DNA double-strand break (DSB) signaling and repair are critical for genome integrity. They rely on highly coordinated processes including posttranslational modifications of proteins. Here we show that Pellino1 (Peli1) is a DSB-responsive ubiquitin ligase required for the accumulation of DNA damage response proteins and efficient homologous recombination (HR) repair. Peli1 is activated by ATM-mediated phosphorylation. It is recruited to DSB sites in ATM- and γH2AX-dependent manners. Interaction of Peli1 with phosphorylated histone H2AX enables it to bind to and mediate the formation of K63-linked ubiquitination of NBS1, which subsequently results in feedback activation of ATM and promotes HR repair. Collectively, these results provide a DSB-responsive factor underlying the connection between ATM kinase and DSB-induced ubiquitination.

Histone H2AX undergoes a phosphorylation switch from pTyr142 (H2AX-pY142) to pSer139 (γH2AX) in the DNA damage response (DDR); however, the functional role of H2AX-pY142 remains elusive. Here, we report a new layer of regulation involving transcription-coupled H2AX-pY142 in the DDR. We found that constitutive H2AX-pY142 generated by Williams-Beuren syndrome transcription factor (WSTF) interacts with RNA polymerase II (RNAPII) and is associated with RNAPII-mediated active transcription in proliferating cells. Also, removal of pre-existing H2AX-pY142 by ATM-dependent EYA1/3 phosphatases disrupts this association and requires for transcriptional silencing at transcribed active damage sites. The following recovery of H2AX-pY142 via translocation of WSTF to DNA lesions facilitates transcription-coupled homologous recombination (TC-HR) in the G1 phase, whereby RAD51 loading, but not RPA32, utilizes RNAPII-dependent active RNA transcripts as donor templates. We propose that the WSTF-H2AX-RNAPII axis regulates transcription and TC-HR repair to maintain genome integrity.

Microhomology-mediated end joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52-independent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast.

Chronic skin inflammation including psoriasis is a multisystem disease, affecting more than 5% of the general population. Here we show that Pellino 1 (Peli1), a signal-responsive ubiquitin E3 ligase, is highly up-regulated in human psoriatic skin lesions and that increased Peli1 expression correlates with the immunopathogenesis of psoriasis-like chronic skin inflammatory disease. Interestingly, Peli1 directly interacts with interferon regulatory factor 4 (IRF4, a transcription factor that plays pivotal roles in proliferation and cytokine production) and induces lysine 63-mediated ubiquitination. Peli1-mediated IRF4 ubiquitination appears to be a common systemic signaling mechanism shared by lesional keratinocytes, dendritic cells, macrophages, and T cells, generating a feedback relationship between keratinocyte and Th17 cell responses. Conversely, inhibition of Peli1 interferes with IRF4 induction and attenuates immunopathogenic signaling in the psoriasis. In summary, Peli1-mediated ubiquitination is a common immunopathogenic intercellular signaling in psoriasis-like chronic skin inflammatory microenvironment. Thus, targeting Peli1 could be used as a potential strategy for psoriasis treatment.