Regulation of the DNA Damage Response and Gene Expression by the Dot1L Histone Methyltransferase and the 53Bp1 Tumour Suppressor

Abstract

BackgroundDot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined.Methodology/Principal FindingsUsing the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L−/− and wild type cells are equally resistant to ionising radiation, whereas 53Bp1−/−/Dot1L−/− cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1−/− cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.Conclusions/SignificanceTaken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.

Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3

“…1A and Table S1). Others have used this type of triple-quadrupole MS platform, which has >10 4 dynamic range and the ability to quantify >200 analytical targets in a single run (25), for studying changes in histone modifications (26)(27)(28). We next sought to extend this method to all stable-isotope states of the H3K27-K36 peptide created by SILAC (29).…”

The BioStudies database

“…The study S‐EPMC3044716 (http://www.ebi.ac.uk/biostudies/studies/S-EPMC3044716) relates to the PLoS ONE paper: “Regulation of the DNA damage response and gene expression by the Dot1L histone methyltransferase and the 53Bp1 tumour suppressor” (FitzGerald et al , 2011). The authors have submitted their two transcriptomics datasets to ArrayExpress (Accession MEXP‐2721, E‐MEXP‐2722) and have also referred to the DOT1L locus in Ensembl (ENSGALG00000000843).…”

“…The authors have submitted their two transcriptomics datasets to ArrayExpress (Accession MEXP‐2721, E‐MEXP‐2722) and have also referred to the DOT1L locus in Ensembl (ENSGALG00000000843). Some additional information was included with the paper as supplementary material: primer sequences (Table S1 in FitzGerald et al , 2011; http://www.ebi.ac.uk/biostudies/files/S-EPMC3044716/pone.0014714.s009.docx) and lists of over‐represented genes grouped by GO term (http://www.ebi.ac.uk/biostudies/files/S-EPMC3044716/pone.0014714.s010.pdf). All this information is aggregated in a single BioStudies record (see figure below).…”

Target irradiation induced bystander effects between stem-like and non stem-like cancer cells

“…53BP1 is a direct reader of a DSB-specific histone code, which translates multiple site-specific histone modifications into efficient checkpoint signaling and DSB repair [19]. Since the 53BP1 foci number is believed to be in parallel with the DSB number in cells [20,21], detecting more foci in bystander cells than in controls provides sound evidence of the existence of RIBE. In clinic cancer radiotherapy, there often exists "out of field" effect such as RIBE [11].…”