Top Epigenetics articles: March 2015

Want to know what exciting Epigenetics research was published recently? We have scoured the latest literature to bring you your favorite papers published throughout the last month.

MiR-2 family regulates insect metamorphosis by controlling the juvenile hormone signaling pathway

MicroRNAs regulate metamorphosis in the cockroach Blatella germanica.

​MicroRNAs (miRNAs) perform a variety of functions in insect biology, ranging from cell proliferation and apoptosis, to oogenesis and development. In the cockroach Blatella germanica, miRNAs have been shown to have a role in metamorphosis, with miRNA depletion preventing metamorphosis altogether. However, which miRNAs are involved in metamorphosis regulation, and which targets they act on, is still unknown. 

In this study, Jesus Lozano and colleagues from Pompeu Fabra University in Barcelona sought to further understand the precise regulatory roles of miRNAs in insect metamorphosis. The authors found that: 

  • Depleting Dicer in female penultimate instar nymphs of Blatella germanica​ prevents nymph-to-adult metamorphosis.
  • Dicer depletion results in a reduction of mature miRNAs and increased expression of Krüppel homolog 1 (Kr-h1), a juvenile hormone-dependent transcription factor involved in metamorphosis repression. 
  • Depleting Kr-h1 mRNA in Dicer knockdown insects rescues metamorphosis, indicating that Kr-h1 is the main or only factor affected by miRNA depletion.
  • Kr-h1 contains functional binding sites for miR-2 family RNAs, including miR-2, miR-13a and miR-13b.
  • Treating insects in the last instar with a miR-2 inhibitor prevents metamorphosis, whereas treating Dicer knockdown insects with a miR-2 mimic allows metamorphosis to proceed.

These data indicate that miR-2 family miRNAs permit metamorphosis by scavenging Kr-h1 transcripts during metamorphosis. This highlights an elegant role for a specific miRNA family in regulating insect metamorphosis.

Read the full paper at PNAS, March 2015.

Are you doing miRNA research? Learn about our new multiplex technology for miRNA profiling.

Hepatitis C virus RNA functionally sequesters miR-122

Hepatitis C virus acts as a sponge to sequester host miR-122

Binding of miR-122, an abundant liver miRNA, to hepatitis C virus (HCV) RNA is required for viral replication due to its role in stimulating protein translation and protecting the HCV RNA from degradation.

In this study, a team led by Charles Rice from Rockefeller University sought to understand the global effects of HCV infection on endogenous miRNA targets. By producing global miRNA:target interaction maps during HCV infection, the authors found that:

  • Human Argonaute (AGO) protein binds to HCV 5'UTR at miR-122 sites.
  • During infection, there is a decrease in AGO binding to the human transcriptome and de-repression of miR-122 targets.
  • A quantitive model of miR-122 sequestration by HCV RNA demonstrated that a 90% reduction in miR-122 by HCV could de-repress mRNA targets by up to 4.5-fold for mRNAs expressed at low levels.
  • Swapping the tropism of the HCV relieved miR-122 sequestration and redirected HCV to sequester different miRNAs.

This paper demonstrates that HCV acts to sequester miR-122, and that this leads to de-repression of miR-122 targets in the host transcriptome. The authors speculate that miR-122 sequestration in chronic HCV infection might be a molecular link between HCV infection and liver dysfunction.

Read the full paper at Cell, March 2015.

N6-methyladenosine marks primary microRNAs for processing

m6A is a novel regulator of miRNA processing

miRNA biogenesis involves binding of pre-miRNAs to the RNA binding protein DGCR8, followed by recruitment of the type III RNase Drosha. To correctly process pre-miRNAs, DGCR8 must recognize the pre-miRNA hairpin structure as opposed to other RNA secondary structures.

