Showing papers in "Genes & Development in 2005"

Shelterin: the protein complex that shapes and safeguards human telomeres

Abstract: Added by telomerase, arrays of TTAGGG repeats specify the ends of human chromosomes. A complex formed by six telomere-specific proteins associates with this sequence and protects chromosome ends. By analogy to other chromosomal protein complexes such as condensin and cohesin, I will refer to this complex as shelterin. Three shelterin subunits, TRF1, TRF2, and POT1 directly recognize TTAGGG repeats. They are interconnected by three additional shelterin proteins, TIN2, TPP1, and Rap1, forming a complex that allows cells to distinguish telomeres from sites of DNA damage. Without the protective activity of shelterin, telomeres are no longer hidden from the DNA damage surveillance and chromosome ends are inappropriately processed by DNA repair pathways. How does shelterin avert these events? The current data argue that shelterin is not a static structural component of the telomere. Instead, shelterin is emerging as a protein complex with DNA remodeling activity that acts together with several associated DNA repair factors to change the structure of the telomeric DNA, thereby protecting chromosome ends. Six shelterin subunits: TRF1, TRF2, TIN2, Rap1, TPP1, and POT1.

Smad transcription factors

Abstract: Smad transcription factors lie at the core of one of the most versatile cytokine signaling pathways in metazoan biology-the transforming growth factor-beta (TGFbeta) pathway. Recent progress has shed light into the processes of Smad activation and deactivation, nucleocytoplasmic dynamics, and assembly of transcriptional complexes. A rich repertoire of regulatory devices exerts control over each step of the Smad pathway. This knowledge is enabling work on more complex questions about the organization, integration, and modulation of Smad-dependent transcriptional programs. We are beginning to uncover self-enabled gene response cascades, graded Smad response mechanisms, and Smad-dependent synexpression groups. Our growing understanding of TGFbeta signaling through the Smad pathway provides general principles for how animal cells translate complex inputs into concrete behavior.

Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing

Abstract: Dicer is the enzyme that cleaves double-stranded RNA (dsRNA) into 21–25-nt-long species responsible for sequence-specific RNA-induced gene silencing at the transcriptional, post-transcriptional, or translational level. We disrupted the dicer-1 (dcr-1) gene in mouse embryonic stem (ES) cells by conditional gene targeting and generated Dicer-null ES cells. These cells were viable, despite being completely defective in RNA interference (RNAi) and the generation of microRNAs (miRNAs). However, the mutant ES cells displayed severe defects in differentiation both in vitro and in vivo. Epigenetic silencing of centromeric repeat sequences and the expression of homologous small dsRNAs were markedly reduced. Re-expression of Dicer in the knockout cells rescued these phenotypes. Our data suggest that Dicer participates in multiple, fundamental biological processes in a mammalian organism, ranging from stem cell differentiation to the maintenance of centromeric heterochromatin structure and centromeric silencing.

Proapoptotic Bak Is Sequestered by Mcl-1 and Bcl-xL, but Not Bcl-2, Until Displaced by BH3-only Proteins

