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1.  Chromatin Modifications as Determinants of Muscle Stem Cell Quiescence and Chronological Aging 
Cell reports  2013;4(1):189-204.
The ability to maintain quiescence is critical for the long-term maintenance of a functional stem cell pool. To date, the epigenetic and transcriptional characteristics of quiescent stem cells and how they change with age remain largely unknown. In this study, we explore the chromatin features of adult skeletal muscle stem cells, or satellite cells (SCs), which reside predominantly in a quiescent state in fully developed limb muscles of both young and aged mice. Using a ChIP-seq approach to obtain global epigenetic profiles of quiescent SCs (QSCs), we show that QSCs possess a permissive chromatin state in which few genes are epigenetically repressed by Polycomb group (PcG)-mediated histone 3 lysine 27 trimethylation (H3K27me3), and a large number of genes encoding regulators that specify nonmyogenic lineages are demarcated by bivalent domains at their transcription start sites (TSSs). By comparing epigenetic profiles of QSCs from young and old mice, we also provide direct evidence that, with age, epigenetic changes accumulate and may lead to a functional decline in quiescent stem cells. These findings highlight the importance of chromatin mapping in understanding unique features of stem cell identity and stem cell aging.
PMCID: PMC4103025  PMID: 23810552
2.  Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns 
Cell reports  2012;2(2):419-431.
Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.
PMCID: PMC4103158  PMID: 22902405
3.  Negative Feedback in Genetic Circuits Confers Evolutionary Resilience and Capacitance 
Cell reports  2014;7(6):1789-1795.
Natural selection for specific functions places limits upon the amino acid substitutions a protein can accept. Mechanisms that expand the range of tolerable amino acid substitutions include chaperones that can rescue destabilized proteins and additional, stability enhancing substitutions. Here, we present an alternative mechanism that is simple and uses a frequently encountered network motif. Computational and experimental evidence show that the self-correcting, negative feedback gene regulation motif increases repressor expression in response to deleterious mutations and thereby precisely restores repression of a target gene. Furthermore, this ability to rescue repressor function is observable across the Eubacteria Kingdom through the greater accumulation of amino acid substitutions in negative feedback transcription factors compared to genes they control. We propose that negative feedback represents a self-contained genetic canalization mechanism that preserves phenotype while permitting access to a wider range of functional genotypes.
PMCID: PMC4103627  PMID: 24910431
4.  The Ubiquitin Receptor S5a/Rpn10 Links Centrosomal Proteasomes with Dendrite Development in the Mammalian Brain 
Cell reports  2013;4(1):19-30.
Proteasomes drive the selective degradation of protein substrates with covalently linked ubiquitin chains in eukaryotes. Although proteasomes are distributed throughout the cell, specific biological functions of the proteasome in distinct subcellular locales remain largely unknown. We report that proteasomes localized at the centrosome regulate the degradation of local ubiquitin conjugates in mammalian neurons. We find that the proteasomal subunit S5a/Rpn10, a ubiquitin receptor that selects substrates for degradation, is essential for proteasomal activity at centrosomes in neurons and thereby promotes the elaboration of dendrite arbors in the rodent brain in vivo. We also find that the helix-loop-helix protein Id1 disrupts the interaction of S5a/Rpn10 with the proteasomal lid and thereby inhibits centrosomal proteasome activity and dendrite elaboration in neurons. Together, our findings define a novel function for a specific pool of proteasomes at the neuronal centrosome and identify a biological function for S5a/Rpn10 in the mammalian brain.
PMCID: PMC4103748  PMID: 23831032
5.  Muscle Cells Provide Instructions for Planarian Regeneration 
Cell reports  2013;4(4):633-641.
Regeneration requires both potential and instructions for tissue replacement. In planarians, pluripotent stem cells have the potential to produce all new tissue. The identities of the cells that provide regeneration instructions are unknown. Here, we report that position control genes (PCGs) that control regeneration and tissue turnover are expressed in a subepidermal layer of nonneoblast cells. These subepidermal cells coexpress many PCGs. We propose that these subepidermal cells provide a system of body coordinates and positional information for regeneration, and identify them to be muscle cells of the planarian body wall. Almost all planarian muscle cells express PCGs, suggesting a dual function: contraction and control of patterning. PCG expression is dynamic in muscle cells after injury, even in the absence of neoblasts, suggesting that muscle is instructive for regeneration. We conclude that planarian regeneration involves two highly flexible systems: pluripotent neoblasts that can generate any new cell type and muscle cells that provide positional instructions for the regeneration of any body region.
