Samer Hussein - Academia.edu (original) (raw)

Papers by Samer Hussein

ABSTRACTMRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain re... more ABSTRACTMRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain recognizing H3K36me3 in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing and DNA repair by homologous recombination. In order to get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the chromodomain, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase and PALB2/BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR-tagging of endogenou...

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Frontiers in Cell and Developmental Biology

Nucleic Acids Research

Src associated in mitosis (SAM68) plays major roles in regulating RNA processing events, such as ... more Src associated in mitosis (SAM68) plays major roles in regulating RNA processing events, such as alternative splicing and mRNA translation, implicated in several developmental processes. It was previously shown that SAM68 regulates the alternative splicing of the mechanistic target of rapamycin (mTor), but the mechanism regulating this process remains elusive. Here, we report that SAM68 interacts with U1 small nuclear ribonucleoprotein (U1 snRNP) to promote splicing at the 5 splice site in intron 5 of mTor. We also show that this direct interaction is mediated through U1A, a core-component of U1snRNP. SAM68 was found to bind the RRM1 domain of U1A through its C-terminal tyrosine rich region (YY domain). Deletion of the U1A-SAM68 interaction domain or mutation in SAM68-binding sites in intron 5 of mTor abrogates U1A recruitment and 5 splice site recognition by the U1 snRNP, leading to premature intron 5 termination and polyadenylation. Taken together, our results provide the first mechanistic study by which SAM68 modulates alternative splicing decision, by affecting U1 snRNP recruitment at 5 splice sites.

Cell research, Jan 17, 2017

Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefi... more Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefits equivalent to prolonged fasting or caloric restriction. However, our understanding of the underlying mechanisms of IF-mediated metabolic benefits is limited. Here we show that isocaloric IF improves metabolic homeostasis against diet-induced obesity and metabolic dysfunction primarily through adipose thermogenesis in mice. IF-induced metabolic benefits require fasting-mediated increases of vascular endothelial growth factor (VEGF) expression in white adipose tissue (WAT). Furthermore, periodic adipose-VEGF overexpression could recapitulate the metabolic improvement of IF in non-fasted animals. Importantly, fasting and adipose-VEGF induce alternative activation of adipose macrophage, which is critical for thermogenesis. Human adipose gene analysis further revealed a positive correlation of adipose VEGF-M2 macrophage-WAT browning axis. The present study uncovers the molecular mechanism ...

Can J Chem, 2000

Acid-catalyzed hydrolysis of acetophenone dimethyl acetal (ADMA) and trimethyl orthobenzoate (TMO... more Acid-catalyzed hydrolysis of acetophenone dimethyl acetal (ADMA) and trimethyl orthobenzoate (TMOB) is retarded by cyclodextrins (CDs): α-CD, β-CD, hb-β-CD = "hydroxypropyl-β-cyclodextrin", and γ-CD. The observed first order rate constants (kobs) vary with [CD] in the manner expected for 1:1 binding between the substrates and the CDs. Similar behaviour was found recently for the hydrolysis of benzaldehyde dimethyl acetal (BDMA). With β-CD and hp-β-CD, the binding of all three substrates (BDMA, ADMA, TMOB) is strong and the CD-bound forms have very little reactivity. By contrast, substrate binding by α-CD is much weaker, and the CD-bound forms have appreciable, though reduced, reactivity. Substrate binding by γ-CD is also relatively weak, but the bound substrates have very low reactivities. The hydrolysis reactions of ADMA, TMOB, and BDMA have been evaluated as kinetic probes of the binding of guests to CD hosts. For α-CD, β-CD, and hp-β-CD, the addition of guests reduces the amount of free CD and thereby alleviates retardation of the hydrolysis by the CD. The resultant increases in hydrolysis rates can be analyzed to provide estimates of CD-guest dissociation constants, KG. For aliphatic alcohols and ketones binding to β-CD and hp-β-CD, all three probe reactions provide values of KG that agree well with each other and with literature values determined by other methods. The approach does not work well with α-CD because of its much weaker binding of the kinetic probes and their less pronounced dependence of kobs on [α-CD]. In the case of γ;-CD, the approach cannot be used because added guests cause a further lowering in the rate of hydrolysis, suggesting the formation of an unreactive ternary (substrate·CD·guest) complex.Key words: acetal, hydrolysis, cyclodextrin, host-guest, binding.

