243 research outputs found
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Assembly, remodelled
Biochemical assays reveal that nucleosome maturation and chromatin remodelling by the motor protein Chd1 are distinct, separable enzymatic activities
Multiplexed Illumina sequencing libraries from picogram quantities of DNA
Background: High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. Results: We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. Conclusions: Application of this method allows whole genome studies from samples where material or yields are limiting
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H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex
The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a ‘stairstep’ pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity. DOI: http://dx.doi.org/10.7554/eLife.02833.00
East African lake evidence for Pliocene millennial-scale climate variability
Late Cenozoic climate history in Africa was punctuated by episodes of variability, characterized by the appearance and disappearance of large freshwater lakes within the East African Rift Valley. In the Baringo-Bogoria basin, a well-dated sequence of diatomites and fluviolacustrine sediments documents the precessionally forced cycling of an extensive lake system between 2.70 Ma and 2.55 Ma. One diatomite unit was studied, using the oxygen isotope composition of diatom silica combined with X-ray fluorescence spectrometry and taxonomic assemblage changes, to explore the nature of climate variability during this interval. Data reveal a rapid onset and gradual decline of deepwater lake conditions, which exhibit millennial-scale cyclicity of ∼1400–1700 yr, similar to late Quaternary Dansgaard-Oeschger events. These cycles are thought to reflect enhanced precipitation coincident with increased monsoonal strength, suggesting the existence of a teleconnection between the high latitudes and East Africa during this period. Such climatic variability could have affected faunal and floral evolution at the time
Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5% of yeast genes
Eukaryotic Rvb1p and Rvb2p are two highly conserved proteins related to the helicase subset of the AAA+ family of ATPases. Conditional mutants in both genes show rapid changes in the transcription of over 5% of yeast genes, with a similar number of genes being repressed and activated. Both Rvb1p and Rvb2p are required for maintaining the induced state of many inducible promoters. ATP binding and hydrolysis by Rvb1p and Rvb2p is individually essential in vivo and the two proteins are associated with each other in a high molecular weight complex that shows ATP-dependent chromatin remodeling activity in vitro. Our findings show that Rvb1p and Rvb2p are essential components of a chromatin remodeling complex and determine genes regulated by the complex
DAF-16/FOXO employs the chromatin remodeller SWI/SNF to promote stress resistance and longevity
Organisms are constantly challenged by stresses and privations and require adaptive responses for their survival. The transcription factor DAF-16/FOXO is central nexus in these responses, but despite its importance little is known about how it regulates its target genes. Proteomic identification of DAF-16/FOXO binding partners in Caenorhabditis elegans and their subsequent functional evaluation by RNA interference (RNAi) revealed several candidate DAF-16/FOXO cofactors, most notably the chromatin remodeller SWI/SNF. DAF-16/FOXO and SWI/SNF form a complex and globally colocalize at DAF-16/FOXO target promoters. We show that specifically for gene-activation, DAF-16/FOXO depends on SWI/SNF, facilitating SWI/SNF recruitment to target promoters, in order to activate transcription by presumed remodelling of local chromatin. For the animal, this translates into an essential role of SWI/SNF for DAF-16/FOXO-mediated processes, i.e. dauer formation, stress resistance, and the promotion of longevity. Thus we give insight into the mechanisms of DAF-16/FOXO-mediated transcriptional regulation and establish a critical link between ATP-dependent chromatin remodelling and lifespan regulation
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High-resolution Xist binding maps reveal 2-step spreading during X-inactivation
The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes1-3. During XCI, Xist coats the future inactive X (Xi)4 and recruits Polycomb Repressive Complex 2 (PRC2) to the X-inactivation center (Xic)5. How Xist spreads silencing on a 150 Mb scale is unclear. Here we generate high-resolution maps of Xist binding on the X chromosome across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism, initially targeting gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), suggesting co-migration of Xist and PRC2. Interestingly, when the Xi is acutely stripped off Xist in post-XCI cells, Xist recovers quickly within both gene-rich and -poor domains on a time-scale of hours instead of days, suggesting a previously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms: During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and -poor regions
Inhibition of GATA2 restrains cell proliferation and enhances apoptosis and chemotherapy mediated apoptosis in human GATA2 overexpressing AML cells
GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality. Loss and gain of GATA2 expression has been implicated in myelodysplastic syndrome and acute myeloid leukemia (AML) yet the precise biological impact of GATA2 expression on human AML cell fate decisions remains ambiguous. Herein, we performed large-scale bioinformatics that demonstrated relatively frequent GATA2 overexpression in AML patients as well as select human AML (or AML-like) cell lines. By using shRNAi to target GATA2 in these AML cell lines, and an AML cell line expressing normal levels of GATA2, we found that inhibition of GATA2 caused attenuated cell proliferation and enhanced apoptosis exclusively in AML cell lines that overexpress GATA2. We proceeded to pharmacologically inhibit GATA2 in concert with AML chemotherapeutics and found this augmented cell killing in AML cell lines that overexpress GATA2, but not in an AML cell line expressing normal levels of GATA2. These data indicate that inhibition of GATA2 enhances chemotherapy-mediated apoptosis in human AML cells overexpressing GATA2. Thus, we define novel insights into the oncogenic role of GATA2 in human AML cells and suggest the potential utilization of transient GATA2 therapeutic targeting in AML
Bridging Alone: Religious Conservatism, Marital Homogamy, and Voluntary Association Membership
This study characterizes social insularity of religiously conservative American married couples by examining patterns of voluntary associationmembership. Constructing a dataset of 3938 marital dyads from the second wave of the National Survey of Families and Households, the author investigates whether conservative religious homogamy encourages membership in religious voluntary groups and discourages membership in secular voluntary groups. Results indicate that couples’ shared affiliation with conservative denominations, paired with beliefs in biblical authority and inerrancy, increases the likelihood of religious group membership for husbands and wives and reduces the likelihood of secular group membership for wives, but not for husbands. The social insularity of conservative religious groups appears to be reinforced by homogamy—particularly by wives who share faith with husbands
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Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming
Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2, and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes
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