PMCID: PMC4101538  PMID: 23954785
6.  Acquired Dependence Of Acute Myeloid Leukemia On The DEAD-BOX RNA Helicase DDX5 
Cell reports  2014;7(6):1887-1899.
Acute myeloid leukemia (AML) therapy involves compounds that are cytotoxic to both normal and cancer cells and relapsed AML is resistant to subsequent chemotherapy. Thus agents are needed that selectively kill AML cells with minimal toxicity. Here we report that AML is dependent on DDX5 and that inhibiting DDX5 expression slows AML cell proliferation in vitro and AML progression in vivo, but is not toxic to cells from normal bone marrow. Inhibition of DDX5 expression in AML cells induces apoptosis via induction of reactive oxygen species (ROS). This apoptotic response can be blocked either by BCL2 overexpression or treatment with the ROS scavenger N-Acetyl-L-cysteine (NAC). Combining DDX5 knockdown with a BCL2 family inhibitor cooperate to induce cell death in AML cells. By inhibiting DDX5 expression in vivo we show that DDX5 is dispensable for normal hematopoiesis and tissue homeostasis. These results validate DDX5 as a potential target for blocking AML.
PMCID: PMC4100070  PMID: 24910429
Acute Myeloid Leukemia; DEAD-Box helicase; BCL2; shRNA; cancer therapy validation
7.  The Hippo transducer TAZ interacts with the SWI/SNF complex to regulate breast epithelial lineage commitment 
Cell reports  2014;6(6):1059-1072.
Lineage-committed cells of many tissues exhibit substantial plasticity in contexts such as wound healing and tumorigenesis, but the regulation of this process is not well understood. Here, we identified the Hippo transducer WWTR1/TAZ in a screen of transcription factors able to prompt lineage switching of mammary epithelial cells. Forced expression of TAZ in luminal cells induces them to adopt basal characteristics, and depletion of TAZ in basal/myoepithelial cells leads to luminal differentiation. In human and mouse tissues, TAZ is active only in basal cells and is critical for basal cell maintenance during homeostasis. Accordingly, loss of TAZ affects mammary gland development, leading to an imbalance of luminal and basal populations as well as branching defects. Mechanistically, TAZ interacts with components of the SWI/SNF complex to modulate lineage-specific gene expression. Collectively, these findings uncover a new role for Hippo signaling in the determination of lineage identity through recruitment of chromatin remodeling complexes.
PMCID: PMC4011189  PMID: 24613358
WWTR1/TAZ; Hippo pathway; cellular plasticity; SWI/SNF; differentiation; mammary gland; lineage commitment
8.  Path to the clinic: Assessment of iPSC-based cell therapies in vivo in a non-human primate model 
Cell reports  2014;7(4):1298-1309.
Induced pluripotent stem cell (iPSC)-based cell therapies have a great potential for regenerative medicine, but are also potentially associated with tumorigenic risks. Current rodent models are not the optimal predictors of efficiency and safety for clinical application. Therefore, we developed a clinically relevant non-human primate model to assess the tumorigenic potential and in vivo efficacy of both undifferentiated and differentiated iPSCs in the autologous settings without immunosuppression. Undifferentiated autologous iPSCs indeed formed mature teratomas in a dose-dependent manner. However, tumor formation was accompanied by an inflammatory reaction. On the other hand, iPSC-derived mesodermal stromal-like cells formed new bone in vivo without any evidence of teratoma formation. We therefore show for the first time in a large animal model that closely resembles human physiology that undifferentiated autologous iPSCs form teratomas, and that iPSC-derived progenitor cells can give rise to a functional tissue in vivo.
PMCID: PMC4058889  PMID: 24835994
9.  Calcineurin is universally involved in vesicle endocytosis at neuronal and non-neuronal secretory cells 
Cell reports  2014;7(4):982-988.
Calcium influx triggers and accelerates endocytosis in nerve terminals and non-neuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. At three preparations where controversies arose, including large calyx-type synapses, conventional cerebellar synapses and neuroendocrine chromaffin cells containing large dense-core vesicles, we reported that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell types, developmental stages, or endocytic forms (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at relatively small calcium influx, but did not inhibit endocytosis at large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the controversies in pharmacological studies. We therefore suggest including calcineurin as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis.
