UNG is the major uracil-DNA glycosylase in mammalian cells and is involved in both error-free base excision repair of genomic uracil and mutagenic uracil-processing at the antibody genes. However, the regulation of UNG in these different processes is currently not well understood. The UNG gene encodes two isoforms, UNG1 and UNG2, each possessing unique N-termini that mediate translocation to the mitochondria and the nucleus, respectively. A strict subcellular localization of each isoform has been widely accepted despite a lack of models to study them individually. To determine the roles of each isoform, we generated and characterized several UNG isoform-specific mouse and human cell lines. We identified a distinct UNG1 isoform variant that is targeted to the cell nucleus where it supports antibody class switching and repairs genomic uracil. We propose that the nuclear UNG1 variant, which in contrast to UNG2 lacks a PCNA-binding motif, may be specialized to act on ssDNA through its ability to bind RPA. RPA-coated ssDNA regions include both transcribed antibody genes that are targets for deamination by AID and regions in front of the moving replication forks. Our findings provide new insights into the function of UNG isoforms in adaptive immunity and DNA repair.

To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining ( NHEJ ) is the DNA repair process that fixes double‐strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination ( CSR ). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC 4, DNA ligase 4, DNA ‐ PK cs, Artemis, and XLF . Paralog of XRCC 4 and XLF ( PAXX ) is the recently described accessory NHEJ factor that structurally resembles XRCC 4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom‐generated and commercially available NHEJ ‐deficient human haploid HAP 1 cells, PAXX Δ , XRCC 4 Δ , and XLF Δ . In our studies, HAP 1 PAXX Δ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild‐type controls. By contrast, XRCC 4 Δ and XLF Δ HAP 1 cells possessed significant DNA repair defects measured as sensitivity to double‐strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR , we generated and characterized Paxx −/− and Aid −/− murine lymphoid CH 12F3 cells. CSR to IgA was nearly at wild‐type levels in the Paxx −/− cells and completely ablated in the absence of activation‐induced cytidine deaminase ( AID ). In addition, Paxx −/− CH 12F3 cells were hypersensitive to zeocin when compared to wild‐type controls. We concluded that Paxx ‐deficient mammalian cells maintain robust NHEJ and CSR .

Exploiting the properties of stem cells by microRNA (miRNA) profiling offers an attractive approach to identify new regulators of stem cell fate. Although numerous miRNA have been screened from hematopoietic stem cells (HSC), the targets corresponding to many of these miRNA have not yet been fully elucidated. By miRNA profiling in a subpopulation of CD34+ cells isolated from peripheral blood, we have identified eight clusters of miRNA that were differentially expressed. Further analysis of one of the clusters by bioinformatics revealed that a miRNA, miR-181a*, which is highly expressed in the adherent CD34+ cells, affects the expression levels of Nanog, a stem cell surrogate marker. We show specifically by reporter assay and mutational analysis that miR-181a* targets a seedless 3′ compensatory site in the 3′UTR of Nanog and affects gene expression. We demonstrate that inhibiting miR-181a* upregulates the Nanog expression level, in addition to an increase in alkaline phosphatase activity. Our studies suggest that miR-181a* may be important in controlling the expression level of Nanog in a subpopulation of CD34+ cells.

Background and Objectives: Hematopoietic stem cells (HSC) are the most widely studied and characterized adult stem cells, which play an essential role in sustaining the formation of blood and immune system. The ease of their manipulation, the lack of serious ethical issues, and, in the autologous setting, the absence of their immunogenicity, have made them an attractive tool for developing stem cell-based therapies. Exploiting the properties of HSC by microRNA (miRNA) profiling offers an attractive approach to identify new regulators of stem cell fate. Although numerous miRNA have been screened from hematopoietic stem cells (HSC), the targets corresponding to many of these miRNA have not yet been fully elucidated. Therefore the objective of this study is to generate a miRNA profile from a subpopulation of adherent CD34+ HSC isolated from G- colony-stimulating factor mobilized peripheral blood aiming to understand the role of selected miRNA in regulating HSC stemness. Methods: CD34+ cells from patients' blood were isolated using a CD34+ isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. miRNA profiling of adherent and nonadherent CD34+ cells was done using TaqMan Array MicroRNA Cards. Nanog expression levels was tested using a dual-luciferase reporter construct for miR-181a* or its mutant variant and Nanog 3′ UTR mRNA. Results: In this study, we have identified eight clusters of miRNA that were differentially expressed in an adherent subpopulation of CD34+ stem cells. Further analysis of one of the clusters by bioinformatics revealed that a miRNA, miR-181a*, which is highly expressed in the adherent CD34+ cells, affects the expression levels of Nanog, a stem cell surrogate marker. We show specifically by reporter assay and mutational analysis that miR-181a* targets a seedless 3′ compensatory site in the 3′UTR of Nanog and affects gene expression. We demonstrate that inhibiting miR-181a* upregulates the Nanog expression level, in addition to an increase in alkaline phosphatase activity. Conclusions: In conclusion, our results highlight a new stem cell-related target for the miR-181 family and show that miR-181a* directly targets Nanog in a subpopulation of CD34+ stem cells suggesting a possible role of miR-181a* in regulation of adherent CD34+ HSC cells stemness.