Poly(ADP-Ribose) Polymerase-1 Lacking Enzymatic Activity Is Not Compatible with Mouse Development.

Poly(ADP-ribose) polymerase-1 (PARP1) binds DNA lesions to catalyse poly(ADP-ribosyl)ation (PARylation) using NAD+ as a substrate. PARP1 plays multiple roles in cellular activities, including DNA repair, transcription, cell death, and chromatin remodelling. However, whether these functions are governed by the enzymatic activity or scaffolding function of PARP1 remains elusive. In this study, we inactivated in mice the enzymatic activity of PARP1 by truncating its C-terminus that is essential for ART catalysis (PARP1ΔC/ΔC, designated as PARP1-ΔC). The mutation caused embryonic lethality between embryonic day E8.5 and E13.5, in stark contrast to PARP1 complete knockout (PARP1-/-) mice, which are viable. Embryonic stem (ES) cell lines can be derived from PARP1ΔC/ΔC blastocysts, and these mutant ES cells can differentiate into all three germ layers, yet, with a high degree of cystic structures, indicating defects in epithelial cells. Intriguingly, PARP1-ΔC protein is expressed at very low levels compared to its full-length counterpart, suggesting a selective advantage for cell survival. Noticeably, PARP2 is particularly elevated and permanently present at the chromatin in PARP1-ΔC cells, indicating an engagement of PARP2 by non-enzymatic PARP1 protein at the chromatin. Surprisingly, the introduction of PARP1-ΔC mutation in adult mice did not impair their viability; yet, these mutant mice are hypersensitive to alkylating agents, similar to PARP1-/- mutant mice. Our study demonstrates that the catalytically inactive mutant of PARP1 causes the developmental block, plausibly involving PARP2 trapping.

PARP1: Liaison of Chromatin Remodeling and Transcription.

Poly(ADP-ribosyl)ation (PARylation) is a covalent post-translational modification and plays a key role in the immediate response of cells to stress signals. Poly(ADP-ribose) polymerase 1 (PARP1), the founding member of the PARP superfamily, synthesizes long and branched polymers of ADP-ribose (PAR) onto acceptor proteins, thereby modulating their function and their local surrounding. PARP1 is the most prominent of the PARPs and is responsible for the production of about 90% of PAR in the cell. Therefore, PARP1 and PARylation play a pleotropic role in a wide range of cellular processes, such as DNA repair and genomic stability, cell death, chromatin remodeling, inflammatory response and gene transcription. PARP1 has DNA-binding and catalytic activities that are important for DNA repair, yet also modulate chromatin conformation and gene transcription, which can be independent of DNA damage response. PARP1 and PARylation homeostasis have also been implicated in multiple diseases, including inflammation, stroke, diabetes and cancer. Studies of the molecular action and biological function of PARP1 and PARylation provide a basis for the development of pharmaceutic strategies for clinical applications. This review focuses primarily on the role of PARP1 in the regulation of chromatin remodeling and transcriptional activation.

Cell Type-Specific Role of RNA Nuclease SMG6 in Neurogenesis.

SMG6 is an endonuclease, which cleaves mRNAs during nonsense-mediated mRNA decay (NMD), thereby regulating gene expression and controling mRNA quality. SMG6 has been shown as a differentiation license factor of totipotent embryonic stem cells. To investigate whether it controls the differentiation of lineage-specific pluripotent progenitor cells, we inactivated Smg6 in murine embryonic neural stem cells. Nestin-Cre-mediated deletion of Smg6 in mouse neuroprogenitor cells (NPCs) caused perinatal lethality. Mutant mice brains showed normal structure at E14.5 but great reduction of the cortical NPCs and late-born cortical neurons during later stages of neurogenesis (i.e., E18.5). Smg6 inactivation led to dramatic cell death in ganglionic eminence (GE) and a reduction of interneurons at E14.5. Interestingly, neurosphere assays showed self-renewal defects specifically in interneuron progenitors but not in cortical NPCs. RT-qPCR analysis revealed that the interneuron differentiation regulators Dlx1 and Dlx2 were reduced after Smg6 deletion. Intriguingly, when Smg6 was deleted specifically in cortical and hippocampal progenitors, the mutant mice were viable and showed normal size and architecture of the cortex at E18.5. Thus, SMG6 regulates cell fate in a cell type-specific manner and is more important for neuroprogenitors originating from the GE than for progenitors from the cortex.

Nbs1-mediated DNA damage repair pathway regulates haematopoietic stem cell development and embryonic haematopoiesis.

OBJECTIVES: DNA damages pose threats to haematopoietic stem cells (HSC) maintenance and haematopoietic system homeostasis. Quiescent HSCs in adult mouse bone marrow are resistant to DNA damage, while human umbilical cord blood-derived proliferative HSCs are prone to cell death upon ionizing radiation. Murine embryonic HSCs proliferate in foetal livers and divide symmetrically to generate HSC pool. How murine embryonic HSCs respond to DNA damages is not well-defined. MATERIALS AND METHODS: Mice models with DNA repair molecule Nbs1 or Nbs1/p53 specifically deleted in embryonic HSCs were generated. FACS analysis, in vitro and in vivo HSC differentiation assays, qPCR, immunofluorescence and Western blotting were used to delineate roles of Nbs1-p53 signaling in HSCs and haematopoietic progenitors. RESULTS: Nbs1 deficiency results in persistent DNA breaks in embryonic HSCs, compromises embryonic HSC development and finally results in mouse perinatal lethality. The persistent DNA breaks in Nbs1 deficient embryonic HSCs render cell cycle arrest, while driving a higher rate of cell death in haematopoietic progenitors. Although Nbs1 deficiency promotes Atm-Chk2-p53 axis activation in HSCs and their progenies, ablation of p53 in Nbs1 deficient HSCs accelerates embryonic lethality. CONCLUSIONS: Our study discloses that DNA double-strand repair molecule Nbs1 is essential in embryonic HSC development and haematopoiesis. Persistent DNA damages result in distinct cell fate in HSCs and haematopoietic progenitors. Nbs1 null HSCs tend to be maintained through cell cycle arrest, while Nbs1 null haematopoietic progenitors commit cell death. The discrepancies are mediated possibly by different magnitude of p53 signaling.

