All tags Epigenetics Top Epigenetics articles: November 2014

Top Epigenetics articles: November 2014

We have summarized our favorite papers from November, to help you keep on top of the epigenetics literature.

Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis

Cancer-causing miRNAs produced in secreted vessels from cancer cells

Exosomes are tiny vesicles containing RNA and proteins that are secreted by all cells. When produced by cancer cells, exosomes are suspected to promote tumorigenesis in nontumorigenic cells. MicroRNAs (miRNAs) are one mechanism by which this may occur, as there is some evidence that miRNAs are present in exosomes. However, it is unclear how they could be processed to become functional.​

Raghu Kalluri and colleagues from The University of Texas MD Anderson Cancer Center investigated the processing of miRNAs in breast cancer exosomes. They hypothesized that cancer-derived exosomes would contain the cellular machinery to process pre-miRNAs into functional miRNAs.

The researchers found that:

  • Cancer exosomes are enriched for miRNAs, pre-miRNAs and the core RNA induced silencing complex-loading complex proteins. 
  • Cancer exosomes process pre-miRNAs to generate mature miRNAs.
  • In nontumorigenic cells, cancer-derived exosomes induce tumor formation.
  • Exosomes from the blood of cancer patients contain dicer and process pre-miRNAs to generate mature miRNAs.

The findings indicate that cancer cells are able to manipulate surrounding cells by releasing tumor-inducing miRNAs. Similar to a dandelion spreading its seeds, cancer cells may seed surrounding cells with the disease.

Read full report in Cancer Cell, November 2014.

Abcam products used: anti-CD9 (ab2215), anti-TSG101 (ab83), anti-CD43 (ab9088), anti-PTEN (ab32199), anti-GADPH (ab9483), anti-TRBP (ab72110), anti-AGO2/elF2C2 (ab32381), anti-LAMP1 (ab25630), anti-HGS (ab56468), anti-ARFGEF2 (ab75001) and anti-biotin (ab53494) antibodies.

MicroRNA silencing for cancer therapy targeted to the tumor microenvironment

A new platform for the targeted delivery of anti-cancer miRNA therapy

Dysregulation of miRNA expression is involved in the formation of cancer, and may tumors depend on certain miRNAs (called oncomiRs) for their proliferation. Inhibition of oncomiRs using antisense oligomers (antimiRs) is an emerging therapeutic strategy. However, physiological and cellular barriers hinder antimiR delivery into targeted cells.

A research team led by Dr Christopher Cheng at Yale University has developed a delivery platform for antimiRs that targets the acidic tumor microenvironment, whilst evading systemic clearance by the liver and facilitating cell entry via a non-endocytic pathway. The delivery platform consists of a peptide with low pH-induced transmembrane structure (pHLIP), to which a peptide nucleic acid antimiR of choice is attached.

The team found that:

  • The pHLIP peptide localized to tumors of lymphoid origin in mouse models of lymphoma.
  • Intravenous administration of pHLIP-anti155 into lymphoma mouse model, in which tumorigenesis is dependent on miR-155, resulted in a significant reduction in tumor growth and suppressed metastatic spread of neoplastic lymphocytes.
  • pHLIP-anti155 avoided clearance by the liver; pHLIP-anti155 showed 10-fold reduction in liver accumulation compared with anti155 alone.

OncomiRs are proving to be potent anticancer targets, and this new approach for the delivery of antimRs has great potential for the treatment of oncomiR-dependent tumors in the future.

Read the full report in Nature, November 2014.

Learn more about the tumor microenvironment.

Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers

Architecture of initiation is shared at promoters and enhancers

Promoters and enhancers act as control mechanisms for transcriptional regulation, responding to various stimuli to coordinate transcription initiation of the appropriate genes. However, promoters and enhancers have distinct functions, with enhancers boosting the recruitment of the pre-initiation complex to promoters, which in turn bind the pre-initiation complex.

