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Hydractinia is a colonial marine hydroid that shows remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, Hydractinia symbiolongicarpus and Hydractinia echinata , placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell-type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia 's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia 's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from nonself., (© 2024 Schnitzler et al.; Published by Cold Spring Harbor Laboratory Press.)

Introduction: The majority of studies on oxidative phosphorylation in immune cells have been performed in mouse models, necessitating human translation. To understand the impact of oxidative phosphorylation (OXPHOS) deficiency on human immunity, we studied children with primary mitochondrial disease (MtD)., Methods: scRNAseq analysis of peripheral blood mononuclear cells was performed on matched children with MtD (N = 4) and controls (N = 4). To define B cell function we performed phage display immunoprecipitation sequencing on a cohort of children with MtD (N = 19) and controls (N = 16)., Results: Via scRNAseq, we found marked reductions in select populations involved in the humoral immune response, especially antigen presenting cells, B cell and plasma populations, with sparing of T cell populations. MTRNR2L8 , a marker of bioenergetic stress, was significantly elevated in populations that were most depleted. mir4485 , a miRNA contained in the intron of MTRNR2L8 , was co-expressed. Knockdown studies of mir4485 demonstrated its role in promoting survival by modulating apoptosis. To determine the functional consequences of our findings on humoral immunity, we studied the antiviral antibody repertoire in children with MtD and controls using phage display and immunoprecipitation sequencing. Despite similar viral exposomes, MtD displayed antiviral antibodies with less robust fold changes and limited polyclonality., Discussion: Overall, we show that children with MtD display perturbations in the B cell repertoire which may impact humoral immunity and the ability to clear viral infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gordon-Lipkin, Banerjee, Franco, Tarasenko, Kruk, Thompson, Gildea, Zhang, Wolfsberg, NISC Comparative Sequencing Program, Flegel and McGuire.)

The epithelial and interstitial stem cells of the freshwater polyp Hydra are the best-characterized stem cell systems in any cnidarian, providing valuable insight into cell type evolution and the origin of stemness in animals. However, little is known about the transcriptional regulatory mechanisms that determine how these stem cells are maintained and how they give rise to their diverse differentiated progeny. To address such questions, a thorough understanding of transcriptional regulation in Hydra is needed. To this end, we generated extensive new resources for characterizing transcriptional regulation in Hydra , including new genome assemblies for Hydra oligactis and the AEP strain of Hydra vulgaris , an updated whole-animal single-cell RNA-seq atlas, and genome-wide maps of chromatin interactions, chromatin accessibility, sequence conservation, and histone modifications. These data revealed the existence of large kilobase-scale chromatin interaction domains in the Hydra genome that contain transcriptionally coregulated genes. We also uncovered the transcriptomic profiles of two previously molecularly uncharacterized cell types: isorhiza-type nematocytes and somatic gonad ectoderm. Finally, we identified novel candidate regulators of cell type-specific transcription, several of which have likely been conserved at least since the divergence of Hydra and the jellyfish Clytia hemisphaerica more than 400 million years ago., (© 2023 Cazet et al.; Published by Cold Spring Harbor Laboratory Press.)

Although genomic research has predominantly relied on phenotypic ascertainment of individuals affected with heritable disease, the falling costs of sequencing allow consideration of genomic ascertainment and reverse phenotyping (the ascertainment of individuals with specific genomic variants and subsequent evaluation of physical characteristics). In this research modality, the scientific question is inverted: investigators gather individuals with a genomic variant and test the hypothesis that there is an associated phenotype via targeted phenotypic evaluations. Genomic ascertainment research is thus a model of predictive genomic medicine and genomic screening. Here, we provide our experience implementing this research method. We describe the infrastructure we developed to perform reverse phenotyping studies, including aggregating a super-cohort of sequenced individuals who consented to recontact for genomic ascertainment research. We assessed 13 studies completed at the National Institutes of Health (NIH) that piloted our reverse phenotyping approach. The studies can be broadly categorized as (1) facilitating novel genotype-disease associations, (2) expanding the phenotypic spectra, or (3) demonstrating ex vivo functional mechanisms of disease. We highlight three examples of reverse phenotyping studies in detail and describe how using a targeted reverse phenotyping approach (as opposed to phenotypic ascertainment or clinical informatics approaches) was crucial to the conclusions reached. Finally, we propose a framework and address challenges to building collaborative genomic ascertainment research programs at other institutions. Our goal is for more researchers to take advantage of this approach, which will expand our understanding of the predictive capability of genomic medicine and increase the opportunity to mitigate genomic disease., Competing Interests: Declaration of interests L.G.B. is a member of the Illumina Medical Ethics Board and receives honoraria from Cold Spring Harbor Laboratory Press, royalties from Invitae, Inc., and research support from Merck, Inc., (Published by Elsevier Inc.)

To address the void in the availability of high-quality proteomic data traversing the animal tree, we have implemented a pipeline for generating de novo assemblies based on publicly available data from the NCBI Sequence Read Archive, yielding a comprehensive collection of proteomes from 100 species spanning 21 animal phyla. We have also created the Animal Proteome Database (AniProtDB), a resource providing open access to this collection of high-quality metazoan proteomes, along with information on predicted proteins and protein domains for each taxonomic classification and the ability to perform sequence similarity searches against all proteomes generated using this pipeline. This solution vastly increases the utility of these data by removing the barrier to access for research groups who do not have the expertise or resources to generate these data themselves and enables the use of data from nontraditional research organisms that have the potential to address key questions in biomedicine., (Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2021.)

