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Publications in Biomedical Research by NOMIS researchers

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

NOMIS Researcher(s)

Published in

December 1, 2016

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1038/nature20565

Disease Tolerance Trick or Treat: Give Your Brain Something Good to Eat

NOMIS Researcher(s)

Published in

September 8, 2016

The reprioritization of feeding motivations during disease is proposed to optimize host defense strategies against infection. Now, Wang et al. identify that sickness-induced anorexia differentially shapes the metabolic requirements of cellular stress adaptations, leading to opposite impact on disease tolerance upon bacterial versus viral infections.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.cell.2016.08.034

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition

NOMIS Researcher(s)

Published in

September 1, 2016

Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat-dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G4C2 repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.15252/embr.201541724

Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants

NOMIS Researcher(s)

Published in

July 15, 2016

Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein proposed to function in various RNA metabolic pathways, including transcription regulation, pre-mRNA splicing, RNA transport and microRNA processing. Mutations in the FUS gene were identified in patients with amyotrophic lateral sclerosis (ALS), but the pathomechanisms by which these mutations cause ALS are not known. Here, we show that FUS interacts with the minor spliceosome constituent U11 snRNP, binds preferentially to minor introns and directly regulates their removal. Furthermore, a FUS knockout in neuroblastoma cells strongly disturbs the splicing of minor intron-containing mRNAs, among them mRNAs required for action potential transmission and for functional spinal motor units. Moreover, an ALS-associated FUS mutant that forms cytoplasmic aggregates inhibits splicing of minor introns by trapping U11 and U12 snRNAs in these aggregates. Collectively, our findings suggest a possible pathomechanism for ALS in which mutated FUS inhibits correct splicing of minor introns in mRNAs encoding proteins required for motor neuron survival.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.15252/embj.201593791

Cooperative microbial tolerance behaviors in host-microbiota mutualism

NOMIS Researcher(s)

Published in

June 2, 2016

Animal defense strategies against microbes are most often thought of as a function of the immune system, the primary function of which is to sense and kill microbes through the execution of resistance mechanisms. However, this antagonistic view creates complications for our understanding of beneficial host-microbe interactions. Pathogenic microbes are described as employing a few common behaviors that promote their fitness at the expense of host health and fitness. Here, a complementary framework is proposed to suggest that, in addition to pathogens, beneficial microbes have evolved behaviors to manipulate host processes in order to promote their own fitness and do so through the promotion of host health and fitness. In this Perspective, I explore the idea that patterns or behaviors traditionally ascribed to pathogenic microbes are also employed by beneficial microbes to promote host tolerance defense strategies. Such strategies would promote host health without having a negative impact on microbial fitness and would thereby yield cooperative evolutionary dynamics that are likely required to drive mutualistic co-evolution of hosts and microbes.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.cell.2016.05.049

Understanding the molecular mechanisms of reprogramming

NOMIS Researcher(s)

Published in

May 6, 2016

Despite the profound and rapid advancements in reprogramming technologies since the generation of the first induced pluripotent stem cells (iPSCs) in 2006[1], the molecular basics of the process and its implications are still not fully understood. Recent work has suggested that a subset of TFs, so called “Pioneer TFs”, play an important role during the stochastic phase of iPSC reprogramming [2-6]. Pioneer TFs activities differ from conventional transcription factors in their mechanism of action. They bind directly to condensed chromatin and elicit a series of chromatin remodeling events that lead to opening of the chromatin. Chromatin decondensation by pioneer factors progressively occurs during cell division and in turn exposes specific gene promoters in the DNA to which TFs can now directly bind to promoters that are readily accessible[2, 6]. Here, we will summarize recent advancements on our understanding of the molecular mechanisms underlying reprogramming to iPSC as well as the implications that pioneer Transcription Factor activities might play during different lineage conversion processes.

