Hot spots of DNA double-strand breaks and genomic contacts of human rDNA units are involved in epigenetic regulation [rDNA units]
ABSTRACT: 4C procedure was used for analysis of genomic contacts of rDNA units in HEK 293T cells. The primers for 4C were selected inside IGS. Our data indicate that mostly rDNA units exhibit close proximity with pericentromeric regions in different chromosomes. We also detected the contacts within a rDNA unit and between rDNA units. Examination of rDNA genome-wide contacts in HEK 293T cells using 4C approach.
DNA double-strand breaks (DSBs) are involved in many cellular mechanisms, including replication, transcription, and genome rearrangements. The recent observation that hot spots of DSBs in human chromosomes delimit DNA domains that possess coordinately expressed genes suggests a strong relationship between the organization of transcription patterns and hot spots of DSBs. In this study, we performed mapping of hot spots of DSBs in a human 43-kb ribosomal DNA (rDNA) repeated unit. We observed that ...[more]
Project description:Coordinated ribosomal protein (RP) gene expression is crucial for cellular viability, but the transcriptional network controlling this regulon has only been well characterized in the yeast Saccharomyces cerevisiae. We have used whole-genome transcriptional and location profiling to establish that, in Candida albicans, the RP regulon is controlled by the Myb-domain protein Tbf1 working in conjunction with Cbf1. These two factors bind both the promoters of RP genes and the rDNA locus; Tbf1 activates transcription at these loci and is essential. Orthologs of Tbf1 bind TTAGGG telomeric repeats in most eukaryotes, and TTAGGG cis-elements are present upstream of RP genes in plants and fungi, suggesting that Tbf1 was involved in both functions in ancestral eukaryotes. In all Hemiascomycetes, Rap1 substituted Tbf1 at telomeres and in the S. cerevisiae lineage this substitution also occurred independently at RP genes, illustrating the extreme adaptability and flexibility of transcriptional regulatory networks. Keywords: Expression profiling Expression profiling of tetracycline addition time-course in the tetO-TBF1/tbf1 conditional mutant.
Project description:We used ChIP-seq to map the binding of of C-terminally FLAG3-tagged PhoB to the Escherichia coli K-12 genome during growth in MOPS minimal media with low phosphate or high phosphate (0.2 mM or 1.32 mM, respectively). As a control, we performed ChIP-seq in an untagged strain. We also used ChIP-seq to map Sigma 70 (associates with initiating RNA polymerase) binding across the E. coli K-12 genome in wild-type, ΔphoB, Δhns, and Δhns ΔphoB strains grown in low phosphate medium to determine whether PhoB modulates recruitment of RNA polymerase to promoters and whether this is modulated by H-NS.
Project description:Recent studies have suggested that elevated expression of aldoketoreductase (AKR) 1C1 or 1C2 in tumour cells is associated with increased resistance to DNA damaging agents such as cisplatin and doxorubicin. However, it has not been shown whether selection of tumour cells for resistance to DNA-damaging anthracyclines actually results in increased expression of AKRs and increased conversion of anthracyclines to 10-fold less toxic 13-hydroxy metabolites. It is also unclear whether the induction of aldokeoreductases is temporally correlated with the onset of anthracycline resistance and whether there is a direct relationship between the level of AKR expression or activity and the magnitude of drug resistance. Through microarray profiling of MCF-7 breast cancer cells selected for progressive resistance to doxorubicin or epirubicin, we have identified several genes whose expression has been correlated with both the onset and magnitude of drug resistance, including a “1C” AKR. AKR 1C overexpression was verified by quantitative PCR. Also associated with the onset of anthracycline resistance were genes involved in drug transport (ABCB1), cell signaling and transcription (RDC1, CXCR4), cell proliferation or apoptosis (BMP7, CAV1), ROS protection (TXNRD1, MT2A), and structural or immune system proteins (IFI30, STMN1). Consistent with the role of AKRs in anthracycline resistance, doxorubicin- and epirubicin-resistant breast tumour cells exhibited 2.2-fold and 6.1-fold higher levels of the 13-hydroxy metabolite of doxorubicin (doxorubicinol) than wildtype MCF-7 cells. In addition, an inhibitor of AKR 1C2 (5- cholanic acid) almost completely restored sensitivity to doxorubicin in Abcb1-deficient doxorubicin-resistant cells, while having no effect on Abcb1-expressing epirubicin-resistant cells. Taken together, our findings strongly suggest the involvement of multiple genes in the acquisition of anthracycline resistance in breast tumor cells---in particular redox genes such as the 1C AKRs. Keywords: Drug resistance of breast cancer cells In order to identify genes whose expression strongly correlates with the acquisition or magnitude of drug resistance or are temporally related with the acquisition of resistance, we selected MCF-7 breast tumor cells for survival in increasing concentrations (doses) of doxorubicin or epirubicin. Panels of cells exhibiting progressive resistance to either doxorubicin (MCF-7DOX-2) or epirubicin (MCF-7EPI) were obtained. Cells were also “selected” in the absence of drug at each step during selection to serve as co-cultured control (MCF-7CC) cells. In this study, we have used cDNA microarray analysis of these cell lines to identify a variety of “redox” genes whose expression can be correlated with the acquisition or magnitude of drug resistance in MCF-7DOX-2 and MCF-7EPI cells, including a “1C” aldoketoreductase (AKR).
Project description:We mapped the genome-wide binding of C-terminally FLAG-tagged AraC in S. enterica subsp. enterica serovar Typhimurium strain 14028s using ChIP coupled with deep sequencing (ChIP-seq). We identified five putative target loci for AraC: upstream of araB/araC, araE, araJ, STM14_0178, and within sseD.
Project description:We mapped the genome-wide binding of sigma 70 in E. coli K-12 MG1655 and an hns mutant that is otherwise isogenic using ChIP coupled with deep sequencing (ChIP-seq). We show that intragenic binding of sigma 70 is increased in the hns mutant.
Project description:We mapped the genome-wide binding of the flagellar regulators FlhD, FlhC, and FliA in FLAG-tagged derivatives of E. coli K-12 MG1655 using ChIP coupled with deep sequencing (ChIP-seq). We identify new binding sites for each factor.