Human ChIP-seq grade GAPDH TSS primer pair

Catalog Number

Format

Price

C17011047-50
(pp-1047-050)

50 µl

$35.00

Other format

C17011047-500

500 µl

This primer pair specifically amplifies a genomic region containing the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The primers are thoroughly tested and optimized for routine SYBR® Green Real-Time qPCR assay following ChIP and for ChIP-sequencing library validation (e.g. before and after ChIP-seq library preparation).

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Digging Deeper into Breast Cancer Epigenetics: Insights from ChemicalInhibition of Histone Acetyltransferase TIP60 .
Idrissou, Mouhamed and Lebert, Andre and Boisnier, Tiphanie and Sanchez,Anna and Houfaf Khoufaf, Fatma Zohra and Penault-Llorca, Frédérique andBignon, Yves-Jean and Bernard-Gallon, Dominique
Breast cancer is often sporadic due to several factors. Among them, the deregulation of epigenetic proteins may be involved. TIP60 or KAT5 is an acetyltransferase that regulates gene transcription through the chromatin structure. This pleiotropic protein acts in several cellular pathways by acetylating proteins. RNA...

TIP60/P400/H4K12ac Plays a Role as a Heterochromatin Back-up Skeleton inBreast Cancer.
Idrissou, Mouhamed and Boisnier, Tiphanie and Sanchez, Anna and Khoufaf,Fatma Zohra Houfaf and Penault-Llorca, Frederique and Bignon, Yves-Jean andBernard-Gallon, Dominique
BACKGROUND/AIM: In breast cancer, initiation of carcinogenesis leads to epigenetic dysregulation, which can lead for example to the loss of the heterochromatin skeleton SUV39H1/H3K9me3/HP1 or the supposed secondary skeleton TIP60/P400/H4K12ac/BRD (2/4), which allows the maintenance of chromatin integrity and plastic...

Saliva as a Blood Alternative for Genome-Wide DNA Methylation Profiling by Methylated DNA Immunoprecipitation (MeDIP) Sequencing
Staunstrup N.H. et al.
Background: Interrogation of DNA methylation profiles hold promise for improved diagnostics, as well as the delineation of the aetiology for common human diseases. However, as the primary tissue of the disease is often inaccessible without complicated and inconvenient interventions, there is an increasing interest i...

Emerging Role of One-Carbon Metabolism and DNA Methylation Enrichment on δ-Containing GABAA Receptor Expression in the Cerebellum of Subjects with Alcohol Use Disorders (AUD
Gatta E. et al.
Background Cerebellum is an area of the brain particularly sensitive to the effects of acute and chronic alcohol consumption. Alcohol exposure decreases cerebellar Purkinje cell output by increasing GABA release from Golgi cells onto extrasynaptic α6/δ-containing GABAA receptors located on glutamate...

Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples
Bak S.T. et al.
We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), co...

Genome-wide DNA methylation profiling with MeDIP-seq using archived dried blood spots
Nicklas H. Staunstrup et al.
Background In utero and early-life experienced environmental exposures are suggested to play an important role in many multifactorial diseases potentially mediated through lasting effects on the epigenome. As the epigenome in addition remains modifiable throughout life, identifying specific disease-relevant biomarke...

