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Papers by Gregor Gilfillan

Endogenous RNAs Modulate MicroRNA Sorting to Exosomes and Transfer to Acceptor Cells

Cell Reports, 2014

MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, spe... more MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs) as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs) for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences.

Background: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) ... more Background: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1-20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. Results: We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells.

The EMBO Journal, 2001

In Drosophila, dosage compensation is controlled by the male-speci®c lethal (MSL) complex consist... more In Drosophila, dosage compensation is controlled by the male-speci®c lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is speci®cally localized on the male X chromosome to increase its expression~2-fold. We recently proposed a model for the targeted assembly of the MSL complex, in which initial binding occurs at~35 dispersed chromatin entry sites, followed by spreading in cis into¯anking regions. Here, we analyze one of the chromatin entry sites, the roX1 gene, to determine which sequences are suf®cient to recruit the MSL complex. We found association and spreading of the MSL complex from roX1 transgenes in the absence of detectable roX1 RNA synthesis from the transgene. We mapped the recruitment activity to a 217 bp roX1 fragment that shows male-speci®c DNase hypersensitivity and can be preferentially cross-linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is suf®cient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kilobases into neighboring regions.

Microbiology, 1998

Candida dubhiensis is a recently identified species which is implicated in oral candidosis in HIV... more Candida dubhiensis is a recently identified species which is implicated in oral candidosis in HIV-infected and AIDS patients. The species shares many phenotypic characteristics with, and is phylogenetically closely related to, Candida albicans. In this study the phylogenetic relationship between these two species was investigated and a comparison of putative virulence factors was performed. Four isolates of C. dubhiensis from different clinical sources were chosen for comparison with two reference C. albicans strains. First, the distinct phylogenetic position of C. dubhiensis was further established by the comparison of the sequence of its small rRNA subunit with representative Candida species. The C. dubhiensis isolates formed true unconstricted hyphae under most induction conditions tested but failed to produce true hyphae when induced using N-acetylglucosamine. Oral C. dubhiensis isolates were more adherent to human buccal epithelial cells than the reference C. albicans isolates when grown in glucose and equally adherent when grown in galactose. The C. dubhiensis isolates were sensitive to f luconazole, itraconazole, ketoconazole and amphotericin B. Homologues of seven tested C. albicans secretory aspartyl proteinase (SAP) genes were detected in C. dubhiensis by Southern analysis. In vivo virulence assays using a systemic mouse model suggest that C. dubhiensis is marginally less virulent than C. albicans. These data further confirm the distinct phenotypic and genotypic nature of C. dubhiensis and suggest that this species may be particularly adapted to colonization of the oral cavity.

X-Linked Congenital Adrenal Hypoplasia with Hypogonadotropic Hypogonadism Caused by an Inversion Disrupting a Conserved Noncoding Element Upstream of the NR0B1 ( DAX1 ) Gene

The Journal of Clinical Endocrinology & Metabolism, 2009

X-linked congenital adrenal hypoplasia with hypogonadotropic hypogonadism (AHCH) is known to be c... more X-linked congenital adrenal hypoplasia with hypogonadotropic hypogonadism (AHCH) is known to be caused by coding mutations in the nuclear receptor subfamily 0, group B, member 1 (NR0B1) gene, encoding the transcriptional repressor dosage-sensitive sex-reversal adrenal hypoplasia critical region on the X chromosome protein 1 (DAX1). Four males in a family were affected by AHCH. Our aim was to locate the genetic cause of their disease, knowing that they had no mutation in the obvious candidate gene, NR0B1. Linkage analysis of the X chromosome and mutational screening of conserved noncoding regions upstream of NR0B1 were performed. To functionally characterize the genetic defect, studies of transcription and expression of DAX1 and steroidogenic factor 1 (SF-1) were done. A 60 Mb inversion on the X chromosome with one of the inversion breakpoints located in a conserved noncoding region 4 kb upstream of NR0B1 was detected. The inversion causes relocation of a putative SF-1 binding site implicated in murine gonadal development. A reporter construct lacking this enhancer element upstream of NR0B1 was unresponsive to SF-1 transcriptional activation. Immunohistochemistry suggested that the inversion leads to SF-1 silencing in the patients' testes both in childhood and in adult life. We report a noncoding mutation causing AHCH, an inversion resulting in a phenotype similar to what is caused by intragenic NR0B1 null mutations. The inversion seems to disrupt and/or relocate regulatory sites crucial in DAX1 expression.

