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Common alterations to brain DNA methylation in Alzheimer’s disease.
Alzheimer’s Disease (AD) represents an enormous burden to society, with conventional Genome Wide Association Studies (GWAS) showing that the picture is far from complete. However, by taking advantage of reduced representation bisulfite (RRBS) assays, Epigenome Wide Association Studies (EWAS) have begun to fill the void.
Scientists from the Broad Institute of MIT and Harvard and the University of Exeter (UK) have teamed up to analyze a collection of 708 brains. By assessing the methylation states of AD patients, compared to matched controls, they found:
A sister study, published within the same Nature Neuroscience issue, provides a replication of the results in independent cohorts and brain regions over a combined total of 1,200 patients. Interestingly, they found the same associations in four of the eleven validated genes: ANK1, RHBDF2, RPL13 and CDH23. The large sample sizes suggest that DNA methylation may have role in the onset of AD, given their observance in pre-symptomatic patients.
See more in Nature Neuroscience, August 2014.
Fixing matters of the prenatal alcohol exposure heart.
While primarily known for its effects on the developing brain, prenatal alcohol exposure (PAE) also leaves an impact on the developing heart. Proper Gata4 expression is crucial for cardiovascular development and abnormalities in the regulation of Gata4 expression result in cardiovascular malformations.
A team from Chongqing (China) previously found that PAE may cause histone H3K9 hyperacetylation and over-expression of Gata4 in their mouse model. However, the mechanism behind this remained unclear until their most recent experiments. Here is what the new analysis uncovered:
Ultimately, the findings suggest that P300 and PCAF regulate the expression of Gata4 upon PAE. Thus, anacardic acid appears to protect against PAE induced alterations to cardiac development by inhibiting the binding of transcriptional regulators to the promoter region of Gata4.
Find the complete report in PLoS One, August 2014.
Abcam products used: H3K9 (ab4441).
Epigenetic variation influences individual identity.
It has become clear that variation in DNA sequence leads to individual differences in brain function. A similar role for epigenetic variation in humans has yet to be established. The gene SLC6A4 encodes a serotonin transporter that affects brain function and behavior through regulation of synaptic serotonin signaling. Genetic variation at this locus is well established to affect behavior; however, epigenetic variation and its effect remain unclear.
Yuliya Nikolova and colleagues at Duke University investigated the 20 CpG sites nearest to the transcriptional start site of SLC6A4 in an 80 adolescent discovery group, and an additional 96 individuals. The authors examined the percentage of methylation of these cytosines. Their chosen behavioral phenotype was threat-related amygdala reactivity (TRAR) using blood oxygen level-dependent functional magnetic resonance imaging because of its relevance to normal and pathogenic emotionality.
The researchers found that:
This research suggests that methylation of the proximal promoter reduces SLC6A4 gene expression leading to decreased serotonin transporter levels and therefore reduced regional serotonin reuptake, affecting the amygdala. These data provide a concrete example of common, individual epigenetic variation affecting phenotype similar to genetic variation, and points to the importance of epigenetics in individual identity.
Read the full report in Nature Neuroscience, August 2014.
Potential for targeted epigenetic leukemia therapy.
T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy with poor prognosis due in part to the lack of non-toxic therapies. Finding epigenetic targets has been the focus of recent research on the disorder. The histone H3K27 methyltransferase, Polycomb repressive complex 2 (PRC2), has previously been shown to have a tumor suppressor role in T-ALL; however, its precise role remains unclear.
Aristotelis Tsirigos, Charles Mullighan, Iannis Aifantis and a team from the Howard Hughes and Whitehead institutes sought to investigate the potential de-regulation of H3K27me3 demethylases in T-ALL. They focused on tetratricopeptide repeat X-linked protein (UTX) and Jumonji D3 (JMJD3). Using the NOTCH1 mutation mouse model (which is a defining feature of T-ALL) and human cell lines the researchers found:
JMJD3 and UTX act in opposition; with JMJD3 functioning to initiate and maintain T-ALL while UTX acts as a tumor suppressor and is mutated in the disorder. Inhibition of JMJD3 with a small molecule recapitulates Jmjd3 knockdown and may lead to effective epigenetic therapy.
See the full report in Nature Letters, August 2014.
Optimized ChIP protocol for woody samples.