PMCID: PMC4070379  PMID: 24835995
10.  Mitochondrial Pyruvate Carrier 2 Hypomorphism in Mice Leads to Defects in Glucose-Stimulated Insulin Secretion 
Cell reports  2014;7(6):2042-2053.
Carrier-facilitated pyruvate transport across the inner mitochondrial membrane plays an essential role in anabolic and catabolic intermediary metabolism. The mitochondrial pyruvate carrier 2 (Mpc2) is believed to be a component of the complex that facilitates mitochondrial pyruvate import. Complete MPC2 deficiency resulted in embryonic lethality in mice. However, a second mouse line expressing an N-terminal truncated MPC2 protein (Mpc2Δ16) was viable, but exhibited reduced capacity for mitochondrial pyruvate oxidation. Metabolic studies demonstrated exaggerated blood lactate concentrations after pyruvate, glucose, or insulin challenge in Mpc2Δ16 mice. Additionally, compared to WT controls, Mpc2Δ16 mice exhibited normal insulin sensitivity, but elevated blood glucose after bolus pyruvate or glucose injection. This was attributable to reduced glucose-stimulated insulin secretion and was corrected by sulfonylurea KATP channel inhibitor administration. Collectively, these data are consistent with a role for MPC2 in mitochondrial pyruvate import and suggest that Mpc2 deficiency results in defective pancreatic beta cell glucose sensing.
PMCID: PMC4074444  PMID: 24910426
11.  Isolation and Molecular Characterization of Circulating Melanoma Cells 
Cell reports  2014;7(3):645-653.
Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice, rapidly declining after B-RAF inhibitor treatment. CTCs were shed early from localized tumors and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled comparison of RNA sequencing profiles with the matched primary tumor. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. In patients with metastatic melanoma, CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF targeted therapy. Together, the capture of CTCs and their molecular characterization provide insight into the hematogenous spread of melanoma.
PMCID: PMC4079008  PMID: 24746818
12.  Pten loss induces autocrine FGF signaling to promote skin tumorigenesis 
Cell reports  2014;6(5):818-826.
Inactivation of the Pten tumor suppressor negatively regulates the PI3K-mTOR pathway. In a model of cutaneous squamous cell carcinoma (SCC), we demonstrate that deletion of Pten strongly elevates Fgf10 protein levels without increasing Fgf10 transcription in vitro and in vivo. The translational activation of Fgf10 by Pten deletion is reversed by genetic disruption of the mTORC1 complex, which also prevents skin tumorigenesis in Pten mutants. We further show that ectopic expression of Fgf10 causes skin papillomas, while Pten deletion-induced skin tumors are inhibited by epidermal deletion of Fgfr2. Collectively, our data identify autocrine activation of FGF signaling as an essential mechanism in promoting Pten-deficient skin tumors.
PMCID: PMC4080841  PMID: 24582960
13.  Recurrent Somatic Structural Variations Contribute to Tumorigenesis in Pediatric Osteosarcoma 
Cell reports  2014;7(1):104-112.
Osteosarcoma is a neoplasm of mesenchymal origin with features of osteogenic differentiation. Patients with recurrent or metastatic disease have a very poor prognosis. To define the landscape of somatic mutations in pediatric osteosarcoma, we performed whole-genome sequencing of DNA from 20 osteosarcoma tumor samples and matched normal tissue (obtained from 19 patients) in the discovery cohort as well as 14 samples from 13 patients in the validation cohort. Our results demonstrate that pediatric osteosarcoma is characterized by multiple somatic chromosomal lesions, including structural variations (SVs) and copy number alterations (CNAs). Moreover, single nucleotide variations (SNVs) exhibit a pattern of localized hypermutation called “kataegis” in 50% of the tumors. Despite these regions of kataegis across the osteosarcoma genomes, we detected relatively few recurrent SNVs, and only when SVs were included did we identify the major pathways that are mutated in osteosarcoma. We identified p53 pathway lesions in all 19 patient’s tumors in the discovery cohort, 9 of which were translocations in the first intron of the TP53 gene, leading to gene inactivation. This mechanism of p53 gene inactivation is unique to osteosarcoma among pediatric cancers. In an additional cohort of 32 patients, TP53 gene alterations were identified in 29 of those tumors. Beyond TP53, the RB1, ATRX and DLG2 genes showed recurrent somatic alterations (SNVs and/or SVs) in 29–53% of the tumors. These data highlight the power of whole-genome sequencing in identifying recurrent somatic alterations in cancer genomes that may be missed using other methods.