Adeno-associated viral (AAV) vectors have emerged as the preferred platform for in vivo gene transfer because of their combined efficacy and safety. However, insertional mutagenesis with the subsequent development of hepatocellular carcinomas (HCCs) has been recurrently noted in newborn mice treated with high doses of AAV, and more recently, the association of wild-type AAV integrations in a subset of human HCCs has been documented. Here, we address, in a comprehensive, prospective study, the long-term risk of tumorigenicity in young adult mice following delivery of single-stranded AAVs targeting liver. HCC incidence in mice treated with therapeutic and reporter AAVs was low, in contrast to what has been previously documented in mice treated as newborns with higher doses of AAV. Specifically, HCCs developed in 6 out 76 of AAV-treated mice, and a pathogenic integration of AAV was found in only one tumor. Also, no evidence of liver tumorigenesis was found in juvenile AAV-treated mucopolysaccharidosis type VI (MPS VI) cats followed as long as 8 years after vector administration. Together, our results support the low risk of tumorigenesis associated with AAV-mediated gene transfer targeting juvenile/young adult livers, although constant monitoring of subjects enrolled in AAV clinical trial is advisable., Competing Interests: A.A. is founder and consultant of InnovaVector srl. The other authors have no competing interests to declare., (© 2020 The Authors.)

How individuals perceive uncertainties in sequencing results may affect their clinical utility. The purpose of this study was to explore perceptions of uncertainties in carrier results and how they relate to psychological well-being and health behavior. Post-reproductive adults (N = 462) were randomized to receive carrier results from sequencing through either a web platform or a genetic counselor. On average, participants received two results. Group differences in affective, evaluative, and clinical uncertainties were assessed from baseline to 1 and 6 months; associations with test-specific distress and communication of results were assessed at 6 months. Reductions in affective uncertainty (∆x̅ = 0.78, 95% CI: 0.53, 1.02) and evaluative uncertainty (∆x̅ = 0.69, 95% CI: 0.51, 0.87) followed receipt of results regardless of randomization arm at 1 month. Participants in the web platform arm reported greater clinical uncertainty than those in the genetic counselor arm at 1 and 6 months; this was corroborated by the 1,230 questions asked of the genetic counselor and residual questions reported by those randomized to the web platform. Evaluative uncertainty was associated with a lower likelihood of communicating results to health care providers. Clinical uncertainty was associated with a lower likelihood of communicating results to children. Learning one's carrier results may reduce perceptions of uncertainties, though web-based return may lead to less reduction in clinical uncertainty in the short term. These findings warrant reinforcement of clinical implications to minimize residual questions and promote appropriate health behavior (communicating results to at-risk relatives in the case of carrier results), especially when testing alternative delivery models., (© Society of Behavioral Medicine 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Single-cell RNA sequencing (scRNA-seq) of human primary tissues is a rapidly emerging tool for investigating human health and disease at the molecular level. However, optimal processing of solid tissues presents a number of technical and logistical challenges, especially for tissues that are only available at autopsy, which includes pancreatic islets, a tissue that is highly relevant to diabetes. To assess the possible effects of different sample preparation protocols on fresh islet samples, we performed a detailed comparison of scRNA-seq data generated with islets isolated from a human donor but processed according to four treatment strategies, including fixation and cryopreservation. We found significant and reproducible differences in the proportion of cell types identified, and more minor effects on cell-specific patterns of gene expression. Fresh islets from a second donor confirmed gene expression signatures of alpha and beta subclusters. These findings may well apply to other tissues, emphasizing the need for careful consideration when choosing processing methods, comparing results between different studies, and/or interpreting data in the context of multiple cell types from preserved tissue., (Published by Oxford University Press 2019. This work is written by US Government employees and is in the public domain in the US.)

For over a thousand years, the common goldfish ( Carassius auratus ) was raised throughout Asia for food and as an ornamental pet. As a very close relative of the common carp ( Cyprinus carpio ), goldfish share the recent genome duplication that occurred approximately 14 million years ago in their common ancestor. The combination of centuries of breeding and a wide array of interesting body morphologies provides an exciting opportunity to link genotype to phenotype and to understand the dynamics of genome evolution and speciation. We generated a high-quality draft sequence and gene annotations of a "Wakin" goldfish using 71X PacBio long reads. The two subgenomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged substantially across seven tissues sampled. Loss of sequence identity and/or exons determined the divergence of the expression levels across all tissues, while loss of conserved noncoding elements determined expression variance between different tissues. This assembly provides an important resource for comparative genomics and understanding the causes of goldfish variants.

The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM-integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these "dead" enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.

It is unclear how standing genetic variation affects the prognosis of prostate cancer patients. To provide one controlled answer to this problem, we crossed a dominant, penetrant mouse model of prostate cancer to Diversity Outbred mice, a collection of animals that carries over 40 million SNPs. Integration of disease phenotype and SNP variation data in 493 F1 males identified a metastasis modifier locus on Chromosome 8 (LOD = 8.42); further analysis identified the genes Rwdd4, Cenpu, and Casp3 as functional effectors of this locus. Accordingly, analysis of over 5,300 prostate cancer patient samples revealed correlations between the presence of genetic variants at these loci, their expression levels, cancer aggressiveness, and patient survival. We also observed that ectopic overexpression of RWDD4 and CENPU increased the aggressiveness of two human prostate cancer cell lines. In aggregate, our approach demonstrates how well-characterized genetic variation in mice can be harnessed in conjunction with systems genetics approaches to identify and characterize germline modifiers of human disease processes., (Published by Elsevier Inc.)