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			'name' => 'Digging Deeper into Breast Cancer Epigenetics: Insights from ChemicalInhibition of Histone Acetyltransferase TIP60 .',
			'authors' => 'Idrissou, Mouhamed and Lebert, Andre and Boisnier, Tiphanie and Sanchez,Anna and Houfaf Khoufaf, Fatma Zohra and Penault-Llorca, Frédérique andBignon, Yves-Jean and Bernard-Gallon, Dominique',
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			'date' => '2020-10-01',
			'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32960142',
			'doi' => '10.1089/omi.2020.0104',
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			'name' => 'TIP60/P400/H4K12ac Plays a Role as a Heterochromatin Back-up Skeleton inBreast Cancer.',
			'authors' => 'Idrissou, Mouhamed and Boisnier, Tiphanie and Sanchez, Anna and Khoufaf,Fatma Zohra Houfaf and Penault-Llorca, Frederique and Bignon, Yves-Jean andBernard-Gallon, Dominique',
			'description' => '<p>BACKGROUND/AIM: In breast cancer, initiation of carcinogenesis leads to epigenetic dysregulation, which can lead for example to the loss of the heterochromatin skeleton SUV39H1/H3K9me3/HP1 or the supposed secondary skeleton TIP60/P400/H4K12ac/BRD (2/4), which allows the maintenance of chromatin integrity and plasticity. This study investigated the relationship between TIP60, P400 and H4K12ac and their implications in breast tumors. MATERIALS AND METHODS: Seventy-seven patients diagnosed with breast cancer were included in this study. Chromatin immunoprecipitation (ChIP) assay was used to identify chromatin modifications. Western blot and reverse transcription and quantitative real-time PCR were used to determine protein and gene expression, respectively. RESULTS: We verified the variation in H4K12ac enrichment and the co-localization of H4K12ac and TIP60 on the euchromatin and heterochromatin genes, respectively, by ChIP-qPCR and ChIP-reChIP, which showed an enrichment of H4K12ac on specific genes in tumors compared to the adjacent healthy tissue and a co-localization of H4K12ac with TIP60 in different breast tumor types. Furthermore, RNA and protein expression of TIP60 and P400 was investigated and overexpression of TIP60 and P400 mRNA was associated with tumor aggressiveness. CONCLUSION: There is a potential interaction between H4K12ac and TIP60 in heterochromatin or euchromatin in breast tumors.</p>',
			'date' => '2020-01-01',
			'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33099470',
			'doi' => '10.21873/cgp.20223',
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			'authors' => 'Staunstrup N.H. et al.',
			'description' => '<p><b></b>Background: Interrogation of DNA methylation profiles hold promise for improved diagnostics, as well as the delineation of the aetiology for common human diseases. However, as the primary tissue of the disease is often inaccessible without complicated and inconvenient interventions, there is an increasing interest in peripheral surrogate tissues. Whereas most work has been conducted on blood, saliva is now becoming recognized as an interesting alternative due to the simple and non-invasive manner of collection allowing for self-sampling. Results: In this study we have evaluated if saliva samples are suitable for DNA methylation studies using methylated DNA immunoprecipitation coupled to next-generation sequencing (MeDIP-seq). This was done by comparing the DNA methylation profile in saliva against the benchmark profile of peripheral blood from three individuals. We show that the output, quality, and depth of paired-end 50 bp sequencing reads are comparable between saliva and peripheral blood and, moreover, that the distribution of reads along genomic regions are similar and follow canonical methylation patterns. Conclusion: In summary, we show that high-quality MeDIP-seq data can be generated using saliva, thus supporting the future use of saliva in the generation of DNA methylation information at annotated genes, non-RefSeq genes, and repetitive elements relevant to human disease.</p>',
			'date' => '2017-10-19',
			'pmid' => 'http://www.mdpi.com/2075-4655/1/3/14',
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			'authors' => 'Gatta E. et al.',
			'description' => '<section class="abstract">
<section class="sec">
<div class="title -title">Background</div>
<p>Cerebellum is an area of the brain particularly sensitive to the effects of acute and chronic alcohol consumption. Alcohol exposure decreases cerebellar Purkinje cell output by increasing GABA release from Golgi cells onto extrasynaptic &alpha;<sub>6</sub>/&delta;-containing GABA<sub>A</sub> receptors located on glutamatergic granule cells. Here, we studied whether chronic alcohol consumption induces changes in GABA<sub>A</sub> receptor subunit expression and whether these changes are associated with alterations in epigenetic mechanisms via DNA methylation.</p>
</section>
<section class="sec">
<div class="title -title">Methods</div>
<p>We used a cohort of postmortem cerebellum from control and chronic alcoholics, here defined as alcohol use disorders subjects (n=25/group). <em>S</em>-adenosyl-methionine/<em>S</em>-adenosyl-homocysteine were measured by high-performance liquid chromatography. mRNA levels of various genes were assessed by reverse transcriptase-quantitative polymerase chain reaction. Promoter methylation enrichment was assessed using methylated DNA immunoprecipitation and hydroxy-methylated DNA immunoprecipitation assays.</p>
</section>
<section class="sec">
<div class="title -title">Results</div>
<p>mRNAs encoding key enzymes of 1-carbon metabolism that determine the <em>S</em>-adenosyl-methionine/<em>S</em>-adenosyl-homocysteine ratio were increased, indicating higher &ldquo;methylation index&rdquo; in alcohol use disorder subjects. We found that increased methylation of the promoter of the &delta; subunit GABA<sub>A</sub> receptor was associated with reduced mRNA and protein levels in the cerebellum of alcohol use disorder subjects. No changes were observed in &alpha;<sub>1</sub>- or &alpha;<sub>6</sub>-containing GABA<sub>A</sub> receptor subunits. The expression of DNA-methyltransferases (1, 3A, and 3B) was unaltered, whereas the mRNA level of TET1, which participates in the DNA demethylation pathway, was decreased. Hence, increased methylation of the &delta; subunit GABA<sub>A</sub> receptor promoter may result from alcohol-induced reduction of DNA demethylation.</p>
