ACS Pharmacology & Translational Science, 2021
Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, cha... more Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, characterized by genetic duplication of the critical myelin gene Peripheral Myelin Protein 22 (PMP22). PMP22 overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of PMP22 expression is a major target of therapeutic discovery for CMT1A, we sought to establish unbiased approaches that allow the identification of therapeutic agents for this disease. Using genome editing, we generated a coincidence reporter assay that accurately monitors Pmp22 transcript levels in the S16 rat Schwann cell line, while reducing reporter-based false positives. A quantitative high-throughput screen (qHTS) of 42 577 compounds using this assay revealed diverse novel chemical classes that reduce endogenous Pmp22 transcript levels. Moreover, some of these classes show pharmacological specificity in reducing Pmp22 over another major myelin-associated gene, Mpz (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of Pmp22 transcripts translates to reduced PMP22 protein levels, we edited the S16 genome to generate a reporter assay that expresses a PMP22-HiBiT fusion protein using CRISPR/Cas9. Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and posttranslational regulation of PMP22 with titration-based screening (e.g., qHTS), which could be efficiently incorporated into drug discovery campaigns for CMT1A.
SummaryResearchers have presumed that the “inactive” X chromosome (Xi) has little impact, in tran... more SummaryResearchers have presumed that the “inactive” X chromosome (Xi) has little impact, in trans, on the “active” X (Xa). To test this, we quantified Xi and Xa gene expression in individuals with one Xa and zero to three Xi’s. Our linear modeling revealed modular Xi and Xa transcriptomes and significant Xi-driven expression changes for 38% (162/423) of expressed X-chromosome genes. By integrating allele-specific analyses, we found that modulation of Xa transcript levels by Xi contributes to many of these Xi-driven changes (≥ 121 genes). By incorporating metrics of evolutionary constraint, we identified 10 X-chromosome genes most likely to drive sex differences in common disease, and sex chromosome aneuploidy syndromes. We conclude that human X chromosomes are regulated both in cis, through Xi-wide transcriptional attenuation, and in trans, through positive or negative modulation of individual Xa genes by Xi. The sum of cis and trans effects differs widely among genes.
Determination of the chromosomal location and genomic structure of the Hedgehog-Interacting Prote... more Determination of the chromosomal location and genomic structure of the Hedgehog-Interacting Protein gene, and analysis of its role in Holoprosencephaly Hedgehog-Interacting Protein (HIP) is a novel component of the Sonic Hedgehog (SHH) signaling pathway. Recently, defects in this pathway have been shown to cause holoprosencephaly (HPE), which is the most common birth defect of the brain and face in humans. In animal models knockout mice with homozygous null mutations for Shh displayed abnormalities consistent with HPE. Hip has been shown to function as a co-receptor and attenuate Hedgehog signaling by cell-surface binding of the Hedgehog protein and is hypothesized to act as part of a negative regulatory feedback loop. Although Hip is an important factor in the Shh signaling pathway, its potential role in HPE had not been examined in humans. Here, we report the complete gene structure of the human HIP gene and present its mutational analysis in HPE patients. No mutations were found ...
Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (... more Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (phos-phatase and tensin homolog deleted on chromosome 10) tumor suppressor, but it is not known if these two genetic lesions act together to transform cells. To answer this question, we infected PTEN–/ – neural precursor cells with a retrovirus encoding EGFRvIII, which is a con-stitutively activated receptor. EGFRvIII PTEN–/ – cells formed highly mitotic tumors with nuclear pleomor-phism, necrotic areas, and glioblastoma markers. The transformed cells showed increased cell proliferation, centrosome amplification, colony formation in soft agar, self-renewal, expression of the stem cell marker CD133, and resistance to oxidative stress and ionizing radia-tion. The RAS/mitogen-activated protein kinase (ERK) and phosphoinositide 3-kinase/protein kinase B (PI3K/ Akt) pathways were activated, and checkpoint kinase 1 (Chk1), the DNA damage regulator, was phosphor-ylated at S280 by Akt, suppressi...
After nearly two decades of improvements, the current human reference genome (GRCh38) is the most... more After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our effor...
Highlights d Comprehensive multiomic maps of EndoC-bH1 human b cell line and primary islets d Seq... more Highlights d Comprehensive multiomic maps of EndoC-bH1 human b cell line and primary islets d Sequence motifs enriched in EndoC-specific enhancers reflect its precursor state d Identification of regulatory hubs preserved between EndoC-bH1 and human islets d Identified SNP alleles (including T2D GWAS) altering cisregulatory signatures
SUMMARYEndoC-βH1 is emerging as a critical human beta cell model to study the genetic and environ... more SUMMARYEndoC-βH1 is emerging as a critical human beta cell model to study the genetic and environmental etiologies of beta cell function, especially in the context of diabetes. Comprehensive knowledge of its molecular landscape is lacking yet required to fully take advantage of this model. Here, we report extensive chromosomal (spectral karyotyping), genetic (genotyping), epigenetic (ChIP-seq, ATAC-seq), chromatin interaction (Hi-C, Pol2 ChIA-PET), and transcriptomic (RNA-seq, miRNA-seq) maps of this cell model. Integrated analyses of these maps define known (e.g.,PDX1, ISL1) and putative (e.g.,PCSK1, mir-375) beta cell-specific chromatin interactions and transcriptionalcis-regulatory networks, and identify allelic effects oncis-regulatory element use and expression.Importantly, comparative analyses with maps generated in primary human islets/beta cells indicate substantial preservation of chromatin looping, but also highlight chromosomal heterogeneity and fetal genomic signatures i...
