Bmi1 suppresses protein synthesis and promotes proteostasis in hematopoietic stem cells.

The polycomb complex component Bmi1 promotes the maintenance of stem cells in multiple postnatal tissues, partly by negatively regulating the expression of p16Ink4a and p19Arf, tumor suppressors associated with cellular senescence. However, deficiency for p16Ink4a and p19Arf only partially rescues the function of Bmi1-deficient stem cells. We conditionally deleted Bmi1 from adult hematopoietic cells and found that this slowly depleted hematopoietic stem cells (HSCs). Rather than inducing senescence, Bmi1 deficiency increased HSC division. The increased cell division was caused partly by increased Aristaless-related homeobox (ARX) transcription factor expression, which also increased ribosomal RNA expression. However, ARX deficiency did not rescue HSC depletion. Bmi1 deficiency also increased protein synthesis, protein aggregation, and protein ubiquitylation independent of its effects on cell division and p16Ink4a, p19Arf, and ARX expression. Bmi1 thus promotes HSC quiescence by negatively regulating ARX expression and promotes proteostasis by suppressing protein synthesis. This highlights a new connection between the regulation of stem cell maintenance and proteostasis.

Prdm16 is required for the maintenance of neural stem cells in the postnatal forebrain and their differentiation into ependymal cells.

We and others showed previously that PR domain-containing 16 (Prdm16) is a transcriptional regulator required for stem cell function in multiple fetal and neonatal tissues, including the nervous system. However, Prdm16 germline knockout mice died neonatally, preventing us from testing whether Prdm16 is also required for adult stem cell function. Here we demonstrate that Prdm16 is required for neural stem cell maintenance and neurogenesis in the adult lateral ventricle subventricular zone and dentate gyrus. We also discovered that Prdm16 is required for the formation of ciliated ependymal cells in the lateral ventricle. Conditional Prdm16 deletion during fetal development using Nestin-Cre prevented the formation of ependymal cells, disrupting cerebrospinal fluid flow and causing hydrocephalus. Postnatal Prdm16 deletion using Nestin-CreERT2 did not cause hydrocephalus or prevent the formation of ciliated ependymal cells but caused defects in their differentiation. Prdm16 was required in neural stem/progenitor cells for the expression of Foxj1, a transcription factor that promotes ependymal cell differentiation. These studies show that Prdm16 is required for adult neural stem cell maintenance and neurogenesis as well as the formation of ependymal cells.

Ascorbate regulates haematopoietic stem cell function and leukaemogenesis.

Stem-cell fate can be influenced by metabolite levels in culture, but it is not known whether physiological variations in metabolite levels in normal tissues regulate stem-cell function in vivo. Here we describe a metabolomics method for the analysis of rare cell populations isolated directly from tissues and use it to compare mouse haematopoietic stem cells (HSCs) to restricted haematopoietic progenitors. Each haematopoietic cell type had a distinct metabolic signature. Human and mouse HSCs had unusually high levels of ascorbate, which decreased with differentiation. Systemic ascorbate depletion in mice increased HSC frequency and function, in part by reducing the function of Tet2, a dioxygenase tumour suppressor. Ascorbate depletion cooperated with Flt3 internal tandem duplication (Flt3ITD) leukaemic mutations to accelerate leukaemogenesis, through cell-autonomous and possibly non-cell-autonomous mechanisms, in a manner that was reversed by dietary ascorbate. Ascorbate acted cell-autonomously to negatively regulate HSC function and myelopoiesis through Tet2-dependent and Tet2-independent mechanisms. Ascorbate therefore accumulates within HSCs to promote Tet activity in vivo, limiting HSC frequency and suppressing leukaemogenesis.

Pak2 kinase promotes cellular senescence and organismal aging.

Cellular senescence defines an irreversible cell growth arrest state linked to loss of tissue function and aging in mammals. This transition from proliferation to senescence is typically characterized by increased expression of the cell-cycle inhibitor p16INK4a and formation of senescence-associated heterochromatin foci (SAHF). SAHF formation depends on HIRA-mediated nucleosome assembly of histone H3.3, which is regulated by the serine/threonine protein kinase Pak2. However, it is unknown if Pak2 contributes to cellular senescence. Here, we show that depletion of Pak2 delayed oncogene-induced senescence in IMR90 human fibroblasts and oxidative stress-induced senescence of mouse embryonic fibroblasts (MEFs), whereas overexpression of Pak2 accelerated senescence of IMR90 cells. Importantly, depletion of Pak2 in BubR1 progeroid mice attenuated the onset of aging-associated phenotypes and extended life span. Pak2 is required for expression of genes involved in cellular senescence and regulated the deposition of newly synthesized H3.3 onto chromatin in senescent cells. Together, our results demonstrate that Pak2 is an important regulator of cellular senescence and organismal aging, in part through the regulation of gene expression and H3.3 nucleosome assembly.

A role for Gcn5 in replication-coupled nucleosome assembly.