</section>
<section class="sec">
<div class="title -title">Conclusion</div>
<p>Together, these results support the hypothesis that aberrant DNA methylation pathways may be involved in cerebellar pathophysiology of alcoholism. Furthermore, this work provides novel evidence for a central role of DNA methylation mechanisms in the alcohol-induced neuroadaptive changes of human cerebellar GABA<sub>A</sub> receptor function.</p>
</section>
</section>',
			'date' => '2017-08-19',
			'pmid' => 'https://academic.oup.com/ijnp/article/doi/10.1093/ijnp/pyx075/4085582/Emerging-role-of-one-carbon-metabolism-and-DNA',
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			'name' => 'Digging Deeper into Breast Cancer Epigenetics: Insights from ChemicalInhibition of Histone Acetyltransferase TIP60 .',
			'authors' => 'Idrissou, Mouhamed and Lebert, Andre and Boisnier, Tiphanie and Sanchez,Anna and Houfaf Khoufaf, Fatma Zohra and Penault-Llorca, Frédérique andBignon, Yves-Jean and Bernard-Gallon, Dominique',
			'description' => '<p>Breast cancer is often sporadic due to several factors. Among them, the deregulation of epigenetic proteins may be involved. TIP60 or KAT5 is an acetyltransferase that regulates gene transcription through the chromatin structure. This pleiotropic protein acts in several cellular pathways by acetylating proteins. RNA and protein expressions of TIP60 were shown to decrease in some breast cancer subtypes, particularly in triple-negative breast cancer (TNBC), where a low expression of TIP60 was exhibited compared with luminal subtypes. In this study, the inhibition of the residual activity of TIP60 in breast cancer cell lines was investigated by using two chemical inhibitors, TH1834 and NU9056, first on the acetylation of the specific target, lysine 4 of histone 3 (H3K4) by immunoblotting, and second, by chromatin immunoprecipitation (ChIP)-qPCR (-quantitative Polymerase Chain Reaction). Subsequently, significant decreases or a trend toward decrease of H3K4ac in the different chromatin compartments were observed. In addition, the expression of 48 human nuclear receptors was studied with TaqMan Low-Density Array in these breast cancer cell lines treated with TIP60 inhibitors. The statistical analysis allowed us to comprehensively characterize the androgen receptor and receptors in TNBC cell lines after TH1834 or NU9056 treatment. The understanding of the residual activity of TIP60 in the evolution of breast cancer might be a major asset in the fight against this disease, and could allow TIP60 to be used as a biomarker or therapeutic target for breast cancer progression in the future.</p>',
			'date' => '2020-10-01',
			'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32960142',
			'doi' => '10.1089/omi.2020.0104',
			'modified' => '2021-02-19 17:39:52',
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			'name' => 'TIP60/P400/H4K12ac Plays a Role as a Heterochromatin Back-up Skeleton inBreast Cancer.',
			'authors' => 'Idrissou, Mouhamed and Boisnier, Tiphanie and Sanchez, Anna and Khoufaf,Fatma Zohra Houfaf and Penault-Llorca, Frederique and Bignon, Yves-Jean andBernard-Gallon, Dominique',
			'description' => '<p>BACKGROUND/AIM: In breast cancer, initiation of carcinogenesis leads to epigenetic dysregulation, which can lead for example to the loss of the heterochromatin skeleton SUV39H1/H3K9me3/HP1 or the supposed secondary skeleton TIP60/P400/H4K12ac/BRD (2/4), which allows the maintenance of chromatin integrity and plasticity. This study investigated the relationship between TIP60, P400 and H4K12ac and their implications in breast tumors. MATERIALS AND METHODS: Seventy-seven patients diagnosed with breast cancer were included in this study. Chromatin immunoprecipitation (ChIP) assay was used to identify chromatin modifications. Western blot and reverse transcription and quantitative real-time PCR were used to determine protein and gene expression, respectively. RESULTS: We verified the variation in H4K12ac enrichment and the co-localization of H4K12ac and TIP60 on the euchromatin and heterochromatin genes, respectively, by ChIP-qPCR and ChIP-reChIP, which showed an enrichment of H4K12ac on specific genes in tumors compared to the adjacent healthy tissue and a co-localization of H4K12ac with TIP60 in different breast tumor types. Furthermore, RNA and protein expression of TIP60 and P400 was investigated and overexpression of TIP60 and P400 mRNA was associated with tumor aggressiveness. CONCLUSION: There is a potential interaction between H4K12ac and TIP60 in heterochromatin or euchromatin in breast tumors.</p>',
			'date' => '2020-01-01',
			'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33099470',
			'doi' => '10.21873/cgp.20223',
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			'name' => 'Saliva as a Blood Alternative for Genome-Wide DNA Methylation Profiling by Methylated DNA Immunoprecipitation (MeDIP) Sequencing',
			'authors' => 'Staunstrup N.H. et al.',
			'description' => '<p><b></b>Background: Interrogation of DNA methylation profiles hold promise for improved diagnostics, as well as the delineation of the aetiology for common human diseases. However, as the primary tissue of the disease is often inaccessible without complicated and inconvenient interventions, there is an increasing interest in peripheral surrogate tissues. Whereas most work has been conducted on blood, saliva is now becoming recognized as an interesting alternative due to the simple and non-invasive manner of collection allowing for self-sampling. Results: In this study we have evaluated if saliva samples are suitable for DNA methylation studies using methylated DNA immunoprecipitation coupled to next-generation sequencing (MeDIP-seq). This was done by comparing the DNA methylation profile in saliva against the benchmark profile of peripheral blood from three individuals. We show that the output, quality, and depth of paired-end 50 bp sequencing reads are comparable between saliva and peripheral blood and, moreover, that the distribution of reads along genomic regions are similar and follow canonical methylation patterns. Conclusion: In summary, we show that high-quality MeDIP-seq data can be generated using saliva, thus supporting the future use of saliva in the generation of DNA methylation information at annotated genes, non-RefSeq genes, and repetitive elements relevant to human disease.</p>',