Research field(s)
Health Sciences, Biomedical Research, Biochemistry & Molecular Biology

DOI
10.1016/j.bbrc.2015.11.120

Establishment of human iPSC-based models for the study and targeting of glioma initiating cells

NOMIS Researcher(s)

Published in

February 22, 2016

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in â1/490% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1038/ncomms10743

Nuclear envelope rupture is induced by actin-based nucleus confinement

NOMIS Researcher(s)

Published in

January 1, 2016

Repeated rounds of nuclear envelope (NE) rupture and repair have been observed in laminopathy and cancer cells and result in intermittent loss of nucleus compartmentalization. Currently, the causes of NE rupture are unclear. Here, we show that NE rupture in cancer cells relies on the assembly of contractile actin bundles that interact with the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex. We found that the loss of actin bundles or the LINC complex did not rescue nuclear lamina defects, a previously identified determinant of nuclear membrane stability, but did decrease the number and size of chromatin hernias. Finally, NE rupture inhibition could be rescued in cells treated with actin-depolymerizing drugs by mechanically constraining nucleus height. These data suggest a model of NE rupture where weak membrane areas, caused by defects in lamina organization, rupture because of an increase in intranuclear pressure from actin-based nucleus confinement.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1083/jcb.201603053

Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

NOMIS Researcher(s)

Published in

January 1, 2015

Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins.We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization.

Research field(s)
Health Sciences, Biomedical Research, Biochemistry & Molecular Biology

DOI
10.1038/cddis.2015.18

Sulfonation pathway inhibitors block reactivation of latent HIV-1

NOMIS Researcher(s)

Published in

December 1, 2014

Long-lived pools of latently infected cells are a significant barrier to the development of a cure for HIV-1 infection. A better understanding of the mechanisms of reactivation from latency is needed to facilitate the development of novel therapies that address this problem. Here we show that chemical inhibitors of the sulfonation pathway prevent virus reactivation, both in latently infected J-Lat and U1 cell lines and in a primary human CD4+ T cell model of latency. In each of these models, sulfonation inhibitors decreased transcription initiation from the HIV-1 promoter. These inhibitors block transcription initiation at a step that lies downstream of nucleosome remodeling and affects RNA polymerase II recruitment to the viral promoter. These results suggest that the sulfonation pathway acts by a novel mechanism to regulate efficient virus transcription initiation during reactivation from latency, and further that augmentation of this pathway could be therapeutically useful.

Research field(s)
Health Sciences, Biomedical Research, Virology

DOI
10.1016/j.virol.2014.08.016

CHD1 and CHD2 are positive regulators of HIV-1 gene expression

NOMIS Researcher(s)

Published in

October 8, 2014

Background: Retroviruses encode a very limited number of proteins and therefore must exploit a wide variety of host proteins for completion of their lifecycle. Methods: We performed an insertional mutagenesis screen to identify novel cellular regulators of retroviral replication. Results: This approach identified the ATP-dependent chromatin remodeler, chromodomain helicase DNA-binding protein 2 (CHD2), as well as the highly related CHD1 protein, as positive regulators of both MLV and HIV-1 replication in rodent and human cells. RNAi knockdown of either CHD2 or the related CHD1 protein, in human cells resulted in a block to infection by HIV-1, specifically at the level of transcription. Conclusions: These results demonstrate that CHD1 and CHD2 can act as positive regulators of HIV-1 gene expression.

Research field(s)
Health Sciences, Biomedical Research, Virology

DOI
10.1186/1743-422X-11-180

Reprogramming by lineage specifiers: Blurring the lines between pluripotency and differentiation

NOMIS Researcher(s)

Published in

October 1, 2014

The generation of human induced pluripotent stem cells (iPS) has raised enormous expectations within the biomedical community due to their potential vast implications in regenerative and personalized medicine. However, reprogramming to iPS is still not fully comprehended. Difficulties found in ascribing specific molecular patterns to pluripotent cells (PSCs), and inherent inter-line and intra-line variability between different PSCs need to be resolved. Additionally, and despite multiple assumptions, it remains unclear whether the current in vitro culturing conditions for the maintenance and differentiation of PSCs do indeed recapitulate the developmental processes observed in vivo. As a consequence, basic questions such as what is the actual nature of PSCs remain unanswered and different theories have emerged in regards to the identity of these valuable cell population. Here we discuss on the published theories for defining PSC identity, the implications that the different postulated models have for the reprogramming field as well as speculate on potential future directions that might be opened once a precise knowledge on the nature of PSCs is accomplished.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.gde.2014.09.009

