JADE1 is dispensable for the brain development in mice.

In mammalian brain development, WNT signaling balances proliferation and differentiation of neural progenitor cells, and is essential for the maintenance of regular brain development. JADE1 is a candidate transcription co-factor essential for DNA replication, cell division, and cell cycle regulation. In 293T cells, JADE1 is stabilized by von Hippel-Lindau protein pVHL, promotes the ß-catenin ubiquitination and thus blunts canonical WNT signaling. Furthermore, JADE1 inhibits ß-catenin-induced ectopic axis formation in Xenopus embryos. However, JADE1's role in mammalian brain development remains unknown. Here, we generated a new Jade1 knockout mouse line using CRISPR-Cas9 technology. We found that JADE1 null resulted in decreased survival rate, reduced body weight and brain weight in mice. However, histological analysis revealed a normal brain development. Furthermore, Jade1 null neural progenitor cells proliferated normally in vivo and in vitro. RNA-seq analysis further showed that JADE1 loss did not affect the cerebral cortex gene expression. Our findings indicate that JADE1 is dispensable for developing the cerebral cortex in mice.

NBS1 interacts with Notch signaling in neuronal homeostasis.

NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.

The E3 ubiquitin ligase APC/CCdh1 degrades MCPH1 after MCPH1-ßTrCP2-Cdc25A-mediated mitotic entry to ensure neurogenesis.

Mutations of microcephalin (MCPH1) can cause the neurodevelopmental disorder primary microcephaly type 1. We previously showed that MCPH1 deletion in neural stem cells results in early mitotic entry that distracts cell division mode, leading to exhaustion of the progenitor pool. Here, we show that MCPH1 interacts with and promotes the E3 ligase ßTrCP2 to degrade Cdc25A independent of DNA damage. Overexpression of ßTrCP2 or the knockdown of Cdc25A remedies the high mitotic index and rescues the premature differentiation of Mcph1-deficient neuroprogenitors in vivo MCPH1 itself is degraded by APC/CCdh1, but not APC/CCdc20, in late mitosis and G1 phase. Forced MCPH1 expression causes cell death, underlining the importance of MCPH1 turnover after mitosis. Ectopic expression of Cdh1 leads to premature differentiation of neuroprogenitors, mimicking differentiation defects of Mcph1-knockout neuroprogenitors. The homeostasis of MCPH1 in association with the ubiquitin-proteasome system ensures mitotic entry independent of cell cycle checkpoint. This study provides a mechanistic understanding of how MCPH1 controls neural stem cell fate and brain development.

PGC1α protects against hepatic steatosis and insulin resistance via enhancing IL10-mediated anti-inflammatory response.

Inflammatory responses are pivotal incidences in hepatic metabolic derangements. However, the underlying mechanism remains elusive. The present study aimed to evaluate the role of peroxisome proliferator-activated receptor-gamma, coactivator 1 alpha (PGC1α) in IL10-mediated anti-inflammatory response, and its role in hepatic steatosis and insulin resistance. Hepatocyte-specific PGC1α knock-in (LivPGC1α) mice and the control mice were fed high-fat diet (HFD) for 8 weeks. IL-10 neutralizing antibody was injected into the liver of PGC1α mice. A variety of biological and histological approaches were applied to assess hepatic function. We demonstrated that hepatic PGC1α expression was significantly reduced in mice fed HFD. LivPGC1α livers exhibited enhanced gene expressions involving mitochondrial function, and favored an accelerated lipid metabolism upon HFD. Meanwhile, LivPGC1α mice revealed improved hepatic steatosis and insulin resistance. Mechanistically, PGC1α bound and activated the promotor region of IL-10, thereby attenuating inflammatory response in the liver. Administration of IL10 neutralizing antibody to LivPGC1α mice abolished PGC1α-mediated anti-inflammatory effects in mice. Further, IL-10 neutralizing antibody intervention aggravated hepatic steatosis and insulin resistance in LivPGC1α mice. Taken together, our data indicated that hepatic-specific overexpression of PGC1α exerts a beneficial role in the regulation of hepatic steatosis and insulin resistance via enhancing IL10-mediated anti-inflammatory response. Pharmacological activation of PGC1α-IL10 axis may be promising for the treatment of fatty liver diseases.

