ALBERT

Description: Publication date: 26 December 2013 Source: Cell Reports, Volume 5, Issue 6 Author(s): Verity F. Oliver , Maria Franchina , Andrew E. Jaffe , Kari E. Branham , Mohammad Othman , John R. Heckenlively , Anand Swaroop , Betsy Campochiaro , Brendan J. Vote , Jamie E. Craig , Richard Saffery , David A. Mackey , Jiang Qian , Donald J. Zack , Alex W. Hewitt , Shannath L. Merbs Age-related macular degeneration (AMD) is a leading cause of visual impairment worldwide. Aberrant DNA methylation within the promoter of IL17RC in peripheral blood mononuclear cells has recently been reported in AMD. To validate this association, we examined DNA methylation of the IL17RC promoter in peripheral blood. First, we used Illumina Human Methylation450 Bead Arrays, a widely accepted platform for measuring global DNA methylation. Second, methylation status at multiple sites within the IL17RC promoter was determined by bisulfite pyrosequencing in two cohorts. Third, a methylation-sensitive quantitative PCR-based assay was performed on a subset of samples. In contrast to previous findings, we did not find evidence of differential methylation between AMD cases and age-matched controls. We conclude that hypomethylation within the IL17RC gene promoter in peripheral blood is not suitable for use as a clinical biomarker of AMD. This study highlights the need for considerable replication of epigenetic association studies prior to clinical application. Graphical abstract Teaser Age-related macular degeneration (AMD) etiology remains unclear, but both environmental and genetic factors are important. Modifications to DNA methylation can impact gene expression in response to environmental stimuli. Recently, AMD was associated with the aberrant methylation of the IL17RC gene in blood. In this study, Hewitt, Merbs, and colleagues analyzed peripheral blood-cell DNA from controls and three additional AMD cohorts, and they found no differential IL17RC promoter methylation between the two groups. These results suggest that the methylation status of the IL17RC gene promoter is not suitable for use as a clinical biomarker of AMD.

Description: Publication date: Available online 26 December 2013 Source: Cell Reports Author(s): Xuan Ye , Qian Cai β site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is the major β secretase for generating β-amyloid (Aβ) peptides. The acidic environment of endosomes is optimal for β secretase activity. However, the mechanisms regulating BACE1 traffic from endosomes to lysosomes for degradation are largely unknown. Here, using snapin -deficient mice combined with gene rescue experiments, we reveal that Snapin, as a dynein motor adaptor for late endosomes, mediates BACE1 retrograde transport. hAPP mutant live neurons and mouse brains exhibited BACE1 accumulation within the altered late endocytic organelles and defective lysosomal targeting due to reduced Snapin-dynein coupling. Deleting snapin or disrupting Snapin-dynein coupling reduces BACE1 transport to lysosomes for degradation, thus enhancing APP processing. Overexpressing Snapin in hAPP neurons reduces β site cleavage of APP by enhancing BACE1 turnover. Altogether, our study provides mechanistic insights into the complex regulation of BACE1 level and activity and turnover through retrograde transport, thus controlling Aβ generation in neurons. Graphical abstract Teaser The primary constituent of Alzheimer’s disease plaques is β-amyloid (Aβ) peptide generated by sequential proteolysis of amyloid precursor protein (APP) by β and γ secretases. β site APP-cleaving enzyme 1 (BACE1) is the major neuronal β secretase for Aβ generation. A long-standing question is how BACE1 levels and β secretase activity are regulated. In this study, Ye and Cai reveal a cellular pathway that controls Aβ production in neurons by regulating BACE1 retrograde transport to lysosomes for degradation.