Abstract: How the Bcl-2 family of proteins regulate programmed cell death triggered by developmental cues and in response to multiple stress signals is of intense interest (Adams 2003; Danial and Korsmeyer 2004). Whereas cell survival is promoted by Bcl-2 itself and several close relatives (Bcl-xL, Bcl-w, Mcl-1, and A1), which bear three or four conserved Bcl-2 homology (BH) regions, apoptosis is driven by two other subfamilies. The initial signal for cell death is conveyed by the diverse group of BH3-only proteins, including Bad, Bid, Bim, Puma, and Noxa, which have in common only the small BH3 interaction domain (Huang and Strasser 2000). However, Bax or Bak (multidomain proteins containing BH1-BH3) are required for commitment to cell death (Lindsten et al. 2000; Cheng et al. 2001; Wei et al. 2001; Zong et al. 2001). When activated, they can permeabilize the outer membrane of mitochondria and release proapoptogenic factors (e.g., cytochrome c) needed to activate the caspases that dismantle the cell (Adams 2003; Danial and Korsmeyer 2004; Green and Kroemer 2004). Interactions between members of these three factions of the Bcl-2 family dictate whether a cell lives or dies. When BH3-only proteins have been activated, for example, in response to DNA damage, they can bind via their BH3 domain to a groove on their prosurvival relatives (Sattler et al. 1997). How the BH3-only and Bcl-2-like proteins control the activation of Bax and Bak, however, remains poorly understood (Adams 2003; Danial and Korsmeyer 2004). Most attention has focused on Bax. This soluble monomeric protein (Hsu et al. 1997; Wolter et al. 1997) normally has its membrane-targeting domain inserted into its groove, probably accounting for its cytosolic localization (Suzuki et al. 2000; Schinzel et al. 2004). Several unrelated peptides/proteins have been proposed to modulate Bax activity (for review, see Lucken-Ardjomande and Martinou 2005), but their physiological relevance remains to be established. Alternatively, Bax may be activated via direct engagement by certain BH3-only proteins, the best documented being the active truncated form of Bid, tBid (Wei et al. 2000; Kuwana et al. 2002; Roucou et al. 2002). As discussed elsewhere (Adams 2003), the oldest model, in which Bcl-2 directly engages Bax (Oltvai et al. 1993), has become problematic because Bcl-2 is membrane bound while Bax is cytosolic, and their interaction seems highly dependent on certain detergents used for cell lysis (Hsu and Youle 1997). Nevertheless, it is well established that the BH3 region of Bax can mediate association with Bcl-2 (Zha and Reed 1997; Wang et al. 1998) and that Bcl-2 prevents the oligomerization of Bax, even though no heterodimers can be detected (Mikhailov et al. 2001). Thus, whether the prosurvival proteins restrain Bax activation directly or indirectly remains uncertain (see Discussion). Although Bax and Bak seem in most circumstances to be functionally equivalent (Lindsten et al. 2000; Wei et al. 2001), substantial differences in their regulation would be expected from their distinct localization in healthy cells. Unlike Bax, which is largely cytosolic, Bak resides in complexes on the outer membrane of mitochondria and on the endoplasmic reticulum of healthy cells (Wei et al. 2000; Zong et al. 2003). Nevertheless, on receipt of cytotoxic signals, both Bax and Bak change conformation, and Bax translocates to the organellar membranes, where both Bax and Bak then form homo-oligomers that can associate, leading to membrane permeabilization (Hsu et al. 1997; Wolter et al. 1997; Antonsson et al. 2001; Wei et al. 2001; Mikhailov et al. 2003). Since Bak, unlike Bax, is normally located at its site of action, how is it kept in check to prevent inappropriate cell death? We were prompted to investigate Bak regulation by recent evidence that it can form complexes with Mcl-1 (Cuconati et al. 2003) and that Mcl-1 is degraded at an early stage of apoptosis (Cuconati et al. 2003; Nijhawan et al. 2003). Here we report evidence from binding and functional studies that Bak is subject to negative regulation specifically by Mcl-1 and Bcl-xL but not other prosurvival family members. Thus, contrary to expectation, the prototypic guardian Bcl-2 is unable to prevent Bak activation. We show that stimuli from DNA damage drive BH3-only proteins to displace Bak from Mcl-1 and Bcl-xL, allowing Bak to self-associate and trigger apoptosis. We also report that the association of Noxa with Mcl-1 can trigger Mcl-1 degradation. Our demonstration that a subset of prosurvival family members controls Bak may explain the varied phenotypes observed on disruption of the prosurvival genes (Ranger et al. 2001) and has important implications for current efforts to develop drugs that regulate apoptosis by targeting the Bcl-2 family (Cory et al. 2003).

Embryonic stem cell differentiation: emergence of a new era in biology and medicine

Abstract: The discovery of mouse embryonic stem (ES) cells >20 years ago represented a major advance in biology and experimental medicine, as it enabled the routine manipulation of the mouse genome. Along with the capacity to induce genetic modifications, ES cells provided the basis for establishing an in vitro model of early mammalian development and represented a putative new source of differentiated cell types for cell replacement therapy. While ES cells have been used extensively for creating mouse mutants for more than a decade, their application as a model for developmental biology has been limited and their use in cell replacement therapy remains a goal for many in the field. Recent advances in our understanding of ES cell differentiation, detailed in this review, have provided new insights essential for establishing ES cell-based developmental models and for the generation of clinically relevant populations for cell therapy.

Transcription switches for protoxylem and metaxylem vessel formation

Abstract: Land plants evolved xylem vessels to conduct water and nutrients, and to support the plant. Microarray analysis with a newly established Arabidopsis in vitro xylem vessel element formation system and promoter analysis revealed the possible involvement of some plant-specific NAC-domain transcription factors in xylem formation. VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 can induce transdifferentiation of various cells into metaxylem- and protoxylem-like vessel elements, respectively, in Arabidopsis and poplar. A dominant repression of VND6 and VND7 specifically inhibits metaxylem and protoxylem vessel formation in roots, respectively. These findings suggest that these genes are transcription switches for plant metaxylem and protoxylem vessel formation.