			'date' => '2017-10-19',
			'pmid' => 'http://www.mdpi.com/2075-4655/1/3/14',
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			'authors' => 'Gatta E. et al.',
			'description' => '<section class="abstract">
<section class="sec">
<div class="title -title">Background</div>
<p>Cerebellum is an area of the brain particularly sensitive to the effects of acute and chronic alcohol consumption. Alcohol exposure decreases cerebellar Purkinje cell output by increasing GABA release from Golgi cells onto extrasynaptic &alpha;<sub>6</sub>/&delta;-containing GABA<sub>A</sub> receptors located on glutamatergic granule cells. Here, we studied whether chronic alcohol consumption induces changes in GABA<sub>A</sub> receptor subunit expression and whether these changes are associated with alterations in epigenetic mechanisms via DNA methylation.</p>
</section>
<section class="sec">
<div class="title -title">Methods</div>
<p>We used a cohort of postmortem cerebellum from control and chronic alcoholics, here defined as alcohol use disorders subjects (n=25/group). <em>S</em>-adenosyl-methionine/<em>S</em>-adenosyl-homocysteine were measured by high-performance liquid chromatography. mRNA levels of various genes were assessed by reverse transcriptase-quantitative polymerase chain reaction. Promoter methylation enrichment was assessed using methylated DNA immunoprecipitation and hydroxy-methylated DNA immunoprecipitation assays.</p>
</section>
<section class="sec">
<div class="title -title">Results</div>
<p>mRNAs encoding key enzymes of 1-carbon metabolism that determine the <em>S</em>-adenosyl-methionine/<em>S</em>-adenosyl-homocysteine ratio were increased, indicating higher &ldquo;methylation index&rdquo; in alcohol use disorder subjects. We found that increased methylation of the promoter of the &delta; subunit GABA<sub>A</sub> receptor was associated with reduced mRNA and protein levels in the cerebellum of alcohol use disorder subjects. No changes were observed in &alpha;<sub>1</sub>- or &alpha;<sub>6</sub>-containing GABA<sub>A</sub> receptor subunits. The expression of DNA-methyltransferases (1, 3A, and 3B) was unaltered, whereas the mRNA level of TET1, which participates in the DNA demethylation pathway, was decreased. Hence, increased methylation of the &delta; subunit GABA<sub>A</sub> receptor promoter may result from alcohol-induced reduction of DNA demethylation.</p>
</section>
<section class="sec">
<div class="title -title">Conclusion</div>
<p>Together, these results support the hypothesis that aberrant DNA methylation pathways may be involved in cerebellar pathophysiology of alcoholism. Furthermore, this work provides novel evidence for a central role of DNA methylation mechanisms in the alcohol-induced neuroadaptive changes of human cerebellar GABA<sub>A</sub> receptor function.</p>
</section>
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			'id' => '3108',
			'name' => 'Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples',
			'authors' => 'Bak S.T. et al.',
			'description' => '<p>We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques. Overall, we found that DNA quality was inversely correlated with storage time and DNA quality was insufficient for Illumina methylation arrays; data from methylated DNA immunoprecipitation, clonal bisulfite sequencing, and pyrosequencing of BDNF and ST6GALNAC1 suggested that the original methylation pattern is indeed preserved. Proof-of-principle experiments predicting sex based on the methylation status of the X-inactivated SLC9A7 gene, or genotype differences of the Y and X chromosomes, showed consistency between predicted and actual sex for a subset of FFPE samples. In conclusion, even though DNA from FFPE samples is of low quality and technically challenging, it is likely that a subset of samples can provide reliable data given that the methodology used is designed for small DNA fragments. We propose that simple PCR-based quality control experiments at the genetic and DNA methylation level, carried out at the beginning of any given project, can be used to enrich for the best-performing FFPE samples. The apparent preservation of genetic and DNA methylation patterns in archival FFPE samples may bring along new perspectives for the identification of genetic and epigenetic changes associated with brain-manifested diseases.</p>',
			'date' => '2016-12-19',
			'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/27995571',
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			'name' => 'Genome-wide DNA methylation profiling with MeDIP-seq using archived dried blood spots',
			'authors' => 'Nicklas H. Staunstrup et al.',
			'description' => '<p><strong>Background</strong> In utero and early-life experienced environmental exposures are suggested to play an important role in many multifactorial diseases potentially mediated through lasting effects on the epigenome. As the epigenome in addition remains modifiable throughout life, identifying specific disease-relevant biomarkers may prove challenging. This has led to an increased interest in epigenome-wide association studies using dried blood spots (DBS) routinely collected in perinatal screening programs. Such programs are in place in numerous countries around the world producing large and unique biobanks. However, availability of this biological material is highly limited as each DBS is made only from a few droplets of blood and storage conditions may be suboptimal for epigenetic studies. Furthermore, as relevant markers may reside outside gene bodies, epigenome-wide interrogation is needed.</p>