Scientific reports, Jan 20, 2016

The ventricular conduction system (VCS) orchestrates the harmonious contraction in every heartbea... more The ventricular conduction system (VCS) orchestrates the harmonious contraction in every heartbeat. Defects in the VCS are often associated with life-threatening arrhythmias and also promote adverse remodeling in heart disease. We have previously established that the Irx3 homeobox gene regulates rapid electrical propagation in the VCS by modulating the transcription of gap junction proteins Cx40 and Cx43. However, it is unknown whether other factors contribute to the conduction defects observed in Irx3 knockout (Irx3(-/-)) mice. In this study, we show that during the early postnatal period, Irx3(-/-) mice develop morphological defects in the VCS which are temporally dissociated from changes in gap junction expression. These morphological defects were accompanied with progressive changes in the cardiac electrocardiogram including right bundle branch block. Hypoplastic VCS was not associated with increased apoptosis of VCS cardiomyocytes but with a lack of recruitment and maturation o...

Nature, Jan 17, 2015

In this Article, the address listed as Nicole Cloonan's present address (QIMR Berghofer Medical R... more In this Article, the address listed as Nicole Cloonan's present address (QIMR Berghofer Medical Research Institute, Queensland 4006, Australia) should have been listed as her other affiliation with superscript 16, because the work she did was split equally between the two institutions. This has been corrected in the online versions of the paper.

BioData Mining, 2014

Background: Human genomic variations, including single nucleotide polymorphisms (SNPs) and copy n... more Background: Human genomic variations, including single nucleotide polymorphisms (SNPs) and copy number variations (CNVs), are associated with several phenotypic traits varying from mild features to hereditary diseases. Several genome-wide studies have reported genomic variants that correlate with gene expression levels in various tissue and cell types. Results: We studied human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) measuring the SNPs and CNVs with Affymetrix SNP 6 microarrays and expression values with Affymetrix Exon microarrays. We computed the linear relationships between SNPs and expression levels of exons, transcripts and genes, and the associations between gene CNVs and gene expression levels. Further, for a few of the resulted genes, the expression value was associated with both CNVs and SNPs. Our results revealed altogether 217 genes and 584 SNPs whose genomic alterations affect the transcriptome in the same cells. We analyzed the enriched pathways and gene ontologies within these groups of genes, and found out that the terms related to alternative splicing and development were enriched. Conclusions: Our results revealed that in the human pluripotent stem cells, the expression values of several genes, transcripts and exons were affected due to the genomic variation.

PLoS ONE, 2012

The kidney filtration barrier is formed by the combination of endothelial cells, basement membran... more The kidney filtration barrier is formed by the combination of endothelial cells, basement membrane and epithelial cells called podocytes. These specialized actin-rich cells form long and dynamic protrusions, the foot processes, which surround glomerular capillaries and are connected by specialized intercellular junctions, the slit diaphragms. Failure to maintain the filtration barrier leads to massive proteinuria and nephrosis. A number of proteins reside in the slit diaphragm, notably the transmembrane proteins Nephrin and Neph1, which are both able to act as tyrosine phosphorylated scaffolds that recruit cytoplasmic effectors to initiate downstream signaling. While association between tyrosine-phosphorylated Neph1 and the SH2/SH3 adaptor Grb2 was shown in vitro to be sufficient to induce actin polymerization, in vivo evidence supporting this finding is still lacking. To test this hypothesis, we generated two independent mouse lines bearing a podocyte-specific constitutive inactivation of the Grb2 locus. Surprisingly, we show that mice lacking Grb2 in podocytes display normal renal ultra-structure and function, thus demonstrating that Grb2 is not required for the establishment of the glomerular filtration barrier in vivo. Moreover, our data indicate that Grb2 is not required to restore podocyte function following kidney injury. Therefore, although in vitro experiments suggested that Grb2 is important for the regulation of actin dynamics, our data clearly shows that its function is not essential in podocytes in vivo, thus suggesting that Grb2 rather plays a secondary role in this process.