Description: Publication date: Available online 26 December 2013 Source: Cell Reports Author(s): Daniel F. Markgraf , Robin W. Klemm , Mirco Junker , Hans K. Hannibal-Bach , Christer S. Ejsing , Tom A. Rapoport Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae , LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption. Graphical abstract Teaser Eukaryotic cells store neutral lipids, such as triacylglycerol (TAG), in lipid droplets (LDs). Rapoport and colleagues show that, in S. cerevisiae , during LD growth, TAG synthesis from diacylglycerol (DAG) is caused by the LD-localized acyl-transferase Dga1p. During LD breakdown, a multispanning ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs, suppresses a futile cycle between TAG and DAG on LDs, and promotes the efficient utilization of DAG in the ER.

Description: Publication date: Available online 26 December 2013 Source: Cell Reports Author(s): Pawel Niewiadomski , Jennifer H. Kong , Robert Ahrends , Yan Ma , Eric W. Humke , Sohini Khan , Mary N. Teruel , Bennett G. Novitch , Rajat Rohatgi Gli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. We describe a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity. Graphical abstract Teaser The Gli transcription factors are final executioners of the Hedgehog signaling pathway, both in normal development and in Hedgehog-driven cancers. In this report, Niewiadomski, Rohatgi, and colleagues uncover a multisite phosphorylation mechanism that regulates the activity of the Gli proteins. The pattern of Gli protein phosphorylation can encode multiple states of Gli activity, rather than simply regulating an ON/OFF switch. This “phospho-code” can control whether Gli proteins are truncated into transcriptional repressors or converted to weak or strong transcriptional activators.

Description: Publication date: Available online 26 December 2013 Source: Cell Reports Author(s): Justin D. Lathia , Meizhang Li , Maksim Sinyuk , Alvaro G. Alvarado , William A. Flavahan , Kevin Stoltz , Ann Mari Rosager , James Hale , Masahiro Hitomi , Joseph Gallagher , Qiulian Wu , Jody Martin , Jason G. Vidal , Ichiro Nakano , Rikke H. Dahlrot , Steinbjørn Hansen , Roger E. McLendon , Andrew E. Sloan , Shideng Bao , Anita B. Hjelmeland , Christian T. Carson , Ulhas P. Naik , Bjarne Kristensen , Jeremy N. Rich Stem cells reside in niches that regulate the balance between self-renewal and differentiation. The identity of a stem cell is linked with the ability to interact with its niche through adhesion mechanisms. To identify targets that disrupt cancer stem cell (CSC) adhesion, we performed a flow cytometry screen on patient-derived glioblastoma (GBM) cells and identified junctional adhesion molecule A (JAM-A) as a CSC adhesion mechanism essential for self-renewal and tumor growth. JAM-A was dispensable for normal neural stem/progenitor cell (NPC) function, and JAM-A expression was reduced in normal brain versus GBM. Targeting JAM-A compromised the self-renewal of CSCs. JAM-A expression negatively correlated to GBM patient prognosis. Our results demonstrate that GBM-targeting strategies can be identified through screening adhesion receptors and JAM-A represents a mechanism for niche-driven CSC maintenance. Graphical abstract Teaser Lathia, Rich, and colleagues now use high-throughput flow cytometry analysis of patient-derived glioblastoma specimens to identify unique cell surface receptors that regulate tumor progression. The authors identified junctional adhesion molecule A (JAM-A) as a cancer stem cell maintenance factor and a prognostic glioblastoma biomarker. These findings reveal a role for JAM-A in glioblastoma progression and demonstrate the utility of flow-cytometry-based screening approaches.

