Top Epigenetics articles: June 2014

Struggling to keep up with the literature? Want to stay on top of the hottest research this summer? Check out Abcam’s must read papers for June.

A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice

Chromatin Switch Decides DNA Repair Pathway

Histone H3 lysine 36 (H3K36) modification has been associated with DNA double-strand break (DSB) repair in both yeast and human cells, however it is still unclear how the decision between two alternative repair mechanisms, non-homologous end joining (NHEJ) or homologous recombination (HR), gets made.

Dr. Timothy Humphrey and his cohorts at the University of Oxford investigated how H3K36, based on its modification state, may act as a molecular switch to influence DSB repair pathway selection. H3K36 residues may either be methylated or acetylated, and the scientists hypothesized that the different marks could play a role in the inverse relationship between NHEJ and HR.

The researchers studied DSB repair in fission yeast, with a special focus on H3K36 modifications, and here is what they discovered:

  • Set2 methyltransferase is critical for NHEJ, but suppresses HR
  • The histone acetyltransferase Gcn5 promotes HR and suppresses NHEJ
  • H3K36 methylation and acetylation inhibit each other
  • H3K36 modification and DSB repair pathway choice is associated with cell cycle, with NHEJ occurring in G1, and HR during S and G2 phases
  • H3K36 modification state shifts chromatin accessibility to DSB repair factors

The data revealed what the authors termed a ‘H3K36 chromatin switch’; a mechanism where Set2-dependent H3K36 methylation leads to NHEJ and Gcn5-dependent H3K36 acetylation encourages HR, and coordinates the DSB repair pathway choice in fission yeast.

See the full report in Nature Communications, June 2014.

Genome-Wide Binding of MBD2 Reveals Strong Preference for Highly Methylated Loci

MBD2 Preferentially Binds to Highly Methylated DNA Regions

Researchers from Radboud University in The Netherlands gained new understanding of epigenetic regulation by mapping out the binding behavior of methyl binding domain 2 (MBD2) across the entire genome in MCF-7 breast cancer cells.

MBD2 is a member of the NuRD complex that is thought to repress gene expression when the complex interacts with methylated DNA. Armed with a tagged and stably expressed version of MBD2, ChIP sequencing (ChIP-seq) and whole genome bisulfite sequencing (WGBS) Dr. Hendrik Stunnenberg and his team created an in-depth portrait of the function and role of MDB2 binding. Here are some of the highlights:

  • MBD2 prefers to bind at CpG Island (CGI) promoter regions
  • Methylation density is important, as MBD2 was found most often in highly methylated loci
  • MBD2 binding sites were identified roughly 1 kb downstream of the transcription start sites (TSS) for subset of ~400 active CGI promoters
  • RNA PolII distribution, and therefore transcription, is affected by MBD2 binding downstream of active promoters
  • Tagged MBD2 was shown to differentially bind hypermethylated areas in MCF7’s when compared to normal breast cells

In addition to characterizing genome-wide MBD2 binding patterns, the author’s work revealed increased DNA methylation levels in primary breast cancer samples, suggesting potential role for MBD2 in breast cancer

Find the entire report in PLoS One, June 2014.

A Supercharged Fluorescent Protein as a Versatile Probe for Homogenous DNA Detection and Methylation Analysis

Supercharged Fluorescent Proteins Enable Ultra-sensitive DNA Methylation Detection

The advent of supercharged proteins that are stable and capable of delivering macromolecules into cells, led a scientific team at Hunan University to adapt this new fluorescent biosensing technology for epigenetic applications. The platform is based on supercharged green fluorescent protein (ScGFP), and uses efficient quenching of ScGFP/DNA-quencher nanoscale complex and toehold-mediated strand displacement to generate a reporter signal, which creates a sensitive assay for DNA detection and DNA methylation analysis.

Dr. Zhou Nie and fellow researchers coupled bisulfite conversion with their new ScGFP reporter assay in order to achieve site-specific DNA methylation detection. The group found that the ScGFP assay had very high sensitivity and could detect even single-base mismatches. The new ScGFP-based assay was then used to assess the DNA methylation state in genomic DNA from human colon carcinoma samples, and achieved site-specific resolution using only small amounts of sample DNA (down to 4 amol methylated DNA).

