Novel sequential ChIP and simplified basic ChIP protocols for promoter co-occupancy and target gene identification in human embryonic stem cells

Medeiros, Ricardo B.; Papenfuss, Kate J; Hoium, Brian; Coley, Kristen; Jadrich, Joy; Goh, Saik-Kia; Elayaperumal, Anuratha; Herrera, Julio E.; Resnik, Ernesto; Ni, Hsiao‐Tzu

doi:10.1186/1472-6750-9-59

Cited by 18 publications

(14 citation statements)

References 68 publications

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“…Remarkably, this extended beyond Oct4 co-bound sites, with an increase in Nanog levels observed at many binding sites lacking Oct4 ( Figure 5B). Consequently, it seems unlikely that this effect is due to Oct4 competing with Nanog for binding sites, in keeping with previous reports of simultaneous binding of Oct4 and Nanog to regulatory regions as detected by sequential ChIP (Medeiros et al, 2009). A previous study examining the localisation of Nanog and Sox2 24 hours after transcriptional depletion of Oct4 found that Sox2 and Nanog binding was reduced at many Oct4 co-bound sites following silencing of Oct4 (King and Klose, 2017).…”

Section: Discussionsupporting

confidence: 81%

Auxin-degron system identifies immediate mechanisms of Oct4

Bates

Alves

Silva

2020

Preprint

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The pluripotency factor Oct4 is essential for the maintenance of naïve pluripotent stem cells in vitro and in vivo. However, the specific role of Oct4 in this process remains unknown. Here, we developed a rapid protein-level Oct4 depletion system that demonstrates that the immediate downstream response to loss of Oct4 is reduced expression of key pluripotency factors. Our data show a requirement for Oct4 for the efficient transcription of several key pluripotency factors, and suggest that expression of trophectoderm markers is a subsequent event. Additionally, we find that Nanog is competent to bind to the genome in the absence of Oct4, and this binding is in fact enhanced. Globally, however, active enhancer associated histone mark H3K27ac is depleted. Our work establishes that while Oct4 is required for the maintenance of the naïve transcription factor network, at a normal ESC level it antagonises this network through inhibition of Nanog binding.

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Section: Discussionsupporting

confidence: 81%

Auxin-degron system identifies immediate mechanisms of Oct4

Bates

Alves

Silva

2020

Preprint

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“…Myc-mediated transcriptional regulation requires other cooperating transcription factors to regulate target genes (10, 11, 14). For example, Myc cooperates with other stem cell transcription factors to regulate genes found in embryonic stem cells (15). Myc cooperates with E2F1 to regulate genes involved in nucleotide metabolism and with the hypoxia inducible factor 1 (HIF-1) to regulate genes involved in glucose metabolism (11, 16, 17).…”

Section: Introductionmentioning

confidence: 99%

MYC-Induced Cancer Cell Energy Metabolism and Therapeutic Opportunities

Dang

Gao

2009

Clinical Cancer Research

766

687

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Although cancers have altered glucose metabolism, termed the Warburg effect, which describes the increased uptake and conversion of glucose to lactate by cancer cells under adequate oxygen tension, changes in the metabolism of glutamine and fatty acid have also been documented. The MYC oncogene, which contributes to the genesis of many human cancers, encodes a transcription factor c-Myc, which links altered cellular metabolism to tumorigenesis. c-Myc regulates genes involved in the biogenesis of ribosomes and mitochondria, and regulation of glucose and glutamine metabolism. With E2F1, c-Myc induces genes involved in nucleotide metabolism and DNA replication, and microRNAs that homeostatically attenuate E2F1 expression. With the hypoxia inducible transcription factor HIF-1, ectopic c-Myc cooperatively induces a transcriptional program for hypoxic adaptation. Myc regulates gene expression either directly, such as glycolytic genes including lactate dehydrogenase A (LDHA), or indirectly, such as repression of microRNAs miR-23a/b to increase glutaminase (GLS) protein expression and glutamine metabolism. Ectopic MYC expression in cancers, therefore, could concurrently drive aerobic glycolysis and/or oxidative phosphorylation to provide sufficient energy and anabolic substrates for cell growth and proliferation in the context of the tumor microenvironment. Collectively, these studies indicate that Myc-mediated altered cancer cell energy metabolism could be translated for the development of new anticancer therapies. (Clin Cancer Res 2009;15(21):6479-83)

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“…Since H3K27me2 is not a clear mark of constitutive heterochromatin, we checked whether H3K27me2 histone tail modification was associated with activation or repression of PlTalpha2 gene by sequential chromatin immunoprecipitation (Re-ChIP). This technique has been used to address, in a qualitative manner, whether two proteins can simultaneously co-occupy a stretch of DNA (Furlan-Magaril et al, 2009;Medeiros et al, 2009). We applied the Re-ChIP protocol to analyze chromatin modifications on core promoter of PlTalpha2 gene at two developmental stages: the morula stage, when the gene is not expressed, and the gastrula stage, when the gene is transcriptionally active only in the neural territory (Gianguzza et al, 1995).…”

Section: Post Translational Modifications (Ptms) and Pltalpha2 Gene Amentioning

confidence: 99%

Chromatin dynamics of the developmentally regulatedP. lividus neural alpha tubulin gene

Emanuele¹,

Costa²,

Ragusa³

et al. 2011

Int. J. Dev. Biol.

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Over 40 years ago, Allfrey and colleagues (1964) suggested that two histone modifications, namely acetylation and methylation, might regulate RNA synthesis. Nowadays it is universally accepted that activation of gene expression strictly depends on enzymatic mechanisms able to dynamically modify chromatin structure. Here, using techniques including DNaseI hypersensitive site analysis, chomatin immunoprecipitation and quantitative PCR analysis, we have analyzed the dynamics of histone post-translation modifications involved in developmentally/spatially controlled activation of the sea urchin PlTalpha2 tubulin gene. We have demonstrated that only when the PlTalpha2 core promoter chromatin is acetylated on H3K9, tri-methylated on H3K4 and not di-methylated on H3K27, RNA pol II can be enrolled. In contrast, we have shown that when chromatin is methylated both on H3K9 (me2/3) and H3K27 (me2) and mono methylated on H3K4 the promoter is not accessible to RNA pol II. Our results suggest that, during P. lividus embryogenesis, both HAT/HDAC and HMT/HDM activities, which are able to regulate accessibility of the PlTalpha2 basal promoter to RNA polymerase II, are coordinately switched-on.

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Novel sequential ChIP and simplified basic ChIP protocols for promoter co-occupancy and target gene identification in human embryonic stem cells

Cited by 18 publications

References 68 publications

Auxin-degron system identifies immediate mechanisms of Oct4

Auxin-degron system identifies immediate mechanisms of Oct4

MYC-Induced Cancer Cell Energy Metabolism and Therapeutic Opportunities

Chromatin dynamics of the developmentally regulatedP. lividus neural alpha tubulin gene

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