Hedgehog-Interacting Protein (HIP) is a novel component of the Sonic Hedgehog (SHH) Research Inst... more Hedgehog-Interacting Protein (HIP) is a novel component of the Sonic Hedgehog (SHH) Research Institute, signaling pathway. Recently, defects in this pathway have been shown to cause holopro-National Institutes of Health, sencephaly (HPE), which is the most common birth defect of the brain and face in humans. Bethesda, MD, USA In animal models knockout mice with homozygous null mutations for Shh displayed abnor-2 Physical Mapping Core Facility, malities consistent with HPE. Hip has been shown to function as a co-receptor and atte-National Human Genome nuate Hedgehog signaling by cell-surface binding of the Hedgehog protein and is hypothe-Research Institute, sized to act as part of a negative regulatory feedback loop. Although Hip is an important National Institutes of Health, factor in the Shh signaling pathway, its potential role in HPE had not been examined in Bethesda, MD, USA humans. Here, we report the complete gene structure of the human HIP gene and present 3 Department of Molecular and Cellular Biology, The Biolabs, its mutational analysis in HPE patients. No mutations were found either in the entire coding Harvard University, Cambridge, region or 1 kb upstream of the transcriptional start site (representing the putative promotor) MA, USA suggesting this gene may not be involved in HPE pathogenesis.
Muntjac chromosomes <p>Comparative mapping and sequencing was used to characterize the sites of a... more Muntjac chromosomes <p>Comparative mapping and sequencing was used to characterize the sites of ancestral chromosomal fusions in the Indian muntjac genome.</p>
Adult bone marrow-derived (BMD) cells could be used to repair damaged organs and tissues, but the... more Adult bone marrow-derived (BMD) cells could be used to repair damaged organs and tissues, but the intrinsic plasticity of these cells has been questioned by results of in-vitro studies suggesting that such cells might fuse with other cells giving the appearance of differentiation. We aimed to determine whether fusion events are important in vivo. To test whether BMD cells can colonise an epithelial tissue and differentiate there without fusion, we did in-situ hybridisation with Y and X chromosome probes labelled with 35-sulphur or digoxigenin, or labelled fluorescently. We did immunohistochemistry with anticytokeratin 13 along with fluorescence in-situ hybridisation to identify Y-chromosome positive buccal epithelial cells in cheek scrapings obtained from five females who had received either a bone-marrow transplant or an allogeneic mobilised peripheral-blood progenitor-cell transplant (enriched in CD34+ cells) from male donors. When examined 4-6 years after male-to-female marrow-cell transplantation, all female recipients had Y-chromosome-positive buccal cells (0.8-12.7%). In more than 9700 cells studied, we detected only one XXXY-positive cell (0.01%) and one XXY cell (0.01%), both of which could have arisen when an XY cell fused with an XX cell. Male BMD cells migrate into the cheek and differentiate into epithelial cells, an occurrence that does not depend on fusion of BMD cells to recipient cells. This finding might be an example of transdifferentiation of haemopoietic or stromal progenitor cells. Plasticity of BMD cells could be useful in regenerative medicine.
We report a Nigerian family with a late-onset autosomal dominant neuropathy consistent with Charc... more We report a Nigerian family with a late-onset autosomal dominant neuropathy consistent with Charcot-Marie-Tooth disease. Electrophysiological examination of the index patient confirmed a severe demyelinating neuropathy with secondary axonal features. Sequence analysis of the myelin protein zero (MPZ) gene identified a C-to-G transversion at nucleotide position 234, resulting in a serine-to-tryptophan mutation in codon 78 (S78W) of the translated protein. The presence of this novel missense mutation suggests a diagnosis of Charcot-Marie-Tooth disease type 1B. Our study confirms the worldwide distribution of this disorder and extends the genetic spectrum of mutations in the MPZ gene.
