Most of our genome is often written off as “junk DNA”, meaning it doesn’t encode for genes that get made into proteins. This so-called junk consists of accumulated DNA elements like viral DNA that got stuck in our genome millions of years ago or highly repetitive sequences of DNA that have seemingly no purpose— or so we thought. In recent years, researchers have been discovering that a lot of this DNA isn’t actually junk at all, and while it might not make protein, this genetic “dark matter” can play roles in regulating expression of protein-coding genes or have important cellular functions. Similar to the mysterious functions of the non-coding genome, is the not-well understood transcription factor DUX4, for which researchers in Dr. Stephen Tapscott’s lab continue to uncover more and more unexpected roles. DUX4 first gets activated during early embryonic development where it plays critical roles regulating gene expression, but then gets silenced throughout the rest of embryogenesis and remains silenced in most cell types of the body. Inappropriate activation of DUX4 contributes to a range of diseases including cancer, but most notably is the cause of facioscapulohumeral muscular dystrophy (FSHD) if mis-expressed in muscle cells.
Recently, the Tapscott lab uncovered that activation of DUX4 during embryonic development correlates with expression of a highly repetitive tandem repeat, human satellite II (HSATII), which has often been considered to be part of the junk DNA category. HSATII is found in pericentric regions of chromosomes (near the middle of the chromosome) and is present on nearly half of our chromosomes yet remains silenced. De-repression of HSATII can have devastating consequences to the cell for two reasons, one due to the loss of repression at HSATII genomic loci and second, due to the accumulation of HSATII single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), which is observed in cancer and diseases like FSHD. HSATII DNA and aggregation of its RNA can sequester important regulatory nuclear proteins that control transcription, and thus prevent them from doing their job. The Tapscott lab previously found that this dsRNA accumulation and sequestration of nuclear proteins contributes to DUX4-induced cellular toxicity. These peculiar findings raised even more questions— how is DUX4 regulating HSATII? And what are the cellular consequences of HSATII activation and expression? Dr. Tessa Arends, a postdoctoral fellow in the Tapscott lab, wanted to find the answers to such questions. In a recently published Journal of Cell Biology study, Arends investigated the role of DUX4 in activation of HSATII and how this activation promotes cellular toxicity in the context of FSHD.
Using a human myoblast (muscle cell) cell line with inducible DUX4 expression, Dr. Arends examined which proteins were getting trapped by transcriptionally active HSATII DNA upon inducing DUX4 expression. “HSATII exists as huge tandem blocks of repeats. We found that just activation of HSATII causes sequestration of polycomb repressor components,” Dr. Arends states. Specifically, she identified one of the sequestered proteins was RNF2, the catalytic subunit of the polycomb repressor complex 1 (PRC1), which is an important transcriptional repressor. Additionally, the authors found sequestration of lysine methyltransferases KDM2A and KDM2B, where KDM2B, in particular, has been shown to associate with components of PRC1, including RNF2, to form a non-canonical PRC1 complex. Importantly, simultaneous depletion of KDM2A/B impaired RNF2 accumulation at HSATII DNA, demonstrating the role of these proteins in recruiting RNF2 to HSATII loci. “When we identified that this polycomb repressor complex was being sequestered, this was a real ‘aha’ moment,” describes Dr. Arends. To elicit transcriptional repression, PRC1 adds a specific type of chromatin modification– monoubiquitination of histone H2A.X at lysine 119 (H2AK119Ub) for those chromatin aficionados out there– to the regulatory regions of its target genes. The researchers reasoned that if the PRC1 complex members were being sequestered by HSATII DNA, they wouldn’t be able to deposit this mark and silence their target genes very well. Consistent with this hypothesis, the authors found that in DUX4-expressing cells with RNF2 aggregation, global levels of this PRC1-deposited chromatin modification were reduced.
The ubiquitin mark added to chromatin by PRC1 is known to play roles in DNA damage responses. Dr. Arends then wondered if global loss of this modification due to PRC1 sequestration might leave the cells more susceptible to DNA damage. Here, Arends admits “I’m not a DNA damage expert,” so to explore her hypothesis, she turned to someone who is an expert, Dr. Richard Adeyemi in the Basic Sciences Division. She adds that for this part of the project, Dr. Adeyemi “was a great resource we were able to bounce ideas off of and was critical in helping us figure out exactly which DNA damage factors to look at, particularly when it came to demonstrating these cells have replication stress.” With help from the Adeyemi lab, the authors found that these cells indeed have defective DNA damage signaling following DUX4 expression which is due to aggregation of RNF2 and loss of H2AK119Ub. Loss of this DNA damage signaling had widespread effects on these cells. Arends found that cell cycle progression was impaired following DNA damage and that persistent DNA damage led to replication stress which also correlated with genomic instability.
This study identified a previously unknown role of DUX4 in inducing DNA damage via activation of HSATII which leads to sequestration of important transcriptional repressors that are critical for DNA damage response signaling. Arends notes that perhaps one of the most striking things this study demonstrates is the innate ability of these large repetitive genomic regions to sequester enough proteins to globally alter chromatin states. Struck by these findings, Arends intends to continue her work investigating HSATII to understand how exactly it is regulated and whether it might be able to be used as a therapeutic target for diseases where cells show activation of HSATII like in cancers or FSHD. Additionally, she’s interested in understanding why evolution has kept this so-called junk DNA around and whether it might have specific functions in both development and cancer.
This work was supported by the National Institutes of Health, Friends of FSH Research and the Ovarian Cancer Research Alliance.
Fred Hutch/UW/Seattle Children’s Cancer Consortium members Drs. Stephen Tapscott and Richard Adeyemi contributed to this work.
Arends T, Tsuchida H, Adeyemi RO, Tapscott SJ. DUX4-induced HSATII transcription causes KDM2A/B-PRC1 nuclear foci and impairs DNA damage response. J Cell Biol. 2024.