Function of a foxp3 cis-element in protecting regulatory T cell identity

NOMIS Researcher(s)

Published in

August 14, 2014

The homeostasis of multicellular organisms requires terminally differentiated cells to preserve their lineage specificity. However, it is unclear whether mechanisms exist to actively protect cell identity in response to environmental cues that confer functional plasticity. Regulatory T (Treg) cells, specified by the transcription factor Foxp3, are indispensable for immune system homeostasis. Here, we report that conserved noncoding sequence 2 (CNS2), a CpG-rich Foxp3 intronic cis-element specifically demethylated in mature Tregs, helps maintain immune homeostasis and limit autoimmune disease development by protecting Treg identity in response to signals that shape mature Treg functions and drive their initial differentiation. In activated Tregs, CNS2 helps protect Foxp3 expression from destabilizing cytokine conditions by sensing TCR/NFAT activation, which facilitates the interaction between CNS2 and Foxp3 promoter. Thus, epigenetically marked cis-elements can protect cell identity by sensing key environmental cues central to both cell identity formation and functional plasticity without interfering with initial cell differentiation. © 2014 Elsevier Inc.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.cell.2014.07.030

Reprogramming of human fibroblasts to pluripotency with lineage specifiers

NOMIS Researcher(s)

Published in

September 5, 2013

Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency. © 2013 Elsevier Inc.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.stem.2013.06.019

Adding an unnatural covalent bond to proteins through proximity-enhanced bioreactivity

NOMIS Researcher(s)

Published in

September 1, 2013

Natural proteins often rely on the disulfide bond to covalently link side chains. Here we genetically introduce a new type of covalent bond into proteins by enabling an unnatural amino acid to react with a proximal cysteine. We demonstrate the utility of this bond for enabling irreversible binding between an affibody and its protein substrate, capturing peptide-protein interactions in mammalian cells, and improving the photon output of fluorescent proteins. © 2013 Nature America, Inc. All rights reserved.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1038/nmeth.2595

ZASC1 knockout mice exhibit an early bone marrow-specific defect in murine leukemia virus replication

NOMIS Researcher(s)

Published in

April 30, 2013

Background: ZASC1 is a zinc finger-containing transcription factor that was previously shown to bind to specific DNA binding sites in the Moloney murine leukemia virus (Mo-MuLV) promoter and is required for efficient viral mRNA transcription (J. Virol. 84:7473-7483, 2010). Methods. To determine whether this cellular factor influences Mo-MuLV replication and viral disease pathogenesis in vivo, we generated a ZASC1 knockout mouse model and completed both early infection and long term disease pathogenesis studies. Results: Mice lacking ZASC1 were born at the expected Mendelian ratio and showed no obvious physical or behavioral defects. Analysis of bone marrow samples revealed a specific increase in a common myeloid progenitor cell population in ZASC1-deficient mice, a result that is of considerable interest because osteoclasts derived from the myeloid lineage are among the first bone marrow cells infected by Mo-MuLV (J. Virol. 73: 1617-1623, 1999). Indeed, Mo-MuLV infection of neonatal mice revealed that ZASC1 is required for efficient early virus replication in the bone marrow, but not in the thymus or spleen. However, the absence of ZASC1 did not influence the timing of subsequent tumor progression or the types of tumors resulting from virus infection. Conclusions: These studies have revealed that ZASC1 is important for myeloid cell differentiation in the bone marrow compartment and that this cellular factor is required for efficient Mo-MuLV replication in this tissue at an early time point post-infection. © 2013 Seidel et al.; licensee BioMed Central Ltd.