Perspective: machines for RNAi

Abstract: RNA silencing pathways convert the sequence information in long RNA, typically double-stranded RNA, into ∼21-nt RNA signaling molecules such as small interfering RNAs (siRNAs) and microRNAs (miRNAs). siRNAs and miRNAs provide specificity to protein effector complexes that repress mRNA transcription or translation, or catalyze mRNA destruction. Here, we review our current understanding of how small RNAs are produced, how they are loaded into protein complexes, and how they repress gene expression.

The role of the Rho GTPases in neuronal development

Abstract: Our brain serves as a center for cognitive function and neurons within the brain relay and store information about our surroundings and experiences. Modulation of this complex neuronal circuitry allows us to process that information and respond appropriately. Proper development of neurons is therefore vital to the mental health of an individual, and perturbations in their signaling or morphology are likely to result in cognitive impairment. The development of a neuron requires a series of steps that begins with migration from its birth place and initiation of process outgrowth, and ultimately leads to differentiation and the formation of connections that allow it to communicate with appropriate targets. Over the past several years, it has become clear that the Rho family of GTPases and related molecules play an important role in various aspects of neuronal development, including neurite outgrowth and differentiation, axon pathfinding, and dendritic spine formation and maintenance. Given the importance of these molecules in these processes, it is therefore not surprising that mutations in genes encoding a number of regulators and effectors of the Rho GTPases have been associated with human neurological diseases. This review will focus on the role of the Rho GTPases and their associated signaling molecules throughout neuronal development and discuss how perturbations in Rho GTPase signaling may lead to cognitive disorders.

Bacterial outer membrane vesicles and the host–pathogen interaction

Abstract: Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with and intoxicate host cells. Vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria. Vesicle production occurs in infected tissues and is influenced by environmental factors. Vesicles play roles in establishing a colonization niche, carrying and transmitting virulence factors into host cells, and modulating host defense and response. Vesicle-mediated toxin delivery is a potent virulence mechanism exhibited by diverse Gram-negative pathogens. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease.

Poly(ADP-ribosyl)ation by PARP-1: `PAR-laying' NAD+ into a nuclear signal

Abstract: Poly(ADP-ribose) (PAR) and the PAR polymerases (PARPs) that catalyze its synthesis from donor nicotinamide adenine dinucleotide (NAD+) molecules have received considerable attention in the recent literature. Poly(ADP-ribosyl)ation (PARylation) plays diverse roles in many molecular and cellular processes, including DNA damage detection and repair, chromatin modification, transcription, cell death pathways, insulator function, and mitotic apparatus function. These processes are critical for many physiological and pathophysiological outcomes, including genome maintenance, carcinogenesis, aging, inflammation, and neuronal function. This review highlights recent work on the biochemistry, molecular biology, physiology, and pathophysiology of PARylation, focusing on the activity of PARP-1, the most abundantly expressed member of a family of PARP proteins. In addition, connections between nuclear NAD+ metabolism and nuclear signaling through PARP-1 are discussed.

p53 isoforms can regulate p53 transcriptional activity

Abstract: The recently discovered p53-related genes, p73 and p63, express multiple splice variants and N-terminally truncated forms initiated from an alternative promoter in intron 3. To date, no alternative promoter and multiple splice variants have been described for the p53 gene. In this study, we show that p53 has a gene structure similar to the p73 and p63 genes. The human p53 gene contains an alternative promoter and transcribes multiple splice variants. We show that p53 variants are expressed in normal human tissue in a tissue-dependent manner. We determine that the alternative promoter is conserved through evolution from Drosophila to man, suggesting that the p53 family gene structure plays an essential role in the multiple activities of the p53 family members. Consistent with this hypothesis, p53 variants are differentially expressed in human breast tumors compared with normal breast tissue. We establish that p53 can bind differentially to promoters and can enhance p53 target gene expression in a promoter-dependent manner, while 133p53 is dominant-negative toward full-length p53, inhibiting p53-mediated apoptosis. The differential expression of the p53 isoforms in human tumors may explain the difficulties in linking p53 status to the biological properties and drug sensitivity of human cancer.

Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9.