The mouse homolog of the human MEN1 gene, which is defective in a dominant familial cancer syndro... more The mouse homolog of the human MEN1 gene, which is defective in a dominant familial cancer syndrome, multiple endocrine neoplasia type 1 (MEN1), has been identified and characterized. The mouse Men1 transcript contains an open reading frame encoding a protein of 611 amino acids which has 97% identity and 98% similarity to human menin. Sequence of the entire Men1 gene (9.3 kb) was assembled, revealing 10 exons, with exon 1 being non-coding; a polymorphic tetranucleotide repeat was located in the 5Ј-flanking region. The exon-intron organization and the size of the coding exons 2-9 were well conserved between the human and mouse genes. Fluorescence in situ hybridization localized the Men1 gene to mouse Chromosome (Chr) 19, a region known to be syntenic to human Chr 11q13, the locus for the MEN1 gene. Northern analysis indicated two messages-2.7 kb and 3.1 kb-expressed in all stages of the embryo analyzed and in all eight adult tissues tested. The larger transcript differs from the smaller by the inclusion of an unspliced intron 1. Whole-mount in situ hybridization of 10.5-day and 11.5-day embryos showed ubiquitous expression of Men1 RNA. Western analysis with antibodies raised against a conserved C-terminal peptide identified an approximately 67-kDa protein in the lysates of adult mouse brain, kidney, liver, pancreas, and spleen tissues, consistent with the size of human menin. The levels of mouse menin do not appear to fluctuate during the cell cycle.
Chromosomal region 13q21-q22 harbors a putative breast cancer susceptibility gene and has been im... more Chromosomal region 13q21-q22 harbors a putative breast cancer susceptibility gene and has been implicated as a common site for somatic deletions in a variety of malignant tumors. We have built a complete physical clone contig for a region between D13S1308 and AFM220YE9 based on 18 yeast artificial chromosome and 81 bacterial artificial chromosome (BAC) clones linked together by 22 genetic markers and 61 other sequence tagged sites. Combining data from 47 sequenced BACs (as of June 2001), we have assembled in silico an integrated 5.7-Mb genomic map with 90% sequence coverage. This area contains eight known genes, two hypothetical proteins, 24 additional Unigene clusters, and approximately 100 predicted genes and exons. We have determined the cDNA and genomic sequence, and tissue expression profiles for the KIAA1008 protein (homologous to the yeast mitotic control protein dis3+), KLF12 (AP-2 repressor), progesterone induced blocking factor 1, zinc finger transcription factor KLF5, and LIM domain only-7, and for the hypothetical proteins FLJ22624 and FLJ21869. Mutation screening of the five known genes in 19 breast cancer families has revealed numerous polymorphisms, but no deleterious mutations. These data provide a basis and resources for further analyses of this chromosomal region in the development of cancer.
The -synuclein protein is highly homologous to the ␣-synuclein protein for which two mutations w... more The -synuclein protein is highly homologous to the ␣-synuclein protein for which two mutations were reported in some familial cases of Parkinson disease. It has been shown that both ␣and -synucleins may be able to inhibit phospholipase D2 selectively. We have observed that the -synuclein gene (HGMW-approved symbol, SNCB) is highly expressed in brain including the substantia nigra, the main region of neuronal degeneration in patients with Parkinson disease. We have determined the intron-exon structure of the -synuclein gene and established sequencing assays that will facilitate the search for mutations in the -synuclein gene in patients with Parkinson disease or other neurodegenerative disorders.
animal kingdom, from crustaceans to primates (Boman Defensins, a family of antimicrobial peptides... more animal kingdom, from crustaceans to primates (Boman Defensins, a family of antimicrobial peptides isoet al., 1994). These peptides characteristically have a lated from several mammalian species, have a probroad spectrum of antibiotic activity in vitro, supportposed functional role in innate host defense. In huing a similar functional role in vivo (Bevins, 1994; Bomans, certain defensin genes are expressed in phagoman et al., 1991; Zasloff, 1992). The various individual cytic cells of hematopoietic origin, while others are antimicrobial peptides differ significantly in primary expressed in Paneth cells, epithelial cells of the small sequence, although several families can be distinintestine. In this study, we determined the chromoguished (Boman, 1995). Each family is defined by consomal localization of the human defensin (HD) genes served structural features of the peptides and, where expressed in Paneth cells, HD-5 and HD-6. Analysis of information is available, is supported by the organizaa panel of human/hamster hybrids localized both HDtion of their respective genes. However, the evolution of 5 and HD-6 to chromosome 8. Southern blot analysis antimicrobial gene families is incompletely understood. of DNA from cell lines that contain either chromosome As concerns mount regarding clinically relevant micro-8 deletions or duplications further localized these two organisms acquiring resistance to conventional antibigenes to 8p21-pter. Fluorescence in situ hybridization otics, research on antimicrobial peptides and other molanalysis of metaphase chromosomes using an HD-5 ecules of innate host defense has gained significance. probe further supported the regional map assignment. Defensins are one family of antimicrobial peptides Previous studies had localized the hematopoietic found in many mammalian species (for reviews see genes to chromosome 8p23, and the current work is
Hyper-lgE syndrome with recurrent infections (HIES) is a primary immunodeficiency disease charact... more Hyper-lgE syndrome with recurrent infections (HIES) is a primary immunodeficiency disease characterized by recurrent s k~n and lung abscesses and extreme elevations of serum IgE, but also involving dentition, bones, and connective tissue. Although the etiology of HlES is unknown, autosomal dominant inheritance has been observed in multiple kindreds. A 1 7 year old male with sporadic HIES, autism, and mild mental retardation was found to have a supernumerary marker chromosome in peripheral blood lymphocytes and skin fibroblasts. Microdissection and FlSH analysis of the marker chromosome showed that it was derived from a small interstitial deletion of one homologue of chromosome 4q21. Lack of hybridization of probes speciflc for tel0meres and alphoid centromeres, including a centromere 4 speclfic probe, established that the marker was an analphoid ring chromosome. Comparative genotyping of transformed B-cell subclones with (M+) and without (M-) the marker chromosome showed loss of the maternal alleles in M-cells between markers D4S1569 and D4S3010. FlSH using YAC clones from 4q21 confirmed the size and location of the interstit~al deletion. Thus our patient's phenotypes were associated with de novo formation of a marker chromosome containing 15-20 cM of DNA deleted from h i s maternally derived chromosome 4. Proximal chromosome 4q therefore is a candidate region for disease genes for both HlES and autism. Identification of genes disrupted or lost dur~ng the formation of the marker chromosome as well a s linkage studies in kindreds with HlES or autism may help us to understand the etiology of these complex phenotypes.