Nature, 2014

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the t... more Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Nature Communications, 2014

Reprogramming of somatic cells to induced pluripotent stem cells involves a dynamic rearrangement... more Reprogramming of somatic cells to induced pluripotent stem cells involves a dynamic rearrangement of the epigenetic landscape. To characterize this epigenomic roadmap, we have performed MethylC-seq, ChIP-seq (H3K4/K27/K36me3) and RNA-Seq on samples taken at several time points during murine secondary reprogramming as part of Project Grandiose. We find that DNA methylation gain during reprogramming occurs gradually, while loss is achieved only at the ESC-like state. Binding sites of activated factors exhibit focal demethylation during reprogramming, while ESC-like pluripotent cells are distinguished by extension of demethylation to the wider neighbourhood. We observed that genes with CpG-rich promoters demonstrate stable low methylation and strong engagement of histone marks, whereas genes with CpG-poor promoters are safeguarded by methylation. Such DNA methylation-driven control is the key to the regulation of ESC-pluripotency genes, including Dppa4, Dppa5a and Esrrb. These results reveal the crucial role that DNA methylation plays as an epigenetic switch driving somatic cells to pluripotency.

Nature Communications, 2014

MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells... more MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells (iPSCs), however, exactly how miRNA expression changes support the transition to pluripotency requires further investigation. Here we use a murine secondary reprogramming system to sample cellular trajectories towards iPSCs or a novel pluripotent 'F-class' state and perform small RNA sequencing. We detect sweeping changes in an early and a late wave, revealing that distinct miRNA milieus characterize alternate states of pluripotency. miRNA isoform expression is common but surprisingly varies little between cell states. Referencing other omic data sets generated in parallel, we find that miRNA expression is changed through transcriptional and post-transcriptional mechanisms. miRNA transcription is commonly regulated by dynamic histone modification, while DNA methylation/demethylation consolidates these changes at multiple loci. Importantly, our results suggest that a novel subset of distinctly expressed miRNAs supports pluripotency in the F-class state, substituting for miRNAs that serve such roles in iPSCs.

Nature Communications, 2014

The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogra... more The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quantitative mass spectrometry-based analysis to probe in-depth dynamic proteome changes during somatic cell reprogramming. Our data reveal defined waves of proteome resetting, with the first wave occurring 48 h after the activation of the reprogramming transgenes and involving specific biological processes linked to the c-Myc transcriptional network. A second wave of proteome reorganization occurs in a later stage of reprogramming, where we characterize the proteome of two distinct pluripotent cellular populations. In addition, the overlay of our proteome resource with parallel generated-omics data is explored to identify post-transcriptionally regulated proteins involved in key steps during reprogramming.

