Epigenetics articles of the month: September 2015

Read our selection of the most exciting Epigenetics research papers published this month.

Gain-of-function p53 mutants co-opt chromatin pathways to drive cancer growth

Chromatin modifiers are necessary for p53 gain-of-function oncogenic ability

p53 acts as a powerful tumor suppressor in normal cells. However, certain missense mutations can cause p53 to gain tumorigenic potential. Although there is evidence that gain-of-function (GOF) p53 acts through transcriptional regulation of oncogenic pathways, the individual pathways targeted by GOF p53 have not been established.​

To further understand the oncogenic function of p53 GOF, a team led by Shelley Berger from the University of Pennsylvania in Philadelphia investigated the role of chromatin pathways. They found that:

  • GOF p53 binds and activates genes related to histone modification, including genes encoding MLL1 and MLL2 histone methyltransferases, and MOZ histone acetyltransferase.
  • GOF p53 mouse embryonic fibroblasts (MEFs) show higher H3K9 acetylation and H3K4 trimethylation compared with control.
  • Reduced MLL1 expression in p53 GOF cancer cells reduces cell growth and inhibition of MLL1 activity prevents proliferation of p53 GOF MEFs.

The results of this study reveal that GOF p53 mutations contribute to cell proliferation through chromatin pathways, particularly involving the histone methyltransferase MLL1. This raises the possibility that p53 GOF cancers may be treated using epigenetic therapeutics in the future.

Read the full article in Nature, September 2015.

Find out more about p53.

HNRNPA2B1 is a mediator of m6A-dependent nuclear RNA processing events

RNA binding protein HNRNPA2B1 is an m6A reader that regulates miRNA processing

N6-methyladenosine (m6A) is the most common RNA methylation and has been implicated in processes such as miRNA processing. However, a reader of this mark that mediates nuclear RNA processing has not previously been identified.

Claudio Alarcόn and colleagues from Rockefeller University and Columbia University in New York identified the RNA binding protein HNRNPA2B1 as a reader of m6A. They found that:

  • HNRNPA2B1 recognizes and binds to RNA sequences bearing m6A.
  • HNRNPA2B1 regulates common splicing events to METTL3, an enzyme responsible for deposition of m6A marks.
  • Depletion of HNRNPA2B1 reduces abundance of mature miRNAs and leads to accumulation of pri-miRNA transcripts, suggesting that it regulates pri-miRNA processing.
  • HNRNPA2B1 depletion results in miRNA processing defects that are comparable with depletion of METTL3.

These results identify HNRNPA2B1 as a nuclear reader of the m6A mark. The data support a model in which HNRNPA2B1 acts downstream of METTL3/m6A to regulate processing of pri-miRNAs.

Read the full paper in Cell, September 2015.

Polycomb regulates mesoderm cell fate-specification in embryonic stem cells through activation and repression mechanisms

Mel18 is required to specify PRC1 function in a context- and stage-specific manner

Polycomb repressive complexes, PRC1 and PRC2, are important epigenetic regulators in embryonic and adult stem cells. The core subunit composition of the polycomb complexes can vary to regulate distinct biological processes; however, the mechanisms behind this specificity are currently unknown.

By focusing on Mel18, one of six polycomb group RING finger protein (PCGF) paralogues, a team led by Luciano Di Croce from the Centre for Genomic Regulation and Pompeu Fabra University in Barcelona found that:

  • Mel18 binds canonical PRC1 (cPRC) target genes in embryonic stem cells to stabilize cPRC and regulate transcription.
  • Mel18 is necessary to generate mesoderm precursor cells and commit them to the cardiomyocyte lineage.
  • Mel18 is required for positive regulation of genes related to mesoderm specification but represses genes associated with late cardiac development, pluripotency, and ectoderm and endoderm cell fate.
  • Along with Ring1B, Rybp and Cbx2, Mel18 directly represses negative regulators of the BMP pathway in mesoderm precursor cells.

