Publications by authors named "John R Seavitt"

20 Publications

  • Page 1 of 1

COPB2 loss of function causes a coatopathy with osteoporosis and developmental delay.

Am J Hum Genet 2021 09 26;108(9):1710-1724. Epub 2021 Aug 26.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2 mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2 mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2 mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.
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http://dx.doi.org/10.1016/j.ajhg.2021.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8456174PMC
September 2021

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

PLoS Genet 2020 12 28;16(12):e1009190. Epub 2020 Dec 28.

Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.
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http://dx.doi.org/10.1371/journal.pgen.1009190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822523PMC
December 2020

A global Slc7a7 knockout mouse model demonstrates characteristic phenotypes of human lysinuric protein intolerance.

Hum Mol Genet 2020 08;29(13):2171-2184

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Lysinuric protein intolerance (LPI) is an inborn error of cationic amino acid (arginine, lysine, ornithine) transport caused by biallelic pathogenic variants in SLC7A7, which encodes the light subunit of the y+LAT1 transporter. Treatments for the complications of LPI, including growth failure, renal disease, pulmonary alveolar proteinosis, autoimmune disorders and osteoporosis, are limited. Given the early lethality of the only published global Slc7a7 knockout mouse model, a viable animal model to investigate global SLC7A7 deficiency is needed. Hence, we generated two mouse models with global Slc7a7 deficiency (Slc7a7em1Lbu/em1Lbu; Slc7a7Lbu/Lbu and Slc7a7em1(IMPC)Bay/em1(IMPC)Bay; Slc7a7Bay/Bay) using CRISPR/Cas9 technology by introducing a deletion of exons 3 and 4. Perinatal lethality was observed in Slc7a7Lbu/Lbu and Slc7a7Bay/Bay mice on the C57BL/6 and C57BL/6NJ inbred genetic backgrounds, respectively. We noted improved survival of Slc7a7Lbu/Lbu mice on the 129 Sv/Ev × C57BL/6 F2 background, but postnatal growth failure occurred. Consistent with human LPI, these Slc7a7Lbu/Lbu mice exhibited reduced plasma and increased urinary concentrations of the cationic amino acids. Histopathological assessment revealed loss of brush border and lipid vacuolation in the renal cortex of Slc7a7Lbu/Lbu mice, which combined with aminoaciduria suggests proximal tubular dysfunction. Micro-computed tomography of L4 vertebrae and skeletal radiographs showed delayed skeletal development and suggested decreased mineralization in Slc7a7Lbu/Lbu mice, respectively. In addition to delayed skeletal development and delayed development in the kidneys, the lungs and liver were observed based on histopathological assessment. Overall, our Slc7a7Lbu/Lbu mouse model on the F2 mixed background recapitulates multiple human LPI phenotypes and may be useful for future studies of LPI pathology.
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http://dx.doi.org/10.1093/hmg/ddaa107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399531PMC
August 2020

Human and mouse essentiality screens as a resource for disease gene discovery.

Nat Commun 2020 01 31;11(1):655. Epub 2020 Jan 31.

Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany.

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery.
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http://dx.doi.org/10.1038/s41467-020-14284-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994715PMC
January 2020

Bi-allelic Variants in TONSL Cause SPONASTRIME Dysplasia and a Spectrum of Skeletal Dysplasia Phenotypes.

Am J Hum Genet 2019 03 14;104(3):422-438. Epub 2019 Feb 14.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.
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http://dx.doi.org/10.1016/j.ajhg.2019.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408318PMC
March 2019

Identification of genes required for eye development by high-throughput screening of mouse knockouts.

Commun Biol 2018 21;1:236. Epub 2018 Dec 21.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.
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http://dx.doi.org/10.1038/s42003-018-0226-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303268PMC
December 2018

Rapid and Integrative Discovery of Retina Regulatory Molecules.

