Critical parameters for efficient sonication and improved chromatin immunoprecipitation of high molecular weight proteins

Solubilization of cross-linked cells followed by chromatin shearing is essential for successful chromatin immunoprecipitation (ChIP). However, this task, typically accomplished by ultrasound treatment, may often become a pitfall of the process, due to inconsistent results obtained between different experiments under seemingly identical conditions. To address this issue we systematically studied ultrasound-mediated cell lysis and chromatin shearing, identified critical parameters of the process and formulated a generic strategy for rational optimization of ultrasound treatment. We also demonstrated that whereas ultrasound treatment required to shear chromatin to within a range of 100–400 bp typically degrades large proteins, a combination of brief sonication and benzonase digestion allows for the generation of similarly sized chromatin fragments while preserving the integrity of associated proteins. This approach should drastically improve ChIP efficiency for this class of proteins

DNMT inhibitors reverse a specific signature of aberrant promoter DNA methylation and associated gene silencing in AML

<b>Background</b>. Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are neoplastic disorders of hematopoietic stem cells. DNA methyltransferase inhibitors (DNMTi), 5-azacytidine (AzaC) and 5-aza-2’-deoxycytidine (Decitabine), benefit some MDS/AML patients. However, the role of DNMTi-induced DNA hypomethylation in regulation of gene expression in AML is unclear.<p></p> <b>Results. </b> We compared the effects of AzaC on DNA methylation and gene expression using whole-genome single-nucleotide bisulfite-sequencing (WGBS) and RNA-sequencing in OCI-AML3 (AML3) cells. For data analysis, we used an approach recently developed for discovery of differential patterns of DNA methylation associated with changes in gene expression, that is tailored to single-nucleotide bisulfite-sequencing data (Washington University Interpolated Methylation Signatures (WIMSi)). By this approach, a subset of genes upregulated by AzaC was found to be characterized by AzaC-induced signature methylation loss flanking the transcription start site. These genes are enriched for genes increased in methylation and decreased in expression in AML3 cells compared to normal hematopoietic stem and progenitor cells. Moreover, these genes are preferentially upregulated by Decitabine in human primary AML blasts, and control cell proliferation, death and development. <p></p> <b>Conclusions.</b> Our WGBS and WIMSi data analysis approach has identified a set of genes whose is methylation and silencing in AML is reversed by DNMTi. These genes are good candidates for direct regulation by DNMTi, and their reactivation by DNMTi may contribute to therapeutic activity. This study also demonstrates the ability of WIMSi to reveal relationships between DNA methylation and gene expression, based on single-nucleotide bisulfite-sequencing and RNA-seq data.<p></p&gt

Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Background: Histone modification H4K20me3 and its methyltransferase SUV420H2 have been implicated in suppression of tumorigenesis. The underlying mechanism is unclear, although H4K20me3 abundance increases during cellular senescence, a stable proliferation arrest and tumor suppressor process, triggered by diverse molecular cues, including activated oncogenes. Here, we investigate the function of H4K20me3 in senescence and tumor suppression. Results: Using immunofluorescence and ChIP-seq we determine the distribution of H4K20me3 in proliferating and senescent human cells. Altered H4K20me3 in senescence is coupled to H4K16ac and DNA methylation changes in senescence. In senescent cells, H4K20me3 is especially enriched at DNA sequences contained within specialized domains of senescence-associated heterochromatin foci (SAHF), as well as specific families of non-genic and genic repeats. Altered H4K20me3 does not correlate strongly with changes in gene expression between proliferating and senescent cells; however, in senescent cells, but not proliferating cells, H4K20me3 enrichment at gene bodies correlates inversely with gene expression, reflecting de novo accumulation of H4K20me3 at repressed genes in senescent cells, including at genes also repressed in proliferating cells. Although elevated SUV420H2 upregulates H4K20me3, this does not accelerate senescence of primary human cells. However, elevated SUV420H2/H4K20me3 reinforces oncogene-induced senescence-associated proliferation arrest and slows tumorigenesis in vivo. Conclusions: These results corroborate a role for chromatin in underpinning the senescence phenotype but do not support a major role for H4K20me3 in initiation of senescence. Rather, we speculate that H4K20me3 plays a role in heterochromatinization and stabilization of the epigenome and genome of pre-malignant, oncogene-expressing senescent cells, thereby suppressing epigenetic and genetic instability and contributing to long-term senescence-mediated tumor suppression

Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion.