PMCID: PMC4096827  PMID: 24703847
14.  Metabolic resource allocation in individual microbes determines ecosystem interactions and spatial dynamics 
Cell reports  2014;7(4):1104-1115.
The inter-species exchange of metabolites plays a key role in the spatio-temporal dynamics of microbial communities. This raises the question whether ecosystem-level behavior of structured communities can be predicted using genome-scale models of metabolism for multiple organisms. We developed a modeling framework that integrates dynamic flux balance analysis with diffusion on a lattice, and applied it to engineered consortia. First, we predicted, and experimentally confirmed, the species-ratio to which a 2-species mutualistic consortium converges, and the equilibrium composition of a newly engineered 3-member community. We next identified a specific spatial arrangement of colonies, which gives rise to what we term the “eclipse dilemma”: does a competitor placed between a colony and its cross-feeding partner benefit or hurt growth of the original colony? Our experimentally validated finding, that the net outcome is beneficial, highlights the complex nature of metabolic interactions in microbial communities, while at the same time demonstrating their predictability.
PMCID: PMC4097880  PMID: 24794435
15.  SRC-2 Is an Essential Coactivator for Orchestrating Metabolism and Circadian Rhythm 
Cell reports  2014;6(4):633-645.
Synchrony of the mammalian circadian clock is achieved by complex transcriptional and translational feedback loops centered on the BMAL1: CLOCK heterodimer. Modulation of circadian feedback loops is essential for maintaining rhythmicity, yet the role of transcriptional coactivators in driving BMAL1:CLOCK transcriptional networks is largely unexplored. Here, we show diurnal hepatic steroid receptor coactivator 2 (SRC-2) recruitment to the genome that extensively overlaps with the BMAL1 cistrome during the light phase, targeting genes that enrich for circadian and metabolic processes. Notably, SRC-2 ablation impairs wheel-running behavior, alters circadian gene expression in several peripheral tissues, alters the rhythmicity of the hepatic metabolome, and deregulates the synchronization of cell-autonomous metabolites. We identify SRC-2 as a potent coregulator of BMAL1:CLOCK and find that SRC-2 targets itself with BMAL1:CLOCK in a feedforward loop. Collectively, our data suggest that SRC-2 is a transcriptional coactivator of the BMAL1:CLOCK oscillators and establish SRC-2 as a critical positive regulator of the mammalian circa-dian clock.
PMCID: PMC4096300  PMID: 24529706
16.  5S Ribosomal RNA Is an Essential Component of a Nascent Ribosomal Precursor Complex that Regulates the Hdm2-p53 Checkpoint 
Cell reports  2013;4(1):87-98.
Recently, we demonstrated that RPL5 and RPL11 act in a mutually dependent manner to inhibit Hdm2 and stabilize p53 following impaired ribosome biogenesis. Given that RPL5 and RPL11 form a preribosomal complex with noncoding 5S ribosomal RNA (rRNA) and the three have been implicated in the p53 response, we reasoned they may be part of an Hdm2-inhibitory complex. Here, we show that small interfering RNAs directed against 5S rRNA have no effect on total or nascent levels of the noncoding rRNA, though they prevent the reported Hdm4 inhibition of p53. To achieve efficient inhibition of 5S rRNA synthesis, we targeted TFIIIA, a specific RNA polymerase III cofactor, which, like depletion of either RPL5 or RPL11, did not induce p53. Instead, 5S rRNA acts in a dependent manner with RPL5 and RPL11 to inhibit Hdm2 and stabilize p53. Moreover, depletion of any one of the three components abolished the binding of the other two to Hdm2, explaining their common dependence. Finally, we demonstrate that the RPL5/RPL11/5S rRNA preribosomal complex is redirected from assembly into nascent 60S ribosomes to Hdm2 inhibition as a consequence of impaired ribosome biogenesis. Thus, the activation of the Hdm2-inhibitory complex is not a passive but a regulated event, whose potential role in tumor suppression has been recently noted.
PMCID: PMC3928573  PMID: 23831031
17.  PTEN C-terminal Deletion Causes Genomic Instability and Tumor Development 
Cell reports  2014;6(5):844-854.
