-
Hank H Qi,
Madathia Sarkissian,
Gang-Qing Hu,
Zhibin Wang, Arindam Bhattacharjee,
D Benjamin Gordon,
Michelle Gonzales,
Fei Lan,
Pat P Ongusaha,
Maite Huarte,
Nasser K Yaghi,
Huijun Lim,
Benjamin A Garcia,
Leonardo Brizuela,
Keji Zhao,
Thomas M Roberts,
Yang Shi
[show abstract]
[hide abstract]
ABSTRACT: X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability. Causal mutations have been found in approximately 90 X-linked genes; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of approximately 7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.
Nature 07/2010; 466(7305):503-7. · 36.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: While whole-genome resequencing remains expensive, genomic partitioning provides an affordable means of targeting sequence efforts towards regions of high interest. There are several competitive methods for targeted capture; these include molecular inversion probes, microdroplet-segregated multiplex PCR, and on-array or in-solution capture-by-hybridization. Enrichment of the human exome by array hybridization has been successfully applied to pinpoint the causative allele of Mendelian disorders. This protocol focuses on the application of Agilent 1 M arrays for capture-by-hybridization and sequencing on the Illumina platform, although the library preparation method may be adaptable to other vendors' array platforms and sequencing technologies.
Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.] 07/2010; Chapter 18:Unit 18.3.
-
Hernán A Burbano,
Emily Hodges,
Richard E Green,
Adrian W Briggs,
Johannes Krause,
Matthias Meyer,
Jeffrey M Good,
Tomislav Maricic,
Philip L F Johnson,
Zhenyu Xuan,
Michelle Rooks, Arindam Bhattacharjee,
Leonardo Brizuela,
Frank W Albert,
Marco de la Rasilla,
Javier Fortea,
Antonio Rosas,
Michael Lachmann,
Gregory J Hannon,
Svante Pääbo
[show abstract]
[hide abstract]
ABSTRACT: It is now possible to perform whole-genome shotgun sequencing as well as capture of specific genomic regions for extinct organisms. However, targeted resequencing of large parts of nuclear genomes has yet to be demonstrated for ancient DNA. Here we show that hybridization capture on microarrays can successfully recover more than a megabase of target regions from Neandertal DNA even in the presence of approximately 99.8% microbial DNA. Using this approach, we have sequenced approximately 14,000 protein-coding positions inferred to have changed on the human lineage since the last common ancestor shared with chimpanzees. By generating the sequence of one Neandertal and 50 present-day humans at these positions, we have identified 88 amino acid substitutions that have become fixed in humans since our divergence from the Neandertals.
Science 05/2010; 328(5979):723-5. · 31.20 Impact Factor
-
Juan Manuel Rosa-Rosa,
Francisco Javier Gracia-Aznárez,
Emily Hodges,
Guillermo Pita,
Michelle Rooks,
Zhenyu Xuan, Arindam Bhattacharjee,
Leonardo Brizuela,
José M Silva,
Gregory J Hannon,
Javier Benitez
[show abstract]
[hide abstract]
ABSTRACT: The classical candidate-gene approach has failed to identify novel breast cancer susceptibility genes. Nowadays, massive parallel sequencing technology allows the development of studies unaffordable a few years ago. However, analysis protocols are not yet sufficiently developed to extract all information from the huge amount of data obtained.
In this study, we performed high throughput sequencing in two regions located on chromosomes 3 and 6, recently identified by linkage studies by our group as candidate regions for harbouring breast cancer susceptibility genes. In order to enrich for the coding regions of all described genes located in both candidate regions, a hybrid-selection method on tiling microarrays was performed.
We developed an analysis pipeline based on SOAP aligner to identify candidate variants with a high real positive confirmation rate (0.89), with which we identified eight variants considered candidates for functional studies. The results suggest that the present strategy might be a valid second step for identifying high penetrance genes.
PLoS ONE 01/2010; 5(4):e9976. · 4.09 Impact Factor
-
Emily Hodges,
Andrew D Smith,
Jude Kendall,
Zhenyu Xuan,
Kandasamy Ravi,
Michelle Rooks,
Michael Q Zhang,
Kenny Ye, Arindam Bhattacharjee,
Leonardo Brizuela,
W Richard McCombie,
Michael Wigler,
Gregory J Hannon,
James B Hicks
[show abstract]
[hide abstract]
ABSTRACT: DNA methylation stabilizes developmentally programmed gene expression states. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Presently, few methods enable quantitative, large-scale, single-base resolution mapping of DNA methylation states in desired regions of a complex mammalian genome. Here, we present an approach that combines array-based hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in genomic regions spanning hundreds of thousands of bases. This single molecule strategy enables methylation variable positions to be quantitatively examined with high sampling precision. Using bisulfite capture, we assessed methylation patterns across 324 randomly selected CpG islands (CGI) representing more than 25,000 CpG sites. A single lane of Illumina sequencing permitted methylation states to be definitively called for >90% of target sties. The accuracy of the hybrid-selection approach was verified using conventional bisulfite capillary sequencing of cloned PCR products amplified from a subset of the selected regions. This confirmed that even partially methylated states could be successfully called. A comparison of human primary and cancer cells revealed multiple differentially methylated regions. More than 25% of islands showed complex methylation patterns either with partial methylation states defining the entire CGI or with contrasting methylation states appearing in specific regional blocks within the island. We observed that transitions in methylation state often correlate with genomic landmarks, including transcriptional start sites and intron-exon junctions. Methylation, along with specific histone marks, was enriched in exonic regions, suggesting that chromatin states can foreshadow the content of mature mRNAs.