The scientists noted several benefits of the supercharged fluorescent protein system:

  • ScGFP probes are fairly simple and inexpensive to design
  • Assays are highly adaptable to DNA methylation analysis or cancer diagnositics
  • May one daybe applicable to in vitro studies of biomolecules

The authors are confident, based on the performance they observed, that supercharged proteins, like ScGFP will become a highly useful approach to develop biosensor platforms.

Read the full article in Angewandte Chemie International Edition, June 2014.

Single-cell western blotting

Single Cell Western Blots Tackle Cell Population Heterogeneity

Heterogeneity in cell populations confounds many current analysis techniques, and has been a substantial obstacle to scientific research in several areas including cell differentiation, development, cancer and immune response. Recent technological advances have enabled new understanding of the varied genomes and transcriptomes between cells in the same tissue, however researchers still have a need for more optimized tools.

Scientists at the University of California, Berkeley have devised a new method for analyzing complex cell populations, which they refer to as single-cell western blotting or scWestern. The scWestern platform uses an open-microwell array structure that allows about 2,000 individual cells to be assayed in under 4 hours. The team led by Dr. David Schaffer and Dr. Amy Herr show in this Nature Methods article that scWestern has features that addresses several critical issues often encountered when investigating cell populations, including:

  • Optimized for protein analysis in many cells
  • Provides single-cell resolution
  • Offers quantitative, highly specific identification of target proteins
  • Uses antibody labels that do not interfere with protein or cell functions

The authors found during development that scWestens report data about both protein mass, as well as antibody binding which delivers a very high degree of protein specificity. When applied in a single-cell context, scWesterns enable researchers to have a quantitative, multiplexed, bench top tool to investigate cell-to-cell variation in cell functions. Eventually the team expect that scWesterns may be found in applications that incorporate upstream functional or morphological screens or measure individual cell response to therapeutics.

Read the full report at Nature Methods, July 2014.

Increased macroH2A1.1 Expression Correlates with Poor Survival of Triple-Negative Breast Cancer Patients

Elevated macroH2A1.1 Indicates Poor Breast Cancer Prognosis

Epithelial-Mesenchymal Transition (EMT) is thought to have a major role in breast cancer, and epigenetic modifications are a factor in establishing EMT, as well as cancer subclasses. To determine what influence, if any, histone variants have in these transitions, researchers from the Université de Toulouse in France analyzed expression levels of histone macroH2A1 splice variants and searched for correlations with breast cancer status, prognosis, and types.

The group led by Dr. Anne-Claire Lavigne scoured the GEO, EMBL-EBI and publisher databases (may-august 2012), searching for differential expression of macroH2A1 variant mRNAs in breast cancer cells and tumors. The scientists calculated macroH2A1.1/macroH2A1 mRNA ratios and then attempted to relate them to molecular breast cancer subclasses and hallmarks of EMT. Here’s what their analysis revealed:

  • Elevated macroH2A1.1/macroH2A1 mRNA ratios are associated with the claudin-low breast cancer subtype
  • A positive correlation exists between macroH2A1 ratios and molecular indicators of the EMT
  • Untreated Triple Negative Breast Cancers (TNBC) exhibited high macroH2A1.1 mRNA ratios and displayed poor outcomes

The authors conclude that macroH2A1.1 expression level is a key factor in the epigenetic mechanisms that link poor clinical outcomes to specific molecular breast cancer subtypes, and more broadly to the EMT process.

Find the full article in PLoS One, June 2014.

Histone deacetylase (HDAC) 1 and 2 are essential for accurate cell division and the pluripotency of embryonic stem cells

HDAC1 and HDAC2 Critical for Stem Cell Pluripotency

Histone deacetylase 1 and 2 (HDAC1/2) are closely related proteins that modulate gene expression by controlling DNA access through the manipulation of chromatin structure. Previous work has shown that both HDAC1 and HDAC2 need to be eliminated in order to cause phenotypic changes, indicating that they have redundant activities.