Cell Rep 2018 08;24(9):2506-2519

Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA; Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA; Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

Retinal function relies on precisely organized neurons and synapses and a properly patterned vasculature to support them. Alterations in these features can result in vision loss. However, our understanding of retinal organization pathways remains incomplete because of a lack of methods to rapidly identify neuron and vasculature regulators in mammals. Here we developed a pipeline for the identification of neural and synaptic integrity genes by high-throughput retinal screening (INSiGHT) that analyzes candidate expression, vascular patterning, cellular organization, and synaptic arrangement. Using this system, we examined 102 mutant mouse lines and identified 16 unique retinal regulatory genes. Fifteen of these candidates are identified as novel retina regulators, and many (9 of 16) are associated with human neural diseases. These results expand the genetic landscape involved in retinal circuit organization and provide a road map for continued discovery of mammalian retinal regulators and disease-causing alleles.
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http://dx.doi.org/10.1016/j.celrep.2018.07.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170014PMC
August 2018

The International Mouse Phenotyping Consortium (IMPC): a functional catalogue of the mammalian genome that informs conservation.

Conserv Genet 2018 19;19(4):995-1005. Epub 2018 May 19.

1European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD UK.

The International Mouse Phenotyping Consortium (IMPC) is building a catalogue of mammalian gene function by producing and phenotyping a knockout mouse line for every protein-coding gene. To date, the IMPC has generated and characterised 5186 mutant lines. One-third of the lines have been found to be non-viable and over 300 new mouse models of human disease have been identified thus far. While current bioinformatics efforts are focused on translating results to better understand human disease processes, IMPC data also aids understanding genetic function and processes in other species. Here we show, using gorilla genomic data, how genes essential to development in mice can be used to help assess the potentially deleterious impact of gene variants in other species. This type of analyses could be used to select optimal breeders in endangered species to maintain or increase fitness and avoid variants associated to impaired-health phenotypes or loss-of-function mutations in genes of critical importance. We also show, using selected examples from various mammal species, how IMPC data can aid in the identification of candidate genes for studying a condition of interest, deliver information about the mechanisms involved, or support predictions for the function of genes that may play a role in adaptation. With genotyping costs decreasing and the continued improvements of bioinformatics tools, the analyses we demonstrate can be routinely applied.
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http://dx.doi.org/10.1007/s10592-018-1072-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061128PMC
May 2018

Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles.

BMC Biol 2018 06 21;16(1):69. Epub 2018 Jun 21.

Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX, 77030, USA.

Background: The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs).

Results: Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected.

Conclusions: Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.
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http://dx.doi.org/10.1186/s12915-018-0529-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011517PMC
June 2018

Response to "Unexpected mutations after CRISPR-Cas9 editing in vivo".

Nat Methods 2018 04 30;15(4):235-236. Epub 2018 Mar 30.

IMPC Cas9 Working Group and the IMPC Steering Committee and executive director of the IMPC (www.mousephenotype.org).

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http://dx.doi.org/10.1038/nmeth.4559DOI Listing
April 2018

Prevalence of sexual dimorphism in mammalian phenotypic traits.

Nat Commun 2017 06 26;8:15475. Epub 2017 Jun 26.

Mouse Genetics Project, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans.
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http://dx.doi.org/10.1038/ncomms15475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490203PMC
June 2017

Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features.

Am J Hum Genet 2017 Apr 23;100(4):676-688. Epub 2017 Mar 23.

Laboratory for Pediatric Brain Disease, Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego, CA 92093, USA.

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.
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http://dx.doi.org/10.1016/j.ajhg.2017.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384096PMC
April 2017

Three-dimensional microCT imaging of mouse development from early post-implantation to early postnatal stages.

Dev Biol 2016 11 23;419(2):229-236. Epub 2016 Sep 23.

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA; Optical Imaging and Vital Microscopy Core, Baylor College of Medicine, Houston, TX 77030, USA; Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

In this work, we report the use of iodine-contrast microCT to perform high-throughput 3D morphological analysis of mouse embryos and neonates between embryonic day 8.5 to postnatal day 3, with high spatial resolution up to 3µm/voxel. We show that mouse embryos at early stages can be imaged either within extra embryonic tissues such as the yolk sac or the decidua without physically disturbing the embryos. This method enables a full, undisturbed analysis of embryo turning, allantois development, vitelline vessels remodeling, yolk sac and early placenta development, which provides increased insights into early embryonic lethality in mutant lines. Moreover, these methods are inexpensive, simple to learn and do not require substantial processing time, making them ideal for high throughput analysis of mouse mutants with embryonic and early postnatal lethality.
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http://dx.doi.org/10.1016/j.ydbio.2016.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405732PMC
November 2016

High-throughput discovery of novel developmental phenotypes.