<p><strong>Results</strong> Here we demonstrate, as a proof of principle, that genome-wide interrogation of the methylome based on methylated DNA immunoprecipitation coupled with next-generation sequencing (MeDIP-seq) is feasible using a single 3.2 mm DBS punch (60 ng DNA) from filter cards archived for up to 16 years. The enrichment profile, sequence quality and distribution of reads across genetic regions were comparable between samples archived 16 years, 4 years and a freshly prepared control sample.</p>
<p><strong>Conclusions</strong> In summary, we show that high-quality MeDIP-seq data is achievable from neonatal screening filter cards stored at room temperature, thereby providing information on annotated as well as on non-RefSeq genes and repetitive elements. Moreover, the quantity of DNA from one DBS punch proved sufficient allowing for multiple epigenome studies using one single DBS.</p>',
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			'url' => 'files/products/reagents/primer_pairs/Datasheet_GAPDH-TSS_pp1047.pdf',
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			'name' => 'Digging Deeper into Breast Cancer Epigenetics: Insights from ChemicalInhibition of Histone Acetyltransferase TIP60 .',
			'authors' => 'Idrissou, Mouhamed and Lebert, Andre and Boisnier, Tiphanie and Sanchez,Anna and Houfaf Khoufaf, Fatma Zohra and Penault-Llorca, Frédérique andBignon, Yves-Jean and Bernard-Gallon, Dominique',
			'description' => '<p>Breast cancer is often sporadic due to several factors. Among them, the deregulation of epigenetic proteins may be involved. TIP60 or KAT5 is an acetyltransferase that regulates gene transcription through the chromatin structure. This pleiotropic protein acts in several cellular pathways by acetylating proteins. RNA and protein expressions of TIP60 were shown to decrease in some breast cancer subtypes, particularly in triple-negative breast cancer (TNBC), where a low expression of TIP60 was exhibited compared with luminal subtypes. In this study, the inhibition of the residual activity of TIP60 in breast cancer cell lines was investigated by using two chemical inhibitors, TH1834 and NU9056, first on the acetylation of the specific target, lysine 4 of histone 3 (H3K4) by immunoblotting, and second, by chromatin immunoprecipitation (ChIP)-qPCR (-quantitative Polymerase Chain Reaction). Subsequently, significant decreases or a trend toward decrease of H3K4ac in the different chromatin compartments were observed. In addition, the expression of 48 human nuclear receptors was studied with TaqMan Low-Density Array in these breast cancer cell lines treated with TIP60 inhibitors. The statistical analysis allowed us to comprehensively characterize the androgen receptor and receptors in TNBC cell lines after TH1834 or NU9056 treatment. The understanding of the residual activity of TIP60 in the evolution of breast cancer might be a major asset in the fight against this disease, and could allow TIP60 to be used as a biomarker or therapeutic target for breast cancer progression in the future.</p>',
			'date' => '2020-10-01',
			'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32960142',
			'doi' => '10.1089/omi.2020.0104',
			'modified' => '2021-02-19 17:39:52',
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			'name' => 'TIP60/P400/H4K12ac Plays a Role as a Heterochromatin Back-up Skeleton inBreast Cancer.',
			'authors' => 'Idrissou, Mouhamed and Boisnier, Tiphanie and Sanchez, Anna and Khoufaf,Fatma Zohra Houfaf and Penault-Llorca, Frederique and Bignon, Yves-Jean andBernard-Gallon, Dominique',
			'description' => '<p>BACKGROUND/AIM: In breast cancer, initiation of carcinogenesis leads to epigenetic dysregulation, which can lead for example to the loss of the heterochromatin skeleton SUV39H1/H3K9me3/HP1 or the supposed secondary skeleton TIP60/P400/H4K12ac/BRD (2/4), which allows the maintenance of chromatin integrity and plasticity. This study investigated the relationship between TIP60, P400 and H4K12ac and their implications in breast tumors. MATERIALS AND METHODS: Seventy-seven patients diagnosed with breast cancer were included in this study. Chromatin immunoprecipitation (ChIP) assay was used to identify chromatin modifications. Western blot and reverse transcription and quantitative real-time PCR were used to determine protein and gene expression, respectively. RESULTS: We verified the variation in H4K12ac enrichment and the co-localization of H4K12ac and TIP60 on the euchromatin and heterochromatin genes, respectively, by ChIP-qPCR and ChIP-reChIP, which showed an enrichment of H4K12ac on specific genes in tumors compared to the adjacent healthy tissue and a co-localization of H4K12ac with TIP60 in different breast tumor types. Furthermore, RNA and protein expression of TIP60 and P400 was investigated and overexpression of TIP60 and P400 mRNA was associated with tumor aggressiveness. CONCLUSION: There is a potential interaction between H4K12ac and TIP60 in heterochromatin or euchromatin in breast tumors.</p>',
			'date' => '2020-01-01',
			'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33099470',
			'doi' => '10.21873/cgp.20223',
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			'name' => 'Saliva as a Blood Alternative for Genome-Wide DNA Methylation Profiling by Methylated DNA Immunoprecipitation (MeDIP) Sequencing',
			'authors' => 'Staunstrup N.H. et al.',