Background: Endometrial cancer (EC) is the 8th leading cause of cancer death amongst American women. Most ECs are endometrioid, serous, or clear cell carcinomas, or an admixture of histologies. Serous and clear ECs are clinically aggressive tumors for which alternative therapeutic approaches are needed. The purpose of this study was to search for somatic mutations in the tyrosine kinome of serous and clear cell ECs, because mutated kinases can point to potential therapeutic targets., Methods: In a mutation discovery screen, we PCR amplified and Sanger sequenced the exons encoding the catalytic domains of 86 tyrosine kinases from 24 serous, 11 clear cell, and 5 mixed histology ECs. For somatically mutated genes, we next sequenced the remaining coding exons from the 40 discovery screen tumors and sequenced all coding exons from another 72 ECs (10 clear cell, 21 serous, 41 endometrioid). We assessed the copy number of mutated kinases in this cohort of 112 tumors using quantitative real time PCR, and we used immunoblotting to measure expression of these kinases in endometrial cancer cell lines., Results: Overall, we identified somatic mutations in TNK2 (tyrosine kinase non-receptor, 2) and DDR1 (discoidin domain receptor tyrosine kinase 1) in 5.3% (6 of 112) and 2.7% (3 of 112) of ECs. Copy number gains of TNK2 and DDR1 were identified in another 4.5% and 0.9% of 112 cases respectively. Immunoblotting confirmed TNK2 and DDR1 expression in endometrial cancer cell lines. Three of five missense mutations in TNK2 and one of two missense mutations in DDR1 are predicted to impact protein function by two or more in silico algorithms. The TNK2(P761Rfs*72) frameshift mutation was recurrent in EC, and the DDR1(R570Q) missense mutation was recurrent across tumor types., Conclusions: This is the first study to systematically search for mutations in the tyrosine kinome in clear cell endometrial tumors. Our findings indicate that high-frequency somatic mutations in the catalytic domains of the tyrosine kinome are rare in clear cell ECs. We uncovered ten new mutations in TNK2 and DDR1 within serous and endometrioid ECs, thus providing novel insights into the mutation spectrum of each gene in EC.

Background: Quantification of a transcriptional profile is a useful way to evaluate the activity of a cell at a given point in time. Although RNA-Seq has revolutionized transcriptional profiling, the costs of RNA-Seq are still significantly higher than microarrays, and often the depth of data delivered from RNA-Seq is in excess of what is needed for simple transcript quantification. Digital Gene Expression (DGE) is a cost-effective, sequence-based approach for simple transcript quantification: by sequencing one read per molecule of RNA, this technique can be used to efficiently count transcripts while obviating the need for transcript-length normalization and reducing the total numbers of reads necessary for accurate quantification. Here, we present trieFinder, a program specifically designed to rapidly map, parse, and annotate DGE tags of various lengths against cDNA and/or genomic sequence databases., Results: The trieFinder algorithm maps DGE tags in a two-step process. First, it scans FASTA files of RefSeq, UniGene, and genomic DNA sequences to create a database of all tags that can be derived from a predefined restriction site. Next, it compares the experimental DGE tags to this tag database, taking advantage of the fact that the tags are stored as a prefix tree, or "trie", which allows for linear-time searches for exact matches. DGE tags with mismatches are analyzed by recursive calls in the data structure. We find that, in terms of alignment speed, the mapping functionality of trieFinder compares favorably with Bowtie., Conclusions: trieFinder can quickly provide the user an annotation of the DGE tags from three sources simultaneously, simplifying transcript quantification and novel transcript detection, delivering the data in a simple parsed format, obviating the need to post-process the alignment results. trieFinder is available at http://research.nhgri.nih.gov/software/trieFinder/.

Background: Mnemiopsis leidyi is a ctenophore native to the coastal waters of the western Atlantic Ocean. A number of studies on Mnemiopsis have led to a better understanding of many key biological processes, and these studies have contributed to the emergence of Mnemiopsis as an important model for evolutionary and developmental studies. Recently, we sequenced, assembled, annotated, and performed a preliminary analysis on the 150-megabase genome of the ctenophore, Mnemiopsis. This sequencing effort has produced the first set of whole-genome sequencing data on any ctenophore species and is amongst the first wave of projects to sequence an animal genome de novo solely using next-generation sequencing technologies., Description: The Mnemiopsis Genome Project Portal (http://research.nhgri.nih.gov/mnemiopsis/) is intended both as a resource for obtaining genomic information on Mnemiopsis through an intuitive and easy-to-use interface and as a model for developing customized Web portals that enable access to genomic data. The scope of data available through this Portal goes well beyond the sequence data available through GenBank, providing key biological information not available elsewhere, such as pathway and protein domain analyses; it also features a customized genome browser for data visualization., Conclusions: We expect that the availability of these data will allow investigators to advance their own research projects aimed at understanding phylogenetic diversity and the evolution of proteins that play a fundamental role in metazoan development. The overall approach taken in the development of this Web site can serve as a viable model for disseminating data from whole-genome sequencing projects, framed in a way that best-serves the specific needs of the scientific community.

Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3,700,000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors.

An understanding of ctenophore biology is critical for reconstructing events that occurred early in animal evolution. Toward this goal, we have sequenced, assembled, and annotated the genome of the ctenophore Mnemiopsis leidyi. Our phylogenomic analyses of both amino acid positions and gene content suggest that ctenophores rather than sponges are the sister lineage to all other animals. Mnemiopsis lacks many of the genes found in bilaterian mesodermal cell types, suggesting that these cell types evolved independently. The set of neural genes in Mnemiopsis is similar to that of sponges, indicating that sponges may have lost a nervous system. These results present a newly supported view of early animal evolution that accounts for major losses and/or gains of sophisticated cell types, including nerve and muscle cells.