Adam Siepel, John Lis and colleagues at the Cornell University used GRO-cap (global run-on sequencing coupled with enrichment for nascent RNAs with 5'caps) to compare hundreds of thousands of promoters and enhancers with the aim of identifying the nature and organization of the initiation sites.

The researchers found that:

  • Transcription initiation occurs in a bidirectional manner at thousands of enhancers and promoters, both of which share a basic architecture at initiation sites.
  • RNA polymerase II does not randomly initiate at open DNA regions associated with enhancers and divergent promoters, instead these sites facilitate its initiation.
  • It is the post-translational mechanisms, and not the transcriptional initiation steps, that distinguish promoters from enhancers.

The authors provide novel insights into the architecture of both promoters and enhancers across the human genome, as well as the use of post-translational mechanisms to distinguish between the two. They propose an exciting potential relationship between divergent transcription and the origin of new genes.

Read the full report in Nature Genetics, November 2014.

An Argonaute 2 switch regulates circulating miR-210 to coordinate hypoxic adaptation across cells

MiRNA/Argonaute 2 protein network regulates hypoxia adaptation

Hypoxia (low oxygen) is a major stressor in mammals and is associated with a host of diseases and conditions. Adaptation to hypoxia requires a variety of physiological and genetic responses, importantly communication between distant tissues.

One mechanism by which tissues communicate is through miRNAs; one miRNA in particular, miR-210, controls adaptive responses to hypoxia. However, it is unknown whether miR-210 controls adaptation to hypoxia by communicating with distant tissues through secretion into the bloodstream.

Stephen Y. Chan and colleagues at Harvard Medical School investigated the role of secreted miR-210 in communicating between hypoxic source tissue and recipient cells. They also investigated the involvement of post-translational modifications to the Argonaute 2 protein (which produces miRNAs) using antibody-based detection (immunoblotting).

The researchers found that:

  • miRNA-210 is released from human and mouse cells in culture and in vivo during hypoxic conditions.
  • Hypoxia induces the release of miR-210 associated with Argonaute 2.
  • Prolyl-hydroxylation of Argonaute 2 regulates the hypoxic activation and extracellular release of miR-210.
  • Released miR-210 is transported to recipient cells to regulate adaptation.

The identification of this unique molecular communication system provides deeper insight into regulation of hypoxia adaptation. Furthermore, it provides a possible system by which other complex intracellular communications may take place.

Read the full report in Biochimica et Biophysica Acta, November 2014.

Abcam products used: anti-fibronectin (ab2413) and anti-mouse IgG H&L (HRP) (ab6728) antibodies.

Unbiased analysis of potential targets of breast cancer susceptibility loci by Capture Hi-C

Capture Hi-C finds role for gene deserts in breast cancer

Genome-wide association studies (GWAS) often identify variants in gene deserts as being associated with complex diseases, such as breast cancer. These hits may occur because of important regulatory sequences in these regions, which may affect disease-causing genes.

Olivia Fletcher and colleagues at the Institute of Cancer Research, UK, hypothesized that gene deserts harboring risk variants for breast cancer contained enhancer sequences that regulate specific cancer-related genes over long distances. To investigate this, they developed a combination of Capture-C and Hi-C, which they called C Capture Hi-C (CHi-C). This new protocol is essentially Hi-C with an added sequence capture step to enrich for desired regions.

Using this new technique, the researchers found that:

  • Putative regulatory elements in gene deserts interacted with protein-coding genes and long non-coding (Inc) RNAs up to 2.6 megabases away.
  • Target genes included known cancer-related genes such as MYC.
  • A SNP linked to a GWAS hit causes a functional change in the binding of the FOXA1 transcription factor, and thus may be the causative variant.

High-resolution interaction maps of scientific genome regions can be provided by Chi-C. Such interaction maps can be useful in delineating functional roles of disease-associated regions that seem to serve no genomic function.​

Read the full report in Genome Research, November, 2014.

Abcam products used: anti-FOXA1 (ab5089) antibody.

