Volume 34, Issue 3, Pages 298-310 (May 2009) Formation of Dynamic γ-H2AX Domains along Broken DNA Strands Is Distinctly Regulated by ATM and MDC1 and Dependent upon H2AX Densities in Chromatin  Velibor Savic, Bu Yin, Nancy L. Maas, Andrea L. Bredemeyer, Andrea C. Carpenter, Beth A. Helmink, Katherine S. Yang-Iott, Barry P. Sleckman, Craig H. Bassing  Molecular Cell  Volume 34, Issue 3, Pages 298-310 (May 2009) DOI: 10.1016/j.molcel.2009.04.012 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 γ-H2AX Forms along Tcrα Locus DNA Strands Broken during V(D)J Recombination in Primary Cells (A) Organization and orientation of the murine Tcrα locus and γ-H2AX densities at particular locations within and adjacent to Tcrα in wild-type and Tcrβ:Rag2−/− thymocytes. (B) γ-H2AX densities at particular locations within and adjacent to Tcrα in LβP and LAβP thymocytes. (C) H2AX densities at particular locations within and adjacent to Tcrα in LβP, LAβP, and Tcrβ:Rag2−/− thymocytes. Error bars represent SD of three independent experiments. Molecular Cell 2009 34, 298-310DOI: (10.1016/j.molcel.2009.04.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 γ-H2AX Forms along Igκ Locus DNA Strands Broken during V(D)J Recombination in Cell Lines (A) Organization and orientation of the murine Igκ locus and γ-H2AX densities at locations within and adjacent to Igκ in wild-type and Artemis−/−Rag2−/− pre-B cells treated with STI571 for 24 hr. (B) Schematic of the germline Jκ locus and the Jκ SEs and CEs generated by RAG DSBs. The Jκ segments, the BamHI restriction sites, and the Jκ Southern probe are indicated. (C) Southern analysis of Igκ DSBs in wild-type, Artemis−/−Rag2−/−, and Artemis−/− cells treated with STI571 for the indicated number of hours (h). Locations of germline Jκ loci and Jκ CEs are shown. (D) γ-H2AX densities at sequences 50 kb telomeric of Jκ5 in wild-type or Artemis−/−Rag2−/− cells after STI571 treatment for the indicated times. (E) H2AX densities at locations within and adjacent to Igκ in Artemis−/−Rag2−/− cells untreated or treated with STI571 for 72 hr. Error bars represent SD of three independent experiments. Molecular Cell 2009 34, 298-310DOI: (10.1016/j.molcel.2009.04.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 A Dynamic γ-H2AX Domain Is Maintained along Broken DNA Strands (A) Organization and orientation of the murine Igκ locus and γ-H2AX densities at locations within and adjacent to Igκ in Artemis−/− and Artemis−/−Rag2−/− pre-B cells treated with STI571 for 24 hr. (B) γ-H2AX densities at Jκ5 and locations telomeric of Jκ5 in Artemis−/− and Artemis−/−Rag2−/− cells treated with STI571 for the indicated time in hours (h). Error bars have been omitted for clarity. (C) γ-H2AX densities at sequences 50 kb (left) or 400 kb (right) telomeric of Jκ5 compared to the relative fraction of Igκ DSBs induced after STI571 treatment of Artemis−/− cells for the indicated times. (D and E) γ-H2AX (D) and H2AX (E) densities at locations telomeric of Jκ5 in Artemis−/− cells treated with STI571 for 72 hr or with STI71 with endothall for the last 24 hr. Error bars represent SD of three independent experiments. Molecular Cell 2009 34, 298-310DOI: (10.1016/j.molcel.2009.04.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 ATM Generates and Maintains a Dynamic γ-H2AX Domain along Broken DNA Strands (A) γ-H2AX densities at Jκ5 and locations telomeric of Jκ5 in Artemis−/−, Atm−/−Artemis−/−, and Scid pre-B cells treated with STI571 for 72 hr. Scid cells also were treated with the KU55933 ATM inhibitor. (B and C) γ-H2AX densities at Jκ5 and locations telomeric of Jκ5 in Scid (B) and Atm−/−Artemis−/− (C) cells treated with STI571 for the indicated time in hours (h). Error bars have been omitted for clarity. (D) γ-H2AX densities at locations telomeric of Jκ5 in Artemis−/− cells untreated or treated with STI571 for 48 hr, 72 hr, or 72 hr with caffeine for the last 24 hr. Error bars represent SD of three independent experiments. Molecular Cell 2009 34, 298-310DOI: (10.1016/j.molcel.2009.04.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 ATM Generates γ-H2AX at Distances from DSBs in an MDC1-Independent Manner (A) γ-H2AX densities at Jκ5 and locations telomeric of Jκ5 in Artemis−/−Rag2−/−, Atm−/−Artemis−/−, and Atm−/−Mdc1−/− pre-B cells treated with STI571 for 72 hr. (B) γ-H2AX densities at sequences 50 kb (left) and 400 kb (right) telomeric of Jκ5 in Artemis−/−Rag2−/−, Atm−/−Artemis−/−, Atm−/−Mdc1−/−, and Artemis−/− cells treated with STI571 for 72 hr. (C and D) H2AX densities at Jκ5 and locations telomeric of Jκ5 in Artemis−/−, Artemis−/−Rag2−/−, Atm−/−Artemis−/−, and Atm−/−Mdc1−/− cells untreated (C) or treated with STI571 for 72 hr (D). Error bars represent SD of three independent experiments. Molecular Cell 2009 34, 298-310DOI: (10.1016/j.molcel.2009.04.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 H2AX Levels in Chromatin Influence γ-H2AX Densities along Broken DNA Strands (A) γ-H2AX densities at locations telomeric of Jκ5 in Artemis−/−, Artemis−/−H2ax+/−, and Artemis−/−Rag2−/− pre-B cells treated with STI571 for 72 hr. (B) γ-H2AX densities at the same locations in the Artemis−/−H2ax+/− and Artemis−/−Rag2−/− cells from (A) plotted on a different scale. (C) H2AX densities at locations telomeric of Jκ5 in Artemis−/−, Artemis−/−H2ax+/−, and Artemis−/−H2ax−/− pre-B cells treated with STI571 for 72 hr. Error bars represent SD of three independent experiments. Molecular Cell 2009 34, 298-310DOI: (10.1016/j.molcel.2009.04.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Models Depicting How γ-H2AX Forms along DNA Strands Broken in G1 Phase Cells In wild-type cells, the γ-H2AX self-reinforcing mechanism generates and maintains γ-H2AX at high densities near DNA ends, and soluble ATM forms γ-H2AX at lower densities over sequences further away from DSBs. DNA-PKcs may contribute to γ-H2AX formation near DNA ends. In ATM-deficient cells, DNA-PKcs generates γ-H2AX at low densities near DNA ends, but not over sequences further away from DSBs. In MDC1-deficient cells, soluble ATM generates γ-H2AX at low densities both near DNA ends and over sequences further away from DSBs. In H2AX haploinsufficient cells, the γ-H2AX self-reinforcing mechanism is not as effective as in wild-type cells and generates γ-H2AX at low densities near DNA ends, but soluble ATM forms γ-H2AX over sequences further away from DSBs as in wild-type cells. Molecular Cell 2009 34, 298-310DOI: (10.1016/j.molcel.2009.04.012) Copyright © 2009 Elsevier Inc. Terms and Conditions