

Distinct epigenomic features in end-stage failing human hearts

Background— The epigenome refers to marks on the genome, including DNA methylation and histone modifications, that regulate the expression of underlying genes. A consistent profile of gene expression changes in end-stage cardiomyopathy led us to hypothesize that distinct global patterns of the epigenome may also exist. Methods and Results— We constructed genome-wide maps of DNA methylation and histone-3 lysine-36 trimethylation (H3K36me3) enrichment for cardiomyopathic and normal human hearts. More than 506 Mb sequences per library were generated by high-throughput sequencing, allowing us to assign methylation scores to ≈28 million CG dinucleotides in the human genome. DNA methylation was significantly different in promoter CpG islands, intragenic CpG islands, gene bodies, and H3K36me3-enriched regions of the genome. DNA methylation differences were present in promoters of upregulated genes but not downregulated genes. H3K36me3 enrichment itself was also significantly different in coding regions of the genome. Specifically, abundance of RNA transcripts encoded by the DUX4 locus correlated to differential DNA methylation and H3K36me3 enrichment. In vitro, Dux gene expression was responsive to a specific inhibitor of DNA methyltransferase, and Dux siRNA knockdown led to reduced cell viability. Conclusions— Distinct epigenomic patterns exist in important DNA elements of the cardiac genome in human end-stage cardiomyopathy. The epigenome may control the expression of local or distal genes with critical functions in myocardial stress response. If epigenomic patterns track with disease progression, assays for the epigenome may be useful for assessing prognosis in heart failure. Further studies are needed to determine whether and how the epigenome contributes to the development of cardiomyopathy

Author Correction: Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks (Nature Communications, (2018), 9, 1, (2526), 10.1038/s41467-018-04931-0)

In the original version of the Article, the gene symbol for tissue factor pathway inhibitor was inadvertently given as ‘TFP1’ instead of ‘TFPI’. This has now been corrected in both the PDF and HTML versions of the Article

Characterisation of an Arabidopsis mutant with altered greening characteristics

Two plastid-to-nucleus signalling pathways had previously been identified by studies on genomes uncoupled (gun) mutants of Arabidopsis. A pathway responding to the state of plastid protein synthesis was disrupted in gunl mutants. Unlike the wild type, the mutants show expression of nuclear photosynthesis genes in the presence of norflurazon, which results in chloroplast photooxidation, or in the presence of lincomycin, an inhibitor of plastid translation. The aim of this study was to understand the role of plastid protein synthesis in plastid-to-nucleus signalling. Five putative gunl-like mutants from a new collection of Arabidopsis gun mutants with a green fluorescent protein (GFP) reporter gene under the control of a tobacco RbcS (encoding ribulose-1,5-bisphosphate carboxylase small subunit) promoter were examined further. One of the mutant lines, PR48.2N, showed two-fold higher transcript abundance of nuclear photosynthesis genes, RBCS and LHCBJ (encoding light-harvesting chlorophyll a/b-binding protein 1), compared to wild type with or without treatments of norflurazon or lincomycin. Pigment analysis of PR48.2N seedlings illuminated for 16 hours after being subjected to various lengths of dark treatment demonstrated that the mutant line accumulated less chlorophyll than wild type after short periods of darkness (2-4 days) but showed an enhanced ability to green after prolonged dark treatments (5-10 days). Consistent with the enhanced greening ability, transcript abundance of nuclear photosynthesis genes was higher and there was more thylakoid membrane in chloroplasts in greened PR48.2N seedlings after prolonged darkness compared to the wild type. Microarray analysis indicated that a group of transcripts encoding seed storage proteins, oleosins and late embryogenesis abundant proteins showed very low abundance in PR48.2N seedlings. The promoter regions of the genes shared some ciselements possibly involved in regulation by abscisic acid (ABA). However, the ABA content of PR48.2N seedlings was not significantly different to wild type, although the germination of mutant seeds was more sensitive to inhibition by ABA than the wild type. Expression of the GFP reporter gene in the presence of lincomycin and the enhanced greening ability of PR48.2N were shown to be inherited in a recessive manner by examining the segregation of these phenotypes in the F2 progeny of a PR48.2N (Ws) x wild type (Ler) cross. Genetic analysis using F2 individuals from agunl-1 x PR48.2N cross showed that the mutated genes were not allelic but might be interacting because putative double mutants with much higher GFP expression were observed. Preliminary data from PCR-based mapping methods using a small F2 mapping population suggest that the locus providing enhanced greening ability in PR48.2N may be in a region on chromosome V. PR48.2N phenotypes such as expression of the GFP reporter gene in the presence of norflurazon or lincomycin, enhanced greening ability, lower transcript levels of seed protein genes, and hypersensitivity to the ABA inhibitory effect on germination appeared to co-segregate in the mapping population. Although PR48.2N mutant was initially identified in a screen for gunl-like mutants, the evidence presented in this dissertation suggests that the product encoded by the gene mutated in PR48.2N may have a regulatory role in the transition from heterotrophic to photoautotrophic growth during early seedling development