PAR-CLIP analysis uncovers AUF1 impact on target RNA fate and genome integrity

Unbiased method for mapping site-specific protein-RNA interactions

RNA-binding proteins (RBPs) play a central role in regulating post-transcriptional levels of gene expression through a number of mechanisms. One such RBP, AUF1 (AU-rich element binding factor 1), is known to target and degrade a number of mRNA transcripts implicated in cancer, inflammation, stress and aging. AUF1 can interact with a diverse set of targets; however, previously available methods demonstrated bias that could not elucidate the specific interaction sites.

Myriam Gorospe and colleagues at the National Institute of Health mapped the interactions of AUF1 with the entire collection of its target RNAs using a new unbiased approach called photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analysis (PAR-CLIP). This method provides both the identity of AUF1 target transcripts and the sequences at the specific sites where AUF1 interacts. 

The authors were able to obtain highly precise sequence resolution of interactions with mRNAs and non-coding RNAs. They also demonstrated that AUF1 regulates the target transcript fate in a number of ways by primarily recognizing U-/GU-rich and not AU rich regions as previously assumed.

The research revealed that:

  • AUF1 most often associates with highly U- and GU-rich regions in 3' UTRs and introns.
  • AUF1 lowers the steady-state levels of many target mRNAs and long non-coding RNAs.
  • Additionally, some AUF1 target mRNAs encoding DNA-maintenance proteins showed an increase in steady-state levels.

PAR-CLIP analysis employed in this study systematically revealed that AUF1 can interact with mRNAs and long non-coding RNAs to transcriptionally regulate their expression while also modulating stability. Understanding molecular mechanisms of AUF1 action is critical for revealing its role in processes such as aging and cancer.

Read the full report in Nature Communications, November 2014.

Abcam products used: Anti-USP1 antibody (ab84772).

The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice

H3K9me3 mediated imprinting protects against maternal X-chromosome inactivation

In mammals, one of the two X-chromosomes in the female is inactivated to maintain dosage compensation. X-chromosome inactivation is established through the expression of Xist, a long non-coding RNA that acts in cis to cover the the entire X-chromosome. 

RNF12, an E3 ubiquitin ligase, is the primary factor responsible for expression of Xist by the paternal X-chromosome in pre-implantation embryos. However, although RNF12 is abundant in oocytes, Xist expression by the maternal X-chromosome is repressed to maintain the activity of this chromosome.

Atsushi Fukuda and colleagues from the National Research Institute for Child Health and Development in Japan, and Harvard University investigated factors responsible for the repression of maternal Xist. Using a new chromatin immunoprecipitation method that allows chromatin analysis in pre-implantation embryos, they found that:

  • H3K9me3 is enriched at the Xist promoter region of the maternal X-chromosome, preventing Xist activation by RNF12.
  • H3K9me3 at the Xist promoter region is lost in embyronic stem cells, and ES-cloned embryos show RNF12-dependent Xist expression.
  • High incidence (70–80%) of parthenogenote embryonic lethality immediately after implantation was due to a lack of paternal X-inactivation in the trophoectoderm, rather than loss of paternally expressed imprinted genes.

This study reveals the molecular mechanisms underlying the imprinting of X-inactivation and highlights the role of maternal-specific H3K9me3 modification in embryo development.

Read the full report in Nature Communications, November 2014.

Abcam products used: anti-H3K9me3 (ab8898),  anti-H3K9me2 (ab1220), rabbit IgG (ab37415).

Long non-coding RNA PANDA and scaffold-attachment-factor SAFA control senescence entry and exit

SAFA and PANDA control cellular entry and exit from senescence

Fuctional ablation of polycomb group (PcG) proteins, such as PRC1 and PRC2, induces a senescence arrest, yet these proteins lack inherent DNA binding activity. Long non-coding (lnc) RNAs have been shown to direct PRCs to site-selected targets. 

Oliver Bischof and colleagues at the University of Utah used Co-IP, qRT-PCR and western blotting to establish the association between the DNA- and RNA-binding scaffold-attachment-factor A (SAFA) and the lnc RNA, PANDA, with PRC1, PRC2, and transcription factor NF-YA.