In this study, Claudio Alarcón and colleagues from the Laboratory of Systems Cancer Biology at Rockefeller University investigated the mechanisms involved in DGCR8 recognition and binding to pri-miRNAs. By examining miRNA processing in mammalian cells, the authors found that: 

  • Within pri-miRNA sequences, the m6A methylation sequence is overrepresented and the m6A modification is enriched.
  • METTL3 depletion leads to a global downregulation of mature miRNAs and an increse in unprocessed pre-miRNA levels.
  • m6A marks in pri-miRNAs are required for efficient in vitro pri-miRA processing.
  • Depletion of METTL3 reduces the total RNA bound by DGCR8.

Taken together, these results indicate that METTL3 targets m6A to pre-miRNA sequences, and DGCR8 then interacts with m6A methylated RNA. The paper identifies m6A as a novel regulator of miRNA processing, allowing the microprocessor complex composed of DGCR8 and Drosha to recognize its specific substrates. 

Find the full paper in Nature, March 2015.

Read more about microRNA research.

Autism-associated chromatin modifier CHD8 regulates other autism risk genes during human neurodevelopment

CHD8 regulates a network of high risk autism spectrum disorder genes

Loss of function mutations in chromatin modifiers have been shown to be associated with autism spectrum disorder (ASD). In particular, CHD8, a gene encoding chromatin helicase is strongly associated with ASD risk. 

Other high risk ASD genes have been identified, and these have been shown to be co-expressed during neurodevelopment.

In this study, a team led by James Noonan from Yale School of Medicine explored the role of CHD8 in regulating ASD risk genes in human neurodevelopment. Using chromatin immunoprecipitation (ChIP) coupled to high-throughput sequencing (ChIP-seq), the authors mapped CHD8 binding sites in the human midfetal brain, neural stem cells and mouse embryonic cortex.

The authors found that:

  • CHD8 targets are enriched for ASK risk genes in human neural stem cells and human midfetal brain.
  • A conserved set of CHD8-binding sites is enriched for ASD risk genes.
  • CHD8 knockdown leads to significant dysregulation of ASD risk genes in human neural stem cells.

The data presented in this study provides evidence that CHD8 directly regulates a conserved network of ASD risk genes in human and mouse neurodevelopment, and that loss of CHD8 contributes to ASD by perturbing this gene network. 

Read the full paper in Nature Communications, March 2015.

ChIP-nexus enables improved detection of in vivo transcription factor binding footprints

New technique to pinpoint transcription factor binding sites

Understanding how combinations of transcription factors control gene expression requires single-nucleotide mapping of transcription factor binding. Although approaches to achieve this currently exist, ChIP-exo and ChIP-seq are hampered by technical difficulties and resolution issues.

A team led by Julia Zeitlinger at Stower's Institute for Medical Research, Missouri developed ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation), a new technique to improve mapping of genome-wide transcription factor binding. This technique combines the standard ChIP-exo protocol with the library preparation protocol from iCLIP to improve the efficiency of incorporating DNA fragments into the library.

To showcase this technique, the authors tested four proteins and found that:

  • ChIP nexus outperforms ChIP-exo and ChIP-seq, giving high quality data without over amplification.
  • ChIP nexus is able to pinpoint binding sites for the transcription factors Dorsal and Twist.
  • Binding of transcription factor Max is influenced by DNA sequences next to its binding motif, and Max interaction was stronger on one side of its binding motif.

This paper presents a robust and reproducible new technique that will enhance research into in vivo transcription factor specificity. By using this technique to investigate Max binding to DNA, the authors have shown how local DNA features outside of the binding motif can influence the specificity of protein-DNA interactions.

Find the full paper in Nature, March 2015. 

Interested in ChIP techniques? Learn about recent advanced ChIP techniques.


Nuclear architecture dictates HIV-1 integration site selection

HIV-1 integration into the host genome occurs at the edge of the nucleus

A crucial step in the lifecycle of human immunodeficiency virus type 1 (HIV-1) is its integration into the host genome. Evidence from previous work suggests that HIV-1 integrates into certain transcriptionally active genes; however, how HIV-1 selects target genes has previously not been determined.