Chromatin immunoprecipitation (ChIP) is a critical tool for understanding histone and transcription factor relationships with DNA, but while it has been used extensively in many species, including Arabidopsis, a ChIP protocol designed to perform with wood-forming plant tissues and cells was needed.
Vincent Chiang and a team from North Carolina State University set out to optimize the standard ChIP protocol for use with woody tissues. The scientists used stem-differentiating xylem (SDX), which is a wood-forming tissue from the plant Populus trichocarpa, as a model system and then systematically identified and refined sub-optimal areas in the ChIP protocol. Improvements were made to virtually every step of the method including: tissue collection, cross-linking, nuclear isolation, chromatin extraction, DNA fragmentation, immunoprecipitation, DNA purification and sequence analysis.
The new protocol takes about 2.5 days, and delivers a solid 8–10-fold enrichment of transcription factor fragments over background. The collected DNA is also of high quality, and acceptable for common analysis methods including PCR and DNA sequencing.
The researchers demonstrated the utility of the enhanced ChIP protocol by studying genome-wide specific transcription factor-DNA interactions and histone modifications that occur during wood formation. The authors believe that this technique may also be useful for several tissue types, and currently it is the only ChIP system proven to be successful working with wood-forming tissue.
See the complete protocol at Nature Protocols, August 2014.
PAT-ChIP opens up the options for FFPE sample studies.
A recently developed ChIP technique known as pathology tissue-chromatin immunoprecipitation (PAT-ChIP) has made it possible to study chromatin from formalin-fixed and paraffin-embedded (FFPE) tissues. However, the use of FFPE tissue sections is hampered by tissue heterogeneity, which confounds epigenetic analysis. Researchers Saverio Minucci (European Institute of Oncology), Mirco Fanelli (Unversity of Urbino) and colleagues devised an approach to enhance the homogeneity of FFPE cell populations and get the most benefit from the PAT-ChIP system.
The team tested core needle biopsies (CNBs) and laser microdissection (LMD) from FFPE samples as a means of selecting for a more homogenous cell population, and then set out to make them compatible with the PAT-ChIP procedure. Here are some of the results:
The authors conclude that both needle biopsies and laser microdissection can substantially enrich cell populations from FFPE samples. When paired with an optimized PAT-ChIP approach, the homogenous samples provide improved epigenetic profiling that may potentially be used to discover and validate novel epigenetic biomarkers.
See the complete protocol at Epigenetics and Chromatin, August 2014.
Abcam products used: H3K27Ac (ab4729).
H3K27me3 recruited by short DNA sequences.
The chromatin mark H3K27me3 is known to be involved with Polycomb-mediated gene repression, but the mechanism by which it is targeted to a specific loci has yet to be revealed in mammalian cells.
Dirk Schübeler and fellow researchers at the Friedrich Miescher Institute for Biomedical Research applied iterative genome editing to systematically determine the exact DNA sequences that are able to recruit the Polycomb complex PRC2 on its own. The team inserted 28 DNA elements at a specific chromosomal position in mouse ES cells and then measured the amount of H3K27me3 that was deposited. Very short DNA elements (220 nucleotides long) were identified that correctly recreated endogenous H3K27me3 patterns.
The newly discovered Polycomb recruiter sequences tend to be CpG-rich, and the scientists found that either DNA methylation, or presence of an enhancer can interfere with recruitment. The authors suggest that their data supports a model whereby PRC2 is attracted by the local CpG density of specific loci, but may also be blocked when DNA methylation or transcriptional activity is present.
Find the full report at PNAS, August 2014.
MacroH2A2 turnover at ES cell promoters.
The dynamic movement of certain nucleosomes is critical to the epigenetic inheritance of chromatin architecture and the maintenance of pluripotency in embryonic stem (ES) cells. The histone MacroH2A2 is generally thought of as a repressive mark that locks in the epigenetic state of differentiated cells, but it’s role in nucleosome dynamics is much less understood than that of other histone variants.
A research group led by Christopher Lengner (University of Pennsylvania) and Oliver Rando (University of Massachusetts Medical School) used a pulse-chase experimental approach to measure genome-wide histone dynamics in mouse ES cells and somatic tissues, and investigate the dynamics of MacroH2A2. Here is was the investigators found:
The authors found these unanticipated results to provide new insights into the dynamics of MacroH2A2 turnover in pluripotent cells, and set the stage for further investigation into tissue-specific histone dynamics in vivo.
See more details of the study at PLoS Genetics, August 2014.