C57BL/6 is a well-characterized mouse strain that is used extensively for immunological and neuro... more C57BL/6 is a well-characterized mouse strain that is used extensively for immunological and neurological research. The establishment of C57BL/6 ES cell lines has facilitated the study of gene-altered mice in a pure genetic background-however, relatively few such lines exist. Using a defined media supplement, knockout serum replacement (KSR) with knockout DMEM (KSR-KD-MEM), we find that we can readily establish ES cell lines from blastocysts of C57BL/6J mice. Six lines were established, all of which were karyotypically normal and could be maintained in the undifferentiated state on mouse embryonic fibroblast (MEF) feeders. One line was further tested and found to be karyotypically stable and germline competent, both prior to manipulation and after gene targeting. For this cell line, efficiencies of cell cloning and chimera generation were greater when maintained in KSR-KDMEM. Our work suggests that the use of defined serum-free media may facilitate the generation of ES cells from inbred mouse strains. genesis 39:100-104, 2004.
Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. To ... more Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. To fine map such genomic changes by comparative genomic hybridization (CGH), a high resolution (100 kb) chromosome 8 array that can detect single copy changes was developed using Phi29 DNA polymerase amplified BAC (bacterial artificial chromosome) DNA. The BAC array CGH resolved the two known amplified regions (8q21 and 8q24) of a breast cancer cell line (SKBR3) into nine separate regions including six amplicons and three deleted regions, all of which were verified by Fluorescence in situ hybridization. The extent of the gain/loss for each region was validated by qPCR. CGH was performed with a total of 8 breast cancer cell lines, and common regions of genomic amplification/deletion were identified by segmentation analysis. A 1.2-Mb region (125.3-126.5 Mb) and a 1.0-Mb region (128.1-129.1 Mb) in 8q24 were amplified in 7/8 cell lines. A global expression analysis was performed to evaluate expression changes associated with genomic amplification/deletion: a novel gene, TRMT12 (at 125.5 Mb), amplified in 7/8 cell lines, showed highest expression in these cell lines. Further analysis by RT-qPCR using RNA from 30 breast tumors showed that TRMT12 was overexpressed >2 fold in 87% (26/30) of the tumors. TRMT12 is a homologue of a yeast gene encoding a tRNA methyltransferase involved in the posttranscriptional modification of tRNA Phe , and exploring the biological consequence of its altered expression, may reveal novel pathways in tumorigenesis. This article contains Supplementary Material available at
The reported draft human genome sequence includes many contigs that are separated by gaps of unkn... more The reported draft human genome sequence includes many contigs that are separated by gaps of unknown sequence. These gaps may be due to chromosomal regions that are not present in the Escherichia coli libraries used for DNA sequencing because they cannot be cloned efficiently, if at all, in bacteria. Using a yeast artificial chromosome (YAC)/ bacterial artificial chromosome (BAC) library generated in yeast, we found that approximately 6% of human DNA sequences tested transformed E. coli cells less efficiently than yeast cells, and were less stable in E. coli than in yeast. When the ends of several YAC/BAC isolates cloned in yeast were sequenced and compared with the reported draft sequence, major inconsistencies were found with the sequences of those YAC/BAC isolates that transformed E. coli cells inefficiently. Two human genomic fragments were reisolated from human DNA by transformation-associated recombination (TAR) cloning. Re-sequencing of these regions showed that the errors in the draft are the results of both missassembly and loss of specific DNA sequences during cloning in E. coli. These results show that TAR cloning might be a valuable method that could be widely used during the final stages of the Human Genome Project.