Diagnosis by sequencing: correction of misdiagnosis from FSHD2 to LGMD2A by whole-exome analysis

European Journal of Human Genetics, 2012

We studied and validated facioscapulohumeral muscular dystrophy (FSHD) samples from patients with... more We studied and validated facioscapulohumeral muscular dystrophy (FSHD) samples from patients without a D4Z4 contraction (FSHD2 or 'phenotypic FSHD'). For this, we developed non-radioactive protocols to test D4Z4 allele constitution and DNA methylation, and applied these to samples from the Coriell Institute Cell Repository. The D4Z4 sizing showed two related subjects to have classic chromosome 4 contraction-dependent FSHD1. A third sample (GM17726) did not have a short chromosome 4 fragment, and had been assigned as non-4q FSHD (FSHD2). We tested D4Z4 haplotype and methylation for this individual but found both to be inconsistent with this diagnosis. Using exome sequencing, we identified two known pathogenic mutations in CAPN3 (Arg490Gln and Thr184Argfs(*)36), indicating a case of LGMD2A rather than FSHD. Our study shows how a wrong diagnosis can easily be corrected by whole-exome sequencing by constraining the variant analysis to candidate genes after the data have been generated. This new way of 'diagnosis by sequencing' is likely to become common place in genetic diagnostic laboratories. We also publish a digoxigenin-labeled Southern protocol to test D4Z4 methylation. Our data supports hypomethylation as a good epigenetic predictor for FSHD2. The non-radioactive protocol will help to make this assay more accessible to clinical diagnostic laboratories and the wider FSHD research community.European Journal of Human Genetics advance online publication, 29 February 2012; doi:10.1038/ejhg.2012.42.

European Journal of Human Genetics, 2012

Next-generation sequencing (NGS) techniques have already shown their potential in the identificat... more Next-generation sequencing (NGS) techniques have already shown their potential in the identification of mutations underlying rare inherited disorders. We report here the application of linkage analysis in combination with targeted DNA capture and NGS to a Norwegian family affected by an undiagnosed mental retardation disorder with an autosomal recessive inheritance pattern. Linkage analysis identified two loci on chromosomes 9 and 17 which were subject to target enrichment by hybridization to a custom microarray. NGS achieved 20-fold or greater sequence coverage of 83% of all protein-coding exons in the target regions. This led to the identification of compound heterozygous mutations in NAGLU, compatible with the diagnosis of Mucopolysaccharidosis IIIB (MPS IIIB or Sanfilippo Syndrome type B). This diagnosis was confirmed by demonstrating elevated levels of heparan sulphate in urine and low activity of a-N-acetyl-glucosaminidase in cultured fibroblasts. Our findings describe a mild form of MPS IIIB and illustrate the diagnostic potential of targeted NGS in Mendelian disease with unknown aetiology.

BMC Genomics, 2012

Background: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) ... more Background: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1-20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. Results: We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells.

The American Journal of Human Genetics, 2008

PLoS ONE, 2014

Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence co... more Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence composition from samples of ancient or historic origin. Here, we identify a novel artifact in sequences from historic samples of Atlantic cod (Gadus morhua), which forms interrupted palindromes consisting of reverse complementary sequence at the 59 and 39-ends of sequencing reads. The palindromic sequences themselves have specific properties -the bases at the 59-end align well to the reference genome, whereas extensive misalignments exists among the bases at the terminal 39-end. The terminal 39 bases are artificial extensions likely caused by the occurrence of hairpin loops in single stranded DNA (ssDNA), which can be ligated and amplified in particular library creation protocols. We propose that such hairpin loops allow the inclusion of erroneous nucleotides, specifically at the 39-end of DNA strands, with the 59-end of the same strand providing the template. We also find these palindromes in previously published ancient DNA (aDNA) datasets, albeit at varying and substantially lower frequencies. This artifact can negatively affect the yield of endogenous DNA in these types of samples and introduces sequence bias.

