Publications by authors named "Malka Nissim-Rafinia"

26 Publications

  • Page 1 of 1

Pluripotent stem cell-derived models of neurological diseases reveal early transcriptional heterogeneity.

Genome Biol 2021 Mar 4;22(1):73. Epub 2021 Mar 4.

Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel.

Background: Many neurodegenerative diseases develop only later in life, when cells in the nervous system lose their structure or function. In many forms of neurodegenerative diseases, this late-onset phenomenon remains largely unexplained.

Results: Analyzing single-cell RNA sequencing from Alzheimer's disease (AD) and Huntington's disease (HD) patients, we find increased transcriptional heterogeneity in disease-state neurons. We hypothesize that transcriptional heterogeneity precedes neurodegenerative disease pathologies. To test this idea experimentally, we use juvenile forms (72Q; 180Q) of HD iPSCs, differentiate them into committed neuronal progenitors, and obtain single-cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking down several differentially variable genes results in increased aggregate formation, a pathology associated with HD. We further validate the increased transcriptional heterogeneity in CHD8+/- cells, a model for autism spectrum disorder.

Conclusions: Overall, our results suggest that although neurodegenerative diseases develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value.
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http://dx.doi.org/10.1186/s13059-021-02301-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934477PMC
March 2021

Embryonic Stem Cell Differentiation Is Regulated by SET through Interactions with p53 and β-Catenin.

Stem Cell Reports 2020 12;15(6):1260-1274

Department of Genetics, The Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel; The Edmond and Lily Safra Center for Brain Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel. Electronic address:

The multifunctional histone chaperone, SET, is essential for embryonic development in the mouse. Previously, we identified SET as a factor that is rapidly downregulated during embryonic stem cell (ESC) differentiation, suggesting a possible role in the maintenance of pluripotency. Here, we explore SET's function in early differentiation. Using immunoprecipitation coupled with protein quantitation by LC-MS/MS, we uncover factors and complexes, including P53 and β-catenin, by which SET regulates lineage specification. Knockdown for P53 in SET-knockout (KO) ESCs partially rescues lineage marker misregulation during differentiation. Paradoxically, SET-KO ESCs show increased expression of several Wnt target genes despite reduced levels of active β-catenin. Further analysis of RNA sequencing datasets hints at a co-regulatory relationship between SET and TCF proteins, terminal effectors of Wnt signaling. Overall, we discover a role for both P53 and β-catenin in SET-regulated early differentiation and raise a hypothesis for SET function at the β-catenin-TCF regulatory axis.
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http://dx.doi.org/10.1016/j.stemcr.2020.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724474PMC
December 2020

The Chromatin Regulator ZMYM2 Restricts Human Pluripotent Stem Cell Growth and Is Essential for Teratoma Formation.

Stem Cell Reports 2020 12 18;15(6):1275-1286. Epub 2020 Jun 18.

The Azrieli Center for Stem Cells and Genetic Research, The Hebrew University, Jerusalem, Israel; Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel. Electronic address:

Chromatin regulators play fundamental roles in controlling pluripotency and differentiation. We examined the effect of mutations in 703 genes from nearly 70 chromatin-modifying complexes on human embryonic stem cell (ESC) growth. While the vast majority of chromatin-associated complexes are essential for ESC growth, the only complexes that conferred growth advantage upon mutation of their members, were the repressive complexes LSD-CoREST and BHC. Both complexes include the most potent growth-restricting chromatin-related protein, ZMYM2. Interestingly, while ZMYM2 expression is rather low in human blastocysts, its expression peaks in primed ESCs and is again downregulated upon differentiation. ZMYM2-null ESCs overexpress pluripotency genes and show genome-wide promotor-localized histone H3 hyper-acetylation. These mutant cells were also refractory to differentiate in vitro and failed to produce teratomas upon injection into immunodeficient mice. Our results suggest a central role for ZMYM2 in the transcriptional regulation of the undifferentiated state and in the exit-from-pluripotency of human ESCs.
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http://dx.doi.org/10.1016/j.stemcr.2020.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724477PMC
December 2020

Differential DNA methylation of vocal and facial anatomy genes in modern humans.

Nat Commun 2020 03 4;11(1):1189. Epub 2020 Mar 4.

Department of Genetics, The Alexander Silberman Institute of Life Sciences, Faculty of Science, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel.

