People with type 2 diabetes mellitus (T2DM) have a 25-fold higher risk of limb loss than non-diabetics due in large part to impaired wound healing. Here, we show that the impaired wound healing phenotype found in T2D mice is recapitulated in lethally irradiated wild type recipients, whose hematopoiesis is reconstituted with hematopoietic stem cells (HSCs) from T2D mice, indicating an HSC-autonomous mechanism. This impaired wound healing phenotype of T2D mice is due to a Nox-2-dependent increase in HSC oxidant stress that decreases microRNA let-7d-3p, which, in turn, directly upregulates Dnmt1, leading to the hypermethylation of Notch1, PU.1, and Klf4. This HSC-autonomous mechanism reduces the number of wound macrophages and skews their polarization towards M1 macrophages. These findings reveal a novel inflammatory mechanism by which a metabolic disorder induces an epigenetic mechanism in HSCs, which predetermines the gene expression of terminally differentiated inflammatory cells that controls their number and function.

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The checkpoint kinases ATM and ATR are redundantly required for maintenance of stable telomeres in diverse organisms, including budding and fission yeasts, Arabidopsis, Drosophila, and mammals. However, the molecular basis for telomere instability in cells lacking ATM and ATR has not yet been elucidated fully in organisms that utilize both the telomere protection complex shelterin and telomerase to maintain telomeres, such as fission yeast and humans. Here, we demonstrate by quantitative chromatin immunoprecipitation (ChIP) assays that simultaneous loss of Tel1ATM and Rad3ATR kinases leads to a defect in recruitment of telomerase to telomeres, reduced binding of the shelterin complex subunits Ccq1 and Tpz1, and increased binding of RPA and homologous recombination repair factors to telomeres. Moreover, we show that interaction between Tpz1-Ccq1 and telomerase, thought to be important for telomerase recruitment to telomeres, is disrupted in tel1Δ rad3Δ cells. Thus, Tel1ATM and Rad3ATR are redundantly required for both protection of telomeres against recombination and promotion of telomerase recruitment. Based on our current findings, we propose the existence of a regulatory loop between Tel1ATM/Rad3ATR kinases and Tpz1-Ccq1 to ensure proper protection and maintenance of telomeres in fission yeast.

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27 Kip1 . Moreover, expression of DDB1 reduces the level of p27 Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27 Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27 Kip1 .The Cul4A gene is amplified and overexpressed in breast and hepatocellular carcinomas (6, 42). Also, Cul4A is essential for mammalian development (18). It encodes a protein of the cullin family. The cullins are central components of several E3 ubiquitin ligases (11). Cul4A associates with the damaged-DNA binding protein DDB (22,32). DDB consists of two subunits: DDB1 and DDB2. The DDB2 subunit is mutated in xeroderma pigmentosum (complementation group E) (reviewed in reference 35). Cul4A participates in the ubiquitination of the DDB2 subunit of DDB and induces its proteolysis through the ubiquitin-proteasome pathway (22). Recent studies indicated that the DDB1 subunit of DDB functions as an adaptor for substrate binding by Cul4A in a manner similar to how Skp1 functions in the Skp1-cullin1-F-box (SCF) complex (15). However, unlike the case for Skp1, there are instances where DDB1 directly targets a substrate without additional adaptor proteins. For example, Cul4A has been implicated in the proteolysis of the replication licensing protein Cdt1 following DNA damage (14, 44). It was shown that the interaction between Cul4A and Cdt1 is mediated by DDB1 (15). In other examples, Cul4A-DDB1 interacts with additional adaptors to target a specific protein. The DDB1-Cul4A complex associates with hDET1, an ortholog of Arabidopsis De-etiolated-1, and hCOP1, an ortholog of Arabidopsis constitutively photomorphogenic-1 (COP1), to induce proteolysis of the c-Jun protein through the ubiquitin-proteasome pathway (40). In that study, the authors proposed that the hDET1-hCOP1 functioned as the heteromeric substrate adaptor and, in keeping with the SCF E3 ligase,...