Abstract: Histone H3 Lys 9 (H3-K9) methylation is a crucial epigenetic mark for transcriptional silencing. G9a is the major mammalian H3-K9 methyltransferase that targets euchromatic regions and is essential for murine embryogenesis. There is a single G9a-related methyltransferase in mammals, called GLP/Eu-HMTase1. Here we show that GLP is also important for H3-K9 methylation of mouse euchromatin. GLP-deficiency led to embryonic lethality, a severe reduction of H3-K9 mono- and dimethylation, the induction of Mage-a gene expression, and HP1 relocalization in embryonic stem cells, all of which were phenotypes of G9a-deficiency. Furthermore, we show that G9a and GLP formed a stoichiometric heteromeric complex in a wide variety of cell types. Biochemical analyses revealed that formation of the G9a/GLP complex was dependent on their enzymatic SET domains. Taken together, our new findings revealed that G9a and GLP cooperatively exert H3-K9 methyltransferase function in vivo, likely through the formation of higher-order heteromeric complexes.

Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint

Abstract: The DNA damage response pathway is a cellular surveillance system that senses the presence of damaged DNA and elicits an appropriate and effective response to that damage. First identified as a regulator of cell cycle transitions, the DNA damage response pathway has since been shown to regulate a number of other cellular processes, which include DNA repair, apoptosis, and replication fork stabilization (Zhou and Elledge 2000; Cimprich 2003). The importance of this pathway is demonstrated by the conservation of this response from yeast to humans (Zhou and Elledge 2000; Melo and Toczyski 2002) and by several studies that have shown that loss of checkpoint proteins predisposes affected individuals to cancer (Sherr 2004). One critical component of the DNA damage response pathway is the ATR–ATRIP complex. ATR is a phosphatidylinositol kinase-related protein kinase that is thought to function as both a sensor and transducer in the DNA damage response. ATR, and its associated protein ATRIP, respond to a broad spectrum of genotoxic agents that includes ultraviolet light (UV), topoisomerase inhibitors, alkylating agents, and cis-platinum, as well as chemicals that disrupt replication, such as aphidicolin and hydroxyurea (HU) (Zhou and Elledge 2000; Cortez et al. 2001; Melo and Toczyski 2002). Following DNA damage, ATR phosphorylates and activates the checkpoint kinase Chk1 (Melo and Toczyski 2002). In higher eukaryotes, the phosphorylation of Chk1 also requires the activities of the Rad9–Rad1–Hus1 (RHR, aka 9–1–1) complex and Claspin (Melo and Toczyski 2002). The RHR complex is a PCNA-related complex that is loaded on to primed DNA in vitro (Ellison and Stillman 2003; Zou et al. 2003) and is recruited to sites of DNA damage in vivo (Kondo et al. 2001; Melo et al. 2001). Claspin was initially identified as a protein that bound the activated form of Chk1, and it has been shown to bind chromatin throughout S phase (Kumagai and Dunphy 2000; Lee et al. 2003). We and others previously showed that recruitment of ATR and Rad1 to chromatin (Lupardus et al. 2002) and activation of Chk1 (Lupardus et al. 2002; Stokes et al. 2002) requires DNA replication in Xenopus egg extracts following several types of DNA damage. Studies in mammalian cells also indicate that ATR binds UV-damaged chromatin in S phase but not G1 phase (Ward et al. 2004). In addition, other studies show that a replication fork must be established in Saccharomyces cerevisiae for checkpoint activation induced by methylmethane sulfonate (MMS) (Tercero et al. 2003). Taken together, these observations suggest that one or more replication-dependent events are needed to generate the signal that ATR recognizes for many types of DNA damage. Although the exact nature of the biochemical signal(s) responsible for activating the ATR pathway and the replication-dependent steps necessary for its formation are still unclear, evidence from a number of different systems supports a central role for replication protein A (RPA)-coated single-stranded DNA (ssDNA) in the response. In yeast, certain RPA mutants exhibit a checkpoint defect and also adapt more rapidly to DNA damage (Longhese et al. 1996; Pellicioli et al. 1999). In addition, knock-down of the ssDNA-binding protein RPA results in a significant loss of both Chk1 phosphorylation and ATR foci formation following DNA damage in mammalian cells (Zou and Elledge 2003). In Xenopus egg extracts, RPA is also required for the recruitment of ATR to chromatin following treatment with aphidicolin (You et al. 2002) or etoposide (Costanzo et al. 2003) and for the recruitment of ATR to poly(dA)70 ssDNA (Lee et al. 2003). Importantly, in vitro experiments have shown that RPA is sufficient for the binding of ATRIP to ssDNA (Zou and Elledge 2003) and that RPA also facilitates the association of the RHR complex with DNA (Ellison and Stillman 2003; Zou et al. 2003). Interestingly, the amount of ssDNA appears to increase following genotoxic stress, as RPA accumulates on chromatin in Xenopus extracts and mammalian cells treated with UV, MMS, HU, or aphidicolin (Michael et al. 2000; Mimura et al. 2000; Walter 2000; Lupardus et al. 2002; Zou and Elledge 2003). Moreover, in budding yeast, increased amounts of ssDNA have been observed by electron microscopy following HU treatment (Sogo et al. 2002). In the case of DNA damage, the mechanism by which this ssDNA accumulates is not known, nor is it clear if ssDNA accumulation is required for checkpoint activation. In principle, a number of DNA repair (e.g., nucleotide excision repair, base excision repair) and recombination processes could lead to the generation of ssDNA following DNA damage at several points in the cell cycle. Alternatively, during DNA replication, ssDNA could be formed if DNA polymerases are slowed by lesions and the replicative helicase continues to unwind DNA. Indeed, uncoupling of helicase and polymerase activities has been previously observed in the presence of aphidicolin (Walter and Newport 2000), and recent studies have shown that this aphidicolin-induced uncoupling is dependent on the MCM helicase (Pacek and Walter 2004). In this study, we used a cell-free extract system derived from Xenopus eggs (Walter et al. 1998) to examine the mechanism by which ssDNA accumulates following DNA damage. We demonstrate that the appearance and disappearance of a highly unwound form of plasmid DNA that accumulates following aphidicolin treatment (Walter and Newport 2000) correlates with the phosphorylation of Chk1 on Ser 344 (S344). Importantly, this hyperunwound form of DNA was also observed upon replication of plasmid DNA damaged with either UV or cis-platinum. This suggests that DNA damage induces uncoupling of helicase and polymerase activities and that these lesions, as well as aphidicolin, may generate a common checkpoint-activating DNA structure. Moreover, while stalling the replication fork with aphidicolin results in a robust checkpoint response, we find that aphidicolin elicits no checkpoint when the MCM DNA helicase is inactivated. Using plasmids of varying sizes, we also show that functional uncoupling of DNA unwinding and DNA synthesis during S phase may serve to amplify the level of Chk1 phosphorylation that can be achieved at each individual replication fork. Finally, we demonstrate that although DNA unwinding is necessary for checkpoint activation, it is not sufficient and that additional DNA synthesis by Polα is needed. Taken together, these results suggest that functional uncoupling of helicase and polymerase activities is necessary to convert DNA lesions and chemical inhibitors of DNA replication into the signal(s) that activate the ATR-dependent checkpoint.

TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo

Abstract: TGF-beta-activated kinase 1 (TAK1), a member of the MAPKKK family, is thought to be a key modulator of the inducible transcription factors NF-kappaB and AP-1 and, therefore, plays a crucial role in regulating the genes that mediate inflammation. Although in vitro biochemical studies have revealed the existence of a TAK1 complex, which includes TAK1 and the adapter proteins TAB1 and TAB2, it remains unclear which members of this complex are essential for signaling. To analyze the function of TAK1 in vivo, we have deleted the Tak1 gene in mice, with the resulting phenotype being early embryonic lethality. Using embryonic fibroblasts lacking TAK1, TAB1, or TAB2, we have found that TNFR1, IL-1R, TLR3, and TLR4-mediated NF-kappaB and AP-1 activation are severely impaired in Tak1(m/m) cells, but they are normal in Tab1(-/-) and Tab2(-/-) cells. In addition, Tak1(m/m) cells are highly sensitive to TNF-induced apoptosis. TAK1 mediates IKK activation in TNF-alpha and IL-1 signaling pathways, where it functions downstream of RIP1-TRAF2 and MyD88-IRAK1-TRAF6, respectively. However, TAK1 is not required for NF-kappaB activation through the alternative pathway following LT-beta signaling. In the TGF-beta signaling pathway, TAK1 deletion leads to impaired NF-kappaB and c-Jun N-terminal kinase (JNK) activation without impacting Smad2 activation or TGF-beta-induced gene expression. Therefore, our studies suggests that TAK1 acts as an upstream activating kinase for IKKbeta and JNK, but not IKKalpha, revealing an unexpectedly specific role of TAK1 in inflammatory signaling pathways.

A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis.

