Top Epigenetics articles: July 2014

Living life on the research edge? Take a minute to stop and see what’s going on around you. Here’s our selection of top papers this month.

Epigenenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility.

Enhancer H3K4 Dimethylation Linked to Asthma

Worldwide asthma cases have been consistently rising, highlighting the need for an even deeper understanding of the molecular basis of the disease. Asthma sufferers have an overabundance of memory CD4(+) T cells that produce type 2 cytokines (TH2 cells). Researchers at the La Jolla Institute for Allergy & Immunology mapped genome-wide histone modification profiles for T cells in order to determine their role in asthma.

The group, led by corresponding author Pandurangan Vijayanand, surveyed T cell populations isolated from healthy and asthmatic peripheral blood, and used that information to identify enhancers that may have a role in human TH1 and TH2 cell differentiation. Analysis of the results revealed:

  • Disease-specific enhancers behaved differently between the healthy and asthmatic cohorts.
  • During  TH2 cell development, enhancer regions with increased histone H3 Lys4 dimethyl (H3K4me2) marks also had the highest enrichment for asthma-linked single nucleotide polymorphisms (SNPs)
  • Subsequent analysis of the cell-specific enhancers uncovered transcription factors, microRNAs and genes that potentially effect TH2 cell differentiation.

The study confirms the role for TH2 cells in asthma, and establishes the utility of enhancer profiling for the investigation of disease pathogenesis.

See the complete analysis at Nature Immunology, August 2014.

Revealing long noncoding RNA architecture and functions using domain-specific chromatin isolation by RNA purification.

Chromatin, Long Noncoding RNA Interactions Revealed by dChIRP

Using traditional approaches makes it a challenge to investigate the functional domains of long noncoding RNAs (lncRNAs). In this article, the authors from Howard Hughes Medical Institute and Stanford University introduce a new method, named domain-specific chromatin isolation by RNA purification (dChIRP), aimed at better deciphering lncRNA function and architecture.

A team led by Howard Chang used dChIRP to inspect lncRNAs domain by domain, and help them identify functional elements. To demonstrate the technique, the researchers analyzed dChIRP of the Drosophila melanogaster lncRNA, roX1. Here is what they learned:

  • roX1 has a 'three-fingered hand' ribonucleoprotein topology
  • Each of the three RNA fingers binds to both chromatin and the male-specific lethal (MSL) protein complex and prevents male lethality in roX-null flies
  • The roX binding sites correlate with chromosome conformation data, suggesting that roX-bound loci cluster near each other.

The scientists were able to obtain RNA genomic localization signals over 20 fold more robust than previous methods. Based on those results, the authors claim that dChIRP is highly useful for decoding RNA-RNA, RNA-protein and RNA-chromatin interactions at the level of individual RNA domains in living cells with very high precision and sensitivity.

Find more details on the dChIPR method at Nature Biotechnology, July 2014.

Uses our Rabbit polyclonal to actin (ab1801).

Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries

DNA-barcoded Nucleosomes Advance Chromatin Exploration

Delving into the molecular underpinnings of histone post-translational modifications (PTMs) remains a difficult task for epigenetics researchers. Current technologies are adept at either generating large amounts of correlative, genome-scale data; or consist of traditional biochemical approaches that focus on direct and quantitative detection of relationships. A scientific team from Princeton University has developed a new platform that combines the advantages of previous approaches and enhances the biochemical study of chromatin recognition and signaling.

Tom Muir and his colleagues based the new approach on the streamlined semisynthesis of DNA-barcoded nucleosome libraries (DNLs) with specific combinations of PTMs. The DNLs are chromatin immunoprecipitated after treatment with purified chromatin effectors or the nuclear proteome, and then analyzed by multiplexed DNA-barcode sequencing. The new workflow has several advantages including:

  • Nucleosomes provide a more physiologically relevant substrate than histones
  • Has a higher throughput and lower material requirements than single nucleosome experiments
  • Platform is ultrasensitive and can collect thousands of data points detailing the binding preferences of various nuclear factors for PTM profiles.

The authors suggest that their new high throughput and highly sensitive technology will catalyze the understanding of molecular factors operating at the chromatin level.

See the complete DNL platform at Nature Methods, August 2014.

Uses our Rabbit polyclonal to H3 trimethyl K4 (ab8580).

Identification of Proteins Associated with an IFNγ-Responsive Promoter by a Retroviral Expression System for enChIP Using CRISPR.

Development of Retroviral enChIP Using CRISPR

The ability to isolate and analyze the molecular interactions of specific genomic regions is critical to epigenomic investigations. A recent advancement in this area, the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology was developed for improved purification of targeted genomic sites.

In this publication, Hodaka Fuji and a team from Osaka University added a new retroviral expression system for enChIP using CRISPR technology.  The researchers proved that target genomic loci may be purified with very high efficiency, and that any potential off-target sites can be kept to a minimum if the guide RNA (gRNA) design for the target site has an appropriately long unique seed sequence.

The authors combined enChIP with stable isotope labeling using amino acids in cell culture (SILAC) analysis to demonstrate a potential application of the technique. The resulting data showed that:

  • Several proteins had increased interferon (IFN) regulatory factor-1 (IRF-1) promoter region association after IFNγ stimulation.
  • Many identified proteins were not previously reported to have IFNγ-induced gene expression, or interactions with histone deacetylase complexes.
  • The elevated IFNγ-induced, IRF-1 promoter associations were confirmed by standard ChIP assays.

The scientists suggest that the new retroviral enChIP system with CRISPR creates a very useful tool for biochemical genomic studies in live cells, especially for functions like transcription and epigenetic regulation.

Read more about  enChIP at PLoS One, July 2014.

DNA methylation dynamics of the human preimplantation embryo.

