MOLECULAR & CELLULAR ONCOLOGY 2016, VOL. 3, NO. 6, e1244513 (3 pages) http://dx.doi.org/10.1080/23723556.2016.1244513
AUTHOR’S VIEW
Putting the brakes on transcription at damaged chromatin: Do Polycomb silencers do more than modify histones? Younghoon Kee Department of Cell Biology, Microbiology, and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, FL, USA
ABSTRACT
ARTICLE HISTORY
One of the cellular responses to DNA damage is to monitor and execute temporary arrest of RNA synthesis at chromatin lesions. The Polycomb silencer BMI1 is a well-known contributor to this process. We recently described a new mode of BMI1-mediated transcription arrest at lesions that involves UBR5 E3 ligase and FACT histone chaperon.
Received 28 September 2016 Revised 29 September 2016 Accepted 30 September 2016 KEYWORDS
BMI1; FACT; Polycomb
Although it is well known that replication fork movement at damaged chromatin must be properly regulated, increasing evidence suggests that the same is true for progression of transcriptional machineries. Studies indicate that inappropriately regulated RNA polymerase movement along the chromatin can have profound effects on genome–transcriptome integrity.1 Lesion sensing, polymerase arrest, and DNA repair are mechanisms used to ensure that “transcription stress” is resolved in a timely manner. Among several proposed players in these processes are BMI1 and its associated Polycomb proteins. Polycomb genes were initially identified as epigenetic repressors of HOX genes that affect posterior transformation of the fruit fly. Since their discovery, Polycomb proteins have become widely recognized in all metazoans for their conserved transcriptional repressive function throughout development.2,3 Biochemical and genetic studies suggest that there are 2 primary Polycomb repressive complexes (PRCs). PRC2 (containing the EZH2 methyltransferase) induces histone H3 methylation (H3K27-me) that induces local gene silencing. The mode of gene repression by PRC1 (the canonical PRC1 contains BMI1, RNF1, and RNF2, the catalytic RING domain subunit) is less clear although it is generally accepted that PRC1-induced gene silencing primarily involves monoubiquitination of histone H2A at the K119 residue, the only known substrate of the complex.2 Interestingly, more recent studies showed that PRC1 can induce gene silencing independent of histone ubiquitination in the context of Drosophila or mouse embryo development.4,5 These studies suggested that the enzymatic activity of PRC1 may not be essential in gene repression, at least in the early developmental stages. Some studies also suggested that PRC1 induces a compact chromatin state that may also contribute to gene silencing.2 An interesting twist in Polycomb biology is a series of findings that BMI1 and several other Polycomb factors are rapidly recruited to DNA lesions (e.g., double-strand breaks, UV-induced lesions), and that BMI1 function is required for the DNA damage response and maintenance of genome integrity (reviewed in ref. 66). BMI1 is thought to induce the ubiquitination of histone H2A (and H2AX) CONTACT Younghoon Kee © 2016 Taylor & Francis Group, LLC
[email protected]
at lesions, but whether this is the primary mechanism responsible for transcriptional repression at the damaged sites is not clear. Our recent work unexpectedly showed that chromatin recruitment of UBR5 E3 ligase is largely dependent on BMI1 and 2 other PRC1 components, RNF1 and RNF2.7 We found that, similar to BMI1, ablation of UBR5 leads to derepression of transcription at damaged sites, suggesting that UBR5 may be a downstream effector of BMI1 in mediating transcription repression. This phenotype of UBR5depleted cells was rescued by wild-type UBR5 but not by a catalytically inactive mutant, suggesting that UBR5 must ubiquitinate a substrate to repress transcription. Further analysis revealed that UBR5 interacts with the histone chaperone complex FACT, which is a heterodimer of SPT16 and SSRP1. Interestingly, a previous report found that SPT16 is localized to UV lesions and is required for the dynamic exchange of histone H2A and H2B molecules and for RNA Pol II to resume transcription.8 Therefore, an intriguing hypothesis is that UBR5 acts to temporarily suppress FACT activity, an event controlled by BMI1 and PRC1, to allow repair to occur until transcription resumes. How might UBR5 suppress FACT activity? We found that the SPT16 foci at the lesions are enlarged in BMI1- and UBR5-depleted cells, suggesting that BMI1 and UBR5 may suppress the local concentration of FACT at the lesions. We found that UBR5 ubiquitinates SPT16, and postulate that this modification may account for