Nonsense-mediated mRNA decay: a 'nonsense' pathway makes sense in stem cell biology.

Nonsense-mediated mRNA decay (NMD) is a highly conserved post-transcriptional regulatory mechanism of gene expression in eukaryotes. Originally, NMD was identified as an RNA surveillance machinery in degrading 'aberrant' mRNA species with premature termination codons. Recent studies indicate that NMD regulates the stability of natural gene transcripts that play significant roles in cell functions. Although components and action modes of the NMD machinery in degrading its RNA targets have been extensively studied with biochemical and structural approaches, the biological roles of NMD remain to be defined. Stem cells are rare cell populations, which play essential roles in tissue homeostasis and hold great promises in regenerative medicine. Stem cells self-renew to maintain the cellular identity and differentiate into somatic lineages with specialized functions to sustain tissue integrity. Transcriptional regulations and epigenetic modulations have been extensively implicated in stem cell biology. However, post-transcriptional regulatory mechanisms, such as NMD, in stem cell regulation are largely unknown. In this paper, we summarize the recent findings on biological roles of NMD factors in embryonic and tissue-specific stem cells. Furthermore, we discuss the possible mechanisms of NMD in regulating stem cell fates.

DNA Damage Response in Hematopoietic Stem Cell Ageing.

Maintenance of tissue-specific stem cells is vital for organ homeostasis and organismal longevity. Hematopoietic stem cells (HSCs) are the most primitive cell type in the hematopoietic system. They divide asymmetrically and give rise to daughter cells with HSC identity (self-renewal) and progenitor progenies (differentiation), which further proliferate and differentiate into full hematopoietic lineages. Mammalian ageing process is accompanied with abnormalities in the HSC self-renewal and differentiation. Transcriptional changes and epigenetic modulations have been implicated as the key regulators in HSC ageing process. The DNA damage response (DDR) in the cells involves an orchestrated signaling pathway, consisting of cell cycle regulation, cell death and senescence, transcriptional regulation, as well as chromatin remodeling. Recent studies employing DNA repair-deficient mouse models indicate that DDR could intrinsically and extrinsically regulate HSC maintenance and play important roles in tissue homeostasis of the hematopoietic system. In this review, we summarize the current understanding of how the DDR determines the HSC fates and finally contributes to organismal ageing.

SIRT6 Controls Hematopoietic Stem Cell Homeostasis through Epigenetic Regulation of Wnt Signaling.

Proper regulation of Wnt signaling is critical for the maintenance of hematopoietic stem cell (HSC) homeostasis. The epigenetic regulation of Wnt signaling in HSCs remains largely unknown. Here, we report that the histone deacetylase SIRT6 regulates HSC homeostasis through the transcriptional repression of Wnt target genes. Sirt6 deletion promoted HSC proliferation through aberrant activation of Wnt signaling. SIRT6-deficient HSCs exhibited impaired self-renewal ability in serial competitive transplantation assay. Mechanistically, SIRT6 inhibits the transcription of Wnt target genes by interacting with transcription factor LEF1 and deacetylating histone 3 at lysine 56. Pharmacological inhibition of the Wnt pathway rescued the aberrant proliferation and functional defect in SIRT6-deficient HSCs. Taken together, these findings disclose a new link between SIRT6 and Wnt signaling in the regulation of adult stem cell homeostasis and self-renewal capacity.

Dual functions of Nbs1 in the repair of DNA breaks and proliferation ensure proper V(D)J recombination and T-cell development.

Immunodeficiency and lymphoid malignancy are hallmarks of the human disease Nijmegen breakage syndrome (NBS; OMIM 251260), which is caused by NBS1 mutations. Although NBS1 has been shown to bind to the T-cell receptor alpha (TCRα) locus, its role in TCRß rearrangement is unclear. Hypomorphic mutations of Nbs1 in mice and patients result in relatively mild T-cell deficiencies, raising the question of whether the truncated Nbs1 protein might have clouded a certain function of NBS1 in T-cell development. Here we show that the deletion of the entire Nbs1 protein in T-cell precursors (Nbs1(T-del)) results in severe lymphopenia and a hindrance to the double-negative 3 (DN3)-to-DN4 transition in early T-cell development, due to abnormal TCRß coding and signal joints as well as the functions of Nbs1 in T-cell expansion. Chromatin immunoprecipitation (ChIP) analysis of the TCR loci reveals that Nbs1 depletion compromises the loading of Mre11/Rad50 to V(D)J-generated DNA double-strand breaks (DSBs) and thereby affects resection of DNA termini and chromatin conformation of the postcleavage complex. Although a p53 deficiency relieves the DN3→DN4 transition block, neither a p53 deficiency nor ectopic expression of TCRαß rescues the major T-cell loss in Nbs1(T-del) mice. All together, these results demonstrate that Nbs1's functions in both repair of V(D)J-generated DSBs and proliferation are essential for T-cell development.