<p><b>(A)</b> Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H₂O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. <b>(B)</b> Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. <b>(C)</b> Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148023#sec002" target="_blank">Material and Methods</a> section for detailed reaction conditions. <b>(D)</b> Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.</p

Systematic optimization of sonication conditions.

<p><b>Part 1. (A)</b> Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. <b>(B)</b> The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H₂O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. <i>p</i>-value for analysis of variance between sonicated samples is 1.0×10⁻⁵, between all samples 1.4×10⁻⁶. <i>p</i>-values for selected T-tests are shown on the graph. <b>(C)</b> Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H₂O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. <b>(D)</b> The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H₂O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. <i>p</i>-value for analysis of variance between sonicated samples is 3.6×10⁻⁹ (L-power) and 8.2×10⁻¹⁰ (H-power). <i>p</i>-values for selected T-tests are shown on the graph.</p

Placing the HIRA Histone Chaperone Complex in the Chromatin Landscape

The HIRA chaperone complex, comprised of HIRA, UBN1, and CABIN1, collaborates with histone-binding protein ASF1a to incorporate histone variant H3.3 into chromatin in a DNA replication-independent manner. To better understand HIRA’s function and mechanism, we integrated HIRA, UBN1, ASF1a, and histone H3.3 chromatin immunoprecipitation sequencing and gene expression analyses. Most HIRA-binding sites colocalize with UBN1, ASF1a, and H3.3 at active promoters and active and weak/poised enhancers. At promoters, binding of HIRA/UBN1/ASF1a correlates with the level of gene expression. HIRA is required for deposition of histone H3.3 at its binding sites. There are marked differences in nucleosome and coregulator composition at different classes of HIRA-bound regulatory sites. Underscoring this, we report physical interactions between the HIRA complex and transcription factors, a chromatin insulator and an ATP-dependent chromatin-remodeling complex. Our results map the distribution of the HIRA chaperone across the chromatin landscape and point to different interacting partners at functionally distinct regulatory sites

Profiling of transcriptional and epigenetic changes during directed endothelial differentiation of human embryonic stem cells identifies FOXA2 as a marker of early mesoderm commitment

Introduction: Differentiation of vascular endothelial cells (ECs) in clinically relevant numbers for injection into ischaemic areas could offer therapeutic potential in the treatment of cardiovascular conditions, including myocardial infarction, peripheral vascular disease and stroke. While we and others have demonstrated successful generation of functional endothelial-like cells from human embryonic stem cells (hESCs), little is understood regarding the complex transcriptional and epigenetic changes that occur during differentiation, in particular during early commitment to a mesodermal lineage. Methods: We performed the first gene expression microarray study of hESCs undergoing directed differentiation to ECs using a monolayer-based, feeder-free and serum-free protocol. Microarray results were confirmed by quantitative RT-PCR and immunocytochemistry, and chromatin immunoprecipitation (ChIP)-PCR analysis was utilised to determine the bivalent status of differentially expressed genes. Results: We identified 22 transcription factors specific to early mesoderm commitment. Among these factors, FOXA2 was observed to be the most significantly differentially expressed at the hESC–EC day 2 timepoint. ChIP-PCR analysis revealed that the FOXA2 transcription start site is bivalently marked with histone modifications for both gene activation (H3K4me3) and repression (H3K27me3) in hESCs, suggesting the transcription factor may be a key regulator of hESC differentiation. Conclusion: This enhanced knowledge of the lineage commitment process will help improve the design of directed differentiation protocols, increasing the yield of endothelial-like cells for regenerative medicine therapies in cardiovascular disease