Technological advances have greatly increased the availability of human genomic sequencing. However, the capacity to analyze genomic data in a clinically meaningful way lags behind the ability to generate such data. To help address this obstacle, we reviewed all conditions with genetic causes and constructed the Clinical Genomic Database (CGD) (http://research.nhgri.nih.gov/CGD/), a searchable, freely Web-accessible database of conditions based on the clinical utility of genetic diagnosis and the availability of specific medical interventions. The CGD currently includes a total of 2,616 genes organized clinically by affected organ systems and interventions (including preventive measures, disease surveillance, and medical or surgical interventions) that could be reasonably warranted by the identification of pathogenic mutations. To aid independent analysis and optimize new data incorporation, the CGD also includes all genetic conditions for which genetic knowledge may affect the selection of supportive care, informed medical decision-making, prognostic considerations, reproductive decisions, and allow avoidance of unnecessary testing, but for which specific interventions are not otherwise currently available. For each entry, the CGD includes the gene symbol, conditions, allelic conditions, clinical categorization (for both manifestations and interventions), mode of inheritance, affected age group, description of interventions/rationale, links to other complementary databases, including databases of variants and presumed pathogenic mutations, and links to PubMed references (>20,000). The CGD will be regularly maintained and updated to keep pace with scientific discovery. Further content-based expert opinions are actively solicited. Eventually, the CGD may assist the rapid curation of individual genomes as part of active medical care.

Importance: While mutations in glucocerebrosidase (GBA1) are associated with an increased risk for Parkinson disease (PD), it is important to establish whether such mutations are also a common risk factor for other Lewy body disorders., Objective: To establish whether GBA1 mutations are a risk factor for dementia with Lewy bodies (DLB). DESIGN We compared genotype data on patients and controls from 11 centers. Data concerning demographics, age at onset, disease duration, and clinical and pathological features were collected when available. We conducted pooled analyses using logistic regression to investigate GBA1 mutation carrier status as predicting DLB or PD with dementia status, using common control subjects as a reference group. Random-effects meta-analyses were conducted to account for additional heterogeneity., Setting: Eleven centers from sites around the world performing genotyping., Participants: Seven hundred twenty-one cases met diagnostic criteria for DLB and 151 had PD with dementia. We compared these cases with 1962 controls from the same centers matched for age, sex, and ethnicity., Main Outcome Measures: Frequency of GBA1 mutations in cases and controls. RESULTS We found a significant association between GBA1 mutation carrier status and DLB, with an odds ratio of 8.28 (95% CI, 4.78-14.88). The odds ratio for PD with dementia was 6.48 (95% CI, 2.53-15.37). The mean age at diagnosis of DLB was earlier in GBA1 mutation carriers than in noncarriers (63.5 vs 68.9 years; P < .001), with higher disease severity scores., Conclusions and Relevance: Mutations in GBA1 are a significant risk factor for DLB. GBA1 mutations likely play an even larger role in the genetic etiology of DLB than in PD, providing insight into the role of glucocerebrosidase in Lewy body disease.

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1's predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.

ZInC (Zebrafish Insertional Collection, http://research.nhgri.nih.gov/ZInC/) is a web-searchable interface of insertional mutants in zebrafish. Over the last two decades, the zebrafish has become a popular model organism for studying vertebrate development as well as for modeling human diseases. To facilitate such studies, we are generating a genome-wide knockout resource that targets every zebrafish protein-coding gene. All mutant fish are freely available to the scientific community through the Zebrafish International Resource Center (ZIRC). To assist researchers in finding mutant and insertion information, we developed a comprehensive database with a web front-end, the ZInC. It can be queried using multiple types of input such as ZFIN (Zebrafish Information Network) IDs, UniGene accession numbers and gene symbols from zebrafish, human and mouse. In the future, ZInC may include data from other insertional mutation projects as well. ZInC cross-references all integration data with the ZFIN (http://zfin.org/).

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoformα (TRIM5α) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (χHIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34(+) cells. To evaluate whether χHIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34(+) cells were transduced with standard SIV vectors and the other half with χHIV vectors. As compared with SIV vectors, χHIV vectors achieved higher vector integration, and the transgene expression rates were two- to threefold higher in granulocytes and red blood cells and equivalent in lymphocytes and platelets for 2 years. A recipient of χHIV vector-only transduced cells reached up to 40% of transgene expression rates in granulocytes and lymphocytes and 20% in red blood cells. Similar to HIV1 and SIV vectors, χHIV vector frequently integrated into gene regions, especially into introns. In summary, our χHIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This χHIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models.

All nonmammalian vertebrates studied can regenerate inner ear mechanosensory receptors (i.e., hair cells) (Corwin and Cotanche, 1988; Lombarte et al., 1993; Baird et al., 1996), but mammals possess only a very limited capacity for regeneration after birth (Roberson and Rubel, 1994). As a result, mammals experience permanent deficiencies in hearing and balance once their inner ear hair cells are lost. The mechanisms of hair cell regeneration are poorly understood. Because the inner ear sensory epithelium is highly conserved in all vertebrates (Fritzsch et al., 2007), we chose to study hair cell regeneration mechanism in adult zebrafish, hoping the results would be transferrable to inducing hair cell regeneration in mammals. We defined the comprehensive network of genes involved in hair cell regeneration in the inner ear of adult zebrafish with the powerful transcriptional profiling technique digital gene expression, which leverages the power of next-generation sequencing ('t Hoen et al., 2008). We also identified a key pathway, stat3/socs3, and demonstrated its role in promoting hair cell regeneration through stem cell activation, cell division, and differentiation. In addition, transient pharmacological inhibition of stat3 signaling accelerated hair cell regeneration without overproducing cells. Taking other published datasets into account (Sano et al., 1999; Schebesta et al., 2006; Dierssen et al., 2008; Riehle et al., 2008; Zhu et al., 2008; Qin et al., 2009), we propose that the stat3/socs3 pathway is a key response in all tissue regeneration and thus an important therapeutic target for a broad application in tissue repair and injury healing.