Changes in potential regulatory elements are thought to be key drivers of phenotypic divergence. However, identifying changes to regulatory elements that underlie human-specific traits has proven very challenging. Here, we use 63 reconstructed and experimentally measured DNA methylation maps of ancient and present-day humans, as well as of six chimpanzees, to detect differentially methylated regions that likely emerged in modern humans after the split from Neanderthals and Denisovans. We show that genes associated with face and vocal tract anatomy went through particularly extensive methylation changes. Specifically, we identify widespread hypermethylation in a network of face- and voice-associated genes (SOX9, ACAN, COL2A1, NFIX and XYLT1). We propose that these repression patterns appeared after the split from Neanderthals and Denisovans, and that they might have played a key role in shaping the modern human face and vocal tract.
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http://dx.doi.org/10.1038/s41467-020-15020-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055320PMC
March 2020

Correction to: Progerin-Induced Transcriptional Changes in Huntington's Disease Human Pluripotent Stem Cell-Derived Neurons.

Mol Neurobiol 2020 03;57(3):1778

Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel.

In the original version of the paper, the name of one of the contributing authors, Dr. Mundackal S. Divya (orcid:0000-0002-2869-7191).
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http://dx.doi.org/10.1007/s12035-020-01877-7DOI Listing
March 2020

Progerin-Induced Transcriptional Changes in Huntington's Disease Human Pluripotent Stem Cell-Derived Neurons.

Mol Neurobiol 2020 Mar 13;57(3):1768-1777. Epub 2019 Dec 13.

Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel.

Huntington's disease (HD) is a neurodegenerative late-onset genetic disorder caused by CAG expansions in the coding region of the Huntingtin (HTT) gene, resulting in a poly-glutamine (polyQ) expanded HTT protein. Considerable efforts have been devoted for studying HD and other polyQ diseases using animal models and cell culture systems, but no treatment currently exists. Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer an elegant solution for modeling human diseases. However, as embryonic or rejuvenated cells, respectively, these pluripotent stem cells (PSCs) do not recapitulate the late-onset feature of the disease. Here, we applied a robust and rapid differentiation protocol to derive electrophysiologically active striatal GABAergic neurons from human wild-type (WT) and HD ESCs and iPSCs. RNA-seq analyses revealed that HD and WT PSC-derived neurons are highly similar in their gene expression patterns. Interestingly, ectopic expression of Progerin in both WT and HD neurons exacerbated the otherwise non-significant changes in gene expression between these cells, revealing IGF1 and genes involved in neurogenesis and nervous system development as consistently altered in the HD cells. This work provides a useful tool for modeling HD in human PSCs and reveals potential molecular targets altered in HD neurons.
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http://dx.doi.org/10.1007/s12035-019-01839-8DOI Listing
March 2020

AUTS2 isoforms control neuronal differentiation.

Mol Psychiatry 2021 02 5;26(2):666-681. Epub 2019 Apr 5.

Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.

Mutations in AUTS2 are associated with autism, intellectual disability, and microcephaly. AUTS2 is expressed in the brain and interacts with polycomb proteins, yet it is still unclear how mutations in AUTS2 lead to neurodevelopmental phenotypes. Here we report that when neuronal differentiation is initiated, there is a shift in expression from a long isoform to a short AUTS2 isoform. Yeast two-hybrid screen identified the splicing factor SF3B1 as an interactor of both isoforms, whereas the polycomb group proteins, PCGF3 and PCGF5, were found to interact exclusively with the long AUTS2 isoform. Reporter assays showed that the first exons of the long AUTS2 isoform function as a transcription repressor, but the part that consist of the short isoform acts as a transcriptional activator, both influenced by the cellular context. The expression levels of PCGF3 influenced the ability of the long AUTS2 isoform to activate or repress transcription. Mouse embryonic stem cells (mESCs) with heterozygote mutations in Auts2 had an increase in cell death during in vitro corticogenesis, which was significantly rescued by overexpressing the human AUTS2 transcripts. mESCs with a truncated AUTS2 protein (missing exons 12-20) showed premature neuronal differentiation, whereas cells overexpressing AUTS2, especially the long transcript, showed increase in expression of pluripotency markers and delayed differentiation. Taken together, our data suggest that the precise expression of AUTS2 isoforms is essential for regulating transcription and the timing of neuronal differentiation.
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http://dx.doi.org/10.1038/s41380-019-0409-1DOI Listing
February 2021

Spironolactone inhibits the growth of cancer stem cells by impairing DNA damage response.