Description: Publication date: Available online 26 December 2013 Source: Cell Reports Author(s): Jozef Madzo , Hui Liu , Alexis Rodriguez , Aparna Vasanthakumar , Sriram Sundaravel , Donne Bennett D. Caces , Timothy J. Looney , Li Zhang , Janet B. Lepore , Trisha Macrae , Robert Duszynski , Alan H. Shih , Chun-Xiao Song , Miao Yu , Yiting Yu , Robert Grossman , Brigitte Raumann , Amit Verma , Chuan He , Ross L. Levine , Don Lavelle , Bruce T. Lahn , Amittha Wickrema , Lucy A. Godley Hematopoietic stem cell differentiation involves the silencing of self-renewal genes and induction of a specific transcriptional program. Identification of multiple covalent cytosine modifications raises the question of how these derivatized bases influence stem cell commitment. Using a replicative primary human hematopoietic stem/progenitor cell differentiation system, we demonstrate dynamic changes of 5-hydroxymethylcytosine (5-hmC) during stem cell commitment and differentiation to the erythroid lineage. Genomic loci that maintain or gain 5-hmC density throughout erythroid differentiation contain binding sites for erythroid transcription factors and several factors not previously recognized as erythroid-specific factors. The functional importance of 5-hmC was demonstrated by impaired erythroid differentiation, with augmentation of myeloid potential, and disrupted 5-hmC patterning in leukemia patient-derived CD34+ stem/early progenitor cells with TET methylcytosine dioxygenase 2 ( TET2 ) mutations. Thus, chemical conjugation and affinity purification of 5-hmC-enriched sequences followed by sequencing serve as resources for deciphering functional implications for gene expression during stem cell commitment and differentiation along a particular lineage. Graphical abstract Teaser 5-hydroxymethylcytosine (5-hmC) is a DNA modification catalyzed by the TET enzymes. The significance of this modification is not completely defined, but it is known to be dysregulated in hematological malignancies. In this study, Wickrema, Godley, and colleagues demonstrate that 5-hmC plays an important role in hematopoietic stem cell commitment to the erythroid lineage and is associated with activating histone marks at a genome-wide level. TET2 deficiency disrupts 5-hmC patterns and compromises erythroid differentiation.

Description: Publication date: 26 December 2013 Source: Cell Reports, Volume 5, Issue 6 Author(s): Vinod Udayar , Virginie Buggia-Prévot , Rita L. Guerreiro , Gabriele Siegel , Naresh Rambabu , Amanda L. Soohoo , Moorthi Ponnusamy , Barbara Siegenthaler , Jitin Bali , Mikael Simons , Jonas Ries , Manojkumar A. Puthenveedu , John Hardy , Gopal Thinakaran , Lawrence Rajendran Alzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides, which are generated from amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aβ levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Aβ production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled β-secretase endosomal recycling to the plasma membrane and thus affected Aβ production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Aβ levels and show how systems biology approaches can unravel the molecular complexity underlying AD. Graphical abstract Teaser Alzheimer’s disease is a neurodegenerative disorder that results from the buildup of a toxic peptide, Aß. The primary proteins involved in Aß production are membrane associated, but the cellular trafficking systems involved in their regulation are not fully understood. Here, Rajendran and colleagues provide a comprehensive road map outlining the cellular routes involved in Aß production and implicate Rab11 in this process. They also identify a recycling route that replenishes BACE1, an enzyme involved in Aß production, to early endosomes where Aβ production occurs.

Description: Publication date: Available online 26 December 2013 Source: Cell Reports Author(s): Caleb McKinney , Jiri Zavadil , Christopher Bianco , Lora Shiflett , Stuart Brown , Ian Mohr Unlike many viruses that suppress cellular protein synthesis, host mRNA translation and polyribosome formation are stimulated by human cytomegalovirus (HCMV). How HCMV impacts the translationally regulated cellular mRNA repertoire and its contribution to virus biology remains unknown. Using polysome profiling, we show that HCMV presides over the cellular translational landscape, selectively accessing the host genome to extend its own coding capacity and regulate virus replication. Expression of the HCMV UL38 mTORC1-activator partially recapitulates these translational alterations in uninfected cells. The signature of cellular mRNAs translationally stimulated by HCMV resembles pathophysiological states (such as cancer) where translation initiation factor levels or activity increase. In contrast, cellular mRNAs repressed by HCMV include those involved in differentiation and the immune response. Surprisingly, interfering with the virus-induced activation of cellular mRNA translation can either limit or enhance HCMV growth. The unanticipated extent to which HCMV specifically manipulates host mRNA translation may aid in understanding its association with complex inflammatory disorders and cancer. Graphical abstract Teaser Cellular protein synthesis is often inhibited in virus-infected cells to limit production of host defense molecules and foster viral mRNA translation. In contrast, Mohr and colleagues now show that HCMV selectively manipulates which host mRNAs are recruited to or excluded from polyribosomes without globally suppressing ongoing cellular protein synthesis. Thus, by presiding over the cellular translational landscape, HCMV precisely accesses the host genome, effectively extending its own coding capacity to regulate virus replication.

