Project description:HaCat cell cycle experiment: During the somatic cell cycle, DNA and epigenetic modifications in DNA and histones are copied to daughter cells. DNA replication timing is tightly regulated and linked to GC content, chromatin structure, andgene transcription, but how maintenance of histone modifications relates to replication timing and transcription is less understood.The gene expression patters on HaCaT keratinocytes during the cell cycle is studied by a time series analysis of synchroniced cells sampled at 3 hour intervals. We show that genes enriched with the repressive chromatin mark histone H3 lysine 27 tri-methylation are transcribed during DNA replication . The gene expression is related to replication timing, as genes expressed during G1/S transition andearly S phase generally have higher GC content and are replicated earlier than genes expressed during late S phase. These results indicate widespread replication-dependent expression in mammals and support a role for replication in transiently activating transcription of epigenetically silenced genes.
Project description:We compared the mRNAs expression profile of HeLa cells between two phases of the mitotic cell cycle: S and G2/M phases. Results provide insight into the regulation of transcript levels during mitotic cell cycle progression. HeLa cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and cells were taken after 2 hours release (S phase) and 8 hours release (G2/M phase). Keywords: time course Comparison of mRNAs measured at S phase (2 hours release after double thymidine blockade) and at G2/M phase (8 hours release after double thymidine blockade); 3 biological replicates at each of the two time points; two technical replicates with dye swapping per comparison; additional comparison between G2/M (8h) and S (2h).
Project description:We compared the poly(A) tail length status of mRNAs of HeLa cells between two phases of the mitotic cell cycle: S and G2/M phases. Hundreds of mRNAs were found to be regulated by changes in their poly(A) tail length during mitotic cell cycle in a phase specific manner. Many of these differentially polyadenylated mRNAs encode proteins related to cell death, cell cycle and cellular growth and proliferation. HeLa cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and samples were taken after 2 hours release (S phase) and 8 hours release (G2/M phase). For each condition total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 μg of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ºC to isolate mRNAs with short poly(A) tail (<30As, SHORT fraction). Oligo(dT) chromatography: mRNAs were purified independently of their poly(A) tail length with Ambion Poly(A)Purist kit from 20 μg total RNA (ALL fraction). Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261. Keywords: time course Comparison of ALL fraction mRNAs and SHORT fraction mRNAs measured after 2 hours (S phase) and 8 hours release (G2/M) from double thymidine blockade; 3 biological replicates at each of the two time points; two technical replicates with dye swapping per comparison.
Project description:Periodic expression of cell cycle genes highlights the importance of precise temporal control of transcription in regulating cell cycle events. We used HeLa cells enriched for different phases of the cell cycle to identify genes that are expressed in a periodic fashion in specific cell cycle phases. These data were generated for comparison with ChIP-Sequencing data obtained for two subunits of the B-Myb-MuvB complex, B-Myb and LIN9, in HeLa cells. HeLa cells were synchronized at the beginning of S phase by double thymidine block and then released into the cell cycle by washing out the thymidine resulting in tight synchrony at S, G2 and M phases of the cell cycle. Most cells entered mitosis at 8 hours after release as determined by visual inspection. Three independent replicas of double thymidine block and release experiments were performed. RNA was isolated at specific times after release from the thymidine block (0, 2, 4, 6, 8 and 12 hours) and used for generating expression profiles.
Project description:We identified the cell cycle-regulated mRNA transcripts genome-wide in the osteosarcoma derived U2OS cell line. This resulted in 2,140 transcripts mapping to 1,871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identified genomic loci bound by the G2/M transcription factor FOXM1 by Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associated these with cell cycle-regulated genes. FOXM1 was bound to cell cycle-regulated genes with peak expression in both S phase and G2/M phases. ChIP-seq genomic loci were shown to be responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly activates promoters of G2/M phase genes and weakly activates those induced in S phase. Analysis of ChIP-seq data from a panel of cell cycle-transcription factors (E2F1, E2F4, E2F6, and GABPA) from ENCODE and ChIP-seq data for the DREAM complex, found that a set of core cell cycle genes regulated in both U2OS and HeLa cells are bound by multiple cell cycle transcription factors. These data identify the cell cycle regulated genes in a second cancer derived cell line and provide a comprehensive picture of the transcriptional regulatory systems controlling periodic gene expression in the human cell division cycle. Cell cycle-regulated gene expression identified from three double thymidine time courses and one thymidine nocodazole time course.
Project description:M. hominis cells were grown in liquid medium supplied with arginine or thymidine as a carbon source. LC-MS analysis was performed on Ultimate 3000 Nano LC System (Thermo Fisher Scientific) coupled with Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific), an untargeted label-free bottom-up proteomic strategy was used, DDA (Data Dependent Acquisition) approach. Protein identification and label-free quantification were performed with PEAKS software.
Project description:HeLa S3 cells were synchronized in early S phase with a double thymidine block as described in Whitfield et al(2002)Mol.Biol.Cell. This experiment set contains the complete set of 24K arrays for the first double thymidine block synchronization (Thy-Thy1).
Project description:HeLa S3 cells were synchronized in early S phase with a double thymidine block as described in Whitfield et al(2002)Mol.Biol.Cell. This experiment set contains the complete set of 24K arrays for the second double thymidine block synchronization (Thy-Thy2).
Project description:HeLa S3 cells were synchronized in early S phase with a double thymidine block as described in Whitfield et al(2002)Mol.Biol.Cell. This experiment set contains the complete set of 43K arrays for the third double thymidine block synchronization (Thy-Thy3).