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Mr Double Stories.7z.001

E.R., M.N., A.A. and M.S. conceived the study. E.R. created the data set; M.S. and O.M. double-checked the data for accuracy. M.S. wrote the first draft of the manuscript, and all authors contributed to finalizing the paper. All authors reviewed the manuscript and provided input.

Mr Double Stories.7z.001


The DNA damage response (DDR), comprising distinct repair and signalling pathways, safeguards genomic integrity. Protein ubiquitylation is an important regulatory mechanism of the DDR. To study its role in the UV-induced DDR, we characterized changes in protein ubiquitylation following DNA damage using quantitative di-Gly proteomics. Interestingly, we identified multiple sites of histone H1 that are ubiquitylated upon UV-damage. We show that UV-dependent histone H1 ubiquitylation at multiple lysines is mediated by the E3-ligase HUWE1. Recently, it was shown that poly-ubiquitylated histone H1 is an important signalling intermediate in the double strand break response. This poly-ubiquitylation is dependent on RNF8 and Ubc13 which extend pre-existing ubiquitin modifications to K63-linked chains. Here we demonstrate that HUWE1 depleted cells showed reduced recruitment of RNF168 and 53BP1 to sites of DNA damage, two factors downstream of RNF8 mediated histone H1 poly-ubiquitylation, while recruitment of MDC1, which act upstream of histone H1 ubiquitylation, was not affected. Our data show that histone H1 is a prominent target for ubiquitylation after UV-induced DNA damage. Our data are in line with a model in which HUWE1 primes histone H1 with ubiquitin to allow ubiquitin chain elongation by RNF8, thereby stimulating the RNF8-RNF168 mediated DDR.

DNA integrity is constantly threatened by endogenous and exogenous DNA damaging agents. DNA damage interferes with transcription and replication, causing mutations, chromosomal aberrations and cell death, which may eventually induce malignant transformation and aging1. To counteract these deleterious effects cells have evolved an intricate network called the DNA damage response (DDR). This consists of signalling pathways that regulate cell cycle checkpoints and apoptosis, and a set of highly specialized DNA repair mechanisms each capable of repairing a specific subset of DNA lesions. UV-induced helix distorting lesions are typically repaired by nucleotide excision repair (NER)2. Helix distorting lesions located at any position in the genome are recognized by the global genome repair (GG-NER) proteins XPC and the UV-DDB complex. DNA lesions in the transcribed strand of active genes that cause stalling of RNA polymerase II initiate transcription-coupled repair (TC-NER). After damage recognition the DNA helix surrounding the lesion is unwound by TFIIH, which together with XPA verifies the lesion3,4,5,6. Next, RPA stabilizes the repair complex and positions the endonucleases XPG and ERCC1/XPF to excise the damaged DNA7. Before the single stranded DNA gap is filled by DNA synthesis by PCNA and the DNA polymerases δ, ε or κ8 it activates ATR signalling, which subsequently results in phosphorylation of histone H2AX on serine 139 (yH2AX)9,10,11. Phosphorylation of H2AX is a major DDR signalling event initiating the recruitment of many DDR-factors to activate cell cycle checkpoints and stimulate repair. This is induced by many types of genomic insults, such as DNA double strand breaks (DSBs) and stretches of ssDNA following replication fork stalling or NER-mediated excision12,13. MDC1 is directly recruited to yH2AX and functions as a scaffold protein crucial for the recruitment of many downstream DDR factors, such as RNF8, 53BP1 and BRCA110,14,15.

Recently, it was shown that H1 ubiquitylation following double strand break induction was dependent on the E3 ligase RNF832. Also after UV-induced DNA damage RNF8 is activated10, however this activation is dependent on NER. This makes RNF8 a less likely candidate to be responsible for our observed H1 ubiquitylation. Another E3-ligase, previously shown to be able to ubiquitylate histone H1 in vitro, is HUWE1 (HECT, UBA, and WWE domain containing protein 1)43. In vivo, HUWE1 was found to be involved in the ubiquitylation of histone H2AX in both unperturbed conditions and upon replication stress44,45. In addition, HUWE1 regulates the DDR by targeting different proteins involved in cell cycle checkpoint control, homologous recombination and base excision repair, such as Cdc646, BRCA147, TopBP148 and POLB49. To investigate whether HUWE1 is responsible for the UV-induced ubiquitylation of histone H1 we tested the ubiquitylation status of FLAG-H1.2 in cells expressing a doxycycline inducible shRNA targeting HUWE150 (Fig. 2d). Interestingly, HUWE1 knockdown resulted in an almost complete absence of the UV-induced Flag-H1.2 ubiquitylation (Fig. 2b,c). To exclude doxycycline induced effects on H1 ubiquitylation we performed a similar experiment using shControl and shHUWE1 cells, both treated with doxycycline (Supplemental Fig. S2). Also in this experiment the UV-induced H1 ubiquitylation is absent following HUWE1 depletion. Furthermore, the observed UV-independent decrease of H1 ubiquitylation, suggests a possible role for HUWE1 in the constitutive H1 ubiquitylation as well. To further confirm the HUWE1-dependency on ubiquitylation of endogenous H1 species we used quantitative di-Gly proteomics. UV/mock SILAC ratios of the vast majority of ubiquitylated histone H1 peptides were strongly reduced in HUWE1 KO cells as compared to WT HeLa cells (Fig. 2e,f). Of all identified UV-induced ubiquitylation sites, only lysine 85 was not influenced by HUWE1 knock out (KO) (Fig. 2f and Supplemental Table S3), suggesting that probably an additional E3 ligase is involved in H1 ubiquitylation. However, HUWE1 KO severely reduced the levels of histone H1 ubiquitylation on 9 different lysines, indicating that the ubiquitylation levels of histone H1 after UV damage is mainly regulated by HUWE1.

It was enlarged to be a duplex in 1904 designed by Arthur L. Pillsbury. The primary façade is composed of a five-bay front; three associated with the c 1855 structure and two associated with the 1904 structure. Entrance to the two parts is gained through a double porch which divides by a set of double columns which are flanked on their respective ends by large square recessed panel columns. The columns rise from three brick piers capped with clapboard sided bases. The Pillsbury designed addition on the east side has two bays which have a door and two windows on the first story and one single and one double window on the second story. The roof to this part is a low-pitched hip roof with a bay dormer offset on the roof. 041b061a72


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