Tumor suppressor PTEN controls genomic stability and inhibits tumorigenesis. The N-terminal phosphatase domain of PTEN antagonizes the PI3K/AKT pathway, but its C-terminal function is less defined. Here we describe a knock-in mouse model of a nonsense mutation that results in deletion of the entire Pten C-terminal region, referred to as PtenΔC. Mice heterozygous for PtenΔC develop multiple spontaneous tumors, including cancers and B cell lymphoma. Heterozygous deletion of the Pten C-terminal domain also causes genomic instability and common fragile site rearrangement. We found that Pten C terminal disruption induces p53 and its downstream targets. Simultaneous depletion of p53 promotes metastasis without influencing initiation of tumors, suggesting that p53 mainly suppresses tumor progression. Our data highlight the essential role of the PTEN C-terminus in the maintenance of genomic stability and suppression of tumorigenesis.
PMCID: PMC4090077  PMID: 24561254
PTEN; Tumor; Chromosome Instability
18.  Death Induced by CD95 or CD95 Ligand Elimination 
Cell reports  2014;7(1):208-222.
CD95 (Fas/APO-1), when bound by its cognate ligand CD95L, induces cells to die by apoptosis. We now show that elimination of CD95 or CD95L results in a form of cell death that is independent of caspase-8, RIPK1/MLKL, and p53, is not inhibited by Bcl-xL expression, and preferentially affects cancer cells. All tumors that formed in mouse models of low-grade serous ovarian cancer or chemically induced liver cancer with tissue specific deletion of CD95 still expressed CD95, suggesting that cancer cannot form in the absence of CD95. Death induced by CD95R/L elimination (DICE) is characterized by an increase in cell size and production of mitochondrial ROS, and DNA damage. It resembles a necrotic form of mitotic catastrophe. No single drug was found to completely block this form of cell death, and it could also not be blocked by the knockdown of a single gene, making it a promising new way to kill cancer cells.
PMCID: PMC4083055  PMID: 24656822
19.  Wnt signaling is required for long-term memory formation 
Cell reports  2013;4(6):1082-1089.
Wnt signaling regulates synaptic plasticity and neurogenesis in the adult nervous system, suggesting a potential role in behavioral processes. Here, we probed the requirement for Wnt signaling during olfactory memory formation in Drosophila using an inducible RNA interference approach. Interfering with β-catenin expression in the adult mushroom body neurons specifically impaired long-term memory without altering short-term memory. The impairment was reversible, rescued with expression of a wild-type β-catenin transgene, and correlated with a disruption of a cellular long-term memory trace. Inhibition of wingless, a Wnt ligand, and arrow, a Wnt co-receptor, also impaired long-term memory. Wingless expression in wild type flies was transiently elevated in the brain after long-term memory conditioning. Thus, inhibiting three key components of the Wnt signaling pathway in the adult mushroom bodies impairs long-term memory, collectively indicating that this pathway mechanistically underlies this specific form of memory.
PMCID: PMC4083693  PMID: 24035392
20.  Autocrine Effects of Tumor-Derived Complement 
Cell reports  2014;6(6):1085-1095.
We describe a role for the complement system in enhancing cancer growth. Cancer cells secrete complement proteins that stimulate tumor growth upon activation. Complement promotes tumor growth via a direct autocrine effect that is partially independent of tumor-infiltrating cytotoxic T cells. Activated C5aR and C3aR signal through the PI3K/AKT pathway in cancer cells, and silencing the PI3K or AKT gene in cancer cells eliminates the progrowth effects of C5aR and C3aR stimulation. In patients with ovarian or lung cancer, higher tumoral C3 or C5aR mRNA levels were associated with decreased overall survival. These data identify a role for tumor-derived complement proteins in promoting tumor growth, and they therefore have substantial clinical and therapeutic implications.
PMCID: PMC4084868  PMID: 24613353
21.  An Unbiased Analysis Method to Quantify mRNA Localization Reveals Its Correlation with Cell Motility 
Cell reports  2012;1(2):179-184.
Localization of mRNA is a critical mechanism used by a large fraction of transcripts to restrict its translation to specific cellular regions. Although current high- resolution imaging techniques provide ample information, the analysis methods for localization have either been qualitative or employed quantification in non-randomly selected regions of interest. Here, we describe an analytical method for objective quantification of mRNA localization using a combination of two characteristics of its molecular distribution, polarization and dispersion. The validity of the method is demonstrated using single-molecule FISH images of budding yeast and fibroblasts. Live-cell analysis of endogenous β-actin mRNA in mouse fibroblasts reveals that mRNA polarization has a half- life of ~16 min and is cross-correlated with directed cell migration. This novel approach provides insights into the dynamic regulation of mRNA localization and its physiological roles.