Genes, Chromosomes and Cancer, 2013

Copy number changes or reduced expression of the Neuron navigator 3 (NAV3) gene occurs in neurobl... more Copy number changes or reduced expression of the Neuron navigator 3 (NAV3) gene occurs in neuroblastomas and malignancies of epithelial or lymphoid origin. To elucidate whether NAV3 has a role in the tumorigenesis of nervous system tumors in general, we studied central and peripheral nervous system tumors for NAV3 copy number changes. In search for common tumorigenic denominators, we analyzed 113 central and peripheral nervous system tumors, including glial tumors (grades I-IV gliomas), medulloblastomas, and neuroblastomas. NAV3 copy number changes were studied by fluorescence in situ hybridization and correlated to survival analyses. To identify target genes of NAV3 deletion, NAV3 was silenced by siRNA in glioblastoma cell lines and gene expression profiles were analyzed by Agilent 4×44k dual-color microarrays. Selected upregulations were confirmed by immunohistochemistry and quantitative polymerase chain reaction. We found NAV3 amplifications to dominate in neuronally differentiated tumors, whereas glial tumors showed almost equal proportions of NAV3 deletion and amplification. However, Grade IV gliomas had more frequent NAV3 deletions than grades I-III gliomas. Silencing of NAV3 in glioma cell lines led to the upregulation of receptor genes associated with gonadotropin-releasing hormone and Jak-Stat signaling pathways. Kaplan-Meier analysis of the entire clinical tumor material showed association between NAV3 amplifications and favorable prognosis, as well as NAV3 deletions and unfavorable prognosis. With Cox regression model, a hazard ratio of 0.51 was observed for NAV3 amplifications and 1.36 for NAV3 deletions. We conclude that NAV3 may be a potential new prognostic biomarker and a potential therapeutic target.

EMBO Molecular Medicine, 2014

Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiog... more Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor-A (VEGF) biologics, termed VEGF "Sticky-traps," with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky-traps could locally inhibit angiogenesis to at least the same extent as the original VEGF-trap that also gains whole-body access. Stickytraps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky-trap remained localized to various regions of the eye, such as the inner-limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky-trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre-maturity.

Current Opinion in Genetics & Development, 2012

The ground-breaking work of Yamanaka and Thomson showed that forced expression of just four trans... more The ground-breaking work of Yamanaka and Thomson showed that forced expression of just four transcription factors can reprogram mouse and human somatic cells to pluripotency, leading to the discovery of the so-called induced pluripotent stem cells (iPSCs). Similar to embryonic stem cells (ESCs), iPSCs have the ability to permanently self-renew and also give rise to multiple cell types once differentiated. These cells opened up the opportunity to develop human disease models in vitro, drug and toxicity screening tools, as well as a continuous autologous cell source for future cell-based therapies. Therefore, it is not surprising that the methods for generating iPSCs have significantly evolved over the past few years. To date the reprogramming methods include the use of various transfection/transduction systems, small molecules to enhance the reprogramming process, and to adapt to a multitude of different cell type sources. We are now able to convert essentially any somatic cell type into iPSCs with increased efficiency and at higher quality when compared to ESCs. More recently, this field has been expanded to direct reprogramming of one cell type to another, including lineage-specific progenitors. Here, we provide a concise review of methods to generate induced pluripotent stem cells, and discuss the most recent strategies augmenting the reprogramming process and increasing the quality of iPSCs.

Canadian Journal of Chemistry, 2000

Acid-catalyzed hydrolysis of acetophenone dimethyl acetal (ADMA) and trimethyl orthobenzoate (TMO... more Acid-catalyzed hydrolysis of acetophenone dimethyl acetal (ADMA) and trimethyl orthobenzoate (TMOB) is retarded by cyclodextrins (CDs): α-CD, β-CD, hb-β-CD = "hydroxypropyl-β-cyclodextrin", and γ-CD. The observed first order rate constants (kobs) vary with [CD] in the manner expected for 1:1 binding between the substrates and the CDs. Similar behaviour was found recently for the hydrolysis of benzaldehyde dimethyl acetal (BDMA). With β-CD and hp-β-CD, the binding of all three substrates (BDMA, ADMA, TMOB) is strong and the CD-bound forms have very little reactivity. By contrast, substrate binding by α-CD is much weaker, and the CD-bound forms have appreciable, though reduced, reactivity. Substrate binding by γ-CD is also relatively weak, but the bound substrates have very low reactivities. The hydrolysis reactions of ADMA, TMOB, and BDMA have been evaluated as kinetic probes of the binding of guests to CD hosts. For α-CD, β-CD, and hp-β-CD, the addition of guests reduces t...

ABSTRACTMRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain re... more ABSTRACTMRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain recognizing H3K36me3 in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing and DNA repair by homologous recombination. In order to get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the chromodomain, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase and PALB2/BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR-tagging of endogenou...

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Frontiers in Cell and Developmental Biology

Nucleic Acids Research

Src associated in mitosis (SAM68) plays major roles in regulating RNA processing events, such as ... more Src associated in mitosis (SAM68) plays major roles in regulating RNA processing events, such as alternative splicing and mRNA translation, implicated in several developmental processes. It was previously shown that SAM68 regulates the alternative splicing of the mechanistic target of rapamycin (mTor), but the mechanism regulating this process remains elusive. Here, we report that SAM68 interacts with U1 small nuclear ribonucleoprotein (U1 snRNP) to promote splicing at the 5 splice site in intron 5 of mTor. We also show that this direct interaction is mediated through U1A, a core-component of U1snRNP. SAM68 was found to bind the RRM1 domain of U1A through its C-terminal tyrosine rich region (YY domain). Deletion of the U1A-SAM68 interaction domain or mutation in SAM68-binding sites in intron 5 of mTor abrogates U1A recruitment and 5 splice site recognition by the U1 snRNP, leading to premature intron 5 termination and polyadenylation. Taken together, our results provide the first mechanistic study by which SAM68 modulates alternative splicing decision, by affecting U1 snRNP recruitment at 5 splice sites.

Cell research, Jan 17, 2017

Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefi... more Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefits equivalent to prolonged fasting or caloric restriction. However, our understanding of the underlying mechanisms of IF-mediated metabolic benefits is limited. Here we show that isocaloric IF improves metabolic homeostasis against diet-induced obesity and metabolic dysfunction primarily through adipose thermogenesis in mice. IF-induced metabolic benefits require fasting-mediated increases of vascular endothelial growth factor (VEGF) expression in white adipose tissue (WAT). Furthermore, periodic adipose-VEGF overexpression could recapitulate the metabolic improvement of IF in non-fasted animals. Importantly, fasting and adipose-VEGF induce alternative activation of adipose macrophage, which is critical for thermogenesis. Human adipose gene analysis further revealed a positive correlation of adipose VEGF-M2 macrophage-WAT browning axis. The present study uncovers the molecular mechanism ...

Can J Chem, 2000

Acid-catalyzed hydrolysis of acetophenone dimethyl acetal (ADMA) and trimethyl orthobenzoate (TMO... more Acid-catalyzed hydrolysis of acetophenone dimethyl acetal (ADMA) and trimethyl orthobenzoate (TMOB) is retarded by cyclodextrins (CDs): α-CD, β-CD, hb-β-CD = "hydroxypropyl-β-cyclodextrin", and γ-CD. The observed first order rate constants (kobs) vary with [CD] in the manner expected for 1:1 binding between the substrates and the CDs. Similar behaviour was found recently for the hydrolysis of benzaldehyde dimethyl acetal (BDMA). With β-CD and hp-β-CD, the binding of all three substrates (BDMA, ADMA, TMOB) is strong and the CD-bound forms have very little reactivity. By contrast, substrate binding by α-CD is much weaker, and the CD-bound forms have appreciable, though reduced, reactivity. Substrate binding by γ-CD is also relatively weak, but the bound substrates have very low reactivities. The hydrolysis reactions of ADMA, TMOB, and BDMA have been evaluated as kinetic probes of the binding of guests to CD hosts. For α-CD, β-CD, and hp-β-CD, the addition of guests reduces the amount of free CD and thereby alleviates retardation of the hydrolysis by the CD. The resultant increases in hydrolysis rates can be analyzed to provide estimates of CD-guest dissociation constants, KG. For aliphatic alcohols and ketones binding to β-CD and hp-β-CD, all three probe reactions provide values of KG that agree well with each other and with literature values determined by other methods. The approach does not work well with α-CD because of its much weaker binding of the kinetic probes and their less pronounced dependence of kobs on [α-CD]. In the case of γ;-CD, the approach cannot be used because added guests cause a further lowering in the rate of hydrolysis, suggesting the formation of an unreactive ternary (substrate·CD·guest) complex.Key words: acetal, hydrolysis, cyclodextrin, host-guest, binding.