The researchers found that:

  • SAFA and PANDA physically and functionally interact with PRC1, PRC2 and transcription factor NF-YA in a cell-fate dependent manner.
  • PANDA and SAFA predominantly interact in pre-senescent, proliferating cells; however, in senescent cells PANDA expression strongly increased, limiting the expression of proliferation-promoting genes by binding to, and sequestering, the transcription factor NF-YA.

The researchers propose that PANDA may be viewed as a 'locking device' that confines cells in their current proliferative state and that modulating its level of expression can cause entry or exit from senescence. The authors provide insights into the role of PcG proteins in differentiated cells, where their main function appears to be assisting in modulating the expression of certain genes rather than to merely maintain epigenetic memory of their silencing.

Read the full report in Nature Communications, November 2014.

Abcam products used: H3K27-acetyl (ab4729), H4K8-acetyl (ab15823), anti-histone H2A (ab18975).

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​Changing chromatin fiber conformation by nucleosome repositioning

Single-nucleosome repositioning affects chromatin conformation

Nucleosome positioning on DNA is highly dynamic and has the potential to affect all types of chromatin processes. However, the impact of the spacing and position of nucleosomes on chromatin dynamics is still very poorly understood. Since it is extremely difficult to manipulate nucleosomes in living cells, researchers often use computer models to understand local chromatin dynamics. 

Gero Wedemann and colleagues from The University of Applied Sciences, Stralsund, Germany used computer modeling of 101 nucleosome fibers to understand the effect of repositioning single nucleosomes on the properties of chromatin fibers. Their simulations displaced the central nucleosome by adding and removing nucleotide linkers on either side.

They found that:

  • Displacing the central nucleosome promoted bending at this site, thus promoting long-range contacts between nucleosomes.
  • Shifting of the nucleosome by two nucleotides had the largest destabilization effect, whereas ten nucleotides (one helical turn) had very little effect.
  • Higher order chromatin structure can present energy barriers to repositioning of nucleosomes.

These results provide a framework for understanding the effects of nucleosome positioning on chromatin structure. Further, they provide evidence that the activity of chromatin remodeling complexes acting at single nucleosomes have the potential to affect chromatin conformation on a broad scale. 

Read the full report in Biophysical Journal, ​November 2014.

A negative feedback loop of transcription factors specifies alternative dendritic cell chromatin states

Transcription factors drive dendritic cell fate via chromatin remodeling

Dendritic cells (DCs) are a type of immune cell that arise from a shared progenitor, yet they are programmed to perform distinct immunological roles to combat inflammation and infection. This distinction is based on the nuclear chromatin state, which regulates the transcriptional status of that cell, thus driving cell specification. However, the regulatory mechanisms that enable the chromatin states associated with cell-fate decisions are not well understood. 

Ido Amit and colleagues at the Weizmann Institute, Israel, mapped the epigenetic (chromatin) and transcriptional state in two DC subtypes, plasmacytoid (p)DCs and monocyte-derived (mo)DCs, to determine what regulators were required for setting up specific DC identity.

The authors found that:

  • The transcription factor (TF) lrf8 was preferentially enriched in thousands of regulatory genomic regions in pDCs, while another TF, Cebpb, was more strongly enriched in moDCs.
  • Enhancement of these TFs was observed close to cell subtype specific genes, indicating their importance for regulating transcriptional state.
  • Pertubing these TFs altered chromatin state and led to deregulation of cell identity, whilst overexpressing lrf8 in moDCs reprogrammed them towards a pDC identity.
  • lrf8 and Cebpb self-interact, forming a double-negative feedback circuit to ensure the maintenance of the appropriate chromatin state, and thus cell identity, in pDCs and moDCs, respectively.

This study identified specific 'pioneer' transcription factors that regulate chromatin state to program functionally distinct subtypes of immune cells derived from the same hematopoietic origin.

Read the full report in Molecular Cell, November 2014.

Abcam products used: anti-H3K27Ac (ab4729) antibody.

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