Research field(s)
Health Sciences, Biomedical Research, Virology

DOI
10.1186/1743-422X-10-130

Reprogramming toward heart regeneration: Stem cells and beyond

NOMIS Researcher(s)

Published in

March 7, 2013

Finding a cure for cardiovascular disease remains a major unmet medical need. Recent investigations have started to unveil the mechanisms of mammalian heart regeneration. The study of the regenerative mechanisms in lower vertebrate and mammalian animal models has provided clues for the experimental activation of proregenerative responses in the heart. In parallel, the use of endogenous adult stem cell populations alongside the recent application of reprogramming technologies has created major expectations for the development of therapies targeting heart disease. Together, these new approaches are bringing us closer to more successful strategies for the treatment of heart disease. © 2013 Elsevier Inc.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.stem.2013.02.008

Directed Differentiation of Human Pluripotent Cells to Ureteric Bud Kidney Progenitor-Like Cells

NOMIS Researcher(s)

Published in

January 1, 2013

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently, differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately, differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether, our results provide a new platform for the study of kidney diseases and lineage commitment, and open new avenues for the future application of regenerative strategies in the clinic. © 2013 Macmillan Publishers Limited. All rights reserved.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1038/ncb2872

TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons

NOMIS Researcher(s)

Published in

November 2, 2012

Nuclear clearance of TDP-43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but the mechanisms are unclear. Here, we show that TDP-43 knockdown specifically reduces the number and motility of RAB11-positive recycling endosomes in dendrites, while TDP-43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP-43-knockdown neurons and decreased β2-transferrin levels in patient CSF. Whole proteome quantification identified the upregulation of the ESCRT component VPS4B upon TDP-43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP-43 represses VPS4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP-43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP-43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP-43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP-43-induced neurodegeneration by blocking trophic signaling.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.15252/embj.201694221

Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase

NOMIS Researcher(s)

Published in

January 1, 2012

Background: Despite the effectiveness of highly active antiretroviral therapy (HAART), there remains an urgent need to develop new human immunodeficiency virus type 1 (HIV-1) inhibitors with better pharmacokinetic properties that are well tolerated, and that block common drug resistant virus strains. Methods: Here we screened an in-house small molecule library for novel inhibitors of HIV-1 replication. Results: An active compound containing a 3-aminoimidazo[1,2-a]pyridine scaffold was identified and quantitatively characterized as a non-nucleoside reverse transcriptase inhibitor (NNRTI). Conclusions: The potency of this compound coupled with its inexpensive chemical synthesis and tractability for downstream SAR analysis make this inhibitor a suitable lead candidate for further development as an antiviral drug.

Research field(s)
Health Sciences, Biomedical Research, Virology

DOI
10.1186/1743-422X-9-305

Differential requirements for clathrin endocytic pathway components in cellular entry by Ebola and Marburg glycoprotein pseudovirions

NOMIS Researcher(s)

Published in

October 10, 2011

Clathrin-mediated endocytosis was previously implicated as one of the cellular pathways involved in filoviral glycoprotein mediated viral entry into target cells. Here we have further dissected the requirements for different components of this pathway in Ebola versus Marburg virus glycoprotein (GP) mediated viral infection. Although a number of these components were involved in both cases; Ebola GP-dependent viral entry specifically required the cargo recognition proteins Eps15 and DAB2 as well as the clathrin adaptor protein AP-2. In contrast, Marburg GP-mediated infection was independent of these three proteins and instead required beta-arrestin 1 (ARRB1). These findings have revealed an unexpected difference between the clathrin pathway requirements for Ebola GP versus Marburg GP pseudovirion infection. Anthrax toxin also uses a clathrin-, and ARRB1-dependent pathway for cellular entry, indicating that the mechanism used by Marburg GP pseudovirions may be more generally important for pathogen entry. © 2011 Elsevier Inc.

Research field(s)
Health Sciences, Biomedical Research, Virology

DOI
10.1016/j.virol.2011.07.018

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