Scientific reports, Jan 20, 2016

The ventricular conduction system (VCS) orchestrates the harmonious contraction in every heartbea... more The ventricular conduction system (VCS) orchestrates the harmonious contraction in every heartbeat. Defects in the VCS are often associated with life-threatening arrhythmias and also promote adverse remodeling in heart disease. We have previously established that the Irx3 homeobox gene regulates rapid electrical propagation in the VCS by modulating the transcription of gap junction proteins Cx40 and Cx43. However, it is unknown whether other factors contribute to the conduction defects observed in Irx3 knockout (Irx3(-/-)) mice. In this study, we show that during the early postnatal period, Irx3(-/-) mice develop morphological defects in the VCS which are temporally dissociated from changes in gap junction expression. These morphological defects were accompanied with progressive changes in the cardiac electrocardiogram including right bundle branch block. Hypoplastic VCS was not associated with increased apoptosis of VCS cardiomyocytes but with a lack of recruitment and maturation o...

Nature, Jan 17, 2015

In this Article, the address listed as Nicole Cloonan's present address (QIMR Berghofer Medical R... more In this Article, the address listed as Nicole Cloonan's present address (QIMR Berghofer Medical Research Institute, Queensland 4006, Australia) should have been listed as her other affiliation with superscript 16, because the work she did was split equally between the two institutions. This has been corrected in the online versions of the paper.

BioData Mining, 2014

Background: Human genomic variations, including single nucleotide polymorphisms (SNPs) and copy n... more Background: Human genomic variations, including single nucleotide polymorphisms (SNPs) and copy number variations (CNVs), are associated with several phenotypic traits varying from mild features to hereditary diseases. Several genome-wide studies have reported genomic variants that correlate with gene expression levels in various tissue and cell types. Results: We studied human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) measuring the SNPs and CNVs with Affymetrix SNP 6 microarrays and expression values with Affymetrix Exon microarrays. We computed the linear relationships between SNPs and expression levels of exons, transcripts and genes, and the associations between gene CNVs and gene expression levels. Further, for a few of the resulted genes, the expression value was associated with both CNVs and SNPs. Our results revealed altogether 217 genes and 584 SNPs whose genomic alterations affect the transcriptome in the same cells. We analyzed the enriched pathways and gene ontologies within these groups of genes, and found out that the terms related to alternative splicing and development were enriched. Conclusions: Our results revealed that in the human pluripotent stem cells, the expression values of several genes, transcripts and exons were affected due to the genomic variation.

PLoS ONE, 2012

The kidney filtration barrier is formed by the combination of endothelial cells, basement membran... more The kidney filtration barrier is formed by the combination of endothelial cells, basement membrane and epithelial cells called podocytes. These specialized actin-rich cells form long and dynamic protrusions, the foot processes, which surround glomerular capillaries and are connected by specialized intercellular junctions, the slit diaphragms. Failure to maintain the filtration barrier leads to massive proteinuria and nephrosis. A number of proteins reside in the slit diaphragm, notably the transmembrane proteins Nephrin and Neph1, which are both able to act as tyrosine phosphorylated scaffolds that recruit cytoplasmic effectors to initiate downstream signaling. While association between tyrosine-phosphorylated Neph1 and the SH2/SH3 adaptor Grb2 was shown in vitro to be sufficient to induce actin polymerization, in vivo evidence supporting this finding is still lacking. To test this hypothesis, we generated two independent mouse lines bearing a podocyte-specific constitutive inactivation of the Grb2 locus. Surprisingly, we show that mice lacking Grb2 in podocytes display normal renal ultra-structure and function, thus demonstrating that Grb2 is not required for the establishment of the glomerular filtration barrier in vivo. Moreover, our data indicate that Grb2 is not required to restore podocyte function following kidney injury. Therefore, although in vitro experiments suggested that Grb2 is important for the regulation of actin dynamics, our data clearly shows that its function is not essential in podocytes in vivo, thus suggesting that Grb2 rather plays a secondary role in this process.