transcription repression. In summary, this finding revealed an unexpected link among FACT activity, UBR5, and BMI1 in the context of controlling Pol II progression through damaged chromatin (Fig. 1). Clearly, there are still many unanswered questions: (1) Does recruitment of UBR5 to chromatin require the H2AK119-Ub mark induced by PRC1? UBR5 has a ubiquitinassociated (UBA) domain, and it is possible that this is involved in interaction with the ubiquitin mark on histones. Although our data did not indicate that this is the case, further thorough investigation is warranted. (2) Is SPT16 ubiquitination indeed inhibitory to FACT activity, as well as to the nucleosomal dynamics necessary for transcriptional elongation? Identification of the specific ubiquitination sites may be
e1244513-2
Y. KEE
Figure 1. Various models for Polycomb repressive complex 1–induced transcription repression at damaged chromatin. (A). PRC1 containing BMI1 induces ubiquitination at H2A (K119-Ub), which leads to repression of Pol II progression near the damaged regions (the catalytic subunit RNF2 is not shown). There may be a reader of the ubiquitinated histone that mediates RNA Pol II inhibition. ZRF1 was identified as a reader of ubiquitinated H2A to positively regulate the expression of developmental genes, but whether it is also involved in the DNA damage response is unknown. (B). PRC1-induced repression of RNA Pol II elongation involves UBR5 and FACT histone chaperone activity. Whether UBR5 recruitment to the damaged sites requires ubiquitinated H2A is not known.
necessary to answer these questions. (3) How does the role of UBR5 in transcriptional repression relate to a previously known role of UBR5 in suppressing RNF168-mediated ubiquitin signaling? Knockdown of UBR5 (and another E3 ligase, TRIP12) increases the 53BP1 and ubiquitin foci at the damage sites, which in turn negatively affects RNA synthesis.9 While we could not convincingly reconcile the seemingly contradictory effects of this study and our results, there are several notable differences in the experimental conditions. For example, our experiments were performed under UV-damage conditions during which overall RNA synthesis is suppressed, whereas the study by Gudjonson et al. monitored spontaneous 53BP1 foci spots when UBR5 was depleted. It is possible that in the UBR5-depleted cells 53BP1 and SPT16 exhibit opposing activity between DSB signaling and transcription, with SPT16 exerting dominating effects at the UV lesions. There is a clear need for further investigation to explain these differences. (4) Do ATM, ATR, or DNA-PK kinases, which are all known to participate in the transcriptional repression albeit under different contexts, crosstalk with UBR5 in transcriptional
repression, possibly through BMI1? UBR5 was shown to ubiquitinate the ATM-associated protein ATMIN, an event that leads to activation of the ATM signaling.10 Therefore, it is conceivable that a feedback loop may exist between UBR5 and ATM in transcriptional silencing. In summary, future studies will hopefully provide better insight into the role of BMI1 and histone ubiquitination in transcriptional silencing at damaged chromatin and their other putative non-canonical activity in supporting the DNA damage response and genomic integrity.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References 1. Svejstrup JQ. The interface between transcription and mechanisms maintaining genome integrity. Trends Biochem Sci 2010; 35:333-8; PMID:20194025; http://dx.doi.org/10.1016/j.tibs.2010.02.001
MOLECULAR & CELLULAR ONCOLOGY
2. Sparmann A, van Lohuizen M. Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer 2006; 6:846-56; PMID:17060944; http://dx.doi.org/10.1038/nrc1991 3. Di Croce L, Helin K. Transcriptional regulation by Polycomb group proteins. Nat Struct Mol Biol 2013; 20:1147-55; PMID:24096405; http://dx.doi.org/10.1038/nsmb.2669 4. Pengelly AR, Kalb R, Finkl K, Muller J. Transcriptional repression by PRC1 in the absence of H2A monoubiquitylation. Genes Dev 2015; 29:1487-92; PMID:26178786; http://dx.doi.org/10.1101/gad.265439.115 5. Illingworth RS, Moffat M, Mann AR, Read D, Hunter CJ, Pradeepa MM, Adams IR, Bickmore WA. The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development. Genes Dev 2015; 29:1897902; PMID:26385961; http://dx.doi.org/10.1101/gad.268151.115 6. Vissers JH, van Lohuizen M, Citterio E. The emerging role of Polycomb repressors in the response to DNA damage. J Cell Sci 2012; 125:3939-48; PMID:23104738; http://dx.doi.org/10.1242/jcs.107375 7. Sanchez A, De Vivo A, Uprety N, Kim J, Stevens SM, Jr, Kee Y. BMI1UBR5 axis regulates transcriptional repression at damaged chromatin.