ACS Pharmacology & Translational Science, 2021
Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, cha... more Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, characterized by genetic duplication of the critical myelin gene Peripheral Myelin Protein 22 (PMP22). PMP22 overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of PMP22 expression is a major target of therapeutic discovery for CMT1A, we sought to establish unbiased approaches that allow the identification of therapeutic agents for this disease. Using genome editing, we generated a coincidence reporter assay that accurately monitors Pmp22 transcript levels in the S16 rat Schwann cell line, while reducing reporter-based false positives. A quantitative high-throughput screen (qHTS) of 42 577 compounds using this assay revealed diverse novel chemical classes that reduce endogenous Pmp22 transcript levels. Moreover, some of these classes show pharmacological specificity in reducing Pmp22 over another major myelin-associated gene, Mpz (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of Pmp22 transcripts translates to reduced PMP22 protein levels, we edited the S16 genome to generate a reporter assay that expresses a PMP22-HiBiT fusion protein using CRISPR/Cas9. Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and posttranslational regulation of PMP22 with titration-based screening (e.g., qHTS), which could be efficiently incorporated into drug discovery campaigns for CMT1A.
SummaryResearchers have presumed that the “inactive” X chromosome (Xi) has little impact, in tran... more SummaryResearchers have presumed that the “inactive” X chromosome (Xi) has little impact, in trans, on the “active” X (Xa). To test this, we quantified Xi and Xa gene expression in individuals with one Xa and zero to three Xi’s. Our linear modeling revealed modular Xi and Xa transcriptomes and significant Xi-driven expression changes for 38% (162/423) of expressed X-chromosome genes. By integrating allele-specific analyses, we found that modulation of Xa transcript levels by Xi contributes to many of these Xi-driven changes (≥ 121 genes). By incorporating metrics of evolutionary constraint, we identified 10 X-chromosome genes most likely to drive sex differences in common disease, and sex chromosome aneuploidy syndromes. We conclude that human X chromosomes are regulated both in cis, through Xi-wide transcriptional attenuation, and in trans, through positive or negative modulation of individual Xa genes by Xi. The sum of cis and trans effects differs widely among genes.
Determination of the chromosomal location and genomic structure of the Hedgehog-Interacting Prote... more Determination of the chromosomal location and genomic structure of the Hedgehog-Interacting Protein gene, and analysis of its role in Holoprosencephaly Hedgehog-Interacting Protein (HIP) is a novel component of the Sonic Hedgehog (SHH) signaling pathway. Recently, defects in this pathway have been shown to cause holoprosencephaly (HPE), which is the most common birth defect of the brain and face in humans. In animal models knockout mice with homozygous null mutations for Shh displayed abnormalities consistent with HPE. Hip has been shown to function as a co-receptor and attenuate Hedgehog signaling by cell-surface binding of the Hedgehog protein and is hypothesized to act as part of a negative regulatory feedback loop. Although Hip is an important factor in the Shh signaling pathway, its potential role in HPE had not been examined in humans. Here, we report the complete gene structure of the human HIP gene and present its mutational analysis in HPE patients. No mutations were found ...
Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (... more Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (phos-phatase and tensin homolog deleted on chromosome 10) tumor suppressor, but it is not known if these two genetic lesions act together to transform cells. To answer this question, we infected PTEN–/ – neural precursor cells with a retrovirus encoding EGFRvIII, which is a con-stitutively activated receptor. EGFRvIII PTEN–/ – cells formed highly mitotic tumors with nuclear pleomor-phism, necrotic areas, and glioblastoma markers. The transformed cells showed increased cell proliferation, centrosome amplification, colony formation in soft agar, self-renewal, expression of the stem cell marker CD133, and resistance to oxidative stress and ionizing radia-tion. The RAS/mitogen-activated protein kinase (ERK) and phosphoinositide 3-kinase/protein kinase B (PI3K/ Akt) pathways were activated, and checkpoint kinase 1 (Chk1), the DNA damage regulator, was phosphor-ylated at S280 by Akt, suppressi...
After nearly two decades of improvements, the current human reference genome (GRCh38) is the most... more After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our effor...
Highlights d Comprehensive multiomic maps of EndoC-bH1 human b cell line and primary islets d Seq... more Highlights d Comprehensive multiomic maps of EndoC-bH1 human b cell line and primary islets d Sequence motifs enriched in EndoC-specific enhancers reflect its precursor state d Identification of regulatory hubs preserved between EndoC-bH1 and human islets d Identified SNP alleles (including T2D GWAS) altering cisregulatory signatures
SUMMARYEndoC-βH1 is emerging as a critical human beta cell model to study the genetic and environ... more SUMMARYEndoC-βH1 is emerging as a critical human beta cell model to study the genetic and environmental etiologies of beta cell function, especially in the context of diabetes. Comprehensive knowledge of its molecular landscape is lacking yet required to fully take advantage of this model. Here, we report extensive chromosomal (spectral karyotyping), genetic (genotyping), epigenetic (ChIP-seq, ATAC-seq), chromatin interaction (Hi-C, Pol2 ChIA-PET), and transcriptomic (RNA-seq, miRNA-seq) maps of this cell model. Integrated analyses of these maps define known (e.g.,PDX1, ISL1) and putative (e.g.,PCSK1, mir-375) beta cell-specific chromatin interactions and transcriptionalcis-regulatory networks, and identify allelic effects oncis-regulatory element use and expression.Importantly, comparative analyses with maps generated in primary human islets/beta cells indicate substantial preservation of chromatin looping, but also highlight chromosomal heterogeneity and fetal genomic signatures i...