Scientists at the University of Leicester explored the role of HDAC1/2 in stem cells, starting by developing a specialized embryonic stem (ES) cell line model. The double conditional knockout (DKO) (HDAC1Lox/Lox; HDAC2Lox/Lox; CreER) cell line allows them to inactivate HDAC1 and HDAC2 simultaneously with a tamoxifen-inducible Cre/estrogen receptor fusion expressed from the ROSA26 locus. When Dr. Shaun Cowley and his group studied the results from their new ES cell line, here is what they observed:

  • Loss of HDAC1/2 reduced total HDAC activity by 60%, and caused the loss of cell viability after 4 days
  • HDAC1/2 are needed for proper chromosome segregation, as evidenced by increased mitotic defects, chromatin bridges, and micronuclei
  • Microarray analysis uncovered 1,708 differentially expressed genes in Hdac1/2-deleted cells
  • Pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1 had lowered expression, suggesting HDAC1/2 function is important for stem cell self-renewal
  • Viability rescue of DKO cells demonstrated that interference with IP4 binding reduces HDAC1/2 activity in vivo

The research team concludes that HDAC1 and HDAC2 are an essential component in cellular proliferation and stem cell self-renewal by maintaining the gene expression of key pluripotent transcription factors. The authors also suggest that because of the cell death induced by inactivation of HDAC1/2, and subsequent defective DNA replication or mitosis, specific HDAC1/2 inhibitors should make highly effective cancer therapeutic targets.

Find the complete article in PNAS, June 2014.

DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin

The Role of H3K9 Methylation in DSB Repair

When DNA double-strand breaks (DSB) occur, dynamic histone modifications are needed to facilitate their repair. Histone H3 methylated on lysine 9 (H3K9me3) interacts with, and activates the acetyltransferase Tip60 for DSB repair, but the regulation of H3K9 methylation in this mechanism remains unclear.

A research group from the Dana-Farber Cancer Institute at Harvard Medical School delved into the topic of H3K9 in BSB repair, to determine exactly how it works. Dr. Brendan Price and colleagues mapped out the process in their latest PNAS paper:

  • A complex of kap-1, HP1, suv39h1 (an H3K9 methyltransferase) is quickly loaded onto chromatin at DSB sites
  • Suv39h1 methylates H3K9, encouraging additional loading of kap-1/HP1/suv39h1 complex
  • The continuous cycle spreads H3K9me3 and kap-1/HP1/suv39h1 complexes several kilobases from the DSB
  • H3K9me3 activate the Tip60 acetyltransferase, which acetylates both ataxia telangiectasia-mutated (ATM) kinase and histone H4
  • Activated ATM phosphorylates kap-1, releasing the repressive kap-1/HP1/suv39h1 complex from the chromatin
  • ATM activation completes the negative feedback loop, and slows down DSB repair

The authors summarize that dynamic histone methylation and kap-1 phosphorylation during DSB repair constitutes a mechanism that regulates chromatin compaction of heterochromatin, so that DSB repair can take place within a common epigenetic and structural template.

See the entire article in PNAS, June 2014.

Histone H4 tail mediates allosteric regulation of nucleosome remodeling by linker DNA

Histone H4 Tails Keep Nucleosomes Properly Spaced

The ISWI family of chromatin remodeling enzymes control heterochromatin formation and transcriptional silencing by keeping nucleosomes spaced at regular intervals. Nucleosome spacing depends on the length of extranucleosomal linker DNA, however the remainder of the mechanism has yet to be described.

In a newly released Nature article, a team from Harvard University investigated the mechanism of nucleosome remodeling by human ATP-dependent chromatin assembly and remodeling factor (ACF), which is an ISWI enzyme containing Snf2h and Acf1 subunits. Here is what Dr. Xiaowei Zhuang and his fellow researchers found:

  • ACF senses linker DNA length through interactions between its accessory (Acf1) and catalytic subunits (Snf2h) and the nucleosome’s H4 tail
  • AutoN domain mutations within Snf2h destroy linker-length sensitivity during remodeling
  • Linker-length sensitivity can be blocked by exogenous H4-tail peptides or nucleosomal H4 tail deletion
  • Acf1 binds both the H4-tail peptide and DNA, and lengthening the linker DNA reduces the Acf1-H4 tail proximity

The authors report a nucleosome spacing mechanism where linker DNA sensing by Acf1 is then communicated to Snf2h via nucleosomal H4 tails. When nucleosomes contain short linker DNA, Acf1 will preferentially bind to the H4 tail.  This allows AutoN to block Snf2h ATPase activity. With longer linker DNA, Acf1 will shift towards binding linker DNA, and the freed H4 tail then out competes AutoN from ATPase, thereby activating ACF.

The new mechanism makes linker DNA (which creates uniform spacing for heterochromatin structure) and the H4 tail (which identifies chromatin regions for silencing) two critical features in the regulation of nucleosome remodeling.

Read the complete article in Nature, June 2014.