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1.  Improved microarray methods for profiling the yeast knockout strain collection 
Nucleic Acids Research  2005;33(12):e103.
A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3–6% and 15–18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens.
PMCID: PMC1169235  PMID: 15994458
2.  Disparities in Breast Cancer Characteristics and Outcomes by Race/Ethnicity 
Disparities in breast cancer stage and mortality by race/ethnicity in the United States are persistent and well known. However, few studies have assessed differences across racial/ethnic subgroups of women broadly defined as Hispanic, Asian, or Pacific Islander, particularly using more recent data.
Using data from 17 population-based cancer registries in the Surveillance, Epidemiology, and End Results (SEER) Program, we evaluated the relationships between race/ethnicity and breast cancer stage, hormone receptor status, treatment, and mortality. The cohort consisted of 229,594 women 40-79 years of age diagnosed with invasive breast carcinoma between January 2000 and December 2006, including 176,094 non-Hispanic whites, 20,486 blacks, 15,835 Hispanic whites, 14,951 Asians, 1,224 Pacific Islanders and 1,004 American Indians/Alaska Natives.
With respect to statistically significant findings, American Indian/Alaska Native, Asian Indian/Pakistani, black, Filipino, Hawaiian, Mexican, Puerto Rican, and Samoan women had 1.3 to 7.1-fold higher odds of presenting with stage IV breast cancer compared to non-Hispanic white women. Almost all groups were more likely to be diagnosed with estrogen receptor-negative/progesterone receptor-negative (ER-/PR-) disease with black and Puerto Rican women having the highest odds ratios (2.4 and 1.9-fold increases, respectively) compared to non-Hispanic whites. Lastly, black, Hawaiian, Puerto Rican, and Samoan patients had 1.5 to 1.8-fold elevated risks of breast cancer specific mortality.
Breast cancer disparities persist by race/ethnicity, though there is substantial variation within subgroups of women broadly defined as Hispanic or Asian. Targeted, multi-pronged interventions that are culturally appropriate may be important means of reducing the magnitudes of these disparities.
PMCID: PMC3081535  PMID: 21076864
Breast cancer; disparities; race; ethnicity; stage; estrogen receptor; progesterone receptor; mortality
3.  Integrative Analysis of the Caenorhabditis elegans Genome by the modENCODE Project 
Gerstein, Mark B. | Lu, Zhi John | Van Nostrand, Eric L. | Cheng, Chao | Arshinoff, Bradley I. | Liu, Tao | Yip, Kevin Y. | Robilotto, Rebecca | Rechtsteiner, Andreas | Ikegami, Kohta | Alves, Pedro | Chateigner, Aurelien | Perry, Marc | Morris, Mitzi | Auerbach, Raymond K. | Feng, Xin | Leng, Jing | Vielle, Anne | Niu, Wei | Rhrissorrakrai, Kahn | Agarwal, Ashish | Alexander, Roger P. | Barber, Galt | Brdlik, Cathleen M. | Brennan, Jennifer | Brouillet, Jeremy Jean | Carr, Adrian | Cheung, Ming-Sin | Clawson, Hiram | Contrino, Sergio | Dannenberg, Luke O. | Dernburg, Abby F. | Desai, Arshad | Dick, Lindsay | Dosé, Andréa C. | Du, Jiang | Egelhofer, Thea | Ercan, Sevinc | Euskirchen, Ghia | Ewing, Brent | Feingold, Elise A. | Gassmann, Reto | Good, Peter J. | Green, Phil | Gullier, Francois | Gutwein, Michelle | Guyer, Mark S. | Habegger, Lukas | Han, Ting | Henikoff, Jorja G. | Henz, Stefan R. | Hinrichs, Angie | Holster, Heather | Hyman, Tony | Iniguez, A. Leo | Janette, Judith | Jensen, Morten | Kato, Masaomi | Kent, W. James | Kephart, Ellen | Khivansara, Vishal | Khurana, Ekta | Kim, John K. | Kolasinska-Zwierz, Paulina | Lai, Eric C. | Latorre, Isabel | Leahey, Amber | Lewis, Suzanna | Lloyd, Paul | Lochovsky, Lucas | Lowdon, Rebecca F. | Lubling, Yaniv | Lyne, Rachel | MacCoss, Michael | Mackowiak, Sebastian D. | Mangone, Marco | McKay, Sheldon | Mecenas, Desirea | Merrihew, Gennifer | Miller, David M. | Muroyama, Andrew | Murray, John I. | Ooi, Siew-Loon | Pham, Hoang | Phippen, Taryn | Preston, Elicia A. | Rajewsky, Nikolaus | Rätsch, Gunnar | Rosenbaum, Heidi | Rozowsky, Joel | Rutherford, Kim | Ruzanov, Peter | Sarov, Mihail | Sasidharan, Rajkumar | Sboner, Andrea | Scheid, Paul | Segal, Eran | Shin, Hyunjin | Shou, Chong | Slack, Frank J. | Slightam, Cindie | Smith, Richard | Spencer, William C. | Stinson, E. O. | Taing, Scott | Takasaki, Teruaki | Vafeados, Dionne | Voronina, Ksenia | Wang, Guilin | Washington, Nicole L. | Whittle, Christina M. | Wu, Beijing | Yan, Koon-Kiu | Zeller, Georg | Zha, Zheng | Zhong, Mei | Zhou, Xingliang | Ahringer, Julie | Strome, Susan | Gunsalus, Kristin C. | Micklem, Gos | Liu, X. Shirley | Reinke, Valerie | Kim, Stuart K. | Hillier, LaDeana W. | Henikoff, Steven | Piano, Fabio | Snyder, Michael | Stein, Lincoln | Lieb, Jason D. | Waterston, Robert H.