Genome Research 09/2009; 19(9):1593-605. · 13.61 Impact Factor
-
Sarah B Ng,
Emily H Turner,
Peggy D Robertson,
Steven D Flygare,
Abigail W Bigham,
Choli Lee,
Tristan Shaffer,
Michelle Wong, Arindam Bhattacharjee,
Evan E Eichler,
Michael Bamshad,
Deborah A Nickerson,
Jay Shendure
[show abstract]
[hide abstract]
ABSTRACT: Genome-wide association studies suggest that common genetic variants explain only a modest fraction of heritable risk for common diseases, raising the question of whether rare variants account for a significant fraction of unexplained heritability. Although DNA sequencing costs have fallen markedly, they remain far from what is necessary for rare and novel variants to be routinely identified at a genome-wide scale in large cohorts. We have therefore sought to develop second-generation methods for targeted sequencing of all protein-coding regions ('exomes'), to reduce costs while enriching for discovery of highly penetrant variants. Here we report on the targeted capture and massively parallel sequencing of the exomes of 12 humans. These include eight HapMap individuals representing three populations, and four unrelated individuals with a rare dominantly inherited disorder, Freeman-Sheldon syndrome (FSS). We demonstrate the sensitive and specific identification of rare and common variants in over 300 megabases of coding sequence. Using FSS as a proof-of-concept, we show that candidate genes for Mendelian disorders can be identified by exome sequencing of a small number of unrelated, affected individuals. This strategy may be extendable to diseases with more complex genetics through larger sample sizes and appropriate weighting of non-synonymous variants by predicted functional impact.
Nature 09/2009; 461(7261):272-6. · 36.28 Impact Factor
-
Sarah B. Ng,
Emily H. Turner,
Peggy D. Robertson,
Steven D. Flygare,
Abigail W. Bigham,
Choli Lee,
Tristan Shaffer,
Michelle Wong, Arindam Bhattacharjee,
Evan E. Eichler,
Michael Bamshad,
Deborah A. Nickerson,
Jay Shendure
[show abstract]
[hide abstract]
ABSTRACT: Genome-wide association studies suggest that common genetic variants explain only a modest fraction of heritable risk for common diseases, raising the question of whether rare variants account for a significant fraction of unexplained heritability
Nature 08/2009; 461(7261):272-276. · 36.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Complementary techniques that deepen information content and minimize reagent costs are required to realize the full potential of massively parallel sequencing. Here, we describe a resequencing approach that directs focus to genomic regions of high interest by combining hybridization-based purification of multi-megabase regions with sequencing on the Illumina Genome Analyzer (GA). The capture matrix is created by a microarray on which probes can be programmed as desired to target any non-repeat portion of the genome, while the method requires only a basic familiarity with microarray hybridization. We present a detailed protocol suitable for 1-2 microg of input genomic DNA and highlight key design tips in which high specificity (>65% of reads stem from enriched exons) and high sensitivity (98% targeted base pair coverage) can be achieved. We have successfully applied this to the enrichment of coding regions, in both human and mouse, ranging from 0.5 to 4 Mb in length. From genomic DNA library production to base-called sequences, this procedure takes approximately 9-10 d inclusive of array captures and one Illumina flow cell run.
Nature Protocol 01/2009; 4(6):960-74. · 8.36 Impact Factor
-
David S Johnson,
Wei Li,
D Benjamin Gordon, Arindam Bhattacharjee,
Bo Curry,
Jayati Ghosh,
Leonardo Brizuela,
Jason S Carroll,
Myles Brown,
Paul Flicek, [......],
Annie Yang,
Zarmik Moqtaderi,
Heather Hirsch,
Hennady P Shulha,
Yutao Fu,
Zhiping Weng,
Kevin Struhl,
Richard M Myers,
Jason D Lieb,
X Shirley Liu
[show abstract]
[hide abstract]
ABSTRACT: The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols, and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated.