			'description' => '<p><b></b>Background: Interrogation of DNA methylation profiles hold promise for improved diagnostics, as well as the delineation of the aetiology for common human diseases. However, as the primary tissue of the disease is often inaccessible without complicated and inconvenient interventions, there is an increasing interest in peripheral surrogate tissues. Whereas most work has been conducted on blood, saliva is now becoming recognized as an interesting alternative due to the simple and non-invasive manner of collection allowing for self-sampling. Results: In this study we have evaluated if saliva samples are suitable for DNA methylation studies using methylated DNA immunoprecipitation coupled to next-generation sequencing (MeDIP-seq). This was done by comparing the DNA methylation profile in saliva against the benchmark profile of peripheral blood from three individuals. We show that the output, quality, and depth of paired-end 50 bp sequencing reads are comparable between saliva and peripheral blood and, moreover, that the distribution of reads along genomic regions are similar and follow canonical methylation patterns. Conclusion: In summary, we show that high-quality MeDIP-seq data can be generated using saliva, thus supporting the future use of saliva in the generation of DNA methylation information at annotated genes, non-RefSeq genes, and repetitive elements relevant to human disease.</p>',
			'date' => '2017-10-19',
			'pmid' => 'http://www.mdpi.com/2075-4655/1/3/14',
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			'name' => 'Emerging Role of One-Carbon Metabolism and DNA Methylation Enrichment on δ-Containing GABAA Receptor Expression in the Cerebellum of Subjects with Alcohol Use Disorders (AUD',
			'authors' => 'Gatta E. et al.',
			'description' => '<section class="abstract">
<section class="sec">
<div class="title -title">Background</div>
<p>Cerebellum is an area of the brain particularly sensitive to the effects of acute and chronic alcohol consumption. Alcohol exposure decreases cerebellar Purkinje cell output by increasing GABA release from Golgi cells onto extrasynaptic &alpha;<sub>6</sub>/&delta;-containing GABA<sub>A</sub> receptors located on glutamatergic granule cells. Here, we studied whether chronic alcohol consumption induces changes in GABA<sub>A</sub> receptor subunit expression and whether these changes are associated with alterations in epigenetic mechanisms via DNA methylation.</p>
</section>
<section class="sec">
<div class="title -title">Methods</div>
<p>We used a cohort of postmortem cerebellum from control and chronic alcoholics, here defined as alcohol use disorders subjects (n=25/group). <em>S</em>-adenosyl-methionine/<em>S</em>-adenosyl-homocysteine were measured by high-performance liquid chromatography. mRNA levels of various genes were assessed by reverse transcriptase-quantitative polymerase chain reaction. Promoter methylation enrichment was assessed using methylated DNA immunoprecipitation and hydroxy-methylated DNA immunoprecipitation assays.</p>
</section>
<section class="sec">
<div class="title -title">Results</div>
<p>mRNAs encoding key enzymes of 1-carbon metabolism that determine the <em>S</em>-adenosyl-methionine/<em>S</em>-adenosyl-homocysteine ratio were increased, indicating higher &ldquo;methylation index&rdquo; in alcohol use disorder subjects. We found that increased methylation of the promoter of the &delta; subunit GABA<sub>A</sub> receptor was associated with reduced mRNA and protein levels in the cerebellum of alcohol use disorder subjects. No changes were observed in &alpha;<sub>1</sub>- or &alpha;<sub>6</sub>-containing GABA<sub>A</sub> receptor subunits. The expression of DNA-methyltransferases (1, 3A, and 3B) was unaltered, whereas the mRNA level of TET1, which participates in the DNA demethylation pathway, was decreased. Hence, increased methylation of the &delta; subunit GABA<sub>A</sub> receptor promoter may result from alcohol-induced reduction of DNA demethylation.</p>
</section>
<section class="sec">
<div class="title -title">Conclusion</div>
<p>Together, these results support the hypothesis that aberrant DNA methylation pathways may be involved in cerebellar pathophysiology of alcoholism. Furthermore, this work provides novel evidence for a central role of DNA methylation mechanisms in the alcohol-induced neuroadaptive changes of human cerebellar GABA<sub>A</sub> receptor function.</p>
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			'name' => 'Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples',
			'authors' => 'Bak S.T. et al.',
			'description' => '<p>We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques. Overall, we found that DNA quality was inversely correlated with storage time and DNA quality was insufficient for Illumina methylation arrays; data from methylated DNA immunoprecipitation, clonal bisulfite sequencing, and pyrosequencing of BDNF and ST6GALNAC1 suggested that the original methylation pattern is indeed preserved. Proof-of-principle experiments predicting sex based on the methylation status of the X-inactivated SLC9A7 gene, or genotype differences of the Y and X chromosomes, showed consistency between predicted and actual sex for a subset of FFPE samples. In conclusion, even though DNA from FFPE samples is of low quality and technically challenging, it is likely that a subset of samples can provide reliable data given that the methodology used is designed for small DNA fragments. We propose that simple PCR-based quality control experiments at the genetic and DNA methylation level, carried out at the beginning of any given project, can be used to enrich for the best-performing FFPE samples. The apparent preservation of genetic and DNA methylation patterns in archival FFPE samples may bring along new perspectives for the identification of genetic and epigenetic changes associated with brain-manifested diseases.</p>',
			'date' => '2016-12-19',
			'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/27995571',
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			'authors' => 'Nicklas H. Staunstrup et al.',
			'description' => '<p><strong>Background</strong> In utero and early-life experienced environmental exposures are suggested to play an important role in many multifactorial diseases potentially mediated through lasting effects on the epigenome. As the epigenome in addition remains modifiable throughout life, identifying specific disease-relevant biomarkers may prove challenging. This has led to an increased interest in epigenome-wide association studies using dried blood spots (DBS) routinely collected in perinatal screening programs. Such programs are in place in numerous countries around the world producing large and unique biobanks. However, availability of this biological material is highly limited as each DBS is made only from a few droplets of blood and storage conditions may be suboptimal for epigenetic studies. Furthermore, as relevant markers may reside outside gene bodies, epigenome-wide interrogation is needed.</p>