Nature 2016 09 14;537(7621):508-514. Epub 2016 Sep 14.

Department of Molecular Physiology and Biophysics, Houston, Texas 77030, USA.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
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http://dx.doi.org/10.1038/nature19356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295821PMC
September 2016

CRIPT exonic deletion and a novel missense mutation in a female with short stature, dysmorphic features, microcephaly, and pigmentary abnormalities.

Am J Med Genet A 2016 08 2;170(8):2206-11. Epub 2016 Jun 2.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas.

Mutations in CRIPT encoding cysteine-rich PDZ domain-binding protein are rare, and to date have been reported in only two patients with autosomal recessive primordial dwarfism and distinctive facies. Here, we describe a female with biallelic mutations in CRIPT presenting with postnatal growth retardation, global developmental delay, and dysmorphic features including frontal bossing, high forehead, and sparse hair and eyebrows. Additional clinical features included high myopia, admixed hyper- and hypopigmented macules primarily on the face, arms, and legs, and syndactyly of 4-5 toes bilaterally. Using whole exome sequencing (WES) and chromosomal microarray analysis (CMA), we detected a c.8G>A (p.C3Y) missense variant in exon 1 of the CRIPT gene inherited from the mother and a 1,331 bp deletion encompassing exon 1, inherited from the father. The c.8G>A (p.C3Y) missense variant in CRIPT was apparently homozygous in the proband due to the exon 1 deletion. Our findings illustrate the clinical utility of combining WES with copy number variant (CNV) analysis to provide a molecular diagnosis to patients with rare Mendelian disorders. Our findings also illustrate the clinical spectrum of CRIPT related mutations. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ajmg.a.37780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725961PMC
August 2016

The chromatin remodeler Mi-2beta is required for CD4 expression and T cell development.

Immunity 2004 Jun;20(6):719-33

Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.

Changes in chromatin structure underlie the activation or silencing of genes during development. The chromatin remodeler Mi-2beta is highly expressed in thymocytes and is presumed to be a transcriptional repressor because of its presence in the nucleosome remodeling deacetylase (NuRD) complex. Using conditional inactivation, we show that Mi-2beta is required at several steps during T cell development: for differentiation of beta selected immature thymocytes, for developmental expression of CD4, and for cell divisions in mature T cells. We further show that Mi-2beta plays a direct role in promoting CD4 gene expression. Mi-2beta associates with the CD4 enhancer as well as the E box binding protein HEB and the histone acetyltransferase (HAT) p300, enabling their recruitment to the CD4 enhancer and causing histone H3-hyperacetylation to this regulatory region. These findings provide important insights into the regulation of CD4 expression during T cell development and define a role for Mi-2beta in gene activation.
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http://dx.doi.org/10.1016/j.immuni.2004.05.005DOI Listing
June 2004

Unconventional potentiation of gene expression by Ikaros.

J Biol Chem 2002 Apr 17;277(15):13007-15. Epub 2002 Jan 17.

Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.

Ikaros is essential for the normal development and regulated proliferation of lymphoid cells. In lymphocytes, Ikaros exists as an integral component of chromatin-remodeling complexes, including the Mi-2beta/nucleosome remodeling and deacetylation complex (NuRD) complex. It is expected that Ikaros, together with these associated activities effects repression, but here we show that they may also potentiate gene expression in cycling cells. Ikaros cannot activate transcription by itself; instead, it enhances the activity of both weak and strong activators. For this role in potentiation, Ikaros requires its DNA binding and dimerization domains. The DNA binding and dimerization properties of Ikaros are also responsible for its targeting to pericentromeric heterochromatin (PC-HC). Significantly, Ikaros mutants with altered specificity for DNA binding that are unable to localize to PC-HC are incapable of stimulating transcription from reporters bearing their cognate sites. Thus, potentiation of gene expression by Ikaros correlates strongly with its ability to localize to PC-HC in combination with the chromatin remodeler Mi-2beta.
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http://dx.doi.org/10.1074/jbc.M111371200DOI Listing
April 2002
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