Abstract: The Arabidopsis genes, TAS2 and TAS1a, produce structurally similar noncoding transcripts that are transformed into short (21-nucleotide [nt]) and long (24-nt) siRNAs by RNA silencing pathways. Some of these short siRNAs direct the cleavage of protein-coding transcripts, and thus function as trans-acting siRNAs (ta-siRNAs). Using genetic analysis, we defined the pathway by which ta-siRNAs and other short siRNAs are generated from these loci. This process is initiated by the miR173-directed cleavage of a primary poly(A) transcript. The 3' fragment is then transformed into short siRNAs by the sequential activity of SGS3, RDR6, and DCL4: SGS3 stabilizes the fragment, RDR6 produces a complementary strand, and DCL4 cleaves the resulting double-stranded molecule into short siRNAs, starting at the end with the miR173 cleavage site and proceeding in 21-nt increments from this point. The 5' cleavage fragment is also processed by this pathway, but less efficiently. The DCL3-dependent pathway that generates long siRNAs does not require miRNA-directed cleavage and plays a minor role in the silencing of these loci. Our results define the core components of a post-transcriptional gene silencing pathway in Arabidopsis and reveal some of the features that direct transcripts to this pathway.

Wnt signaling in the intestinal epithelium: from endoderm to cancer

Abstract: The Wnt pathway controls cell fate during embryonic development. It also persists as a key regulator of homeostasis in adult self-renewing tissues. In these tissues, mutational deregulation of the Wnt cascade is closely associated with malignant transformation. The intestinal epithelium represents the best-understood example for the closely linked roles of Wnt signaling in homeostatic self-renewal and malignant transformation. In this review, we outline current understanding of the physiological role of Wnt signaling in intestinal biology. From this perspective, we then describe how mutational subversion of the Wnt cascade leads to colorectal cancer.

RNA meets chromatin

Abstract: In the universe of science, two worlds have recently collided-those of RNA and chromatin. The intersection of these two fields has been impending, but evidence for such a meaningful collision has only recently become apparent. In this review, we discuss the implications for noncoding RNAs and the formation of specialized chromatin domains in various epigenetic processes as diverse as dosage compensation, RNA interference-mediated heterochromatin assembly and gene silencing, and programmed DNA elimination. While mechanistic details as to how the RNA and chromatin worlds connect remain unclear, intriguing parallels exist in the overall design and machinery used in model organisms from all eukaryotic kingdoms. The role of potential RNA-binding chromatin-associated proteins will be discussed as one possible link between RNA and chromatin.

Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways

Abstract: Bmi-1 is required for the post-natal maintenance of stem cells in multiple tissues including the central nervous system (CNS) and peripheral nervous system (PNS). Deletion of Ink4a or Arf from Bmi-1-/- mice partially rescued stem cell self-renewal and stem cell frequency in the CNS and PNS, as well as forebrain proliferation and gut neurogenesis. Arf deficiency, but not Ink4a deficiency, partially rescued cerebellum development, demonstrating regional differences in the sensitivity of progenitors to p16Ink4a and p19Arf. Deletion of both Ink4a and Arf did not affect the growth or survival of Bmi-1-/- mice or completely rescue neural development. Bmi-1 thus prevents the premature senescence of neural stem cells by repressing Ink4a and Arf, but additional pathways must also function downstream of Bmi-1.

Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis

Abstract: The regulated phosphorylation of ribosomal protein (rp) S6 has attracted much attention since its discovery in 1974, yet its physiological role has remained obscure. To directly address this issue, we have established viable and fertile knock-in mice, whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues (rpS6P-/-). Here we show that contrary to the widely accepted model, this mutation does not affect the translational control of TOP mRNAs. rpS6P-/- mouse embryo fibroblasts (MEFs) display an increased rate of protein synthesis and accelerated cell division, and they are significantly smaller than rpS6P+/+ MEFs. This small size reflects a growth defect, rather than a by-product of their faster cell division. Moreover, the size of rpS6P-/- MEFs, unlike wild-type MEFs, is not further decreased upon rapamycin treatment, implying that the rpS6 is a critical downstream effector of mTOR in regulation of cell size. The small cell phenotype is not confined to embryonal cells, as it also selectively characterizes pancreatic β-cells in adult rpS6P-/- mice. These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose tolerance.