Science (New York, N.Y.)  2010;330(6012):1775-1787.
We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.
PMCID: PMC3142569  PMID: 21177976
4.  A native chromatin purification system for epigenomic profiling in Caenorhabditis elegans 
Nucleic Acids Research  2009;38(4):e26.
High-resolution mapping of chromatin features has emerged as an important strategy for understanding gene regulation and epigenetic inheritance. We describe an in vivo tagging system coupled to chromatin purification for genome-wide epigenetic profiling in Caenorhabditis elegans. In this system, we coexpressed the Escherichia coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino-acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We found that the fusion BioTag::H3.3 was efficiently biotinylated in vivo. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. We found that embryonic chromatin is differentially extracted with increasing salt concentrations. Interestingly, chromatin that remains insoluble after washing in 600 mM salt is enriched at 5′ and 3′ ends, suggesting the presence of large protein complexes that render chromatin insoluble at transcriptional initiation and termination sites. We also found that H3.3 landscapes from these salt fractions display consistent features that correlate with gene activity: the most highly expressed genes contain the most H3.3. This versatile two-component approach has the potential of facilitating genome-wide chromatin dynamics and regulatory site identification in C. elegans.
PMCID: PMC2831312  PMID: 19966274
5.  dSLAM analysis of genome-wide genetic interactions in Saccharomyces cerevisiae 
Methods (San Diego, Calif.)  2007;41(2):206-221.
Analysis of genetic interactions has been extensively exploited to study gene functions and to dissect pathway structures. One such genetic interaction is synthetic lethality, in which the combination of two non-lethal mutations leads to loss of organism viability. We have developed a dSLAM (heterozygote diploid-based synthetic lethality analysis with microarrays) technology that effectively studies synthetic lethality interactions on a genome-wide scale in the budding yeast Saccharomyces cerevisiae. Typically, a query mutation is introduced en masse into a population of ~6,000 haploid-convertible heterozygote diploid Yeast Knockout (YKO) mutants via integrative transformation. Haploid pools of single and double mutants are freshly generated from the resultant heterozygote diploid double mutant pool after meiosis and haploid selection and studied for potential growth defects of each double mutant combination by microarray analysis of the “molecular barcodes” representing each YKO. This technology has been effectively adapted to study other types of genome-wide genetic interactions including gene-compound synthetic lethality, secondary mutation suppression, dosage-dependent synthetic lethality and suppression.
PMCID: PMC2491713  PMID: 17189863
6.  Histone H3.3 Variant Dynamics in the Germline of Caenorhabditis elegans 
PLoS Genetics  2006;2(6):e97.
Germline chromatin undergoes dramatic remodeling events involving histone variants during the life cycle of an organism. A universal histone variant, H3.3, is incorporated at sites of active transcription throughout the cell cycle. The presence of H3.3 in chromatin indicates histone turnover, which is the energy-dependent removal of preexisting histones and replacement with new histones. H3.3 is also incorporated during decondensation of the Drosophila sperm pronucleus, indicating a direct role in chromatin remodeling upon fertilization. Here we present a system to monitor histone turnover and chromatin remodeling during Caenorhabditis elegans development by following the developmental dynamics of H3.3. We generated worm strains expressing green fluorescent protein– or yellow fluorescent protein–fused histone H3.3 proteins, HIS-71 and HIS-72. We found that H3.3 is retained in mature sperm chromatin, raising the possibility that it transmits epigenetic information via the male germline. Upon fertilization, maternal H3.3 enters both male and female pronuclei and is incorporated into paternal chromatin, apparently before the onset of embryonic transcription, suggesting that H3.3 can be incorporated independent of transcription. In early embryos, H3.3 becomes specifically depleted from primordial germ cells. Strikingly, the X chromosome becomes deficient in H3.3 during gametogenesis, indicating a low level of histone turnover. These results raise the possibility that the asymmetry in histone turnover between the X chromosome and autosomes is established during gametogenesis. H3.3 patterns are similar to patterns of H3K4 methylation in the primordial germ cells and on the X chromosome during gametogenesis, suggesting that histone turnover and modification are coupled processes. Our demonstration of dynamic H3.3 incorporation in nondividing cells provides a mechanistic basis for chromatin changes during germ cell development.
Germ cells carry genetic information from one generation to the next. They are converted to gametes during meiosis, which are then reprogrammed for development in the fertilized egg. Gamete production and developmental reprogramming involve dramatic changes in DNA packaging, but little is understood about how these changes are involved in resetting the developmental program for the whole organism. In spermatogenesis, DNA is stripped and repackaged into highly condensed chromatin. After fertilization, sperm DNA is again repackaged as it dramatically decondenses to fuse with the egg nucleus. These repackaging processes involve the four core histone proteins, which tightly wrap DNA into nucleosome particles. A universal variant form of histone 3, H3.3, is abundant in the germ cells of all plants and animals studied and has been shown to turn over at sites of active transcription in various somatic cells. The authors show that H3.3 displays dynamic turnover throughout germ cell development of the roundworm Caenorhabditis elegans. H3.3 incorporates during the first germline stem cell division, continues through meiosis, and ends up in sperm and eggs. Strikingly, H3.3 becomes depleted from primordial germ cells, and the meiotically silenced X chromosome is deficient in H3.3, which suggests that H3.3 dynamics during meiosis and reprogramming transmit epigenetic information.
PMCID: PMC1484599  PMID: 16846252
7.  Human L1 Retrotransposition: cis Preference versus trans Complementation 
Molecular and Cellular Biology  2001;21(4):1429-1439.
Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the ∼400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.
PMCID: PMC99594  PMID: 11158327

Results 1-7 (7)