From DNA-sensing cGAS-STING, to virus-mutating APOBECs, all life forms defend themselves from DNA invasion. The dangers of foreign DNA are reflected in the complexity and compartmentalization of nucleic acid sensors and effectors. Viruses must adapt to this hostile environment or face extinction. We wanted to understand how integrated viruses, i.e. retroviruses, are silenced in the genome. By reverse transcribing their RNA into dsDNA, and integrating into the host genome – retroviruses essentially become host genes. This ability to evade immune detection establishes lifelong persistence.

 

Therefore, a key biological question is whether retroviral nucleic acid is truly ‘undetected’ in the genome, or whether there is a mechanism which allows the host cell to distinguish integrated foreign (viral) from genomic self DNA? This issue is also of central clinical importance as it is the silenced, integrated HIV proviral pool which defies eradication and mandates lifelong HIV treatment.

 

A recent prominent contribution was our discovery and characterisation of a new epigenetic silencing complex we named ‘HUSH’ (Human Silencing Hub). We showed how HUSH protects the genome from the reverse flow of genetic information: HUSH recognizes and silences newly integrated DNA which is RNA in origin. This is significant as RNA-derived retroelements are the predominant route by which our genome acquires new genetic material and make up more than 40% of our genome, a remarkable figure compared to the coding component of only 2%. Our findings raised the central question of how HUSH identifies and silences invading RNA-derived transgenes i.e. What are the discriminating features of these foreign retroelements that distinguish them from self-DNA?

 

 

 

 

 

 

 

 

 

 

 

 

We found that HUSH distinguishes ‘self’ from ‘non-self’ genomic DNA through the recognition of ‘intronless’ DNA the hallmark of reverse transcription. Retroelements, being RNA-derived lack classical non-coding cellular introns. Long, intronless cDNA is therefore recognised and silenced by HUSH, allowing it to distinguish invading retroelements from host genes.

 

The identification of HUSH provides a solution to how the host genome detects and silences retroelement-derived invaders and identifies the basis for genome immune-surveillance and post-integration genome immunity, making HUSH a new component of our innate immune system.

 

Our work also defines a novel function for cellular introns: to distinguish ’self’ from ‘non-self’ DNA. Remarkably, HUSH represses almost all long cellular cDNAs including cDNAs used for cellular expression, including gene therapy. This is readily explicable as cDNAs are, by definition, the product of reverse transcription and therefore lack cellular introns. Indeed, many of the strategies developed over the last ~50 years to improve gene expression have, somewhat unwittingly, been a battle against HUSH.  HUSH inhibition therefore has the potential to improve gene expression, in vitro e.g. for antibody production and in vivo e.g. for adeno-associated virus gene therapy and to release neo-antigens for cytotoxic T-lymphocyte recognition for immunotherapy. The discovery of HUSH as a unique genome immunosurveillance system has broad implications for all life-forms, with significant therapeutic potential.

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Ongoing work in this area focuses on:

 

Mechanisms of HUSH action – How is transcription linked to HUSH-dependent silencing

How viruses – particularly complex lentiviruses evade HUSH activity

How do some intronless genes escape HUSH-dependent silencing

Therapeutic potential of HUSH inhibition

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Recent Papers on HUSH

 

Seczynska M, Bloor S, Cuesta SM, Lehner PJ. Genome surveillance by HUSH- mediated silencing of intronless mobile elements. Nature. 2022

Jan;601(7893):440-445. doi: 10.1038/s41586-021-04228-1. Epub 2021 Nov 18. PMID:34794168; PMCID: PMC8770142.

 

Dupont L, Bloor S, Williamson JC, Cuesta SM, Shah R, Teixeira-Silva A, Naamati A, Greenwood EJD, Sarafianos SG, Matheson NJ, Lehner PJ. The SMC5/6 complex compacts and silences unintegrated HIV-1 DNA and is antagonized by Vpr. Cell Host Microbe. 2021 May 12;29(5):792-805.e6.

doi: 10.1016/j.chom.2021.03.001. Epub 2021 Apr 2. PMID: 33811831; PMCID: PMC8118623.

 

Prigozhin DM, Douse CH, Farleigh LE, Albecka A, Tchasovnikarova IA, Timms RT, Oda SI, Adolf F, Freund SMV, Maslen S, Lehner PJ, Modis Y. Periphilin self- association underpins epigenetic silencing by the HUSH complex.

Nucleic Acids Res. 2020 Oct 9;48(18):10313-10328. doi: 10.1093/nar/gkaa785. PMID: 32976585; PMCID: PMC7544229.