Fine-Tuning of PGC1α Expression Regulates Cardiac Function and Longevity.

RATIONALE: PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1α) represents an attractive target interfering bioenergetics and mitochondrial homeostasis, yet multiple attempts have failed to upregulate PGC1α expression as a therapy, for instance, causing cardiomyopathy. OBJECTIVE: To determine whether a fine-tuning of PGC1α expression is essential for cardiac homeostasis in a context-dependent manner. METHODS AND RESULTS: Moderate cardiac-specific PGC1α overexpression through a ROSA26 locus knock-in strategy was utilized in WT (wild type) mice and in G3Terc-/- (third generation of telomerase deficient; hereafter as G3) mouse model, respectively. Ultrastructure, mitochondrial stress, echocardiographic, and a variety of biological approaches were applied to assess mitochondrial physiology and cardiac function. While WT mice showed a relatively consistent PGC1α expression from 3 to 12 months old, age-matched G3 mice exhibited declined PGC1α expression and compromised mitochondrial function. Cardiac-specific overexpression of PGC1α (PGC1αOE) promoted mitochondrial and cardiac function in 3-month-old WT mice but accelerated cardiac aging and significantly shortened life span in 12-month-old WT mice because of increased mitochondrial damage and reactive oxygen species insult. In contrast, cardiac-specific PGC1α knock in in G3 (G3 PGC1αOE) mice restored mitochondrial homeostasis and attenuated senescence-associated secretory phenotypes, thereby preserving cardiac performance with age and extending health span. Mechanistically, age-dependent defect in mitophagy is associated with accumulation of damaged mitochondria that leads to cardiac impairment and premature death in 12-month-old WT PGC1αOE mice. In the context of telomere dysfunction, PGC1α induction replenished energy supply through restoring the compromised mitochondrial biogenesis and thus is beneficial to old G3 heart. CONCLUSIONS: Fine-tuning the expression of PGC1α is crucial for the cardiac homeostasis because the balance between mitochondrial biogenesis and clearance is vital for regulating mitochondrial function and homeostasis. These results reinforce the importance of carefully evaluating the PGC1α-boosting strategies in a context-dependent manner to facilitate clinical translation of novel cardioprotective therapies.

Telomeric epigenetic response mediated by Gadd45a regulates stem cell aging and lifespan.

Progressive attrition of telomeres triggers DNA damage response (DDR) and limits the regenerative capacity of adult stem cells during mammalian aging. Intriguingly, telomere integrity is not only determined by telomere length but also by the epigenetic status of telomeric/sub-telomeric regions. However, the functional interplay between DDR induced by telomere shortening and epigenetic modifications in aging remains unclear. Here, we show that deletion of Gadd45a improves the maintenance and function of intestinal stem cells (ISCs) and prolongs lifespan of telomerase-deficient mice (G3Terc-/-). Mechanistically, Gadd45a facilitates the generation of a permissive chromatin state for DDR signaling by inducing base excision repair-dependent demethylation of CpG islands specifically at sub-telomeric regions of short telomeres. Deletion of Gadd45a promotes chromatin compaction in sub-telomeric regions and attenuates DDR initiation at short telomeres of G3Terc-/- ISCs. Treatment with a small molecule inhibitor of base excision repair reduces DDR and improves the maintenance and function of G3Terc-/- ISCs. Taken together, our study proposes a therapeutic approach to enhance stem cell function and prolong lifespan by targeting epigenetic modifiers.

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.

Smg6/Est1 licenses embryonic stem cell differentiation via nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional mechanism that targets aberrant transcripts and regulates the cellular RNA reservoir. Genetic modulation in vertebrates suggests that NMD is critical for cellular and tissue homeostasis, although the underlying mechanism remains elusive. Here, we generate knockout mice lacking Smg6/Est1, a key nuclease in NMD and a telomerase cofactor. While the complete loss of Smg6 causes mouse lethality at the blastocyst stage, inducible deletion of Smg6 is compatible with embryonic stem cell (ESC) proliferation despite the absence of telomere maintenance and functional NMD. Differentiation of Smg6-deficient ESCs is blocked due to sustained expression of pluripotency genes, normally repressed by NMD, and forced down-regulation of one such target, c-Myc, relieves the differentiation block. Smg6-null embryonic fibroblasts are viable as well, but are refractory to cellular reprograming into induced pluripotent stem cells (iPSCs). Finally, depletion of all major NMD factors compromises ESC differentiation, thus identifying NMD as a licensing factor for the switch of cell identity in the process of stem cell differentiation and somatic cell reprograming.