To further understand the relevant factors determining HIV-1 integration sites, a team led by Marina Lusic and Mauro Giacca from the International Centre for Genetic Engineering and Biotechnology, Italy used three-dimensional immuno-DNA fluorescence in situ hybridization (FISH) to look at the position of HIV-1 integration sites in the nucleus of CD4+ T cells. They found that:

  • In healthy cells, the majority of HIV-1 recurrent integration genes (RIGs) were located within 1 µm below the nuclear membrane.
  • In CD4+​ T cells infected with HIV-1, 75.2% of pro-viral FISH signals were 1 µm under the nuclear envelope. 
  • Disruption of viral integrase or integration cofactors, Nup153 and LEDGF/p75, results in a significant decrease in peripheral integration of the virus.
  • RIGS are transcriptionally active genes and 90% of RIGs are located outside of lamina-associated domains.

This paper provides a three-dimensional view of HIV integration into the host genome, and demonstrates that HIV target genes are transcriptionally active and are typically positioned less than 1 µm from the nuclear edge. The authors suggest that it is likely that HIV integrates into the first open chromatin it encounters; a factor likely to be related to the short life of viral integrase.

Read the full paper in Nature, March 2015.

Functional annotation of native enhancer with a Cas9-histone demethylase fusion

Characterizing enhancer elements using Cas9-histone demethylase

​Enhancers are implicated in the regulation of development and cellular function. Although there is a pressing need to functionally annotate cell-type specific enhancers involved in cell function regulation, this has been hindered by a lack of suitable technology.

To combat this, Nicola Kearnes and colleagues from the University of Massachusetts used a nuclease deficient Cas9 (dCas9)-histone demethylase LSD1 fusion to characterize the role of enhancer elements in embryonic stem cell (ESC) fate. They found that:

  • Targeting LSD1 to the distal enhancer of Oct4 resulted in loss of Oct4 expression along with phenotypic changes, whereas targeting Oct4 proximal promoter had no effect on Oct4 expression.
  • Targeting eight pluripotency-specific candidate enhancers in dCas9-LSD1 expressing ESCs revealed four to be critical for ESC state, including Enh1, a previously unannotated ESC-specific enhancer.
  • Enh1 functions in the ESC network by regulating expression of Tbx3, a gene implicated in maintaining pluripotency.
  • Around the target site, there is a 6- to 8-fold loss of H3K4me2, the LSD1 substrate, and a dramatic loss of H3K27ac from enhancers.
  • Specific inhibition of LSD1 prevents the loss of Tbx3 expression in dCas9-LSD1 cells.

These data indicate that dCas9-LSD1 fusion can be used to disrupt specific enahncer activity and that LSD1 enzymatic activity is required for enhancer deactivation. In this study, the authors have described an effective approach for further understanding how cis-regulatory regions function in development and cell regulation.

Read the full paper at Nature Methods, March 2014.

The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells

Neural stem cell diffrerentiation is regulated by long non-coding RNAs

Neural stem cells (NSCs) differentiate to produce intermediate progenitors that divide to become young neurons. Although long non-coding RNAs (lncRNAs) have major biological functions, lncRNAs that control the crucial transition between NSCs and neurogenic progenitors have not been identified.

In this study, Alexander Ramos and colleagues from the University of California, San Francisco investigated the role of a specific lncRNA, Pnky, in neural development. By looking at Pnky function in the embryonic and postnatal brain, the authors found that:

  • Pnky is downregulated during NSC lineage progression in vivo.
  • Increased neurogenesis in Pnky knockdown postnatal NSCs results from a shift towards neuronal lineage commitment and an increase in cell amplification.
  • Pnky is expressed in both the developing mouse and human cortex, and regulates the production of young neurons in vivo.
  • Pnky interacts with splicing regulator PTBP1, a repressor of neuronal differentiation.

The data presented in this study demonstrate that Pnky interacts with PTBP1 to regulate production of neurons from neural stem cells. This paper indicates that an evolutionarily conserved lncRNA can regulate neurogenesis in the embryonic and postnatal brain.

Read the full text in Cell Stem Cell, March 2015.