The data presented in this paper show that Mel18 is required to specify PRC1 function in both a context- and stage-specific manner to maintain proper transcriptional regulation in the early stages of cardiac differentiation.

Read the full paper in Cell Stem Cell, September 2015.

The Daxx/Atrx complex protects tandem repetitive elements during DNA hypomethylation by promoting H3K9 trimethylation

Daxx/Atrx prevent dysfunction of repeat elements in the absence of DNA methylation

A crucial role of DNA methylation is to prevent aberrant transcription and recombination of repeat elements. However, repetitive sequences are also protected during hypomethylation, indicating that alternative mechanisms must also exist.

A team led by Zhou Songyang from Sun Yat-sen University in Guangzhou, China and Baylor College of Medicine in Houston, Texas tested the hypothesis that the Daxx/Atrx complex has a role in protecting repetitive sequences. By studying DNA methyltransferase triple knockout mouse embryonic stem cells (mESCs), they found that:

  • Daxx and Atrx have distinct binding sites on chromatin in wild type mESCs.
  • DNA hypomethylation alters the distribution of Daxx and Atrx on chromatin, with Daxx and Atrx enrichment at sequences bearing repetitive elements.
  • Daxx or Atrx knockout in hypomethylated cell lines leads to an increase in repetitive element transcription.
  • Hypomethylation targets Daxx/Atrx to telomeric and subtelomeric regions to maintain telomeres.
  • Daxx recruits the histone methyltransferase SUV39H1 to deposit repressive epigenetic marks at Daxx/Atrx target sites following hypomethylation.

The data presented in this study demonstrate a role of Atrx and Daxx in preventing repetitive element dysfunction in the absence of DNA methylation. This mechanism is may be essential to safeguard the genome of cell types that display low levels of DNA methylation.

Read the full paper in Cell Stem Cell, September 2015.

Investigating BET inhibitor resistance in leukemia

The following two selected papers have looked at resistance to small molecule inhibitors that target BET proteins including the chromatin reader BRD4. BET inhibitors are promising epigenetic therapies for a range of cancers and are currently being tested in clinical trials in patients with acute myeloid leukemia (AML).

In these two papers, different approaches were taken to investigate the mechanisms behind resistance and sensitivity to BET inhibitors in leukemia cells.

BET inhibitor resistance emerges from leukaemia stem cells

BET inhibitor resistance is conferred through increase in WNT/β-catenin pathway

A team led by Mark Dawson from the Peter MacCallum Cancer Centre and the University of Melbourne, Australia approached this problem by generating cell lines that were resistant the BET inhibitor I-BET. Resistant cell lines were generated by exposing immortalized hematopoetic stem progenitor cells to increasing concentrations of I-BET and selecting resistant cells.

They found that:

  • Cell lines resistant to I-BET display cross-resistance to a chemically distinct BET inhibitor, JQ1, and were resistant to BRD4 knockdown.
  • I-BET resistance emerges from a population of leukemia stem cells both ex vivo and in vivo.
  • As in BET-sensitive cells, BRD4 is globally displaced from chromatin in resistant cells; however, the expression of key BRD4 target genes such as MYC were equally expressed in sensitive cells.
  • The WNT/β-catenin pathway is significantly upregulated in BET-resistant cells and stimulation of this pathway in sensitive cells rapidly confers resistance to BET.
  • Chromatin occupancy of β-catenin increases at sites where BRD4 is displaced from chromatin to sustain expression of MYC.

In this study, the authors have identified a potential mechanism behind BET resistance in leukemia cells. BET inhibitors are an extremely promising cancer drug and this work will enable future research to develop strategies to eliminate resistant cell populations.

Read the full paper in Nature, September 2015.

