Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leukaemia

Background & Summary

Between one half to two thirds of the human genome consists of repetitive sequences, most of which are inactivated transposable elements (TEs). These sequences of viral origin integrated into our genomes along evolution. The enormous proportion of TEs suggests that they have played an important role in shaping our genome [1]. Given that the ENCODE Project estimates that at least 80% of the entire genome has functional annotations –such as active transcription, histone marks or transcription factor (TF) binding [2]– just by doing the math one could think that such DNA ‘fossils’ likely contain some functionality.

TEs can act as cis-regulatory modules controlling gene expression although they are epigenetically silenced during early development [3]. Several studies have shown deregulation of TEs, including endogenous retroviruses (ERVs), in different types of cancer. In this preprint, Deniz and collaborators explore the pathogenic role of TEs in acute myeloid leukemia (AML).

Figure 1. Activation of ERVs with regulatory potential in AML. A) Obtention of DNase-seq data for differentiated cells (monocytes and macrophages), hematopoietic stem cells and AML (both from primary samples and cell lines). B) Heatmap showing that chromatin is specifically open at several families of ERV. C) DNase-seq profiles in AML per family of ERV. D) Example of an ERV element behaving as an AML-specific enhancer. This figure has been modified from Figures 1 and 2 from the original preprint.

Key findings

– AML cells activate several families of ERVs. AML cells coming from both primary samples and established cell lines shared enrichment of open chromatin and enhancer signatures as well as binding of leukemia-related TFs in six families of ERVs (Fig. 1). This is in stark contrast to what happens in differentiated blood cells, such as macrophages and monocytes which do not activate these families (Fig. 1B).

– Individual ERV enhancers regulate gene expression. ERVs containing signatures of open chromatin correlated with higher expression of close genes. Deletion of individual ERV candidates proved their enhancer activity by downregulation of specific genes (Fig. 2A and B).

– Broad epigenomic silencing of ERVs reduced cell proliferation. The authors leveraged the repetitive nature of these TEs to target more than 200 of these sites by CRISPR interference (CRISPRi), a sequence-specific tool for gene silencing. Epigenomic editing altered the epigenomic landscape of targets and reduced proliferation in leukemia cells. The downregulated genes APOC1 or APOE seemed to impair cell growth thus contributing to ameliorate the malignant phenotype (Fig. 2C, D and E).

Figure 2. Perturbation of enhancers at ERVs regulates gene expression and affects proliferation. A) LTR5B element at an intron of ZNF321P shows open chromatin and TF binding. B) Deletion of the ERV in A downregulate target genes ZNF321P and ZNF320. C) dCas9-KRAB binds to hundreds of LTR2B elements targeting epigenomic silencing (CRISPRi). D) CRISPRi effectively edit the epigenomic landscape at target sites. E) Downregulation of target genes mediated by CRISPRi leads to impaired proliferation. This figure has been modified from Figures 4 and 5 from the original preprint.

Why I like this preprint

Considering the epigenomic status of cells and tissues as an accurate footprint of their identity and behaviour could be “too simple, sometimes naïve”. However, there is increasing evidence of diseases and disorders with common chromatin-related features such as enhancer rewiring or changes in global DNA methylation or post-translational modifications of histones. Since we are still not able to understand most of the information that global genetic and epigenetic analysis from patients provide it is very important to find disease hallmarks in order to benefit diagnosis and treatment strategies.

This work links two very interesting questions: ‘what is the epigenetic footprint of leukemia cells?’ and ‘what does a huge and repetitive part of the genome have to do with that?’. I like this preprint because the authors provide a combined answer to these questions and turn a potential genetic limitation such as working with very similar sequences into an interesting approach to study global effects of epigenetic silencing.

 

Question to the authors

– The broad CRISPRi-mediated repression performed in LTR2B elements led to epigenetic changes at target loci and downregulation of several genes. However, the ERV element located between the genes APOC1 and APOE seems to be the responsible for the proliferative phenotype. Do the authors think that the rest of the elements targeted through this approach (especially those downregulating their target genes) also contribute to clonal advantages in AML? And, if so, how?

– How do the authors show that downregulation of APOC1 gene and not that of APOE is responsible for the proliferative phenotype? Is any of those gene consistently upregulated in most AML samples?

– The authors showed that the intronic LTR5b element located at ZNF321P gene regulate its expression as well as that of ZNF320 and ZNF888 genes. How do the authors explain downregulation of ZNF320 (Figure 5F) after the CRISPRi-mediated repression targeting LTR2B elements? Do the authors also see downregulation of ZNF321P and ZNF888?

– Have the authors assess the effect in proliferation of other genes downregulated, such as ZNF320, in the CRISPRi experiment?

 

References

1. de Koning AJ et al. 2011. Repetitive Elements May Comprise Over Two-Thirds of the Human Genome. PLoS Genet. 7(12):e1002384.
2. ENCODE Project Consortium. 2012. An integrated encyclopedia of DNA elements in the human genome. Nature 489:57–74.
3. Chuong EB et al. 2017. Regulatory activities of transposable elements: from conflicts to benefits. Nature Rev Genet. 18, 71-86.

 

Tags: chromatin accessibility, crispr, enhancers, leukemia, transposable elements

doi: https://doi.org/10.1242/prelights.19000

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