Hedgehog-Interacting Protein (HIP) is a novel component of the Sonic Hedgehog (SHH) Research Inst... more Hedgehog-Interacting Protein (HIP) is a novel component of the Sonic Hedgehog (SHH) Research Institute, signaling pathway. Recently, defects in this pathway have been shown to cause holopro-National Institutes of Health, sencephaly (HPE), which is the most common birth defect of the brain and face in humans. Bethesda, MD, USA In animal models knockout mice with homozygous null mutations for Shh displayed abnor-2 Physical Mapping Core Facility, malities consistent with HPE. Hip has been shown to function as a co-receptor and atte-National Human Genome nuate Hedgehog signaling by cell-surface binding of the Hedgehog protein and is hypothe-Research Institute, sized to act as part of a negative regulatory feedback loop. Although Hip is an important National Institutes of Health, factor in the Shh signaling pathway, its potential role in HPE had not been examined in Bethesda, MD, USA humans. Here, we report the complete gene structure of the human HIP gene and present 3 Department of Molecular and Cellular Biology, The Biolabs, its mutational analysis in HPE patients. No mutations were found either in the entire coding Harvard University, Cambridge, region or 1 kb upstream of the transcriptional start site (representing the putative promotor) MA, USA suggesting this gene may not be involved in HPE pathogenesis.
Muntjac chromosomes <p>Comparative mapping and sequencing was used to characterize the sites of a... more Muntjac chromosomes <p>Comparative mapping and sequencing was used to characterize the sites of ancestral chromosomal fusions in the Indian muntjac genome.</p>
Adult bone marrow-derived (BMD) cells could be used to repair damaged organs and tissues, but the... more Adult bone marrow-derived (BMD) cells could be used to repair damaged organs and tissues, but the intrinsic plasticity of these cells has been questioned by results of in-vitro studies suggesting that such cells might fuse with other cells giving the appearance of differentiation. We aimed to determine whether fusion events are important in vivo. To test whether BMD cells can colonise an epithelial tissue and differentiate there without fusion, we did in-situ hybridisation with Y and X chromosome probes labelled with 35-sulphur or digoxigenin, or labelled fluorescently. We did immunohistochemistry with anticytokeratin 13 along with fluorescence in-situ hybridisation to identify Y-chromosome positive buccal epithelial cells in cheek scrapings obtained from five females who had received either a bone-marrow transplant or an allogeneic mobilised peripheral-blood progenitor-cell transplant (enriched in CD34+ cells) from male donors. When examined 4-6 years after male-to-female marrow-cell transplantation, all female recipients had Y-chromosome-positive buccal cells (0.8-12.7%). In more than 9700 cells studied, we detected only one XXXY-positive cell (0.01%) and one XXY cell (0.01%), both of which could have arisen when an XY cell fused with an XX cell. Male BMD cells migrate into the cheek and differentiate into epithelial cells, an occurrence that does not depend on fusion of BMD cells to recipient cells. This finding might be an example of transdifferentiation of haemopoietic or stromal progenitor cells. Plasticity of BMD cells could be useful in regenerative medicine.
We report a Nigerian family with a late-onset autosomal dominant neuropathy consistent with Charc... more We report a Nigerian family with a late-onset autosomal dominant neuropathy consistent with Charcot-Marie-Tooth disease. Electrophysiological examination of the index patient confirmed a severe demyelinating neuropathy with secondary axonal features. Sequence analysis of the myelin protein zero (MPZ) gene identified a C-to-G transversion at nucleotide position 234, resulting in a serine-to-tryptophan mutation in codon 78 (S78W) of the translated protein. The presence of this novel missense mutation suggests a diagnosis of Charcot-Marie-Tooth disease type 1B. Our study confirms the worldwide distribution of this disorder and extends the genetic spectrum of mutations in the MPZ gene.
