Expression profiling of DT40 cells treated with Hydroxyurea compared with DT40 mutated for FANCJ or for WRN and BLM
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ABSTRACT: RNA was extracted from three independent wild type cell populations treated for 7 days with Hydroxyurea and allowed to recover for another seven days, as well as from three untreated parallel controls. Extraction was performed using Trizol. Microarray hybridisations were performed using total RNA and the Affymetrix Chicken Genome array. FANCJ and WRN/BLM mutants have been performed at the same day as the WT \PS_2010\ and HU treated cells at the same day as WT \08_2012\
Project description:RNA-seq was performed for transcriptional analysis of wild-type DT40 cells (Gallus gallus, B-cell line) and a H3.3 knockout line (h3.3a-/-, h3.3b-/-). H3.3 is a H3 histone variant encoded on two genes (H3.3A and H3.3B) in chickens.
Project description:PAx5 is indispensible for the committment if early lymphoid progenitors to the B cell lineage as well as for the development and maintenance of B cells. To better understand the functional importance of Pax5 in the later stages of B cell development and investigate the targets of Pax5 regulation, we established a novel Pax5 deficient DT40 B cell line.
Project description:Cancers with microsatellite instability (MSI) depend on the WRN helicase enzyme to manage issues during DNA replication caused by long stretches of (TA) repeats in the DNA. Targeting WRN is a promising strategy for treating MSI cancers, and drugs that inhibit WRN are being developed. Through a process called fragment-based screening, we developed powerful and specific drugs that block WRN's action. These drugs effectively slowed down the growth of MSI cancer models in lab and animal studies by acting like WRN is absent, leading to DNA breaks at the long TA repeats and causing DNA damage. The development of these potent and specific drugs targeting WRN in MSI cancers proves that this approach can work and also helps us understand more about WRN's role in biology.
Project description:A single replicate of exponentially growing DT40 CL18 chicken B lymphoma cells were harvested and extracted RNA was subjected to Illumina GAIIx paired-end sequencing to determine global gene expression. Single replicate RNA-seq expression analysis of DT40 cells.
Project description:PTIP (Pax2 transactivation domain-interacting protein) is a nuclear protein containing six BRCT domains. It has been shown that PTIP affects gene expression by controlling the activity of the transcription factor Pax2 and histone H3 lysine 4 methyltransferase complexes. In addition to its role in transcriptional regulation, PTIP has been implicated in DNA damage response. To ask if the depletion of PTIP affects the expression level of genes encoding DNA damage response factors , we compared the whole transcripts between wild-type and PTIP deficient chicken DT40 B cell lines. The total RNAs were isolated from wild-type and PTIP deficient cells (PTIP-/-/-) using Sepasol®-RNA I (Nacalai tesque, Japan). The gene expression profiles were examined using Genechip® Chicken Genome Array (Affymetrix Cat #900590), by GeneticLab co. Ltd. Japan, following Genechip® protocol.
Project description:Human WRN and BLM genes are members of the conserved RECQ helicase family. Mutations in these genes are associated with Werner and Bloom syndromes. WRN and BLM proteins are implicated in DNA replication, recombination, repair, telomere maintenance, and transcription. Using microfluidics-assisted display of DNA for replication track analysis (ma-RTA), we show that WRN and BLM contribute additively to normal replication fork progression, and non-additively, in a RAD51-dependent pathway, to resumption of replication after arrest by hydroxyurea (HU), a replication-stalling drug. WRN but not BLM is required to support fork progression after HU. Resumption of replication by forks may be necessary but is not sufficient for timely completion of the cell cycle after HU arrest, as depletion of WRN or BLM compromises fork recovery to a similar degree, but only BLM depletion leads to extensive delay of cell division after HU, as well as more pronounced chromatin bridging. Finally, we show that recovery from HU includes apparent removal of some of the DNA that was synthesized immediately after release from HU, a novel phenomenon that we refer to as nascent strand processing, NSP.
Project description:The transcription factor Bcl6 is required for germinal center formation and deregulated expression of Bcl6 has been observed in lymphomas. To gain insight to the function of Bcl6 in terminal differentiation of B cells to plasma cells and to investigate the targets of Bcl6, we established a Bcl6 deficient DT40 B cell line.