PMCID: PMC4079260  PMID: 22832165
22.  Structural Basis of Actin Filament Nucleation by Tandem W Domains 
Cell reports  2013;3(6):1910-1920.
Spontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl), a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization.
PMCID: PMC3780403  PMID: 23727244
23.  SETD2-Dependent Histone H3K36 Trimethylation Is Required for Homologous Recombination Repair and Genome Stability 
Cell Reports  2014;7(6):2006-2018.
Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.
Graphical Abstract
•A role for SETD2 in DSB resection and homologous recombination repair•Histone H3K36me3 is required for homologous recombination•SETD2 and RAD51 suppress mutations arising from microhomology-mediated end-joining•Mutations affecting H3K36me3 levels may promote tumorigenesis
The SETD2 gene encodes the histone H3K36 trimethyltransferase. Pfister et al. now show that human SETD2-dependent H3K36me3 maintains genome stability by promoting error-free DNA repair through homologous recombination (HR). Upon DNA damage, SETD2-depleted cells exhibit reduced DNA resection, impaired recruitment of early HR factors, and increased utilization of the error-prone microhomology-mediated end-joining repair pathway. Eliminating H3K36me3 by overexpressing the oncogene KDM4A also impairs HR. Thus, H3K36me3 suppresses tumorigenesis by promoting accurate DNA repair.
PMCID: PMC4074340  PMID: 24931610
24.  Distinct Stromal Cell Factor Combinations Can Separately Control Hematopoietic Stem Cell Survival, Proliferation, and Self-Renewal 
Cell Reports  2014;7(6):1956-1967.
Hematopoietic stem cells (HSCs) are identified by their ability to sustain prolonged blood cell production in vivo, although recent evidence suggests that durable self-renewal (DSR) is shared by HSC subtypes with distinct self-perpetuating differentiation programs. Net expansions of DSR-HSCs occur in vivo, but molecularly defined conditions that support similar responses in vitro are lacking. We hypothesized that this might require a combination of factors that differentially promote HSC viability, proliferation, and self-renewal. We now demonstrate that HSC survival and maintenance of DSR potential are variably supported by different Steel factor (SF)-containing cocktails with similar HSC-mitogenic activities. In addition, stromal cells produce other factors, including nerve growth factor and collagen 1, that can antagonize the apoptosis of initially quiescent adult HSCs and, in combination with SF and interleukin-11, produce >15-fold net expansions of DSR-HSCs ex vivo within 7 days. These findings point to the molecular basis of HSC control and expansion.
Graphical Abstract
•HSC viability, mitogenesis, and self-renewal are differentially controlled•Stromal cells produce nonmitogenic factors that directly sustain HSC viability•More adult bone marrow cells can produce HSCs than display HSC activity directly•Nerve growth factor and collagen 1 promote serially transplantable HSCs
Wohrer et al. now show that different factors secreted by stromal cells separately control the survival, proliferation, and self-renewal of hematopoietic stem cells. These factors are thus required in combination to stimulate net expansions of these cells with full retention of their original stem cell properties.
PMCID: PMC4074342  PMID: 24910437
25.  Otx2 and Oct4 Drive Early Enhancer Activation during Embryonic Stem Cell Transition from Naive Pluripotency 
Cell Reports  2014;7(6):1968-1981.
Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency.
Graphical Abstract
•Transcription factor Otx2 drives enhancer activation in differentiating mouse ESCs•Oct4 controls Otx2 expression levels•Otx2 collaborates with Oct4 in enhancer activation•Otx2 contributes to enhancer maintenance and de novo activation
The transcription factor Otx2 plays an important role in neural development and in the exit of embryonic stem cells from the pluripotent ground state. In this study, Yang et al. demonstrate that Otx2 drives enhancer activation and maintenance during the early cell-fate transition away from ground state pluripotency. Otx2 is involved in a regulatory partnership with Oct4 wherein Oct4 initially promotes Otx2 expression. Otx2 subsequently recruits Oct4 to a subset of enhancers and establishes a regulatory chromatin landscape.
PMCID: PMC4074343  PMID: 24931607

Results 1-25 (424)