The mouse homolog of the human MEN1 gene, which is defective in a dominant familial cancer syndro... more The mouse homolog of the human MEN1 gene, which is defective in a dominant familial cancer syndrome, multiple endocrine neoplasia type 1 (MEN1), has been identified and characterized. The mouse Men1 transcript contains an open reading frame encoding a protein of 611 amino acids which has 97% identity and 98% similarity to human menin. Sequence of the entire Men1 gene (9.3 kb) was assembled, revealing 10 exons, with exon 1 being non-coding; a polymorphic tetranucleotide repeat was located in the 5Ј-flanking region. The exon-intron organization and the size of the coding exons 2-9 were well conserved between the human and mouse genes. Fluorescence in situ hybridization localized the Men1 gene to mouse Chromosome (Chr) 19, a region known to be syntenic to human Chr 11q13, the locus for the MEN1 gene. Northern analysis indicated two messages-2.7 kb and 3.1 kb-expressed in all stages of the embryo analyzed and in all eight adult tissues tested. The larger transcript differs from the smaller by the inclusion of an unspliced intron 1. Whole-mount in situ hybridization of 10.5-day and 11.5-day embryos showed ubiquitous expression of Men1 RNA. Western analysis with antibodies raised against a conserved C-terminal peptide identified an approximately 67-kDa protein in the lysates of adult mouse brain, kidney, liver, pancreas, and spleen tissues, consistent with the size of human menin. The levels of mouse menin do not appear to fluctuate during the cell cycle.
Chromosomal region 13q21-q22 harbors a putative breast cancer susceptibility gene and has been im... more Chromosomal region 13q21-q22 harbors a putative breast cancer susceptibility gene and has been implicated as a common site for somatic deletions in a variety of malignant tumors. We have built a complete physical clone contig for a region between D13S1308 and AFM220YE9 based on 18 yeast artificial chromosome and 81 bacterial artificial chromosome (BAC) clones linked together by 22 genetic markers and 61 other sequence tagged sites. Combining data from 47 sequenced BACs (as of June 2001), we have assembled in silico an integrated 5.7-Mb genomic map with 90% sequence coverage. This area contains eight known genes, two hypothetical proteins, 24 additional Unigene clusters, and approximately 100 predicted genes and exons. We have determined the cDNA and genomic sequence, and tissue expression profiles for the KIAA1008 protein (homologous to the yeast mitotic control protein dis3+), KLF12 (AP-2 repressor), progesterone induced blocking factor 1, zinc finger transcription factor KLF5, and LIM domain only-7, and for the hypothetical proteins FLJ22624 and FLJ21869. Mutation screening of the five known genes in 19 breast cancer families has revealed numerous polymorphisms, but no deleterious mutations. These data provide a basis and resources for further analyses of this chromosomal region in the development of cancer.
The -synuclein protein is highly homologous to the ␣-synuclein protein for which two mutations w... more The -synuclein protein is highly homologous to the ␣-synuclein protein for which two mutations were reported in some familial cases of Parkinson disease. It has been shown that both ␣and -synucleins may be able to inhibit phospholipase D2 selectively. We have observed that the -synuclein gene (HGMW-approved symbol, SNCB) is highly expressed in brain including the substantia nigra, the main region of neuronal degeneration in patients with Parkinson disease. We have determined the intron-exon structure of the -synuclein gene and established sequencing assays that will facilitate the search for mutations in the -synuclein gene in patients with Parkinson disease or other neurodegenerative disorders.
animal kingdom, from crustaceans to primates (Boman Defensins, a family of antimicrobial peptides... more animal kingdom, from crustaceans to primates (Boman Defensins, a family of antimicrobial peptides isoet al., 1994). These peptides characteristically have a lated from several mammalian species, have a probroad spectrum of antibiotic activity in vitro, supportposed functional role in innate host defense. In huing a similar functional role in vivo (Bevins, 1994; Bomans, certain defensin genes are expressed in phagoman et al., 1991; Zasloff, 1992). The various individual cytic cells of hematopoietic origin, while others are antimicrobial peptides differ significantly in primary expressed in Paneth cells, epithelial cells of the small sequence, although several families can be distinintestine. In this study, we determined the chromoguished (Boman, 1995). Each family is defined by consomal localization of the human defensin (HD) genes served structural features of the peptides and, where expressed in Paneth cells, HD-5 and HD-6. Analysis of information is available, is supported by the organizaa panel of human/hamster hybrids localized both HDtion of their respective genes. However, the evolution of 5 and HD-6 to chromosome 8. Southern blot analysis antimicrobial gene families is incompletely understood. of DNA from cell lines that contain either chromosome As concerns mount regarding clinically relevant micro-8 deletions or duplications further localized these two organisms acquiring resistance to conventional antibigenes to 8p21-pter. Fluorescence in situ hybridization otics, research on antimicrobial peptides and other molanalysis of metaphase chromosomes using an HD-5 ecules of innate host defense has gained significance. probe further supported the regional map assignment. Defensins are one family of antimicrobial peptides Previous studies had localized the hematopoietic found in many mammalian species (for reviews see genes to chromosome 8p23, and the current work is
Hyper-lgE syndrome with recurrent infections (HIES) is a primary immunodeficiency disease charact... more Hyper-lgE syndrome with recurrent infections (HIES) is a primary immunodeficiency disease characterized by recurrent s k~n and lung abscesses and extreme elevations of serum IgE, but also involving dentition, bones, and connective tissue. Although the etiology of HlES is unknown, autosomal dominant inheritance has been observed in multiple kindreds. A 1 7 year old male with sporadic HIES, autism, and mild mental retardation was found to have a supernumerary marker chromosome in peripheral blood lymphocytes and skin fibroblasts. Microdissection and FlSH analysis of the marker chromosome showed that it was derived from a small interstitial deletion of one homologue of chromosome 4q21. Lack of hybridization of probes speciflc for tel0meres and alphoid centromeres, including a centromere 4 speclfic probe, established that the marker was an analphoid ring chromosome. Comparative genotyping of transformed B-cell subclones with (M+) and without (M-) the marker chromosome showed loss of the maternal alleles in M-cells between markers D4S1569 and D4S3010. FlSH using YAC clones from 4q21 confirmed the size and location of the interstit~al deletion. Thus our patient's phenotypes were associated with de novo formation of a marker chromosome containing 15-20 cM of DNA deleted from h i s maternally derived chromosome 4. Proximal chromosome 4q therefore is a candidate region for disease genes for both HlES and autism. Identification of genes disrupted or lost dur~ng the formation of the marker chromosome as well a s linkage studies in kindreds with HlES or autism may help us to understand the etiology of these complex phenotypes.