<p><strong>Results</strong> Here we demonstrate, as a proof of principle, that genome-wide interrogation of the methylome based on methylated DNA immunoprecipitation coupled with next-generation sequencing (MeDIP-seq) is feasible using a single 3.2 mm DBS punch (60 ng DNA) from filter cards archived for up to 16 years. The enrichment profile, sequence quality and distribution of reads across genetic regions were comparable between samples archived 16 years, 4 years and a freshly prepared control sample.</p>
<p><strong>Conclusions</strong> In summary, we show that high-quality MeDIP-seq data is achievable from neonatal screening filter cards stored at room temperature, thereby providing information on annotated as well as on non-RefSeq genes and repetitive elements. Moreover, the quantity of DNA from one DBS punch proved sufficient allowing for multiple epigenome studies using one single DBS.</p>',
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<p><strong>Results</strong> Here we demonstrate, as a proof of principle, that genome-wide interrogation of the methylome based on methylated DNA immunoprecipitation coupled with next-generation sequencing (MeDIP-seq) is feasible using a single 3.2 mm DBS punch (60 ng DNA) from filter cards archived for up to 16 years. The enrichment profile, sequence quality and distribution of reads across genetic regions were comparable between samples archived 16 years, 4 years and a freshly prepared control sample.</p>
<p><strong>Conclusions</strong> In summary, we show that high-quality MeDIP-seq data is achievable from neonatal screening filter cards stored at room temperature, thereby providing information on annotated as well as on non-RefSeq genes and repetitive elements. Moreover, the quantity of DNA from one DBS punch proved sufficient allowing for multiple epigenome studies using one single DBS.</p>',
	'date' => '2016-07-26',
	'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/27462375',
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View::render() - CORE/Cake/View/View.php, line 473
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			'name' => 'Digging Deeper into Breast Cancer Epigenetics: Insights from ChemicalInhibition of Histone Acetyltransferase TIP60 .',
			'authors' => 'Idrissou, Mouhamed and Lebert, Andre and Boisnier, Tiphanie and Sanchez,Anna and Houfaf Khoufaf, Fatma Zohra and Penault-Llorca, Frédérique andBignon, Yves-Jean and Bernard-Gallon, Dominique',
			'description' => '<p>Breast cancer is often sporadic due to several factors. Among them, the deregulation of epigenetic proteins may be involved. TIP60 or KAT5 is an acetyltransferase that regulates gene transcription through the chromatin structure. This pleiotropic protein acts in several cellular pathways by acetylating proteins. RNA and protein expressions of TIP60 were shown to decrease in some breast cancer subtypes, particularly in triple-negative breast cancer (TNBC), where a low expression of TIP60 was exhibited compared with luminal subtypes. In this study, the inhibition of the residual activity of TIP60 in breast cancer cell lines was investigated by using two chemical inhibitors, TH1834 and NU9056, first on the acetylation of the specific target, lysine 4 of histone 3 (H3K4) by immunoblotting, and second, by chromatin immunoprecipitation (ChIP)-qPCR (-quantitative Polymerase Chain Reaction). Subsequently, significant decreases or a trend toward decrease of H3K4ac in the different chromatin compartments were observed. In addition, the expression of 48 human nuclear receptors was studied with TaqMan Low-Density Array in these breast cancer cell lines treated with TIP60 inhibitors. The statistical analysis allowed us to comprehensively characterize the androgen receptor and receptors in TNBC cell lines after TH1834 or NU9056 treatment. The understanding of the residual activity of TIP60 in the evolution of breast cancer might be a major asset in the fight against this disease, and could allow TIP60 to be used as a biomarker or therapeutic target for breast cancer progression in the future.</p>',
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			'name' => 'TIP60/P400/H4K12ac Plays a Role as a Heterochromatin Back-up Skeleton inBreast Cancer.',
			'authors' => 'Idrissou, Mouhamed and Boisnier, Tiphanie and Sanchez, Anna and Khoufaf,Fatma Zohra Houfaf and Penault-Llorca, Frederique and Bignon, Yves-Jean andBernard-Gallon, Dominique',
			'description' => '<p>BACKGROUND/AIM: In breast cancer, initiation of carcinogenesis leads to epigenetic dysregulation, which can lead for example to the loss of the heterochromatin skeleton SUV39H1/H3K9me3/HP1 or the supposed secondary skeleton TIP60/P400/H4K12ac/BRD (2/4), which allows the maintenance of chromatin integrity and plasticity. This study investigated the relationship between TIP60, P400 and H4K12ac and their implications in breast tumors. MATERIALS AND METHODS: Seventy-seven patients diagnosed with breast cancer were included in this study. Chromatin immunoprecipitation (ChIP) assay was used to identify chromatin modifications. Western blot and reverse transcription and quantitative real-time PCR were used to determine protein and gene expression, respectively. RESULTS: We verified the variation in H4K12ac enrichment and the co-localization of H4K12ac and TIP60 on the euchromatin and heterochromatin genes, respectively, by ChIP-qPCR and ChIP-reChIP, which showed an enrichment of H4K12ac on specific genes in tumors compared to the adjacent healthy tissue and a co-localization of H4K12ac with TIP60 in different breast tumor types. Furthermore, RNA and protein expression of TIP60 and P400 was investigated and overexpression of TIP60 and P400 mRNA was associated with tumor aggressiveness. CONCLUSION: There is a potential interaction between H4K12ac and TIP60 in heterochromatin or euchromatin in breast tumors.</p>',
