Characterization of Epigenetic Histone Activation/Repression Marks in Sequences of Genes by Chromatin Immunoprecipitation-Quantitative Polymerase Chain Reaction (ChIP-qPCR)

Bhatia, Shama; Matthews, Jason; Wells, Peter G.

doi:10.1007/978-1-4939-9182-2_25

Cited by 8 publications

(7 citation statements)

References 11 publications

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“…Chromatin-immunoprecipitation qPCR (ChIP-qPCR) was performed as described previously ( Bhatia et al, 2019 ) with slight modifications. Briefly, since the transcription of TERC started to reduce at 40 days in culture, we chose cells cultured for 40 days as samples for ChIP-qPCR: the single clone-derived GDF11 KO and WT Neuro 2a cells were fixed and cross-linked in 1% formaldehyde for 10 min and quenched by glycine (125 mM) for 5 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Loss of Growth Differentiation Factor 11 Shortens Telomere Length by Downregulating Telomerase Activity

Wang

Zhu

et al. 2021

Front. Physiol.

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Maintenance of telomere length is essential to delay replicative cellular senescence. It is controversial on whether growth differentiation factor 11 (GDF11) can reverse cellular senescence, and this work aims to establish the causality between GDF11 and the telomere maintenance unequivocally. Using CRISPR/Cas9 technique and a long-term in vitro culture model of cellular senescence, we show here that in vitro genetic deletion of GDF11 causes shortening of telomere length, downregulation of telomeric reverse transcriptase (TERT) and telomeric RNA component (TERC), the key enzyme and the RNA component for extension of the telomere, and reduction of telomerase activity. In contrast, both recombinant and overexpressed GDF11 restore the transcription of TERT in GDF11KO cells to the wild-type level. Furthermore, loss of GDF11-induced telomere shortening is likely caused by enhancing the nuclear entry of SMAD2 which inhibits the transcription of TERT and TERC. Our results provide the first proof-of-cause-and-effect evidence that endogenous GDF11 plays a causal role for proliferative cells to maintain telomere length, paving the way for potential rejuvenation of the proliferative cells, tissues, and organs.

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Section: Methodsmentioning

confidence: 99%

Loss of Growth Differentiation Factor 11 Shortens Telomere Length by Downregulating Telomerase Activity

Wang

Zhu

et al. 2021

Front. Physiol.

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“…1 IRMs are any substance that binds to the antibody or beads used in the assay; background (noise) occurs when the intended bio-molecular target ( 2 BMT) is not the only 1 IRM in the immunoprecipitation step. Any nucleic acid chain that is in a stable complex with immuno-reactive molecules is isolated from other components through an immunoprecipitation reaction using an antibody bound to magnetic or agarose beads [ [3] , [4] , [5] , [6] , [7] , [8] ]. Once this DNA is isolated, it can be analyzed by microarray, qPCR, or sequencing.…”

Section: Introductionmentioning

confidence: 99%

A novel method for the normalization of ChIP-qPCR data

Solomon

Allan

2021

MethodsX

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ChIP-qPCR permits the study of protein and chromatin interactions. The general technique can apply to the study of the interactions of protein with RNA, and the methylation state of genomic DNA. While the technique is vital to our understanding of epigenetic processes, there is much confusion around the proper normalization methods. Percent Input has recently emerged as a normalization standard, due to its reproducibility and accuracy. This method relies on the use of a constant volume of ChIP Isolate in each qPCR assay. Researchers may accidentally run qPCR assays with a constant amount of isolate, a common practice for RT-qPCR; however, the traditional Percent Input method cannot accurately normalize these data. We developed a novel method that can normalize these data to provide the same reproducible Percent Input value. Here, we present evidence that this novel method of normalizing ChIP-qPCR data works with real samples. Later, we present a mathematical proof which shows how a Percent Input value calculated from Cq (quantification cycle) values obtained from qPCR run with a constant amount (in nanograms of DNA in ChIP isolate) is equal to the traditional Percent Input calculated from quantification cycle (Cq) values obtained from running a constant volume of ChIP isolate. Increases the number of possible data points per sample End values are the same % Input values as the traditional normalization method

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“…ChIP-qPCR was performed as described previously [ 36 ]. To measure the enrichment of potential RpoE-binding targets in the immunoprecipitated DNA samples, percent input and fold enrichment were performed using SYBR green mix.…”

Section: Methodsmentioning

confidence: 99%

“…The relative target levels were calculated using the ΔCt method. lacZ was used as a negative control, with specific details referring to the ChIP Analysis [ 36 ]. The results were reported as the average enrichment of three biological replicates.…”

Section: Methodsmentioning

confidence: 99%

RpoE Facilitates Stress-Resistance, Invasion, and Pathogenicity of Escherichia coli K1

Bai

et al. 2022

Microorganisms

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Escherichia coli K1 is the most common Gram-negative bacterium that causes neonatal meningitis; thus, a better understanding of its pathogenic molecular mechanisms is critical. However, the mechanisms by which E. coli K1 senses the signals of the host and expresses toxins for survival are poorly understood. As an extracytoplasmic function sigma factor, RpoE controls a wide range of pathogenesis-associated pathways in response to environmental stress. We found that the ΔrpoE mutant strain reduced the binding and invasion rate in human brain microvascular endothelial cells (HBMECs) in vitro, level of bacteremia, and percentage of meningitis in vivo. To confirm the direct targets of RpoE in vivo, we performed qRT-PCR and ChIP-qPCR on known toxic genes. RpoE was found to regulate pathogenic target genes, namely, ompA, cnf1, fimB, ibeA, kpsM, and kpsF directly and fimA, aslA, and traJ indirectly. The expression of these genes was upregulated when E. coli K1 was cultured with antibacterial peptides, whereas remained unchanged in the presence of the ΔrpoE mutant strain. Moreover, RpoE reduced IL-6 and IL-8 levels in E. coli K1-infected HBMECs. Altogether, these findings demonstrate that RpoE mediates the host adaptation capacity of E. coli K1 via a regulatory mechanism on virulence factors.

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Characterization of Epigenetic Histone Activation/Repression Marks in Sequences of Genes by Chromatin Immunoprecipitation-Quantitative Polymerase Chain Reaction (ChIP-qPCR)

Cited by 8 publications

References 11 publications

Loss of Growth Differentiation Factor 11 Shortens Telomere Length by Downregulating Telomerase Activity

Loss of Growth Differentiation Factor 11 Shortens Telomere Length by Downregulating Telomerase Activity

A novel method for the normalization of ChIP-qPCR data

RpoE Facilitates Stress-Resistance, Invasion, and Pathogenicity of Escherichia coli K1

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