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 1.3-7.1-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 1.5-1.8-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

Histone H3.3 Variant Dynamics in the Germline of Caenorhabditis elegans

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

Improved microarray methods for profiling the yeast knockout strain collection

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

A native chromatin purification system for epigenomic profiling in Caenorhabditis elegans

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

H3.3 Is Present throughout Development

<div><p>(A) HIS-71::GFP and (B) HIS-72::GFP fluorescence in living adults; cell types as indicated. Note absence of HIS-72::GFP in intestinal nuclei; small fluorescence particles surrounding the intestinal nuclei are autofluorescent gut granules.</p><p>Note also that (B), the fluorescence of germ cell nuclei is less intense than that of somatic cell nuclei.</p><p>(C) HIS-72::GFP expression in larvae; the bottom animal is an L1 larva.</p><p>(D, E) Formaldeyde-fixed embryo showing HIS-72::GFP colocalization with DAPI staining. Double-headed arrows point to anaphase cells with characteristic bar-shaped structure of metaphase chromosome.</p><p>(F, G) Fluorescence and DIC micrographs of embryos at various stages expressing HIS-72::GFP; the embryo at the right is at the two-cell stage. Scale bars, 10 μm.</p></div

H3.3 Is a Component of Mature Sperm Chromatin

<div><p>(A, B) HIS-72::GFP is present throughout the male gonad starting from the mitotic zone and colocalizes with DAPI-staining; insets show enlarged regions of the gonads.</p><p>(C, D) YFP fluorescence and DIC images of sperm from a dissected male.</p><p>(E–H) Sperm from a dissected and formaldehyde-fixed hermaphrodite showing HIS-72::GFP and DAPI colocalization. Scale bars, 10 μm.</p></div

H3.3 Is Provided Maternally in Oocytes and Maternal H3.3 Is Incorporated into Paternal Chromatin upon Fertilization

<div><p>(A, B) HIS-72::GFP colocalizes with DAPI staining throughout the oogenic gonad. High magnification images of the (C, D) transition zone and (E, F) pachytene region. In the oocyte, HIS-72::GFP is provided maternally in the nuclei. (A, C, E) GFP fluorescence and (B, D, F) DAPI images of gonads dissected from hermaphrodites. (A) and (B) are composite images.</p><p>(G, H) Times-lapse images of a single oocyte at meiosis II as the maternal chromosomes divide to segregate a polar body.</p><p>(I–N) N2 males were crossed to temperature-sensitive <i>fem-1(hc17</i>ts<i>) unc-4(e120)</i> HIS-72::GFP hermaphrodites grown at nonpermissive temperature (23 °C). Upon fertilization, as soon as the nuclear envelope of the two pronuclei are formed, (I, J) maternal HIS-72::GFP is imported into both pronuclei and (K–N) incorporated into paternal chromatin. (I, J) GFP fluorescence and DIC images of a live, in utero one-cell embryo. (K–N) Images of a single, dissected, and methonal-fixed one-cell embryo labeled as shown. The maternal pronucleus can be identified by its proximity to the polar body (J and M), and the male pronucleus by its proximity to posterior-localized PGL-1 protein (N). In (N), PGL-1 localization is cytoplasmic in the zygote, while H3K4me2 is localized to chromatin. Scale bars, 10 μm.</p></div