Endogenous RNAs Modulate MicroRNA Sorting to Exosomes and Transfer to Acceptor Cells

Cell Reports, 2014

MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, spe... more MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs) as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs) for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences.

Background: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) ... more Background: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1-20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. Results: We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells.

The EMBO Journal, 2001

In Drosophila, dosage compensation is controlled by the male-speci®c lethal (MSL) complex consist... more In Drosophila, dosage compensation is controlled by the male-speci®c lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is speci®cally localized on the male X chromosome to increase its expression~2-fold. We recently proposed a model for the targeted assembly of the MSL complex, in which initial binding occurs at~35 dispersed chromatin entry sites, followed by spreading in cis into¯anking regions. Here, we analyze one of the chromatin entry sites, the roX1 gene, to determine which sequences are suf®cient to recruit the MSL complex. We found association and spreading of the MSL complex from roX1 transgenes in the absence of detectable roX1 RNA synthesis from the transgene. We mapped the recruitment activity to a 217 bp roX1 fragment that shows male-speci®c DNase hypersensitivity and can be preferentially cross-linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is suf®cient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kilobases into neighboring regions.

Microbiology, 1998

Candida dubhiensis is a recently identified species which is implicated in oral candidosis in HIV... more Candida dubhiensis is a recently identified species which is implicated in oral candidosis in HIV-infected and AIDS patients. The species shares many phenotypic characteristics with, and is phylogenetically closely related to, Candida albicans. In this study the phylogenetic relationship between these two species was investigated and a comparison of putative virulence factors was performed. Four isolates of C. dubhiensis from different clinical sources were chosen for comparison with two reference C. albicans strains. First, the distinct phylogenetic position of C. dubhiensis was further established by the comparison of the sequence of its small rRNA subunit with representative Candida species. The C. dubhiensis isolates formed true unconstricted hyphae under most induction conditions tested but failed to produce true hyphae when induced using N-acetylglucosamine. Oral C. dubhiensis isolates were more adherent to human buccal epithelial cells than the reference C. albicans isolates when grown in glucose and equally adherent when grown in galactose. The C. dubhiensis isolates were sensitive to f luconazole, itraconazole, ketoconazole and amphotericin B. Homologues of seven tested C. albicans secretory aspartyl proteinase (SAP) genes were detected in C. dubhiensis by Southern analysis. In vivo virulence assays using a systemic mouse model suggest that C. dubhiensis is marginally less virulent than C. albicans. These data further confirm the distinct phenotypic and genotypic nature of C. dubhiensis and suggest that this species may be particularly adapted to colonization of the oral cavity.

X-Linked Congenital Adrenal Hypoplasia with Hypogonadotropic Hypogonadism Caused by an Inversion Disrupting a Conserved Noncoding Element Upstream of the NR0B1 ( DAX1 ) Gene

The Journal of Clinical Endocrinology & Metabolism, 2009

X-linked congenital adrenal hypoplasia with hypogonadotropic hypogonadism (AHCH) is known to be c... more X-linked congenital adrenal hypoplasia with hypogonadotropic hypogonadism (AHCH) is known to be caused by coding mutations in the nuclear receptor subfamily 0, group B, member 1 (NR0B1) gene, encoding the transcriptional repressor dosage-sensitive sex-reversal adrenal hypoplasia critical region on the X chromosome protein 1 (DAX1). Four males in a family were affected by AHCH. Our aim was to locate the genetic cause of their disease, knowing that they had no mutation in the obvious candidate gene, NR0B1. Linkage analysis of the X chromosome and mutational screening of conserved noncoding regions upstream of NR0B1 were performed. To functionally characterize the genetic defect, studies of transcription and expression of DAX1 and steroidogenic factor 1 (SF-1) were done. A 60 Mb inversion on the X chromosome with one of the inversion breakpoints located in a conserved noncoding region 4 kb upstream of NR0B1 was detected. The inversion causes relocation of a putative SF-1 binding site implicated in murine gonadal development. A reporter construct lacking this enhancer element upstream of NR0B1 was unresponsive to SF-1 transcriptional activation. Immunohistochemistry suggested that the inversion leads to SF-1 silencing in the patients' testes both in childhood and in adult life. We report a noncoding mutation causing AHCH, an inversion resulting in a phenotype similar to what is caused by intragenic NR0B1 null mutations. The inversion seems to disrupt and/or relocate regulatory sites crucial in DAX1 expression.