			'date' => '2020-01-01',
			'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33099470',
			'doi' => '10.21873/cgp.20223',
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			'authors' => 'Staunstrup N.H. et al.',
			'description' => '<p><b></b>Background: Interrogation of DNA methylation profiles hold promise for improved diagnostics, as well as the delineation of the aetiology for common human diseases. However, as the primary tissue of the disease is often inaccessible without complicated and inconvenient interventions, there is an increasing interest in peripheral surrogate tissues. Whereas most work has been conducted on blood, saliva is now becoming recognized as an interesting alternative due to the simple and non-invasive manner of collection allowing for self-sampling. Results: In this study we have evaluated if saliva samples are suitable for DNA methylation studies using methylated DNA immunoprecipitation coupled to next-generation sequencing (MeDIP-seq). This was done by comparing the DNA methylation profile in saliva against the benchmark profile of peripheral blood from three individuals. We show that the output, quality, and depth of paired-end 50 bp sequencing reads are comparable between saliva and peripheral blood and, moreover, that the distribution of reads along genomic regions are similar and follow canonical methylation patterns. Conclusion: In summary, we show that high-quality MeDIP-seq data can be generated using saliva, thus supporting the future use of saliva in the generation of DNA methylation information at annotated genes, non-RefSeq genes, and repetitive elements relevant to human disease.</p>',
			'date' => '2017-10-19',
			'pmid' => 'http://www.mdpi.com/2075-4655/1/3/14',
			'doi' => '',
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			'authors' => 'Gatta E. et al.',
			'description' => '<section class="abstract">
<section class="sec">
<div class="title -title">Background</div>
<p>Cerebellum is an area of the brain particularly sensitive to the effects of acute and chronic alcohol consumption. Alcohol exposure decreases cerebellar Purkinje cell output by increasing GABA release from Golgi cells onto extrasynaptic &alpha;<sub>6</sub>/&delta;-containing GABA<sub>A</sub> receptors located on glutamatergic granule cells. Here, we studied whether chronic alcohol consumption induces changes in GABA<sub>A</sub> receptor subunit expression and whether these changes are associated with alterations in epigenetic mechanisms via DNA methylation.</p>
</section>
<section class="sec">
<div class="title -title">Methods</div>
<p>We used a cohort of postmortem cerebellum from control and chronic alcoholics, here defined as alcohol use disorders subjects (n=25/group). <em>S</em>-adenosyl-methionine/<em>S</em>-adenosyl-homocysteine were measured by high-performance liquid chromatography. mRNA levels of various genes were assessed by reverse transcriptase-quantitative polymerase chain reaction. Promoter methylation enrichment was assessed using methylated DNA immunoprecipitation and hydroxy-methylated DNA immunoprecipitation assays.</p>
</section>
<section class="sec">
<div class="title -title">Results</div>
<p>mRNAs encoding key enzymes of 1-carbon metabolism that determine the <em>S</em>-adenosyl-methionine/<em>S</em>-adenosyl-homocysteine ratio were increased, indicating higher &ldquo;methylation index&rdquo; in alcohol use disorder subjects. We found that increased methylation of the promoter of the &delta; subunit GABA<sub>A</sub> receptor was associated with reduced mRNA and protein levels in the cerebellum of alcohol use disorder subjects. No changes were observed in &alpha;<sub>1</sub>- or &alpha;<sub>6</sub>-containing GABA<sub>A</sub> receptor subunits. The expression of DNA-methyltransferases (1, 3A, and 3B) was unaltered, whereas the mRNA level of TET1, which participates in the DNA demethylation pathway, was decreased. Hence, increased methylation of the &delta; subunit GABA<sub>A</sub> receptor promoter may result from alcohol-induced reduction of DNA demethylation.</p>
</section>
<section class="sec">
<div class="title -title">Conclusion</div>
<p>Together, these results support the hypothesis that aberrant DNA methylation pathways may be involved in cerebellar pathophysiology of alcoholism. Furthermore, this work provides novel evidence for a central role of DNA methylation mechanisms in the alcohol-induced neuroadaptive changes of human cerebellar GABA<sub>A</sub> receptor function.</p>
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			'description' => '<p>We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques. Overall, we found that DNA quality was inversely correlated with storage time and DNA quality was insufficient for Illumina methylation arrays; data from methylated DNA immunoprecipitation, clonal bisulfite sequencing, and pyrosequencing of BDNF and ST6GALNAC1 suggested that the original methylation pattern is indeed preserved. Proof-of-principle experiments predicting sex based on the methylation status of the X-inactivated SLC9A7 gene, or genotype differences of the Y and X chromosomes, showed consistency between predicted and actual sex for a subset of FFPE samples. In conclusion, even though DNA from FFPE samples is of low quality and technically challenging, it is likely that a subset of samples can provide reliable data given that the methodology used is designed for small DNA fragments. We propose that simple PCR-based quality control experiments at the genetic and DNA methylation level, carried out at the beginning of any given project, can be used to enrich for the best-performing FFPE samples. The apparent preservation of genetic and DNA methylation patterns in archival FFPE samples may bring along new perspectives for the identification of genetic and epigenetic changes associated with brain-manifested diseases.</p>',
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