Transcriptional plasticity promotes primary and acquired resistance to BET inhibition

WNT signaling is a driver of primary and acquired BET resistance in leukemia

In this research, a team lead by Johannes Zuber from Vienna Biocenter in Austria used a chromatin-focused RNAi screen in AML cells that were sensitive to the BET inhibitor JQ1. By investigating transcriptional profiles in sensitive and resistant leukemias, they found that:

  • Suppression of SUZ12, a PRC2 complex component, promotes JQ1 resistance in AML.
  • PRC2 suppression does not directly regulate BRD4 target genes, but facilitates the derepression of compensatory pathways that reactivate BRD4 targets such as MYC.
  • MYC repression occurs after two hours of JQ1 treatment in both sensitive and resistant leukemia; however, although this repression was durable in sensitive cells, resistant cells showed a rapid rebound of MYC expression.
  • MYC expression rebound is driven by a WNT-dependent enhancer that is formed during acquired resistance and pre-established in primary resistant cells.

This research implicates WNT activation in driving resistance in leukemia. JQ1 resistant leukemia cells inactivate PRC2 and rewire the transcriptional regulation of BRD4 targets. The BRD4 dependency of MYC is bypassed by activation of the WNT signaling pathway rather than by direct regulation by PRC2 suppression.

Read the full paper in Nature, September 2015.

NF-kB2 induces senescence bypass in melanoma via a direct transcriptional activation of EZH2

NK-kB2 regulates EZH2 and prevents senescence

Overexpression of enhancer of Zeste homologue 2 (EZH2) results in altered H3K27 methylation. In cancer, EZH2 has been reported to increase cancer growth and metastasis, and suppress the senescence program. Despite its important role in cancer, EZH2 regulation is not fully understood.

A team led by Thierry Passeron from the Mediterranean Center of Molecular Medicine and the University Hospital of Nice, France sought to further understand regulation of EZH2. By looking at the relationship between NF-kB2 and EZH2 in melanoma metastases, they found that:

  • EZH2 is directly regulated by the NF-kB2 non-canonical pathway.
  • Overexpression of Nf-kB2 increases EZH2 expression in melanoma cells and normal melanocytes.
  • NF-kB2 silencing leads to induction of senescence and this process is blocked by EZH2 overexpression.
  • Silencing of NF-kB-inducing kinase (NIK) downregulates EZH2 and induces senescence.
  • NF-kB2 silencing with siRNA and conditional sh-RNA decreases EZH2, induces senescence and reduces tumor growth.

The results presented in this study demonstrate the crucial role the non-canonical NF-kB pathway has in regulating EZH2 expression and senescence, and highlight the therapeutic potential of inhibiting this pathway in melanoma.

Read the full paper in Oncogene, September 2015.

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

Argonaute is essential for correct miRNA strand selection and miRNA ejection

MicroRNAs (miRNAs) are involved in posttranscriptional gene regulation as part of the miRNA induced silencing complex (miRISC). Transcriptional silencing by miRNAs involves association of a guide miRNA with Argonaute protein, while the complementary passenger miRNA strand is discarded.

To understand the protein and miRNA dynamics associated with miRISC maturation, a team led by Victor Ambros from the University of Massachusetts Medical School characterized a previously identified antimorphic mutant of the Argonaute-like protein ALG-1. This mutated protein, in complex with miRNAs, fails to mature into effector miRISCs.

The team found that:

  • miRISC containing the mutant ALG is unable to mature from processing to effector complexes.
  • Mutant alg-1(anti) fails to interact with miRISC effectors known to be necessary for target repression including AIN1 and poly-A binding proteins.
  • miRNA passenger strands, which are expected to be released during maturation, accumulate to abnormal levels for most miRNAs in alg-1(anti) animals.
  • alg-1(anti) is associated with greater quantities of miRNA passenger strands than wild type.
  • miRNA passenger strand accumulation in alg-1(anti) complexes may reflect the presence of two defects; inefficient ejection of passenger RNA after Dicer cleavage and erroneous miRNA passenger incorporation into alg-1(anti) Argonaute as a guide.

Overall, these results suggest that during miRISC maturation, ALG-1 complexes must recognize structural miRNA features for correct strand selection and miRNA ejection.

Read the full text in PNAS, September 2015.

Find out about recent research into miRNAs as biomarkers for a range of diseases.