Nature, 2014

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the t... more Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Nature Communications, 2014

Reprogramming of somatic cells to induced pluripotent stem cells involves a dynamic rearrangement... more Reprogramming of somatic cells to induced pluripotent stem cells involves a dynamic rearrangement of the epigenetic landscape. To characterize this epigenomic roadmap, we have performed MethylC-seq, ChIP-seq (H3K4/K27/K36me3) and RNA-Seq on samples taken at several time points during murine secondary reprogramming as part of Project Grandiose. We find that DNA methylation gain during reprogramming occurs gradually, while loss is achieved only at the ESC-like state. Binding sites of activated factors exhibit focal demethylation during reprogramming, while ESC-like pluripotent cells are distinguished by extension of demethylation to the wider neighbourhood. We observed that genes with CpG-rich promoters demonstrate stable low methylation and strong engagement of histone marks, whereas genes with CpG-poor promoters are safeguarded by methylation. Such DNA methylation-driven control is the key to the regulation of ESC-pluripotency genes, including Dppa4, Dppa5a and Esrrb. These results reveal the crucial role that DNA methylation plays as an epigenetic switch driving somatic cells to pluripotency.

Nature Communications, 2014

MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells... more MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells (iPSCs), however, exactly how miRNA expression changes support the transition to pluripotency requires further investigation. Here we use a murine secondary reprogramming system to sample cellular trajectories towards iPSCs or a novel pluripotent 'F-class' state and perform small RNA sequencing. We detect sweeping changes in an early and a late wave, revealing that distinct miRNA milieus characterize alternate states of pluripotency. miRNA isoform expression is common but surprisingly varies little between cell states. Referencing other omic data sets generated in parallel, we find that miRNA expression is changed through transcriptional and post-transcriptional mechanisms. miRNA transcription is commonly regulated by dynamic histone modification, while DNA methylation/demethylation consolidates these changes at multiple loci. Importantly, our results suggest that a novel subset of distinctly expressed miRNAs supports pluripotency in the F-class state, substituting for miRNAs that serve such roles in iPSCs.

Nature Communications, 2014

The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogra... more The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quantitative mass spectrometry-based analysis to probe in-depth dynamic proteome changes during somatic cell reprogramming. Our data reveal defined waves of proteome resetting, with the first wave occurring 48 h after the activation of the reprogramming transgenes and involving specific biological processes linked to the c-Myc transcriptional network. A second wave of proteome reorganization occurs in a later stage of reprogramming, where we characterize the proteome of two distinct pluripotent cellular populations. In addition, the overlay of our proteome resource with parallel generated-omics data is explored to identify post-transcriptionally regulated proteins involved in key steps during reprogramming.