Description: Publication date: Available online 26 December 2013 Source: Cell Reports Author(s): Brent Neumann , Massimo A. Hilliard Axonal degeneration arises as a consequence of neuronal injury and is a common hallmark of a number of neurodegenerative diseases. However, the genetic causes and the cellular mechanisms that trigger this process are still largely unknown. Based on forward genetic screening in C. elegans , we have identified the α-tubulin acetyltransferase gene mec-17 as causing spontaneous, adult-onset, and progressive axonal degeneration. Loss of MEC-17 leads to microtubule instability, a reduction in mitochondrial number, and disrupted axonal transport, with altered distribution of both mitochondria and synaptic components. Furthermore, mec-17 -mediated axonal degeneration occurs independently from its acetyltransferase domain; is enhanced by mutation of coel-1 , a tubulin-associated molecule; and correlates with the animal’s body length. This study therefore identifies a critical role for the conserved microtubule-associated protein MEC-17 in preserving axon integrity and preventing axonal degeneration. Graphical abstract Teaser Axonal degeneration arises as a consequence of neuronal injury and is a common feature of neurodegenerative disease. Here, Neumann and Hilliard show that loss of the α-tubulin acetyltransferase protein, MEC-17/ATAT1, leads to spontaneous, adult-onset, and progressive axonal degeneration. MEC-17 is required for maintaining microtubule stability, axonal transport, and the localization of mitochondria and synaptic components. These functions are carried out independently from the proteins’ acetyltransferase domain. Thus, MEC-17 is critical for the maintenance of axonal structure.

Description: Publication date: Available online 26 December 2013 Source: Cell Reports Author(s): Stefan Semrau , Nicola Crosetto , Magda Bienko , Marina Boni , Paolo Bernasconi , Roberto Chiarle , Alexander van Oudenaarden Transcribed gene fusions are key biomarkers in many hematologic and solid tumors, often representing the primary oncogenic driver mutation. Here, we report an experimental and computational pipeline for detecting fusion transcripts using single-molecule RNA FISH and unbiased correlation analysis (FuseFISH). We constructed a genome-wide database of optimal oligonucleotide sequences, enabling quick design of FuseFISH probes against known and novel fusions. We implemented FuseFISH in cell lines, tissue sections, and purified RNA, reliably detecting one BCR-ABL1 positive in 10,000 negative cells. In 34 hematologic samples, we detected BCR-ABL1 transcripts with high specificity and sensitivity. Finally, we measured BCR-ABL1 expression heterogeneity and dynamics in single CML cells exposed to the kinase inhibitor Nilotinib. Our resource and methods are ideal for streamlined validation of fusions newly identified by next-generation sequencing, and they pave the way to studying the impact of fusion expression variability on clinical outcome. Graphical abstract Teaser Recurrent gene fusions play a key role in the pathogenesis of many tumors. In this study, Chiarle, van Oudenaarden, and colleagues report a method to quantify fusion transcripts in purified RNA, cells, and histological specimens using single-molecule RNA fluorescence in situ hybridization (FuseFISH). A user-friendly genomic library of probes and an algorithm for robust and unbiased fusion transcript detection make FuseFISH a powerful tool for routine diagnostics, validation of newly identified fusions, and analysis of intratumor fusion expression heterogeneity.