An essential function for the ATR-activation-domain (AAD) of TopBP1 in mouse development and cellular senescence.

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins - Dpb11(TopBP1), Ddc1(Rad9) and Dna2 - all interact with and activate Mec1(ATR). Each contains an ATR activation domain (ADD) that interacts directly with the Mec1(ATR):Ddc2(ATRIP) complex. Any of the Dpb11(TopBP1), Ddc1(Rad9) or Dna2 ADDs is sufficient to activate Mec1(ATR) in vitro. All three can also independently activate Mec1(ATR) in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development.

UPF3A is dispensable for nonsense-mediated mRNA decay in mouse pluripotent and somatic cells.

Nonsense-mediated mRNA decay (NMD) is a highly conserved regulatory mechanism of post-transcriptional gene expression in eukaryotic cells. NMD plays essential roles in mRNA quality and quantity control and thus safeguards multiple biological processes including embryonic stem cell differentiation and organogenesis. UPF3A and UPF3B in vertebrate species, originated from a single UPF3 gene in yeast, are key factors in the NMD machinery. Although UPF3B is a well-recognized weak NMD-promoting factor, whether UPF3A functions in promoting or suppressing NMD is under debate. In this study, we generated a Upf3a conditional knockout mouse strain and established multiple lines of embryonic stem cells and somatic cells without UPF3A. Through extensive analysis on the expressions of 33 NMD targets, we found UPF3A neither represses NMD in mouse embryonic stem cells, somatic cells, nor in major organs including the liver, spleen, and thymus. Our study reinforces that UPF3A is dispensable for NMD when UPF3B is present. Furthermore, UPF3A may weakly and selectively promote NMD in certain murine organs.

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.

Lessons from the diet: Captivity and sex shape the gut microbiota in an oviparous lizard (Calotes versicolor).

Studies have indicated that the abundance and community structure of gut microbiota are altered by diet. In this study, next-generation sequencing of the 16S rRNA gene amplicon was performed to evaluate variations in the gut microbiota of wild and captive individuals of both sexes of Calotes versicolor. The results showed that there was a significant sex difference in microbial community structure for wild C. versicolor, Bacteroide was the dominant genus in wild females (WF), whereas Ochrobactrum was the dominant genus in wild males (WM). Acinetobacter and Hymenobacter were the dominant genera in WF, while Clostridium was the dominant genus in captive females (CF). The results indicated that differences in diet between wild and captive C. versicolor also resulted in variations in gut microbiota. Thus, it was not surprising that captivity and sex shape the gut microbiota in C. versicolor. In summary, the fundamental information presented about the gut microbiota of both sexes of wild (and captive females) C. versicolor, indicates that the artificial environments are not suitable for the wild C. versicolor.

The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice.

MCPH1 is the first gene identified to be responsible for the human autosomal recessive disorder primary microcephaly (MCPH). Mutations in the N-terminal and central domains of MCPH1 are strongly associated with microcephaly in human patients. A recent study showed that the central domain of MCPH1, which is mainly encoded by exon 8, interacts with E3 ligase ßTrCP2 and regulates the G2/M transition of the cell cycle. In order to investigate the biological functions of MCPH1's central domain, we constructed a mouse model that lacked the central domain of MCPH1 by deleting its exon 8 (designated as Mcph1-Δe8). Mcph1-Δe8 mice exhibited a reduced brain size and thinner cortex, likely caused by a compromised self-renewal capacity and premature differentiation of Mcph1-Δe8 neuroprogenitors during corticogenesis. Furthermore, Mcph1-Δe8 mice were sterile because of a loss of germ cells in the testis and ovary. The embryonic fibroblasts of Mcph1-Δe8 mice exhibited premature chromosome condensation (PCC). All of these findings indicate that Mcph1-Δe8 mice are reminiscent of MCPH1 complete knockout mice and Mcph1-ΔBR1 mice. Our study demonstrates that the central domain of MCPH1 represses microcephaly, and is essential for gonad development in mammals.

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.

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.

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.