Oncogene 2019 04 8;38(17):3103-3118. Epub 2019 Jan 8.

Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem, 9190401, Israel.

The cancer stem cell (CSC) model suggests that a subpopulation of cells within the tumor, the CSCs, is responsible for cancer relapse and metastasis formation. CSCs hold unique characteristics, such as self-renewal, differentiation abilities, and resistance to chemotherapy, raising the need for discovering drugs that target CSCs. Previously we have found that the antihypertensive drug spironolactone impairs DNA damage response in cancer cells. Here we show that spironolactone, apart from inhibiting cancerous cell growth, is also highly toxic to CSCs. Notably, we demonstrate that CSCs have high basal levels of DNA double-strand breaks (DSBs). Mechanistically, we reveal that spironolactone does not damage the DNA but impairs DSB repair and induces apoptosis in cancer cells and CSCs while sparing healthy cells. In vivo, spironolactone treatment reduced the size and CSC content of tumors. Overall, we suggest spironolactone as an anticancer reagent, toxic to both cancer cells and, particularly to, CSCs.
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http://dx.doi.org/10.1038/s41388-018-0654-9DOI Listing
April 2019

PARP1-dependent eviction of the linker histone H1 mediates immediate early gene expression during neuronal activation.

J Cell Biol 2018 02 28;217(2):473-481. Epub 2017 Dec 28.

Department of Genetics, The Institute of Life Sciences and The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel

Neuronal stimulation leads to immediate early gene (IEG) expression through calcium-dependent mechanisms. In recent years, considerable attention has been devoted to the transcriptional responses after neuronal stimulation, but relatively little is known about the changes in chromatin dynamics that follow neuronal activation. Here, we use fluorescence recovery after photobleaching, biochemical fractionations, and chromatin immunoprecipitation to show that KCl-induced depolarization in primary cultured cortical neurons causes a rapid release of the linker histone H1 from chromatin, concomitant with IEG expression. H1 release is repressed by PARP inhibition, PARP1 deletion, a non-PARylatable H1, as well as phosphorylation inhibitions and a nonphosphorylatable H1, leading to hindered IEG expression. Further, H1 is replaced by PARP1 on IEG promoters after neuronal stimulation, and PARP inhibition blocks this reciprocal binding response. Our results demonstrate the relationship between neuronal excitation and chromatin plasticity by identifying the roles of polyadenosine diphosphate ribosylation and phosphorylation of H1 in regulating H1 chromatin eviction and IEG expression in stimulated neurons.
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http://dx.doi.org/10.1083/jcb.201703141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800798PMC
February 2018

Alternative SET/TAFI Promoters Regulate Embryonic Stem Cell Differentiation.

Stem Cell Reports 2017 10 28;9(4):1291-1303. Epub 2017 Sep 28.

The Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem 91904, Israel; The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. Electronic address:

Embryonic stem cells (ESCs) are regulated by pluripotency-related transcription factors in concert with chromatin regulators. To identify additional stem cell regulators, we screened a library of endogenously labeled fluorescent fusion proteins in mouse ESCs for fluorescence loss during differentiation. We identified SET, which displayed a rapid isoform shift during early differentiation from the predominant isoform in ESCs, SETα, to the primary isoform in differentiated cells, SETβ, through alternative promoters. SETα is selectively bound and regulated by pluripotency factors. SET depletion causes proliferation slowdown and perturbed neuronal differentiation in vitro and developmental arrest in vivo, and photobleaching methods demonstrate SET's role in maintaining a dynamic chromatin state in ESCs. This work identifies an important regulator of pluripotency and early differentiation, which is controlled by alternative promoter usage.
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http://dx.doi.org/10.1016/j.stemcr.2017.08.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639460PMC
October 2017

Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells.

Genome Biol 2015 Sep 28;16:213. Epub 2015 Sep 28.

Department of Genetics, The Institute of Life Science and The Edmond and Lily Center for Brain Sciences The Hebrew University of Jerusalem, Jerusalem, 91904, Israel.

Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown.

Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1α, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters.

Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state.
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http://dx.doi.org/10.1186/s13059-015-0760-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587738PMC
September 2015

Differential association of chromatin proteins identifies BAF60a/SMARCD1 as a regulator of embryonic stem cell differentiation.

Cell Rep 2015 Mar 26;10(12):2019-31. Epub 2015 Mar 26.