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis., Competing Interests: The authors have declared that no competing interests exist.

This unit includes a basic protocol with an introduction to the Map Viewer, describing how to perform a simple text-based search of genome annotations to view the genomic context of a gene, navigate along a chromosome, zoom in and out, and change the displayed maps to hide and show information. It also describes some of NCBI's sequence-analysis tools, which are provided as links from the Map Viewer. The alternate protocols describe different ways to query the genome sequence, and also illustrate additional features of the Map Viewer. Alternate Protocol 1 shows how to perform and interpret the results of a BLAST search against the human genome. Alternate Protocol 2 demonstrates how to retrieve a list of all genes between two STS markers. Finally, Alternate Protocol 3 shows how to find all annotated members of a gene family., (2011 by John Wiley & Sons, Inc.)

We describe the creation of a specialized web-accessible database named the Pigment Cell Gene Resource, which contains information on the genetic pathways that regulate pigment cell development and function. This manually curated database is comprised of two sections, an annotated literature section and an interactive transcriptional network diagram. Initially, this database focuses on the transcription factor SOX10, which has essential roles in pigment cell development and function, but the database has been designed with the capacity to expand in the future, allowing inclusion of many more pigmentation genes. Database URL: http://research.nhgri.nih.gov/pigment&#95;cell/

Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag, pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico-generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env, gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration.

Derivation of induced pluripotent stem (iPS) cells requires the expression of defined transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28) in the targeted cells. Lentiviral or standard retroviral gene transfer remains the most robust and commonly used approach. Low reprogramming frequency overall, and the higher efficiency of derivation utilizing integrating vectors compared to more recent nonviral approaches, suggests that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. We provide for the first time an extensive analysis of the lentiviral integration profile in human iPS cells. We identified a total of 78 independent IS in eight recently established iPS cell lines derived from either human fetal fibroblasts or newborn foreskin fibroblasts after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28. The number of IS ranged from 5 to 15 IS per individual iPS clone, and 75 IS could be assigned to a unique chromosomal location. The different iPS clones had no IS in common. Expression analysis as well as extensive bioinformatic analysis did not reveal functional concordance of the lentiviral targeted genes between the different clones. Interestingly, in six of the eight iPS clones, some of the IS were found in pairs, integrated into the same chromosomal location within six base pairs of each other or in very close proximity. Our study supports recent reports that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes.

This unit includes a Basic Protocol with an introduction to the Map Viewer, describing how to perform a simple text-based search of genome annotations to view the genomic context of a gene, navigate along a chromosome, zoom in and out, and change the displayed maps to hide and show information. It also describes some of NCBI's sequence-analysis tools, which are provided as links from the Map Viewer. The Alternate Protocols describe different ways to query the genome sequence, and also illustrate additional features of the Map Viewer. Alternate Protocol 1 shows how to perform and interpret the results of a BLAST search against the human genome. Alternate Protocol 2 demonstrates how to retrieve a list of all genes between two STS markers. Finally, Alternate Protocol 3 shows how to find all annotated members of a gene family., ((c) 2010 by John Wiley & Sons, Inc.)

Background: Recent studies indicate an increased frequency of mutations in the gene encoding glucocerebrosidase (GBA), a deficiency of which causes Gaucher's disease, among patients with Parkinson's disease. We aimed to ascertain the frequency of GBA mutations in an ethnically diverse group of patients with Parkinson's disease., Methods: Sixteen centers participated in our international, collaborative study: five from the Americas, six from Europe, two from Israel, and three from Asia. Each center genotyped a standard DNA panel to permit comparison of the genotyping results across centers. Genotypes and phenotypic data from a total of 5691 patients with Parkinson's disease (780 Ashkenazi Jews) and 4898 controls (387 Ashkenazi Jews) were analyzed, with multivariate logistic-regression models and the Mantel-Haenszel procedure used to estimate odds ratios across centers., Results: All 16 centers could detect two GBA mutations, L444P and N370S. Among Ashkenazi Jewish subjects, either mutation was found in 15% of patients and 3% of controls, and among non-Ashkenazi Jewish subjects, either mutation was found in 3% of patients and less than 1% of controls. GBA was fully sequenced for 1883 non-Ashkenazi Jewish patients, and mutations were identified in 7%, showing that limited mutation screening can miss half the mutant alleles. The odds ratio for any GBA mutation in patients versus controls was 5.43 across centers. As compared with patients who did not carry a GBA mutation, those with a GBA mutation presented earlier with the disease, were more likely to have affected relatives, and were more likely to have atypical clinical manifestations., Conclusions: Data collected from 16 centers demonstrate that there is a strong association between GBA mutations and Parkinson's disease., (2009 Massachusetts Medical Society)

Suppressor of tumorigenicity 14 (St14) encodes matriptase, a serine protease, which regulates processing of profilaggrin to filaggin in vivo. Here, we report that transgenic mice with 1% of wild-type St14 levels (St14(hypo/-)) display aberrant processing of profilaggrin and model human ichthyotic skin phenotypes. Scaling of the skin appears at 1 week of age with underlying epidermal acanthosis and orthohyperkeratosis as well as a CD4+ T-cell dermal infiltrate. Upregulation of antimicrobial peptides occurs when challenged by exposure to the postnatal environment. Direct genomic sequencing of bacterial 16S rRNA genes to query microbial diversity identifies a significant shift in both phylogeny and community structure between St14(hypo/-) mice and control littermates. St14(hypo/-) mice have a selective shift in resident skin microbiota with a decrease of the dominant genus of skin bacteria, Pseudomonas and an accompanying increase of Corynebacterium and Streptococcus. St14(hypo/-) mice provide early evidence that the cutaneous microbiome can be specifically altered by genetic state, which may play an important role in modulating skin disease.