Embyronic CpG Dynamics and Parental Origins

CpG dinucleotides have been observed to show changes in relation to disease but it is relatively static in adults aside from a few environmentally responsive loci. In embryos, however, it is a different case, with an enormous amount of reprogramming occurring. While extensively studied in mice, much less is known about how this process occurs in humans.

The labs of Drs. Alexander Meissner and Kevin Eggan from the Broad Institute of MIT and Harvard (Cambridge) used reduced representation bisuflite sequencing (RRBS) to examine the dynamics of over 1 million CpGs in developing human embryos.  Using this technique they developed genome-scale DNA methylation maps of embryonic development. Here’s what they discovered:

  • Human dynamics are similar to mice on a global scale, in that there is hypomethylation with the few maintained states appearing in gene bodies.
  • Maternally contributed methylation is divergent in species-specific sets of CpG island promoters that are not only in known imprinting control regions (ICRs).
  • Retrotransposons, the genomic ancestors of ICRs, also showed diverse regulation from the maternal contribution.

The authors conclude that parental contributions do take on differences in embryonic development, with paternal demethylation appearing to be a general feature of early mammalian embryonic development and the maternal contributions appearing to take on more species-specific roles.

See the full report in Nature, July 2014.

The DNA methylation landscape of human early embryos.

The Developing Epigenetic Landscape of Human Embryos

DNA methylation is an important player in embryonic development that interacts with the rest of the epigenetic landscape, like histone modifications, to ensure the proper developmental programming. Human preimplantation embryos are in rare supply and as a result, have not yet had their epigenetic landscape examined systematically despite extensive evidence from mice that early embryos undergo dramatic reprogramming.

In order to fill this void, Drs. Jie Qian and Fuchou Tang from Peking University (Beijing) used reduced representation bisulphite sequencing (RRBS) and whole-genome bisulphite sequencing (WGBS) to map the development of early embryos from the zygotic stage through to post- implantation. Here’s what they discovered:

  • In humans the major wave of genome-wide demethylation is finished by the 2-cell stage, as opposed to previous observations in mice.
  • Demethylation of the paternal genome is occurs much faster than that of the maternal genome.
  • The textbook inverse relationship between promoter methylation and gene expression progressively increases during early embryonic development and peaks at post-implantation.
  • Active genes marked by H3K4me3 in their promoter regions are not methylated in the pluripotent embryonic stem cells.
  • Evolutionary younger retrotransposons in the genome show less demethylation compared to older elements in the family.

Overall, this work sheds light on the methylation dynamics of human early embryos, their relationship to a histone modification and how this relates to regulation of gene expression and transposons.

Find the complete article in Nature, July 2014.

Yorkie Promotes Transcription by Recruiting a Histone Methyltransferase Complex

Hippo signaling acts to block activating chromatin marks

Limitation of organ growth by the Hippo signaling pathway is accomplished by downregulation of the transcriptional coactivator Yorkie (Yki) in many species. The Hippo and Warts kinases phosphorylate Yki and promote its cytoplasmic localization. The two WW domains of Yki are required for transcriptional activation via histone H3 lysine 4 methylation. However, the precise method of Yki transcriptional co-activation remains unclear.  

Dr. Kenneth Irvine and colleagues at Rutgers University hypothesized that Yki interacts with histone H3 lysine 4 methyltransferase complexes to fulfil its regulatory role.  Since H3K4me3 promotes gene expression, this may represent the mechanism of action of Yki, and thus the mechanism blocked by Hippo signaling.

Using ChIP and in silco analyses in cultured Drosophila S2 cells and in vivo embryos the authors found that:

  • H3K4me3 is induced by Yki binding
  • Yki binds the Trr histone methyltransferase complex protein NcoA6 through its WW domain
  • Nico6A recruitment by Yki is necessary and sufficient for gene activation
  • Yki and Trr substantially co-localize
  • Nco6A is required for Yki-directed growth and transcription in vivo
  • The human Yki homolog YAP binds NCOA6 in human HEK293T cells

The authors propose a model wherein  Yorkie binds to target genes, recruiting the Trr complex by binding to NcoA6. Trr then methylates H3K4 leading to increased transcription.  The implication of NcoA6 in Hippo signaling may mean that H3K4me3-induced gene expression occurs in pathways that crosstalk via Yki.

Read the full report in Cell Reports, July 2014.

Extreme HOT regions are CpG-dense promoters in C. elegans and humans.

CpG dense regions are a conserved open chromatin signal

Highly Occupied Target (HOT) regions are bound by many transcription factors, too many to be explained by sequence motif binding alone. They are also characterized by open chromatin and are present near active elements, but their precise role remains unclear. These regions are found in many species including C. elegans and humans.

Julie Ahringer and colleagues at Cambridge University examined the role of CpG dinucleotide density in HOT region activity. Unmethylated CpG dense regions are common in active mammalian DNA elements. Methylation of CpGs in other genomic regions is thought to play a role in their silencing. Unmethylated CpGs are bound by the CXXC1 protein. Curiously a homolog of this protein, CFP-1, is found in C. elegans which lack DNA methylation. CFP-1 is required for the H3K4me3, the mark of active genes.

Using the ENCODE database, ChIP-seq, and transgenic C. elegans lines the authors found that:

  • Most HOT regions are ubiquitously active promoters in humans and C. elegans
  • CpGs are enriched in human and C. elegans HOT regions
  • CpGs are associated with nucleosome depletion
  • CFP-1 is targeted to CpG dense, H3K4me3 modified regions

These data support the view that unmethylated CpG dense regions serve as a conserved promoter signal that predates DNA methylation. These regions promote an open chromatin state and CXXC1 homolog targeting and are associated with H3K4me3.

See the full report in Genome Research, July 2014.