p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes

Abstract: Adult mammalian cardiomyocytes are considered terminally differentiated and incapable of proliferation. Consequently, acutely injured mammalian hearts do not regenerate, they scar. Here, we show that adult mammalian cardiomyocytes can divide. One important mechanism used by mammalian cardiomyocytes to control cell cycle is p38 MAP kinase activity. p38 regulates expression of genes required for mitosis in cardiomyocytes, including cyclin A and cyclin B. p38 activity is inversely correlated with cardiac growth during development, and its overexpression blocks fetal cardiomyocyte proliferation. Activation of p38 in vivo by MKK3bE reduces BrdU incorporation in fetal cardiomyocytes by 17.6%. In contrast, cardiac-specific p38α knockout mice show a 92.3% increase in neonatal cardiomyocyte mitoses. Furthermore, inhibition of p38 in adult cardiomyocytes promotes cytokinesis. Finally, mitosis in adult cardiomyocytes is associated with transient dedifferentiation of the contractile apparatus. Our findings establish p38 as a key negative regulator of cardiomyocyte proliferation and indicate that adult cardiomyocytes can divide.

Inactivation of S6 ribosomal protein gene in T lymphocytes activates a p53-dependent checkpoint response

Abstract: Ribosome biogenesis has been associated with regulation of cell growth and cell division, but the molecular mechanisms that integrate the effect of ribosome biogenesis on these processes in mammalian cells remain unknown. To study the effect of impaired ribosome functions in vivo, we conditionally deleted one or two alleles of the 40S ribosomal protein S6 gene in T cells in the mouse. While complete deletion of S6 abrogated T-cell development, hemizygous expression did not have any effect on T-cell maturation in the thymus, but inhibited the accumulation of T cells in the spleen and lymph nodes, as a result of their decreased survival in the peripheral lymphoid organs. Additionally, TCR-mediated stimulation of S6-heterozygous T cells induced a normal increase in their size, but cell cycle progression was impaired. Genetic inactivation of p53 tumor suppressor rescued development of S6-homozygous null thymocytes and proliferative defect of S6-heterozygous T cells. These results demonstrate the existence of a p53-dependent checkpoint mechanism that senses changes in the fidelity of the translational machinery to prevent aberrant cell division or eliminate defective T cells in vivo. Failure to activate this checkpoint response could potentially lead to a development of pathological processes such as tumors and autoimmune diseases.

Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone

Abstract: Extracellular matrix (ECM) mineralization is a physiological process in bone and a pathological one in soft tissues. The mechanisms determining the spatial restriction of ECM mineralization to bone physiologically are poorly understood. Here we show that a normal extracellular phosphate concentration is required for bone mineralization, while lowering this concentration prevents mineralization of any ECM. However, simply raising extracellular phosphate concentration is not sufficient to induce pathological mineralization, this is because of the presence in all ECMs of pyrophosphate, an inhibitor of mineralization. ECM mineralization occurs only in bone because of the exclusive coexpression in osteoblasts of Type I collagen and Tnap, an enzyme that cleaves pyrophosphate. This dual requirement explains why Tnap ectopic expression in cells producing fibrillar collagen is sufficient to induce pathological mineralization. This study reveals that coexpression in osteoblasts of otherwise broadly expressed genes is necessary and sufficient to induce bone mineralization and provides evidence that pathological mineralization can be prevented by modulating extracellular phosphate concentration.

Pax3/Pax7 mark a novel population of primitive myogenic cells during development

Abstract: Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel population of resident Pax3+/Pax7+, muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies.

A systematic RNAi screen for longevity genes in C. elegans

Abstract: We report here the first genome-wide functional genomic screen for longevity genes. We systematically surveyed Caenorhabditis elegans genes using large-scale RNA interference (RNAi), and found that RNAi inactivation of 89 genes extend C. elegans lifespan. Components of the daf-2/insulin-like signaling pathway are recovered, as well as genes that regulate metabolism, signal transduction, protein turnover, and gene expression. Many of these candidate longevity genes are conserved across animal phylogeny. Genetic interaction analyses with the new longevity genes indicate that some act upstream of the daf-16/FOXO transcription factor or the sir2.1 protein deacetylase, and others function independently of daf-16/FOXO and sir2.1, and might define new pathways to regulate lifespan.