HPS is an autosomal recessive disorder characterized by oculocutaneous albinism and prolonged bleeding. Eight human genes are described resulting in the HPS subtypes 1-8. Certain HPS proteins combine to form Biogenesis of Lysosome-related Organelles Complexes (BLOCs), thought to function in the formation of intracellular vesicles such as melanosomes, platelet dense bodies, and lytic granules. Specifically, BLOC-2 contains the HPS3, HPS5 and HPS6 proteins. We used phylogenetic footprinting to identify conserved regions in the upstream sequences of HPS3, HPS5 and HPS6. These conserved regions were verified to have in vitro transcription activation activity using luciferase reporter assays. Transcription factor binding site analyses of the regions identified 52 putative sites shared by all three genes. When analysis was limited to the conserved footprints, seven binding sites were found shared among all three genes: Pax-5, AIRE, CACD, ZF5, Zic1, E2F and Churchill. The HPS3 conserved upstream region was sequenced in four patients with decreased fibroblast HPS3 RNA levels and only one HPS3 mutation in the coding exons and surrounding exon/intron boundaries; no mutation was found. These findings illustrate the power of phylogenetic footprinting for identifying potential regulatory regions in non-coding sequences and define the first putative promoter elements for any HPS genes.

We previously reported that lentiviral vectors derived from the simian immunodeficiency virus (SIV) were efficient at transducing rhesus hematopoietic repopulating cells. To evaluate the persistence of vector-containing and -expressing cells long term, and the safety implications of SIV lentiviral vector-mediated gene transfer, we followed 3 rhesus macaques for more than 4 years after transplantation with transduced CD34+ cells. All 3 animals demonstrated significant vector marking and expression of the GFP transgene in T cells, B cells, and granulocytes, with mean GFP+ levels of 6.7% (range, 3.3%-13.0%), 7.4% (4.2%-13.4%), and 5.6% (3.1%-10.5%), respectively. There was no vector silencing in hematopoietic cells over time. Vector insertion site analysis of granulocytes demonstrated sustained highly polyclonal reconstitution, with no evidence for progression to oligoclonality. A significant number of clones were found to contribute at both 1-year and 3- or 4-year time points. No vector integrations were detected in the MDS1/EVI1 region, in contrast to our previous findings with a gamma-retroviral vector. These data show that lentiviral vectors can mediate stable and efficient long-term expression in the progeny of transduced hematopoietic stem cells, with an integration profile that may be safer than that of standard Moloney murine leukemia virus (MLV)-derived retroviral vectors.

Background: Cross-species gene expression analyses using oligonucleotide microarrays designed to evaluate a single species can provide spurious results due to mismatches between the interrogated transcriptome and arrayed probes. Based on the most recent human and chimpanzee genome assemblies, we developed updated and accessible probe masking methods that allow human Affymetrix oligonucleotide microarrays to be used for robust genome-wide expression analyses in both species. In this process, only data from oligonucleotide probes predicted to have robust hybridization sensitivity and specificity for both transcriptomes are retained for analysis., Results: To characterize the utility of this resource, we applied our mask protocols to existing expression data from brains, livers, hearts, testes, and kidneys derived from both species and determined the effects probe numbers have on expression scores of specific transcripts. In all five tissues, probe sets with decreasing numbers of probes showed non-linear trends towards increased variation in expression scores. The relationships between expression variation and probe number in brain data closely matched those observed in simulated expression data sets subjected to random probe masking. However, there is evidence that additional factors affect the observed relationships between gene expression scores and probe number in tissues such as liver and kidney. In parallel, we observed that decreasing the number of probes within probe sets lead to linear increases in both gained and lost inferences of differential cross-species expression in all five tissues, which will affect the interpretation of expression data subject to masking., Conclusion: We introduce a readily implemented and updated resource for human and chimpanzee transcriptome analysis through a commonly used microarray platform. Based on empirical observations derived from the analysis of five distinct data sets, we provide novel guidelines for the interpretation of masked data that take the number of probes present in a given probe set into consideration. These guidelines are applicable to other customized applications that involve masking data from specific subsets of probes.

Expression profile analysis clusters Gpnmb with known pigment genes, Tyrp1, Dct, and Si. During development, Gpnmb is expressed in a pattern similar to Mitf, Dct and Si with expression vastly reduced in Mitf mutant animals. Unlike Dct and Si, Gpnmb remains expressed in a discrete population of caudal melanoblasts in Sox10-deficient embryos. To understand the transcriptional regulation of Gpnmb we performed a whole genome annotation of 2,460,048 consensus MITF binding sites, and cross-referenced this with evolutionarily conserved genomic sequences at the GPNMB locus. One conserved element, GPNMB-MCS3, contained two MITF consensus sites, significantly increased luciferase activity in melanocytes and was sufficient to drive expression in melanoblasts in vivo. Deletion of the 5'-most MITF consensus site dramatically reduced enhancer activity indicating a significant role for this site in Gpnmb transcriptional regulation. Future analysis of the Gpnmb locus will provide insight into the transcriptional regulation of melanocytes, and Gpnmb expression can be used as a marker for analyzing melanocyte development and disease progression.