Genome Research 04/2008; 18(3):393-403. · 13.61 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Published reports suggest that DNA microarrays identify clinically meaningful subtypes of lung adenocarcinomas not recognizable by other routine tests. This report is an investigation of the reproducibility of the reported tumor subtypes.
Three independent cohorts of patients with lung cancer were evaluated using a variety of DNA microarray assays. Using the integrative correlations method, a subset of genes was selected, the reliability of which was acceptable across the different DNA microarray platforms. Tumor subtypes were selected using consensus clustering and genes distinguishing subtypes were identified using the weighted difference statistic. Gene lists were compared across cohorts using centroids and gene set enrichment analysis.
Cohorts of 31, 72, and 128 adenocarcinomas were generated for a total of 231 microarrays, each with 2,553 reliable genes. Three adenocarcinoma subtypes were identified in each cohort. These were named bronchioid, squamoid, and magnoid according to their respective correlations with gene expression patterns from histologically defined bronchioalveolar carcinoma, squamous cell carcinoma, and large-cell carcinoma. Tumor subtypes were distinguishable by many hundreds of genes, and lists generated in one cohort were predictive of tumor subtypes in the two other cohorts. Tumor subtypes correlated with clinically relevant covariates, including stage-specific survival and metastatic pattern. Most notably, bronchioid tumors were correlated with improved survival in early-stage disease, whereas squamoid tumors were associated with better survival in advanced disease.
DNA microarray analysis of lung adenocarcinomas identified reproducible tumor subtypes which differ significantly in clinically important behaviors such as stage-specific survival.
Journal of Clinical Oncology 12/2006; 24(31):5079-90. · 18.37 Impact Factor
-
Peter C Scacheri,
Orit Rozenblatt-Rosen,
Natasha J Caplen,
Tyra G Wolfsberg,
Lowell Umayam,
Jeffrey C Lee,
Christina M Hughes,
Kalai Selvi Shanmugam, Arindam Bhattacharjee,
Matthew Meyerson,
Francis S Collins
[show abstract]
[hide abstract]
ABSTRACT: RNA interference (RNAi) mediated by short interfering RNAs (siRNAs) is a widely used method to analyze gene function. To use RNAi knockdown accurately to infer gene function, it is essential to determine the specificity of siRNA-mediated RNAi. We have assessed the specificity of 10 different siRNAs corresponding to the MEN1 gene by examining the expression of two additional genes, TP53 (p53) and CDKN1A (p21), which are considered functionally unrelated to menin but are sensitive markers of cell state. MEN1 RNA and corresponding protein levels were all reduced after siRNA transfection of HeLa cells, although the degree of inhibition mediated by individual siRNAs varied. Unexpectedly, we observed dramatic and significant changes in protein levels of p53 and p21 that were unrelated to silencing of the target gene. The modulations in p53 and p21 levels were not abolished on titration of the siRNAs, and similar results were obtained in three other cell lines; in none of the cell lines tested did we see an effect on the protein levels of actin. These data suggest that siRNAs can induce nonspecific effects on protein levels that are siRNA sequence dependent but that these effects may be difficult to detect until genes central to a pivotal cellular response, such as p53 and p21, are studied. We find no evidence that activation of the double-stranded RNA-triggered IFN-associated antiviral pathways accounts for these effects, but we speculate that partial complementary sequence matches to off-target genes may result in a micro-RNA-like inhibition of translation.
Proceedings of the National Academy of Sciences 03/2004; 101(7):1892-7. · 9.68 Impact Factor
-
Christina M Hughes,
Orit Rozenblatt-Rosen,
Thomas A Milne,
Terry D Copeland,
Stuart S Levine,
Jeffrey C Lee,
D Neil Hayes,
Kalai Selvi Shanmugam, Arindam Bhattacharjee,
Christine A Biondi,
Graham F Kay,
Nicholas K Hayward,
Jay L Hess,
Matthew Meyerson
[show abstract]
[hide abstract]
ABSTRACT: The cellular function of the menin tumor suppressor protein, product of the MEN1 gene mutated in familial multiple endocrine neoplasia type 1, has not been defined. We now show that menin is associated with a histone methyltransferase complex containing two trithorax family proteins, MLL2 and Ash2L, and other homologs of the yeast Set1 assembly. This menin-associated complex methylates histone H3 on lysine 4. A subset of tumor-derived menin mutants lacks the associated histone methyltransferase activity. In addition, menin is associated with RNA polymerase II whose large subunit carboxyl-terminal domain is phosphorylated on Ser 5. Men1 knockout embryos and cells show decreased expression of the homeobox genes Hoxc6 and Hoxc8. Chromatin immunoprecipitation experiments reveal that menin is bound to the Hoxc8 locus. These results suggest that menin activates the transcription of differentiation-regulating genes by covalent histone modification, and that this activity is related to tumor suppression by MEN1.
Molecular Cell 03/2004; 13(4):587-97. · 14.18 Impact Factor