Department of Genetics, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. Electronic address:

Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.
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http://dx.doi.org/10.1016/j.celrep.2015.02.064DOI Listing
March 2015

Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells.

Cell Metab 2015 Mar;21(3):392-402

Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem 9190401, Israel; Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 9190401, Israel. Electronic address:

Loss of pluripotency is a gradual event whose initiating factors are largely unknown. Here we report the earliest metabolic changes induced during the first hours of differentiation. High-resolution NMR identified 44 metabolites and a distinct metabolic transition occurring during early differentiation. Metabolic and transcriptional analyses showed that pluripotent cells produced acetyl-CoA through glycolysis and rapidly lost this function during differentiation. Importantly, modulation of glycolysis blocked histone deacetylation and differentiation in human and mouse embryonic stem cells. Acetate, a precursor of acetyl-CoA, delayed differentiation and blocked early histone deacetylation in a dose-dependent manner. Inhibitors upstream of acetyl-CoA caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our results show a metabolic switch causing a loss of histone acetylation and pluripotent state during the first hours of differentiation. Our data highlight the important role metabolism plays in pluripotency and suggest that a glycolytic switch controlling histone acetylation can release stem cells from pluripotency.
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http://dx.doi.org/10.1016/j.cmet.2015.02.002DOI Listing
March 2015

HP1 is involved in regulating the global impact of DNA methylation on alternative splicing.

Cell Rep 2015 Feb 19;10(7):1122-34. Epub 2015 Feb 19.

Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel. Electronic address:

The global impact of DNA methylation on alternative splicing is largely unknown. Using a genome-wide approach in wild-type and methylation-deficient embryonic stem cells, we found that DNA methylation can either enhance or silence exon recognition and affects the splicing of more than 20% of alternative exons. These exons are characterized by distinct genetic and epigenetic signatures. Alternative splicing regulation of a subset of these exons can be explained by heterochromatin protein 1 (HP1), which silences or enhances exon recognition in a position-dependent manner. We constructed an experimental system using site-specific targeting of a methylated/unmethylated gene and demonstrate a direct causal relationship between DNA methylation and alternative splicing. HP1 regulates this gene's alternative splicing in a methylation-dependent manner by recruiting splicing factors to its methylated form. Our results demonstrate DNA methylation's significant global influence on mRNA splicing and identify a specific mechanism of splicing regulation mediated by HP1.
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http://dx.doi.org/10.1016/j.celrep.2015.01.038DOI Listing
February 2015

Non-polyadenylated transcription in embryonic stem cells reveals novel non-coding RNA related to pluripotency and differentiation.

Nucleic Acids Res 2013 Jul 29;41(12):6300-15. Epub 2013 Apr 29.

Hebrew University of Jerusalem, Jerusalem 91904, Israel.

The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention, albeit mostly confined to the polyadenylated fraction of RNA, whereas the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in ESCs using a combination of whole-genome tiling arrays and deep sequencing technologies. In addition to identifying previously characterized and new non-coding RNA members, we describe a group of novel conserved RNAs (snacRNAs: small NPA conserved), some of which are differentially expressed between ESC and neuronal progenitor cells, providing the first evidence of a novel group of potentially functional NPA RNA involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal small nuclear RNAs, which are NPA, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that NPA RNA, both known and novel, play important roles in ESCs.
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http://dx.doi.org/10.1093/nar/gkt316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695530PMC
July 2013

Histone modifications and lamin A regulate chromatin protein dynamics in early embryonic stem cell differentiation.

Nat Commun 2012 Jun 19;3:910. Epub 2012 Jun 19.

Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra campus 91904, Israel.

Embryonic stem cells are characterized by unique epigenetic features including decondensed chromatin and hyperdynamic association of chromatin proteins with chromatin. Here we investigate the potential mechanisms that regulate chromatin plasticity in embryonic stem cells. Using epigenetic drugs and mutant embryonic stem cells lacking various chromatin proteins, we find that histone acetylation, G9a-mediated histone H3 lysine 9 (H3K9) methylation and lamin A expression, all affect chromatin protein dynamics. Histone acetylation controls, almost exclusively, euchromatin protein dynamics; lamin A expression regulates heterochromatin protein dynamics, and G9a regulates both euchromatin and heterochromatin protein dynamics. In contrast, we find that DNA methylation and nucleosome repeat length have little or no effect on chromatin-binding protein dynamics in embryonic stem cells. Altered chromatin dynamics associates with perturbed embryonic stem cell differentiation. Together, these data provide mechanistic insights into the epigenetic pathways that are responsible for chromatin plasticity in embryonic stem cells, and indicate that the genome's epigenetic state modulates chromatin plasticity and differentiation potential of embryonic stem cells.
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http://dx.doi.org/10.1038/ncomms1915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564597PMC
June 2012

H3K9 histone acetylation predicts pluripotency and reprogramming capacity of ES cells.