Diagnosis by sequencing: correction of misdiagnosis from FSHD2 to LGMD2A by whole-exome analysis

European Journal of Human Genetics, 2012

We studied and validated facioscapulohumeral muscular dystrophy (FSHD) samples from patients with... more We studied and validated facioscapulohumeral muscular dystrophy (FSHD) samples from patients without a D4Z4 contraction (FSHD2 or 'phenotypic FSHD'). For this, we developed non-radioactive protocols to test D4Z4 allele constitution and DNA methylation, and applied these to samples from the Coriell Institute Cell Repository. The D4Z4 sizing showed two related subjects to have classic chromosome 4 contraction-dependent FSHD1. A third sample (GM17726) did not have a short chromosome 4 fragment, and had been assigned as non-4q FSHD (FSHD2). We tested D4Z4 haplotype and methylation for this individual but found both to be inconsistent with this diagnosis. Using exome sequencing, we identified two known pathogenic mutations in CAPN3 (Arg490Gln and Thr184Argfs(*)36), indicating a case of LGMD2A rather than FSHD. Our study shows how a wrong diagnosis can easily be corrected by whole-exome sequencing by constraining the variant analysis to candidate genes after the data have been generated. This new way of 'diagnosis by sequencing' is likely to become common place in genetic diagnostic laboratories. We also publish a digoxigenin-labeled Southern protocol to test D4Z4 methylation. Our data supports hypomethylation as a good epigenetic predictor for FSHD2. The non-radioactive protocol will help to make this assay more accessible to clinical diagnostic laboratories and the wider FSHD research community.European Journal of Human Genetics advance online publication, 29 February 2012; doi:10.1038/ejhg.2012.42.

European Journal of Human Genetics, 2012

Next-generation sequencing (NGS) techniques have already shown their potential in the identificat... more Next-generation sequencing (NGS) techniques have already shown their potential in the identification of mutations underlying rare inherited disorders. We report here the application of linkage analysis in combination with targeted DNA capture and NGS to a Norwegian family affected by an undiagnosed mental retardation disorder with an autosomal recessive inheritance pattern. Linkage analysis identified two loci on chromosomes 9 and 17 which were subject to target enrichment by hybridization to a custom microarray. NGS achieved 20-fold or greater sequence coverage of 83% of all protein-coding exons in the target regions. This led to the identification of compound heterozygous mutations in NAGLU, compatible with the diagnosis of Mucopolysaccharidosis IIIB (MPS IIIB or Sanfilippo Syndrome type B). This diagnosis was confirmed by demonstrating elevated levels of heparan sulphate in urine and low activity of a-N-acetyl-glucosaminidase in cultured fibroblasts. Our findings describe a mild form of MPS IIIB and illustrate the diagnostic potential of targeted NGS in Mendelian disease with unknown aetiology.

BMC Genomics, 2012

Background: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) ... more Background: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1-20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. Results: We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells.

The American Journal of Human Genetics, 2008

PLoS ONE, 2014

Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence co... more Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence composition from samples of ancient or historic origin. Here, we identify a novel artifact in sequences from historic samples of Atlantic cod (Gadus morhua), which forms interrupted palindromes consisting of reverse complementary sequence at the 59 and 39-ends of sequencing reads. The palindromic sequences themselves have specific properties -the bases at the 59-end align well to the reference genome, whereas extensive misalignments exists among the bases at the terminal 39-end. The terminal 39 bases are artificial extensions likely caused by the occurrence of hairpin loops in single stranded DNA (ssDNA), which can be ligated and amplified in particular library creation protocols. We propose that such hairpin loops allow the inclusion of erroneous nucleotides, specifically at the 39-end of DNA strands, with the 59-end of the same strand providing the template. We also find these palindromes in previously published ancient DNA (aDNA) datasets, albeit at varying and substantially lower frequencies. This artifact can negatively affect the yield of endogenous DNA in these types of samples and introduces sequence bias.