Sean C. Hensley

Mec1/Tel1 Phosphorylation of the INO80 Chromatin Remodeling Complex Influences DNA Damage Checkpoint Responses

The yeast Mec1/Tel1 kinases, ATM/ATR in mammals, coordinate the DNA damage response by phosphorylating proteins involved in DNA repair and checkpoint pathways. Recently, ATP-dependent chromatin remodeling complexes, such as the INO80 complex, have also been implicated in DNA damage responses, although regulatory mechanisms that direct their function remain unknown. Here, we show that the Ies4 subunit of the INO80 complex is phosphorylated by the Mec1/Tel1 kinases during exposure to DNA-damaging agents. Mutation of Ies4s phosphorylation sites does not significantly affect DNA repair processes, but does influence DNA damage checkpoint responses. Additionally, ies4 phosphorylation mutants are linked to the function of checkpoint regulators, such as the replication checkpoint factors Tof1 and Rad53. These findings establish a chromatin remodeling complex as a functional component in the Mec1/Tel1 DNA damage signaling pathway that modulates checkpoint responses and suggest that posttranslational modification of chromatin remodeling complexes regulates their involvement in distinct processes.

Mechanism of suppression of chromosomal instability by DNA polymerase POLQ.

Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3′ single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.

Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R-loop accumulation

Tudor domain-containing protein 3 (TDRD3) is a major methylarginine effector molecule that reads methyl-histone marks and facilitates gene transcription. However, the underlying mechanism by which TDRD3 functions as a transcriptional coactivator is unknown. We identified topoisomerase IIIB (TOP3B) as a component of the TDRD3 complex. TDRD3 serves as a molecular bridge between TOP3B and arginine-methylated histones. The TDRD3-TOP3B complex is recruited to the c-MYC gene promoter primarily by the H4R3me2a mark, and the complex promotes c-MYC gene expression. TOP3B relaxes negative supercoiled DNA and reduces transcription-generated R loops in vitro. TDRD3 knockdown in cells increases R loop formation at the c-MYC locus, and Tdrd3 null mice exhibit elevated R loop formation at this locus in B cells. Tdrd3 null mice show significantly increased c-Myc/Igh translocation, a process driven by R loop structures. By reducing negative supercoiling and resolving R loops, TOP3B promotes transcription, protects against DNA damage, and reduces the frequency of chromosomal translocations.

Proteomic and pathway analyses reveal a network of inflammatory genes associated with differences in skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with skin tumor promotion susceptibility, a proteomic approach was used to examine and identify proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We identified 19 differentially expressed proteins of which 5 were the calcium-binding proteins annexin A1, parvalbumin α, S100A8, S100A9, and S100A11. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially upregulated in epidermis of DBA/2 versus C57BL/6 mice following topical treatment with two other skin tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that these proteins were components of several networks that included inflammation-associated proteins known to be involved in skin tumor promotion (e.g. TNF-α, NFκB). Follow-up studies revealed that Tnf, Nfkb1, Il22, Il1b, Cxcl1, Cxcl2 and Cxcl5 mRNAs were highly expressed in epidermis of DBA/2 compared with C57BL/6 mice at 24h following treatment with TPA. Furthermore, NFκB (p65) was also highly activated at the same time point (as measured by phosphorylation at ser276) in epidermis of DBA/2 mice compared with C57BL/6 mice. Taken together, the present data suggest that differential expression of genes involved in inflammatory pathways in epidermis may play a key role in genetic differences in susceptibility to skin tumor promotion in DBA/2 and C57BL/6 mice.

Differential expression of multiple anti-apoptotic proteins in epidermis of IGF-1 transgenic mice as revealed by 2-dimensional gel electrophoresis/mass spectrometry analysis.

Overexpression of insulin‐like growth factor‐1 (IGF‐1) has been associated with a number of human tumors, including breast, colon, lung, and prostate cancers. In previous studies, we found that mice overexpressing human IGF‐1 in the basal layer of the epidermis (BK5.IGF‐1 mice) developed skin tumors following treatment with the skin tumor initiator, 7,12‐dimethylbenz[a]anthracene, indicating that IGF‐1 can act as a skin tumor promoter. In the present study, we employed a proteomics approach of two‐dimensional (2‐D) gel electrophoresis and mass spectrometry to profile differentially expressed proteins in skin epidermis between BK5.IGF‐1 transgenic and nontransgenic littermates. Two‐D gels from each of three transgenic and three age/sex matched wild‐type littermates were compared at two different pH ranges. Differentially expressed protein spots were identified by Bio‐Rads PDQuest image analysis, in‐gel digested, and analyzed on a MALDI‐TOF MS system. A total of 23 proteins were identified as differentially expressed, 17 of them overexpressed in transgenic mice. These proteins included 14‐3‐3 sigma, galectin‐7, an apoptosis‐related protein, three heat shock proteins, four calcium binding proteins, three proteases or protease inhibitors, one actin regulatory capping protein, and translation initiation factor 5A. The differential expression of GRP78, alpha enolase, and galectin‐7 was verified by 1‐D western blot analysis. Two‐D western blot analyses of alpha enolase and galectin‐7 further revealed that alpha enolase had more than one protein spot dependent on charge. The current data suggest that some of the differentially expressed proteins may play a role in the tumor promoting action of IGF‐1 in mouse skin.

Abstract 2729: Proteomic analysis reveals a network of inflammatory genes in epidermis associated with skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with tumor promotion susceptibility by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a proteomic approach of two-dimensional (2-D) gel electrophoresis and mass spectrometry was used. In these experiments, we examined epidermal protein lysates and identified proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following TPA treatment. Among 19 differentially expressed proteins identified using this methodology were two calcium-binding proteins, S100A8 and S100A9. Their differential expression was further examined and validated by one or more of the following methods: i) one-dimensional (1-D) Western blot analysis; ii) 2-D Western blot analysis; iii) immunohistochemical analysis; and iv) quantitative real-time PCR. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially up-regulated in epidermis of DBA/2 vs C57BL/6 mice following topical treatment with two other tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that S100A8/A9 could be linked to several inflammatory networks. Further analyses revealed significantly increased expression of several inflammation-related genes including TNF-α, NFκB and IL-22 in epidermis of TPA-treated DBA/2 mice. Follow-up studies confirmed that these three inflammation related genes were upregulated in epidermis of TPA-treated DBA/2 mice compared to similarly treated C57BL/6 mice. These data suggest that differential expression of inflammation related genes in epidermis contributes to TPA-induced inflammation and skin tumor promotion susceptibility in DBA/2 mice. Taken together, our present data provide further insight into potential molecular mechanisms for the differential susceptibility of DBA/2 and C57BL/6 mice in terms of both inflammation and skin tumor promotion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2729. doi:10.1158/1538-7445.AM2011-2729