Biochemical and genetic analysis of the yeast proteome with a movable ORF collection

Abstract: Functional analysis of the proteome is an essential part of genomic research. To facilitate different proteomic approaches, a MORF (moveable ORF) library of 5854 yeast expression plasmids was constructed, each expressing a sequence-verified ORF as a C-terminal ORF fusion protein, under regulated control. Analysis of 5573 MORFs demonstrates that nearly all verified ORFs are expressed, suggests the authenticity of 48 ORFs characterized as dubious, and implicates specific processes including cytoskeletal organization and transcriptional control in growth inhibition caused by overexpression. Global analysis of glycosylated proteins identifies 109 new confirmed N-linked and 345 candidate glycoproteins, nearly doubling the known yeast glycome.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms

Abstract: Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1

Abstract: In human cells, a critical pathway in gene regulation subjects mRNAs with AU-rich elements (AREs) to rapid decay by a poorly understood process. AREs have been shown to directly activate deadenylation, decapping, or 3'-to-5' exonucleolytic decay. We demonstrate that enzymes involved in all three of these mRNA decay processes, as well as 5'-to-3' exonucleolytic decay, associate with the protein tristetraprolin (TTP) and its homolog BRF-1, which bind AREs and activate mRNA decay. TTP and BRF-1 each contain two activation domains that can activate mRNA decay after fusion to a heterologous RNA-binding protein, and inhibit ARE-mediated mRNA decay when overexpressed. Both activation domains employ trans-acting factors to trigger mRNA decay, and the N-terminal activation domain functions as a binding platform for mRNA decay enzymes. Our data suggest that the TTP protein family functions as a molecular link between ARE-containing mRNAs and the mRNA decay machinery by recruitment of mRNA decay enzymes, and help explain how deadenylation, decapping, and exonucleolytic decay can all be independently activated on ARE-containing mRNAs. This describes a potentially regulated step in activation of mRNA decay.

Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping

Abstract: The identity and developmental potential of a human cell is specified by its epigenome that is largely defined by patterns of chromatin modifications including histone acetylation. Here we report high-resolution genome-wide mapping of diacetylation of histone H3 at Lys 9 and Lys 14 in resting and activated human T cells by genome-wide mapping technique (GMAT). Our data show that high levels of the H3 acetylation are detected in gene-rich regions. The chromatin accessibility and gene expression of a genetic domain is correlated with hyperacetylation of promoters and other regulatory elements but not with generally elevated acetylation of the entire domain. Islands of acetylation are identified in the intergenic and transcribed regions. The locations of the 46,813 acetylation islands identified in this study are significantly correlated with conserved noncoding sequences (CNSs) and many of them are colocalized with known regulatory elements in T cells. TCR signaling induces 4045 new acetylation loci that may mediate the global chromatin remodeling and gene activation. We propose that the acetylation islands are epigenetic marks that allow prediction of functional regulatory elements.

RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs

Abstract: Hfq-binding antisense small RNAs of Escherichia coli, SgrS and RyhB, mediate the destabilization of target mRNAs in an RNase E-dependent manner. SgrS, whose expression is induced in response to phosphosugar stress, act on the ptsG mRNA encoding a major glucose transporter, while RyhB, whose expression is induced in response to Fe depletion, acts on several mRNAs encoding Fe-binding proteins. In this report, we addressed the question of how SgrS and RyhB RNAs cooperate with RNase E to destabilize the target mRNAs. We demonstrate that Hfq along with SgrS and RyhB copurified with RNase E but not with truncated RNase E. In addition, we show that RNase E but not other degradosome components copurified with Hfq. Taken together, we conclude that RNase E forms variable ribonucleoprotein complexes with Hfq/small RNAs through its C-terminal scaffold region. These complexes, distinct from the RNA degradosome, may act as specialized RNA decay machines that initiate the degradation of mRNAs targeted by each small RNA. The present finding has uncovered the mechanical basis of mRNA destabilization mediated by bacterial small RNAs. The formation of ribonucleoprotein complexes containing RNases could be a general way by which small RNAs destabilize target mRNAs in both prokaryotes and eukaryotes.

p21 loss compromises the relative quiescence of forebrain stem cell proliferation leading to exhaustion of their proliferation capacity

Abstract: Adult stem cells in various tissues are relatively quiescent. The cell cycle inhibitor p21cip1/waf1 (p21) has been shown to be important for maintaining hematopoietic stem cell quiescence and self-renewal. We examined the role of p21 in the regulation of adult mammalian forebrain neural stem cells (NSCs). We found that p21-/- mice between post-natal age 60-240 d have more NSCs than wild-type (+/+) controls due to higher proliferation rates of p21-/- NSCs. Thereafter, NSCs in p21-/- mice decline and are reduced in number at 16 mo relative to p21+/+ mice. Similarly, both p21-/- and p21+/+ NSCs display self-renewal in vitro; however, p21-/- NSCs display limited in vitro self-renewal (surviving a few passages, then exhausting). Thus, p21 contributes to adult NSC relative quiescence, which we propose is necessary for the life-long maintenance of NSC self-renewal because NSCs may be limited to a finite number of divisions.