Sox10 is a dynamically regulated transcription factor gene that is essential for the development of neural crest-derived and oligodendroglial populations. Developmental genes often require multiple regulatory sequences that integrate discrete and overlapping functions to coordinate their expression. To identify Sox10 cis-regulatory elements, we integrated multiple model systems, including cell-based screens and transposon-mediated transgensis in zebrafish, to scrutinize mammalian conserved, noncoding genomic segments at the mouse Sox10 locus. We demonstrate that eight of 11 Sox10 genomic elements direct reporter gene expression in transgenic zebrafish similar to patterns observed in transgenic mice, despite an absence of observable sequence conservation between mice and zebrafish. Multiple segments direct expression in overlapping populations of neural crest derivatives and glial cells, ranging from pan-Sox10 and pan-neural crest regulatory control to the modulation of expression in subpopulations of Sox10-expressing cells, including developing melanocytes and Schwann cells. Several sequences demonstrate overlapping spatial control, yet direct expression in incompletely overlapping developmental intervals. We were able to partially explain neural crest expression patterns by the presence of head to head SoxE family binding sites within two of the elements. Moreover, we were able to use this transcription factor binding site signature to identify the corresponding zebrafish enhancers in the absence of overall sequence homology. We demonstrate the utility of zebrafish transgenesis as a high-fidelity surrogate in the dissection of mammalian gene regulation, especially those with dynamically controlled developmental expression., Competing Interests: The authors have declared that no competing interests exist.

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

The many layers and structures of the skin serve as elaborate hosts to microbes, including a diversity of commensal and pathogenic bacteria that contribute to both human health and disease. To determine the complexity and identity of the microbes inhabiting the skin, we sequenced bacterial 16S small-subunit ribosomal RNA genes isolated from the inner elbow of five healthy human subjects. This analysis revealed 113 operational taxonomic units (OTUs; "phylotypes") at the level of 97% similarity that belong to six bacterial divisions. To survey all depths of the skin, we sampled using three methods: swab, scrape, and punch biopsy. Proteobacteria dominated the skin microbiota at all depths of sampling. Interpersonal variation is approximately equal to intrapersonal variation when considering bacterial community membership and structure. Finally, we report strong similarities in the complexity and identity of mouse and human skin microbiota. This study of healthy human skin microbiota will serve to direct future research addressing the role of skin microbiota in health and disease, and metagenomic projects addressing the complex physiological interactions between the skin and the microbes that inhabit this environment.

In an initial epigenetic characterization of diffuse large B-cell lymphoma (DLBCL), we evaluated the DNA methylation levels of over 500 CpG islands. Twelve CpG islands (AR, CDKN1C, DLC1, DRD2, GATA4, GDNF, GRIN2B, MTHFR, MYOD1, NEUROD1, ONECUT2 and TFAP2A) showed significant methylation in over 85% of tumors. Interestingly, the methylation levels of a CpG island proximal to FLJ21062 differed between the activated B-cell-like (ABC-DLBCL) and germinal center B-cell-like (GCB-DLBCL) subtypes. In addition, we compared the methylation and expression status of 67 genes proximal (within 500 bp) to the methylation assays. We frequently observed that hypermethylated CpG islands are proximal to genes that are expressed at low or undetectable levels in tumors. However, many of these same genes were also poorly expressed in DLBCL tumors where their cognate CpG islands were hypomethylated. Nevertheless, the proportional reductions in BNIP3, MGMT, RBP1, GATA4, IGSF4, CRABP1 and FLJ21062 expression with increasing methylation suggest that epigenetic processes strongly influence these genes. Lastly, the moderate expression of several genes proximal to hypermethylated CpG tracts suggests that DNA methylation assays are not always accurate predictors of gene silencing. Overall, further investigation of the highlighted CpG islands as potential clinical biomarkers is warranted.

The Encyclopedia of DNA Elements (ENCODE) project aims to identify and characterize all functional elements in a representative chromosomal sample comprising 1% of the human genome. Data generated by members of The ENCODE Project Consortium are housed in a number of public databases, such as the UCSC Genome Browser, NCBI's Gene Expression Omnibus (GEO), and EBI's ArrayExpress. As such, it is often difficult for biologists to gather all of the ENCODE data from a particular genomic region of interest and integrate them with relevant information found in other public databases. The ENCODEdb portal was developed to address this problem. ENCODEdb provides a unified, single point-of-access to data generated by the ENCODE Consortium, as well as to data from other source databases that lie within ENCODE regions; this provides the user a complete view of all known data in a particular region of interest. ENCODEdb Genomic Context searches allow for the retrieval of information on functional elements annotated within ENCODE regions, including mRNA, EST, and STS sequences; single nucleotide polymorphisms, and UniGene clusters. Information is also retrieved from GEO, OMIM, and major genome sequence browsers. ENCODEdb Consortium Data searches allow users to perform compound queries on array-based ENCODE data available both from GEO and from the UCSC Genome Browser. Results are retrieved from a specific genomic area of interest and can be further manipulated in a variety of contexts, including the UCSC Genome Browser and the Galaxy large-scale genome analysis platform. The ENCODEdb portal is freely accessible at http://research.nhgri.nih.gov/ENCODEdb.

As part of the RIKEN mouse encyclopedia project, two cDNA libraries were prepared from melanocyte-derived cell lines, using techniques of full-length clone selection and subtraction/normalization to enrich for rare transcripts. End sequencing showed that these libraries display over 83% complete coding sequence at the 5' end and 96-97% complete coding sequence at the 3' end. Evaluation of the libraries, derived from B16F10Y tumor cells and melan-c cells, revealed that they contain clones for a majority of the genes previously demonstrated to function in melanocyte biology. Analysis of genomic locations for transcripts revealed that the distribution of melanocyte genes is non-random throughout the genome. Three genomic regions identified that showed significant clustering of melanocyte-expressed genes contain one or more genes previously shown to regulate melanocyte development or function. A catalog of genes expressed in these libraries is presented, providing a valuable resource of cDNA clones and sequence information that can be used for identification of new genes important for melanocyte development, function, and disease.