Acetylation of lysine residues at the H3 N terminus is proposed to function in replication-coupled (RC) nucleosome assembly, a process critical for the inheritance of epigenetic information and maintenance of genome stability. However, the role of H3 N-terminal lysine acetylation and the corresponding lysine acetyltransferase (KAT) in RC nucleosome assembly are not known. Here we show that Gcn5, a KAT that functions in transcription, works in parallel with Rtt109, the H3 lysine 56 KAT, to promote RC nucleosome assembly. Cells lacking both Gcn5 and Rtt109 are highly sensitive to DNA damaging agents. Moreover, cells lacking GCN5 or those that express N-terminal H3 mutants are compromised for deposition of new H3 onto replicating DNA and also show reduced binding of H3 to CAF-1, a histone chaperone involved in RC nucleosome assembly. These results demonstrate that Gcn5 regulates RC nucleosome assembly, in part, by promoting H3 association with CAF-1 via H3 acetylation.

The Ddc1-Mec3-Rad17 sliding clamp regulates histone-histone chaperone interactions and DNA replication-coupled nucleosome assembly in budding yeast.

The maintenance of genome integrity is regulated in part by chromatin structure and factors involved in the DNA damage response pathway. Nucleosome assembly is a highly regulated process that restores chromatin structure after DNA replication, DNA repair, and gene transcription. During S phase the histone chaperones Asf1, CAF-1, and Rtt106 coordinate to deposit newly synthesized histones H3-H4 onto replicated DNA in budding yeast. Here we describe synthetic genetic interactions between RTT106 and the DDC1-MEC3-RAD17 (9-1-1) complex, a sliding clamp functioning in the S phase DNA damage and replication checkpoint response, upon treatment with DNA damaging agents. The DNA damage sensitivity of rad17Δ rtt106Δ cells depends on the function of Rtt106 in nucleosome assembly. Epistasis analysis reveals that 9-1-1 complex components interact with multiple DNA replication-coupled nucleosome assembly factors, including Rtt106, CAF-1, and lysine residues of H3-H4. Furthermore, rad17Δ cells exhibit defects in the deposition of newly synthesized H3-H4 onto replicated DNA. Finally, deletion of RAD17 results in increased association of Asf1 with checkpoint kinase Rad53, which may lead to the observed reduction in Asf1-H3 interaction in rad17Δ mutant cells. In addition, we observed that the interaction between histone H3-H4 with histone chaperone CAF-1 or Rtt106 increases in cells lacking Rad17. These results support the idea that the 9-1-1 checkpoint protein regulates DNA replication-coupled nucleosome assembly in part through regulating histone-histone chaperone interactions.

The SCFDia2 ubiquitin E3 ligase ubiquitylates Sir4 and functions in transcriptional silencing.

In budding yeast, transcriptional silencing, which is important to regulate gene expression and maintain genome integrity, requires silent information regulator (Sir) proteins. In addition, Rtt106, a histone chaperone involved in nucleosome assembly, functions in transcriptional silencing. However, how transcriptional silencing is regulated during mitotic cell division is not well understood. We show that cells lacking Dia2, a component of the SCF(Dia2) E3 ubiquitin ligase involved in DNA replication, display defects in silencing at the telomere and HMR locus and that the F-box and C-terminal regions of Dia2, two regions important for Dia2's ubiquitylation activity, are required for proper transcriptional silencing at these loci. In addition, we show that Sir proteins are mislocalized in dia2Δ mutant cells. Mutations in Dia2 and Rtt106 result in a synergistic loss of silencing at the HMR locus and significant elevation of Sir4 proteins at the HMR locus, suggesting that silencing defects in dia2Δ mutant cells are due, at least in part, to the altered levels of Sir4 at silent chromatin. Supporting this idea, we show that SCF(Dia2) ubiquitylates Sir4 in vitro and in vivo. Furthermore, Sir4 binding to silent chromatin is dynamically regulated during the cell cycle, and this regulation is lost in dia2Δ mutant cells. These results demonstrate that the SCF(Dia2) complex is involved in transcriptional silencing, ubiquitylates Sir4, and regulates transcriptional silencing during the cell cycle.

Adiponectin receptors sustain haematopoietic stem cells throughout adulthood by protecting them from inflammation.

How are haematopoietic stem cells (HSCs) protected from inflammation, which increases with age and can deplete HSCs? Adiponectin, an anti-inflammatory factor that is not required for HSC function or haematopoiesis, promotes stem/progenitor cell proliferation after bacterial infection and myeloablation. Adiponectin binds two receptors, AdipoR1 and AdipoR2, which have ceramidase activity that increases upon adiponectin binding. Here we found that adiponectin receptors are non-cell-autonomously required in haematopoietic cells to promote HSC quiescence and self-renewal. Adiponectin receptor signalling suppresses inflammatory cytokine expression by myeloid cells and T cells, including interferon-γ and tumour necrosis factor. Without adiponectin receptors, the levels of these factors increase, chronically activating HSCs, reducing their self-renewal potential and depleting them during ageing. Pathogen infection accelerates this loss of HSC self-renewal potential. Blocking interferon-γ or tumour necrosis factor signalling partially rescues these effects. Adiponectin receptors are thus required in immune cells to sustain HSC quiescence and to prevent premature HSC depletion by reducing inflammation.

Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain.

Neurosphere formation is commonly used as a surrogate for neural stem cell (NSC) function but the relationship between neurosphere-initiating cells (NICs) and NSCs remains unclear. We prospectively identified, and isolated by flow cytometry, adult mouse lateral ventricle subventricular zone (SVZ) NICs as Glast(mid)EGFR(high)PlexinB2(high)CD24(-/low)O4/PSA-NCAM(-/low)Ter119/CD45(-) (GEPCOT) cells. They were highly mitotic and short-lived in vivo based on fate-mapping with Ascl1(CreERT2) and Dlx1(CreERT2). In contrast, pre-GEPCOT cells were quiescent, expressed higher Glast, and lower EGFR and PlexinB2. Pre-GEPCOT cells could not form neurospheres but expressed the stem cell markers Slc1a3-CreER(T), GFAP-CreER(T2), Sox2(CreERT2), and Gli1(CreERT2) and were long-lived in vivo. While GEPCOT NICs were ablated by temozolomide, pre-GEPCOT cells survived and repopulated the SVZ. Conditional deletion of the Bmi-1 polycomb protein depleted pre-GEPCOT and GEPCOT cells, though pre-GEPCOT cells were more dependent upon Bmi-1 for Cdkn2a (p16(Ink4a)) repression. Our data distinguish quiescent NSCs from NICs and make it possible to study their properties in vivo.DOI: http://dx.doi.org/10.7554/eLife.02669.001.

Histone chaperones in nucleosome assembly and human disease.

Nucleosome assembly following DNA replication, DNA repair and gene transcription is critical for the maintenance of genome stability and epigenetic information. Nucleosomes are assembled by replication-coupled or replication-independent pathways with the aid of histone chaperone proteins. How these different nucleosome assembly pathways are regulated remains relatively unclear. Recent studies have provided insight into the mechanisms and the roles of histone chaperones in regulating nucleosome assembly. Alterations or mutations in factors involved in nucleosome assembly have also been implicated in cancer and other human diseases. This review highlights the recent progress and outlines future challenges in the field.

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity.

The process of coordinated DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3-H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three histone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3-H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3-H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.

Histones, histone chaperones and nucleosome assembly.

Chromatin structure governs a number of cellular processes including DNA replication, transcription, and DNA repair. During DNA replication, chromatin structure including the basic repeating unit of chromatin, the nucleosome, is temporarily disrupted, and then reformed immediately after the passage of the replication fork. This coordinated process of nucleosome assembly during DNA replication is termed replication-coupled nucleosome assembly. Disruption of this process can lead to genome instability, a hallmark of cancer cells. Therefore, addressing how replication-coupled nucleosome assembly is regulated has been of great interest. Here, we review the current status of this growing field of interest, highlighting recent advances in understanding the regulation of this important process by the dynamic interplay of histone chaperones and histone modifications.

Thiopurine S-methyltransferase pharmacogenetics: autophagy as a mechanism for variant allozyme degradation.

OBJECTIVE: Thiopurine S-methyltransferase (TPMT)*3A is degraded much more rapidly than is the 'wild-type' enzyme through a ubiquitin-proteasome-dependent process. It also forms aggresomes, suggesting a possible dynamic balance between degradation and aggregation. We set out to identify genes encoding proteins participating in these processes. METHODS: Green fluorescent protein tagged TPMT*3A was expressed in a Saccharomyces cerevisiae gene deletion library, and flow cytometry was used to screen for cells with high fluorescence intensity, indicating the loss of a gene essential for TPMT*3A degradation. RESULTS: Twenty-four yeast genes were identified in functional categories that included ubiquitin-dependent protein degradation, vesicle trafficking, and vacuolar degradation. The presence of genes encoding proteins involved in vesicular transport and vacuolar degradation suggested a possible role in TPMT*3A degradation for autophagy--a process not previously identified as a pharmacogenomic mechanism. In support of that hypothesis, TPMT*3A aggregates increased dramatically in mutants for vacuolar protease and autophagy-related genes. Furthermore, TPMT*3A expression in human cells induced autophagy, and small interfering RNA-mediated knockdown of ATG7, an autophagy-related human protein, enhanced TPMT*3A aggregation but not that of TPMT*3C or wild-type TPMT, indicating that autophagy contributes to TPMT*3A degradation in mammalian cells. We also demonstrated that UBE2G2, the human homologue of the E2 ubiquitin-conjugating enzyme identified during the yeast genetic screen, was involved in TPMT*3A degradation in human cells. CONCLUSION: These results indicate that autophagy should be considered among mechanisms responsible for the effects of pharmacogenetically significant polymorphisms that alter encoded amino acids.

Effects of cholecystokinin-58 on type 1 cholecystokinin receptor function and regulation.

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1 receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.