 

Douse CH, Tchasovnikarova IA, Timms RT, Protasio AV, Seczynska M, Prigozhin DM, Albecka A, Wagstaff J, Williamson JC, Freund SMV, Lehner PJ, Modis Y. TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control. Nature Comms. 2020 Oct 2;11(1):4940. doi: 10.1038/s41467-020-18761-6.

PMID: 33009411; PMCID: PMC7532188.

 

Burr ML, Sparbier CE, Chan KL, Chan YC, Kersbergen A, Lam EYN, Azidis-Yates E, Vassiliadis D, Bell CC, Gilan O, Jackson S, Tan L, Wong SQ, Hollizeck S, Michalak EM, Siddle HV, McCabe MT, Prinjha RK, Guerra GR, Solomon BJ, Sandhu S, Dawson SJ, Beavis PA, Tothill RW, Cullinane C, Lehner PJ, Sutherland KD, Dawson MA. An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer. Cancer Cell. 2019 Oct 14;36(4):385-401.e8. doi: 10.1016/j.ccell.2019.08.008. Epub 2019 Sep

26. PMID: 31564637; PMCID: PMC6876280.

 

Timms RT, Tchasovnikarova IA, Lehner PJ. Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites. Nature Protoc. 2019 Jan;14(1):153-170. doi: 10.1038/s41596-018-0087-5. PMID: 30518911.

 

Robbez-Masson L, Tie CHC, Conde L, Tunbak H, Husovsky C, Tchasovnikarova IA, Timms RT, Herrero J, Lehner PJ, Rowe HM. The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes. Genome Research 2018 Jun;28(6):836-845. doi: 10.1101/gr.228171.117. Epub 2018 May 4. PMID: 29728366; PMCID: PMC5991525.

 

Douse CH, Bloor S, Liu Y, Shamin M, Tchasovnikarova IA, Timms RT, Lehner PJ, Modis Y. Neuropathic MORC2 mutations perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms. Nature Comms. 2018 Feb 13;9(1):651. doi: 10.1038/s41467-018-03045-x. PMID: 29440755; PMCID: PMC5811534.

 

Tchasovnikarova IA, Timms RT, Douse CH, Roberts RC, Dougan G, Kingston RE, Modis Y, Lehner PJ. Hyperactivation of HUSH complex function by Charcot-Marie- Tooth disease mutation in MORC2. Nature Genet. 2017 Jul;49(7):1035-1044. doi: 10.1038/ng.3878. Epub 2017 Jun 5. PMID: 28581500; PMCID: PMC5493197.

 

Timms RT, Tchasovnikarova IA, Antrobus R, Dougan G, Lehner PJ. ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex. Cell Rep. 2016 Oct

11;17(3):653-659. doi: 10.1016/j.celrep.2016.09.050. PMID: 27732843; PMCID: PMC5081395.

 

Timms RT, Tchasovnikarova IA, Lehner PJ. Position-effect variegation revisited: HUSHing up heterochromatin in human cells. Bioessays. 2016 Apr;38(4):333-43. doi: 10.1002/bies.201500184. Epub 2016 Feb 8. PMID: 26853531.

 

Tchasovnikarova IA, Timms RT, Matheson NJ, Wals K, Antrobus R, Göttgens B, Dougan G, Dawson MA, Lehner PJ. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells. Science. 2015 Jun26;348(6242):1481-1485. doi: 10.1126/science.aaa7227. Epub 2015 May 28. PMID: 26022416; PMCID: PMC4487827.

 

 

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Novel epigenetic pathways for silencing extrachromosomal DNA

 

In trying to understand why unintegrated lentiviral (HIV) cDNA is so poorly expressed, we identified a role for the SMC5/6 complex in silencing extrachromosomal DNA. This nuclear surveillance of extrachromosomal unintegrated DNA is antagonised by HIV-Vpr, an important lentiviral accessory gene and the only accessory gene delivered as a viral protein in the incoming virion. The SMC5/6 complex also represses hepatitis B virus and therefore represents an evolutionarily conserved mechanism for silencing non-integrated viruses and viral DNA, with important implications for the role of extrachromosomal, oncogene DNA in driving tumour evolution and its potential for silencing.

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References

 

Dupont L, Bloor S, Williamson JC, Martínez Cuesta S, Shah R, Teixeira-Silva A, Naamati A, Greenwood EJD, Sarafianos DG, Matheson NJ, Lehner PJ. The SMC5/6 complex compacts and silences unintegrated HIV-1 DNA and is antagonised by Vpr. Cell Host & Microbe 2021 Mar 26; doi: 10.1016/j.chom.2021.03.001. PMID: 33811831

 

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