C57BL/6 is a well-characterized mouse strain that is used extensively for immunological and neuro... more C57BL/6 is a well-characterized mouse strain that is used extensively for immunological and neurological research. The establishment of C57BL/6 ES cell lines has facilitated the study of gene-altered mice in a pure genetic background-however, relatively few such lines exist. Using a defined media supplement, knockout serum replacement (KSR) with knockout DMEM (KSR-KD-MEM), we find that we can readily establish ES cell lines from blastocysts of C57BL/6J mice. Six lines were established, all of which were karyotypically normal and could be maintained in the undifferentiated state on mouse embryonic fibroblast (MEF) feeders. One line was further tested and found to be karyotypically stable and germline competent, both prior to manipulation and after gene targeting. For this cell line, efficiencies of cell cloning and chimera generation were greater when maintained in KSR-KDMEM. Our work suggests that the use of defined serum-free media may facilitate the generation of ES cells from inbred mouse strains. genesis 39:100-104, 2004.
Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. To ... more Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. To fine map such genomic changes by comparative genomic hybridization (CGH), a high resolution (100 kb) chromosome 8 array that can detect single copy changes was developed using Phi29 DNA polymerase amplified BAC (bacterial artificial chromosome) DNA. The BAC array CGH resolved the two known amplified regions (8q21 and 8q24) of a breast cancer cell line (SKBR3) into nine separate regions including six amplicons and three deleted regions, all of which were verified by Fluorescence in situ hybridization. The extent of the gain/loss for each region was validated by qPCR. CGH was performed with a total of 8 breast cancer cell lines, and common regions of genomic amplification/deletion were identified by segmentation analysis. A 1.2-Mb region (125.3-126.5 Mb) and a 1.0-Mb region (128.1-129.1 Mb) in 8q24 were amplified in 7/8 cell lines. A global expression analysis was performed to evaluate expression changes associated with genomic amplification/deletion: a novel gene, TRMT12 (at 125.5 Mb), amplified in 7/8 cell lines, showed highest expression in these cell lines. Further analysis by RT-qPCR using RNA from 30 breast tumors showed that TRMT12 was overexpressed >2 fold in 87% (26/30) of the tumors. TRMT12 is a homologue of a yeast gene encoding a tRNA methyltransferase involved in the posttranscriptional modification of tRNA Phe , and exploring the biological consequence of its altered expression, may reveal novel pathways in tumorigenesis. This article contains Supplementary Material available at
The reported draft human genome sequence includes many contigs that are separated by gaps of unkn... more The reported draft human genome sequence includes many contigs that are separated by gaps of unknown sequence. These gaps may be due to chromosomal regions that are not present in the Escherichia coli libraries used for DNA sequencing because they cannot be cloned efficiently, if at all, in bacteria. Using a yeast artificial chromosome (YAC)/ bacterial artificial chromosome (BAC) library generated in yeast, we found that approximately 6% of human DNA sequences tested transformed E. coli cells less efficiently than yeast cells, and were less stable in E. coli than in yeast. When the ends of several YAC/BAC isolates cloned in yeast were sequenced and compared with the reported draft sequence, major inconsistencies were found with the sequences of those YAC/BAC isolates that transformed E. coli cells inefficiently. Two human genomic fragments were reisolated from human DNA by transformation-associated recombination (TAR) cloning. Re-sequencing of these regions showed that the errors in the draft are the results of both missassembly and loss of specific DNA sequences during cloning in E. coli. These results show that TAR cloning might be a valuable method that could be widely used during the final stages of the Human Genome Project.
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