Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

10.3791/59375 ◽  
2019 ◽  
Author(s):  
Takahiro Yamakawa ◽  
Keiichi Itakura
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Mammalian Cells ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay

scholarly journals INSM1 functions as a transcriptional repressor of the neuroD/β2 gene through the recruitment of cyclin D1 and histone deacetylases

2006 ◽  
Vol 397 (1) ◽  
pp. 169-177 ◽  
Author(s):  
Wei-Dong Liu ◽  
Hong-Wei Wang ◽  
Michelle Muguira ◽  
Mary B. Breslin ◽  
Michael S. Lan
Keyword(s):  
Cyclin D1 ◽  
Chromatin Immunoprecipitation ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Mammalian Cells ◽  
Transcriptional Repressor ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Repressor Activity

INSM1/IA-1 (insulinoma-associated 1) is a developmentally regulated zinc-finger transcription factor, exclusively expressed in the foetal pancreas and nervous system, and in tumours of neuroendocrine origin. We have identified an INSM1 binding site in the neuroD/β2 promoter and demonstrated transcriptional repressor activity of INSM1 by transient transfection assay. A chromatin immunoprecipitation assay confirmed that in vivo INSM1 is situated on the promoter region of the neuroD/β2 gene. In an attempt to elucidate the molecular mechanism of transcriptional repression by the INSM1 gene, cyclin D1 was identified as an interacting protein by using a 45-day-old human foetal brain cDNA library and a yeast two-hybrid screen. The physical association between INSM1 and cyclin D1 was confirmed by in vitro and in vivo pull-down assay. Cyclin D1 co-operates with INSM1 and suppresses neuroD/β2 promoter activity. Co-immunoprecipitation of INSM1, cyclin D1 and HDACs (histone deacetylases) in mammalian cells revealed that INSM1 interacts with HDAC-1 and -3 and that this interaction is mediated through cyclin D1. Overexpression of cyclin D1 and HDAC-3 significantly enhanced the transcriptional repression activity of INSM1 on the neuroD/β2 promoter. A further chromatin immunoprecipitation assay confirmed that HDAC-3 occupies this same region of the neuroD/β2 promoter, by forming a transcription complex with INSM1. Thus we conclude that INSM1 recruits cyclin D1 and HDACs, which confer transcriptional repressor activity.

scholarly journals Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Yong Feng ◽  
Yunfei Liao ◽  
Jianming Zhang ◽  
Jacson Shen ◽  
Zengwu Shao ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Transcriptional Analysis ◽  
Luciferase Assay ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Gel Shift Assay ◽  
Gel Shift

Abstract Background Aberrant expression of cyclin-dependent protein kinases (CDK) is a hallmark of cancer. CDK11 plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 and CDK11 transcriptionally regulated genes are largely unknown. Methods In this study, we performed a global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional targets of CDK11. Clinical relevance and function of core-binding factor subunit beta (CBFβ) were further accessed in osteosarcoma. Results We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of CBFβ expression in osteosarcoma cells. The CBFβ promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFβ is a direct transcriptional target of CDK11. High expression of CBFβ is associated with poor outcome in osteosarcoma patients. Expression of CBFβ contributes to the proliferation and metastatic behavior of osteosarcoma cells. Conclusions These data establish CBFβ as a mediator of CDK11p110 dependent oncogenesis and suggest that targeting the CDK11- CBFβ pathway may be a promising therapeutic strategy for osteosarcoma treatment. Graphical Abstract

Studying KLF6 and PEPD ‐gene Promoter by Chromatin Immunoprecipitation Assay

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Zeljka Miletic Lanaghan ◽  
Ireti Eni-Aganga ◽  
Muthukumar Balasubramaniam ◽  
Chandravanu Dash ◽  
Jui Pandhare
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Gene Promoter ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay

scholarly journals MKP-1 mediates glucocorticoid-induced ERK1/2 dephosphorylation and reduction in pancreatic β-cell proliferation in islets from early lactating mothers

2010 ◽  
Vol 299 (6) ◽  
pp. E1006-E1015 ◽  
Author(s):  
José E. Nicoletti-Carvalho ◽  
Camilo Lellis-Santos ◽  
Tatiana S. Yamanaka ◽  
Tatiane C. Nogueira ◽  
Luciana C. Caperuto ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Pancreatic Islets ◽  
Chromatin Immunoprecipitation ◽  
Protein Phosphatase ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
Chromatin Immunoprecipitation Assay ◽  
Isolated Pancreatic Islets ◽  
Immunoprecipitation Assay ◽  
Lactating Mothers

Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. The mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. In addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. In addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phospho-ERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression.

Sign in / Sign up

Export Citation Format

Share Document