When the Transcription Wagon Derails

If you were drinking a cup of hot chocolate and the warmth emanating out of it weren’t heat energy but X-rays, in the equivalent amount of energy, a fatal shadow could be cast upon your existence. The fact that such a small amount of ionising radiation (IR) has the potential to damage DNA and have such profound biological consequences abolishes Crick and Watson’s idyllic and pristine DNA double helix, isolated in an unbreakable bubble of aesthetic charm. Fortunately, our cells are armed with an expansive arsenal to respond to such environmental insults. Perhaps this should not come as a surprise - our world is dominated by photosynthesis and aerobic respiration, processes inevitably linked to chemical alteration of DNA primarily by ultraviolet (UV) radiation and reactive oxygen species (ROS). Evolution of DNA-based organisms could not have occurred without selection for robust DNA repair mechanism (1).

Fig 1. The two subcategories of nucleotide excision repair (NER), global genomic NER (GGNER) and transcription-couple NER (TCNER)

One of such DNA repair methods, nucleotide excision repair (NER) uses a set of enzymes to cut out a damaged section of DNA and replace it with a new one. Malfunctioning genes involved in the global genomic NER (GGNER) pathway can cause a disease where affected individuals are particularly susceptible to DNA damage (2). Until recently, it was thought that all types of inherited bone marrow failure (IBMF) were included in this group of diseases. Tummala et al. challenge this belief and show that IBMF associated with biallelic mutations in the ERCC6L2 gene results in a unique syndrome that is caused primarily by a transcription deficiency rather than a DNA repair defect (3). Indeed, there seems to be an erroneous mechanism in the transcription part of transcription-coupled NER (TCNER), rather than in the GGNER pathway linked to most IBMF syndromes.

Sometimes a biallelic loss-of-function variant arises in our genomes; both alleles collectively cause a gene to be partially or wholly inactivated. Thanks to the advances of genetic sequencing, there has been a deluge of studies identifying loss-of-function alleles associated with particular diseases. Tummala et al. confirmed the presence of such a biallelic loss-of-function variant in the ERCC6L2 gene in IBMF cases. IBMF is characterised by the bone marrow’s inability to produce mature blood cells. The phenotypes are variable, can affect only some or all blood lineages and arise during childhood or later on. It can be caused by many different mutations in many different housekeeping genes. Some types of IBMF have been well characterised, allowing better treatment but many still require elucidation (4).

Using exome and candidate gene sequencing, they identified seven variants of the ERCC6L2 gene from eight cases of IBMF in five families. The loss-of-function variants identified ranged from a single missense mutation to deletion of a chromosomal arm or even deletion or addition of an entire chromosome. As expected, affected individuals displayed a range of symptoms, including hypoplastic anaemia, bone marrow aplasia, Myelodysplastic syndrome (MDS) and Acute Myeloid Leukaemia (AML).

What is the function of the ERCC6L2 gene? Previous observations seem to blow smoke and make it appear that ERCC6L2 is only involved in chromatin remodelling and DNA repair (5). Patient cells showed hypersensitivity to certain DNA damaging agents that induce double-stranded DNA breaks, suggesting ERCC6L2 has a role in NER. An innuendo that our deductions are ever fallible was that these cells were also hyper-sensitive to irofulven, an RNA Pol-II interfering agent that triggers TCNER, a sub-pathway of NER dependent on RNA Pol-II (6). In agreement with this finding, when Tummala et al. treated their BMF patient lymphoblastoid cell lines with DNA damaging and RNA Pol-II interfering agents, they found that the cells not only demonstrated increased sensitivity to DNA damage but also to RNA Pol-II agents which inhibit RNA synthesis and elongation. This casts a doubt on the accepted notion that all IBMF syndromes are caused by DNA repair defects. Rather, it seems that ERCC6L2-associated IBMF is caused by a transcription deficiency.

To answer the salient question of the precise role of ERCC6L2 in transcription and DNA repair, Tummala et al. identified the components interacting with ERCC6L2 required for transcription. One component was particularly strongly associated in a non-cell-specific manner: DNA-dependent protein kinase (DNA-PK). DNA-PK occupies the same gene bodies as RNA Pol-II and promotes correct transcription by dislodging RNA Pol-II from DNA damage sites, ensuring it does not transcribe damaged genes. Surprisingly, although DNA-PK was hyperactive in patient-cells, they also demonstrated a failure to stall transcription elongation. DNA-PK is also involved in resolving R-loops. Again, despite DNA-PK being hyperactive in patient-cells, increased R-loops were observed. Accumulation of R-loops can heighten sensitivity to DNA damage and cause genome instability. As such, the idea that accumulation of R-loops causes defects in TCNER and thus is the underlying cause of the high rate of DNA damage and quiescence seen in IBMF patients is very appealing. The exact mechanism of how ERCC6L2 interacts with DNA-PK to control TCNER is not fully understood. Tummala et al. propose that the ERCC6L2-DNA-PK complex binds to nascent mRNA and prevents them from annealing to DNA. If this mechanism is faulty, R-loops accumulate throughout the genome and cause transcription deficiency when DNA damage is incurred. There is a strong link between AML and ERCC6L2-associated IBMF; deciphering its transcription-associated DNA repair defect could lead to parallel advances in gene therapy or pave the way for new strategies preventing consequences, including cancer, from developing. It is easy for us to idealise perfection and question why the seemingly advanced and complex homo sapiens allows for, even seems to buoy up, such flaws. It may be thoughtful to jog our memories from time to time to remember that although our genomes are protected by an intricate infantry of DNA-repairing soldiers, evolution by Darwinian selection decrees that genetic diversification be maintained through the wheel of mutations. The grassroots of life are a delicate balance between mutation and repair, genomic stability and instability, perfection and imperfection.

Bibliography

1. UV-induced DNA damage and repair: a review. Sinha, RP et al. 2002, Photochem. Photobiol. Sci., Vol. 1, pp. 225-236. 2. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology. Shuck, S et al. 2008, Cell Res, Vol. 18, pp. 64–72. 3. Genome instability is a consequence of transcription deficiency in patients with bone marrow failure harboring biallelic ERCC6L2 variants. Tummala, H et al. 30, 2018, Proc Nat Ac Sc USA, Vol. 115, pp. 7777–7782. 4. The Inherited Bone Marrow Failure Syndromes. Chirnomas, SD and Kupfer, GM. 6, 2014, Pediatr Clin North Am. , Vol. 60, pp. 1291-1310. 5. Bone marrow failure syndrome caused by homozygous frameshift mutation in the ERCC6L2 gene. Järviaho, T et al. 2018, Clin Genet, Vol. 93, pp. 392–395. 6. ERCC6L2 mutations link a distinct bone-marrow-failure syndrome to DNA repair and mitochondrial function. Tummala H, et al. 2014, Am J Hum Genet, Vol. 94, pp. 246–256.