Nuclear Run-On Assay Using Biotin Labeling, Magnetic Bead Capture and Analysis by Fluorescence-Based RT-PCR

Patrone, Giovanna; Puppo, Francesca; Cusano, Roberto; Scaranari, Monica; Ceccherini, Isabella; Puliti, Aldamaria; Ravazzolo, Roberto

doi:10.2144/00295st02

Cited by 103 publications

(74 citation statements)

References 7 publications

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“…To distinguish these two possibilities, the transcriptional activities of wildtype and targeted BCL2 alleles were examined with nuclear run-on transcription analysis using a modified protocol with biotin-16-uridine triphosphate (UTP) (Patrone et al, 2000;Tong et al, 2005;Zhang et al, 2005). Results of nuclear run-on assays showed that the transcriptional activity of the targeted allele was 10.3-fold lower than that of the wild-type allele, indicating the expression discrepancy of the wild-type and the targeted BCL2 alleles was mainly due to the difference in transcriptional activities between the two alleles (DCT ¼ 34.13-30.77E3.36; 2 3.36 ¼ 10.3) (Figure 4).…”

Section: Resultsmentioning

confidence: 99%

“…Nuclear run-on assay The transcriptional activity of wild type and of targeted BCL2 alleles was determined by nuclear run-on transcription analysis according to a modified protocol that uses biotin-16-uridine triphosphate (UTP) (Roche, Sandhofer Strasse, Mannheim, Germany) (Patrone et al, 2000;Tong et al, 2005;Zhang et al, 2005). Briefly, 2-3 Â 10 7 Nalm6-1 cells were washed twice with ice-cold PBS and resuspended in cell lysis buffer containing 10 mM Tris-HCl (pH 7.4), 3 mM MgCl 2 , 10 mM NaCl, 150 mM sucrose and 0.5% NP-40.…”

Section: Construction Of the Bcl2 Targeting Vectormentioning

confidence: 99%

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The BCL2 major breakpoint region (mbr) regulates gene expression

Zhang

Durrin

et al. 2006

Oncogene

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BCL2 expression is finely tuned by a variety of environmental and endogenous stimuli and regulated at both transcriptional and post-transcriptional levels. Our previous investigations demonstrated that the BCL2 major breakpoint region (mbr) in the 3 0 -UTR upregulates reporter gene expression, which implies that this region possessed intrinsic regulatory function. However, the effect of the mbr on BCL2 expression, and the underlying regulatory mechanisms, remain to be elucidated. To assess the direct effect of the mbr on the transcriptional activity of the BCL2 gene, we employed targeted homologous recombination to establish a mbr þ /mbr À heterozygous Nalm-6 cell line and then compared the transcriptional activity and apoptotic effect on transcription between the wild type and targeted alleles. We found that deletion of the mbr significantly decreased the transcriptional activity of the corresponding allele in the mbr þ /mbr À cell. The BCL2 allele deleted of the mbr had a slower response to apoptotic stimuli than did the wild type allele. The regulatory function of the mbr was mediated through SATB1. Overexpression of SATB1 increased BCL2 expression, while knockdown of SATB1 with RNAi decreased BCL2 expression. Our results clearly indicated that the mbr could positively regulate BCL2 gene expression and this regulatory function was closely related to SATB1.

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Section: Resultsmentioning

confidence: 99%

Section: Construction Of the Bcl2 Targeting Vectormentioning

confidence: 99%

The BCL2 major breakpoint region (mbr) regulates gene expression

Zhang

Durrin

et al. 2006

Oncogene

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“…Nuclear run-ons were performed using a slightly modified version of the method described by Patrone et al [12]. PCR was performed as described above.…”

Section: Nuclear Run-onmentioning

confidence: 99%

Detailed molecular analysis of the induction of the L-PK gene by glucose

Eckert

Zhang

Collier

et al. 2008

Biochemical and Biophysical Research Communications

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Glucose has powerful effects on gene expression and participates in the fasted to fed transition of the liver. However, the molecular mechanism of glucose-regulated gene expression has not been completely described. In the present study, we performed a detailed analysis of the molecular events of the insulin-independent glucose response of the liver-type pyruvate kinase (L-PK) gene. L-PK mRNA was increased by glucose at the transcriptional level as determined by real-time RT-PCR, mRNA stability measurements, and nuclear run-on assays. LY294002 and LY303511 inhibited the glucose response of the L-PK gene at the transcriptional level. Histones H3 and H4 associated with the L-PK gene promoter were hyperacetylated and HNF4α was constitutively bound in low and high glucose. Treatment with 20 mM glucose increased recruitment of ChREBP, additional HNF4α, and RNA polymerase II. Glucose stimulated the phosphorylation of the C terminal domain of RNA polymerase II, with increased Ser5 phosphorylation near the transcription start site and increased Ser2 phosphorylation near the termination signal. LY294002 and LY303511 blocked the recruitment of RNA polymerase II to the L-PK gene, reducing the rate of transcription. The results of these studies demonstrate fundamental details of the molecular mechanism of glucose activated gene expression.

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“…Nascent IL-8 mRNA was synthesized, biotin-labeled, and extracted using magnetic bead capture, and analyzed by fluorescence-based RT-PCR, as described [40]. Briefly, 50 lL nuclei were added to transcription buffer containing 1 mM each of ATP, GTP, CTP, and 0.5 mM UTP and 0.5 mM biotin-16-UTP, for 30 min.…”

Section: Nuclear Run-on Assaymentioning

confidence: 99%