Nucleus 2011 Jul-Aug;2(4):300-9. Epub 2011 Jul 1.

Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra campus, Jerusalem, Israel.

The pluripotent genome is characterized by unique epigenetic features and a decondensed chromatin conformation. However, the relationship between epigenetic regulation and pluripotency is not altogether clear. Here, using an enhanced MEF/ESC fusion protocol, we compared the reprogramming potency and histone modifications of different embryonic stem cell (ESC) lines (R1, J1, E14, C57BL/6) and found that E14 ESCs are significantly less potent, with significantly reduced H3K9ac levels. Treatment of E14 ESCs with histone deacetylase (HDAC) inhibitors (HDACi) increased H3K9ac levels and restored their reprogramming capacity. Microarray and H3K9ac ChIP-seq analyses, suggested increased extracellular matrix (ECM) activity following HDACi treatment in E14 ESCs. These data suggest that H3K9ac may predict pluripotency and that increasing pluripotency by HDAC inhibition acts through H3K9ac to enhance the activity of target genes involved in ECM production to support pluripotency.
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http://dx.doi.org/10.4161/nucl.2.4.16767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3260568PMC
January 2013

Photobleaching assays (FRAP & FLIP) to measure chromatin protein dynamics in living embryonic stem cells.

J Vis Exp 2011 Jun 29(52). Epub 2011 Jun 29.

Department of Genetics, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem.

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression¹. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells. ² (,)³
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http://dx.doi.org/10.3791/2696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197042PMC
June 2011

Nuclear lamins: key regulators of nuclear structure and activities.

J Cell Mol Med 2009 Jun 4;13(6):1059-85. Epub 2009 Feb 4.

Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Israel.

The nuclear lamina is a proteinaceous structure located underneath the inner nuclear membrane (INM), where it associates with the peripheral chromatin. It contains lamins and lamin-associated proteins, including many integral proteins of the INM, chromatin modifying proteins, transcriptional repressors and structural proteins. A fraction of lamins is also present in the nucleoplasm, where it forms stable complexes and is associated with specific nucleoplasmic proteins. The lamins and their associated proteins are required for most nuclear activities, mitosis and for linking the nucleoplasm to all major cytoskeletal networks in the cytoplasm. Mutations in nuclear lamins and their associated proteins cause about 20 different diseases that are collectively called laminopathies'. This review concentrates mainly on lamins, their structure and their roles in DNA replication, chromatin organization, adult stem cell differentiation, aging, tumorogenesis and the lamin mutations leading to laminopathic diseases.
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http://dx.doi.org/10.1111/j.1582-4934.2008.00676.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496104PMC
June 2009

Stem cells do play with dice: a statistical physics view of transcription.

Cell Cycle 2009 Jan 16;8(1):43-8. Epub 2009 Jan 16.

The Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.

Embryonic stem cells display wide-spread pervasive transcriptional output. Here, we propose that multiple simultaneous transcriptional states underlay pluripotency.
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http://dx.doi.org/10.4161/cc.8.1.7216DOI Listing
January 2009

Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial.

Lancet 2008 Aug 20;372(9640):719-27. Epub 2008 Aug 20.

Hadassah Hebrew University Hospital, Jerusalem, Israel.

Background: In about 10% of patients worldwide and more than 50% of patients in Israel, cystic fibrosis results from nonsense mutations (premature stop codons) in the messenger RNA (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR). PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR.

Methods: This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. The primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. The trial was registered with who.int/ictrp, and with clinicaltrials.gov, number NCT00237380.

Findings: Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle. Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p<0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). We recorded a response in total chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycle's treatment phase (p<0.0001) and in eight of the 21 patients in the second cycle (p<0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycle's treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). Two patients given PTC124 had constipation without intestinal obstruction, and four had mild dysuria. No drug-related serious adverse events were recorded.

Interpretation: In patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction.
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http://dx.doi.org/10.1016/S0140-6736(08)61168-XDOI Listing
August 2008

Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin.