Genes, Chromosomes and Cancer, 2013

Copy number changes or reduced expression of the Neuron navigator 3 (NAV3) gene occurs in neurobl... more Copy number changes or reduced expression of the Neuron navigator 3 (NAV3) gene occurs in neuroblastomas and malignancies of epithelial or lymphoid origin. To elucidate whether NAV3 has a role in the tumorigenesis of nervous system tumors in general, we studied central and peripheral nervous system tumors for NAV3 copy number changes. In search for common tumorigenic denominators, we analyzed 113 central and peripheral nervous system tumors, including glial tumors (grades I-IV gliomas), medulloblastomas, and neuroblastomas. NAV3 copy number changes were studied by fluorescence in situ hybridization and correlated to survival analyses. To identify target genes of NAV3 deletion, NAV3 was silenced by siRNA in glioblastoma cell lines and gene expression profiles were analyzed by Agilent 4×44k dual-color microarrays. Selected upregulations were confirmed by immunohistochemistry and quantitative polymerase chain reaction. We found NAV3 amplifications to dominate in neuronally differentiated tumors, whereas glial tumors showed almost equal proportions of NAV3 deletion and amplification. However, Grade IV gliomas had more frequent NAV3 deletions than grades I-III gliomas. Silencing of NAV3 in glioma cell lines led to the upregulation of receptor genes associated with gonadotropin-releasing hormone and Jak-Stat signaling pathways. Kaplan-Meier analysis of the entire clinical tumor material showed association between NAV3 amplifications and favorable prognosis, as well as NAV3 deletions and unfavorable prognosis. With Cox regression model, a hazard ratio of 0.51 was observed for NAV3 amplifications and 1.36 for NAV3 deletions. We conclude that NAV3 may be a potential new prognostic biomarker and a potential therapeutic target.

EMBO Molecular Medicine, 2014

Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiog... more Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor-A (VEGF) biologics, termed VEGF "Sticky-traps," with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky-traps could locally inhibit angiogenesis to at least the same extent as the original VEGF-trap that also gains whole-body access. Stickytraps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky-trap remained localized to various regions of the eye, such as the inner-limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky-trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre-maturity.

Current Opinion in Genetics & Development, 2012

The ground-breaking work of Yamanaka and Thomson showed that forced expression of just four trans... more The ground-breaking work of Yamanaka and Thomson showed that forced expression of just four transcription factors can reprogram mouse and human somatic cells to pluripotency, leading to the discovery of the so-called induced pluripotent stem cells (iPSCs). Similar to embryonic stem cells (ESCs), iPSCs have the ability to permanently self-renew and also give rise to multiple cell types once differentiated. These cells opened up the opportunity to develop human disease models in vitro, drug and toxicity screening tools, as well as a continuous autologous cell source for future cell-based therapies. Therefore, it is not surprising that the methods for generating iPSCs have significantly evolved over the past few years. To date the reprogramming methods include the use of various transfection/transduction systems, small molecules to enhance the reprogramming process, and to adapt to a multitude of different cell type sources. We are now able to convert essentially any somatic cell type into iPSCs with increased efficiency and at higher quality when compared to ESCs. More recently, this field has been expanded to direct reprogramming of one cell type to another, including lineage-specific progenitors. Here, we provide a concise review of methods to generate induced pluripotent stem cells, and discuss the most recent strategies augmenting the reprogramming process and increasing the quality of iPSCs.

Canadian Journal of Chemistry, 2000

Acid-catalyzed hydrolysis of acetophenone dimethyl acetal (ADMA) and trimethyl orthobenzoate (TMO... more Acid-catalyzed hydrolysis of acetophenone dimethyl acetal (ADMA) and trimethyl orthobenzoate (TMOB) is retarded by cyclodextrins (CDs): α-CD, β-CD, hb-β-CD = "hydroxypropyl-β-cyclodextrin", and γ-CD. The observed first order rate constants (kobs) vary with [CD] in the manner expected for 1:1 binding between the substrates and the CDs. Similar behaviour was found recently for the hydrolysis of benzaldehyde dimethyl acetal (BDMA). With β-CD and hp-β-CD, the binding of all three substrates (BDMA, ADMA, TMOB) is strong and the CD-bound forms have very little reactivity. By contrast, substrate binding by α-CD is much weaker, and the CD-bound forms have appreciable, though reduced, reactivity. Substrate binding by γ-CD is also relatively weak, but the bound substrates have very low reactivities. The hydrolysis reactions of ADMA, TMOB, and BDMA have been evaluated as kinetic probes of the binding of guests to CD hosts. For α-CD, β-CD, and hp-β-CD, the addition of guests reduces t...