This unit includes an introduction to the Map Viewer, which describes how to perform a simple text-based search of genome annotations to view the genomic context of a gene, navigate along a chromosome, zoom in and out, and change the displayed maps to hide and show information. It also describes some of NCBI's sequence-analysis tools, which are provided as links from the Map Viewer. The Alternate Protocols describe different ways to query the genome sequence, and also illustrate additional features of the Map Viewer. Alternate Protocol 1 shows how to perform and interpret the results of a BLAST search against the human genome. Alternate Protocol 2 demonstrates how to retrieve a list of all genes between two STS markers. Finally, Alternate Protocol 3 shows how to find all annotated members of a gene family.

Mapping DNase I hypersensitive sites is an accurate method of identifying the location of gene regulatory elements, including promoters, enhancers, silencers and locus control regions. Although Southern blots are the traditional method of identifying DNase I hypersensitive sites, the conventional manual method is not readily scalable to studying large chromosomal regions, much less the entire genome. Here we describe DNase-chip, an approach that can rapidly identify DNase I hypersensitive sites for any region of interest, or potentially for the entire genome, by using tiled microarrays. We used DNase-chip to identify DNase I hypersensitive sites accurately from a representative 1% of the human genome in both primary and immortalized cell types. We found that although most DNase I hypersensitive sites were present in both cell types studied, some of them were cell-type specific. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome.

A major goal in genomics is to understand how genes are regulated in different tissues, stages of development, diseases, and species. Mapping DNase I hypersensitive (HS) sites within nuclear chromatin is a powerful and well-established method of identifying many different types of regulatory elements, but in the past it has been limited to analysis of single loci. We have recently described a protocol to generate a genome-wide library of DNase HS sites. Here, we report high-throughput analysis, using massively parallel signature sequencing (MPSS), of 230,000 tags from a DNase library generated from quiescent human CD4+ T cells. Of the tags that uniquely map to the genome, we identified 14,190 clusters of sequences that group within close proximity to each other. By using a real-time PCR strategy, we determined that the majority of these clusters represent valid DNase HS sites. Approximately 80% of these DNase HS sites uniquely map within one or more annotated regions of the genome believed to contain regulatory elements, including regions 2 kb upstream of genes, CpG islands, and highly conserved sequences. Most DNase HS sites identified in CD4+ T cells are also HS in CD8+ T cells, B cells, hepatocytes, human umbilical vein endothelial cells (HUVECs), and HeLa cells. However, approximately 10% of the DNase HS sites are lymphocyte specific, indicating that this procedure can identify gene regulatory elements that control cell type specificity. This strategy, which can be applied to any cell line or tissue, will enable a better understanding of how chromatin structure dictates cell function and fate.

The yeast SPT10 gene encodes a putative histone acetyltransferase (HAT) implicated as a global transcription regulator acting through basal promoters. Here we address the mechanism of this global regulation. Although microarray analysis confirmed that Spt10p is a global regulator, Spt10p was not detected at any of the most strongly affected genes in vivo. In contrast, the presence of Spt10p at the core histone gene promoters in vivo was confirmed. Since Spt10p activates the core histone genes, a shortage of histones could occur in spt10Delta cells, resulting in defective chromatin structure and a consequent activation of basal promoters. Consistent with this hypothesis, the spt10Delta phenotype can be rescued by extra copies of the histone genes and chromatin is poorly assembled in spt10Delta cells, as shown by irregular nucleosome spacing and reduced negative supercoiling of the endogenous 2mum plasmid. Furthermore, Spt10p binds specifically and highly cooperatively to pairs of upstream activating sequence elements in the core histone promoters [consensus sequence, (G/A)TTCCN(6)TTCNC], consistent with a direct role in histone gene regulation. No other high-affinity sites are predicted in the yeast genome. Thus, Spt10p is a sequence-specific activator of the histone genes, possessing a DNA-binding domain fused to a likely HAT domain.

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34(+)cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors., Competing Interests: The authors have declared that no conflicts of interest exist.

Background: In contrast to gene-mapping studies of simple Mendelian disorders, genetic analyses of complex traits are far more challenging, and high quality data management systems are often critical to the success of these projects. To minimize the difficulties inherent in complex trait studies, we have developed GeneLink, a Web-accessible, password-protected Sybase database., Results: GeneLink is a powerful tool for complex trait mapping, enabling genotypic data to be easily merged with pedigree and extensive phenotypic data. Specifically designed to facilitate large-scale (multi-center) genetic linkage or association studies, GeneLink securely and efficiently handles large amounts of data and provides additional features to facilitate data analysis by existing software packages and quality control. These include the ability to download chromosome-specific data files containing marker data in map order in various formats appropriate for downstream analyses (e.g., GAS and LINKAGE). Furthermore, an unlimited number of phenotypes (either qualitative or quantitative) can be stored and analyzed. Finally, GeneLink generates several quality assurance reports, including genotyping success rates of specified DNA samples or success and heterozygosity rates for specified markers., Conclusions: GeneLink has already proven an invaluable tool for complex trait mapping studies and is discussed primarily in the context of our large, multi-center study of hereditary prostate cancer (HPC). GeneLink is freely available at http://research.nhgri.nih.gov/genelink.