J Clin Invest 2007 Mar 8;117(3):683-92. Epub 2007 Feb 8.

Department of Genetics, Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, Israel.

Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here we demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS. Importantly, our results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. Together our results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.
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http://dx.doi.org/10.1172/JCI28523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1783999PMC
March 2007

Splicing modulation as a modifier of the CFTR function.

Prog Mol Subcell Biol 2006 ;44:233-54

Department of Genetics, Life Sciences Institute, Hebrew University, Jerusalem 91904, Israel.

A significant fraction of CF-causing mutations affects pre-mRNA splicing. These mutations can generate both aberrant and correct transcripts, the level of which varies among different patients. An inverse correlation was found between this level and disease severity, suggesting a role for splicing regulation as a genetic modifier. Subsequent studies showed that overexpression of splicing factors modulated the level of correctly spliced RNA, transcribed from minigenes carrying CF-causing splicing mutations. Overexpression of splicing factors also modulated the level of normal CFTR transcripts, transcribed from the endogenous CFTR allele carrying splicing mutations, in CF-derived epithelial cells. Several of the factors promoted higher level of correct CFTR transcripts. The increased level of normal transcripts led to activation of the CFTR channel and restoration of its function. Restoration was also obtained by sodium butyrate, a histone deacetylase inhibitor, known to up-regulate the expression of splicing factors. These results highlight the role of the splicing machinery as a modifier of disease severity in patients carrying splicing mutations and shed a new light on the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.
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http://dx.doi.org/10.1007/978-3-540-34449-0_10DOI Listing
January 2007

The splicing machinery is a genetic modifier of disease severity.

Trends Genet 2005 Sep;21(9):480-3

Department of Genetics, The Life Sciences Institute, The Hebrew University, Jerusalem, Israel.

Disease severity correlates with the level of correctly spliced RNA transcribed from genes carrying splicing mutations and with the ratio of alternatively spliced isoforms. Hence, a role for splicing regulation as a genetic modifier has been suggested. Here we discuss recent experiments that provide direct evidence that changes in the level of splicing factors modulate the splicing pattern of disease-associated genes. Importantly, modulation of the splicing pattern led to regulation of the protein function and modification of disease severity.
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http://dx.doi.org/10.1016/j.tig.2005.07.005DOI Listing
September 2005

Restoration of the cystic fibrosis transmembrane conductance regulator function by splicing modulation.

EMBO Rep 2004 Nov;5(11):1071-7

Department of Genetics, Life Sciences Institute, Hebrew University, Jerusalem 91904, Israel.

A significant fraction of disease-causing mutations affects pre-mRNA splicing. These mutations can generate both aberrant and correct transcripts, the level of which varies among different patients. An inverse correlation was found between this level and disease severity, suggesting a role for splicing regulation as a genetic modifier. Overexpression of splicing factors increased the level of correctly spliced RNA, transcribed from minigenes carrying disease-causing splicing mutations. However, whether this increase could restore the protein function was unknown. Here, we demonstrate that overexpression of Htra2-beta1 and SC35 increases the level of normal cystic fibrosis transmembrane conductance regulator (CFTR) transcripts in cystic-fibrosis-derived epithelial cells carrying the 3849+10 kb C --> T splicing mutation. This led to activation of the CFTR channel and restoration of its function. Restoration was also obtained by sodium butyrate, a histone deacetylase inhibitor, known to upregulate the expression of splicing factors. These results highlight the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.
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http://dx.doi.org/10.1038/sj.embor.7400273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299168PMC
November 2004

Splicing regulation as a potential genetic modifier.

Trends Genet 2002 Mar;18(3):123-7

Dept of Genetics, The Life Sciences Institute, The Hebrew University, 91904, Jerusalem, Israel.

Inherited diseases are associated with profound phenotypic variability, which is affected strongly by genetic modifiers. The splicing machinery could be one such modifying system, through a mechanism involving splicing motifs and their interaction with a complex repertoire of splicing factors. Mutations in splicing motifs and changes in levels of splicing factors can result in different splicing patterns. Changes in the level of normal transcripts or in the relative pattern of different mRNA isoforms affect disease expression, leading to phenotypic variability. Here, we discuss the splicing machinery in terms of its significance in disease severity and its potential role as a genetic modifier.
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http://dx.doi.org/10.1016/s0168-9525(01)02619-1DOI Listing
March 2002