e1244513-3
Proc Natl Acad Sci U S A 2016; 113:11243-8; PMID:27647897; http:// dx.doi.org/10.1073/pnas.1610735113 8. Dinant C, Ampatziadis-Michailidis G, Lans H, Tresini M, Lagarou A, Grosbart M, Theil AF, van Cappellen WA, Kimura H, Bartek J, et al. Enhanced chromatin dynamics by FACT promotes transcriptional restart after UV-induced DNA damage. Mol Cell 2013; 51:469-79; PMID:23973375; http://dx.doi.org/ 10.1016/j.molcel.2013.08.007 9. Gudjonsson T, Altmeyer M, Savic V, Toledo L, Dinant C, Grofte M, Bartkova J, Poulsen M, Oka Y, Bekker-Jensen S, et al. TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes. Cell 2012; 150:697-709; PMID:22884692; http://dx.doi. org/10.1016/j.cell.2012.06.039 10. Zhang T, Cronshaw J, Kanu N, Snijders AP, Behrens A. UBR5mediated ubiquitination of ATMIN is required for ionizing radiation-induced ATM signaling and function. Proc Natl Acad Sci U S A 2014; 111:12091-6; PMID:25092319; http://dx.doi.org/10.1073/ pnas.1400230111
AUTHOR’S VIEW
Putting the brakes on transcription at damaged chromatin: Do Polycomb silencers do more than modify histones? Younghoon Kee Department of Cell Biology, Microbiology, and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, FL, USA
ABSTRACT
ARTICLE HISTORY
One of the cellular responses to DNA damage is to monitor and execute temporary arrest of RNA synthesis at chromatin lesions. The Polycomb silencer BMI1 is a well-known contributor to this process. We recently described a new mode of BMI1-mediated transcription arrest at lesions that involves UBR5 E3 ligase and FACT histone chaperon.
Received 28 September 2016 Revised 29 September 2016 Accepted 30 September 2016 KEYWORDS
BMI1; FACT; Polycomb
Although it is well known that replication fork movement at damaged chromatin must be properly regulated, increasing evidence suggests that the same is true for progression of transcriptional machineries. Studies indicate that inappropriately regulated RNA polymerase movement along the chromatin can have profound effects on genome–transcriptome integrity.1 Lesion sensing, polymerase arrest, and DNA repair are mechanisms used to ensure that “transcription stress” is resolved in a timely manner. Among several proposed players in these processes are BMI1 and its associated Polycomb proteins. Polycomb genes were initially identified as epigenetic repressors of HOX genes that affect posterior transformation of the fruit fly. Since their discovery, Polycomb proteins have become widely recognized in all metazoans for their conserved transcriptional repressive function throughout development.2,3 Biochemical and genetic studies suggest that there are 2 primary Polycomb repressive complexes (PRCs). PRC2 (containing the EZH2 methyltransferase) induces histone H3 methylation (H3K27-me) that induces local gene silencing. The mode of gene repression by PRC1 (the canonical PRC1 contains BMI1, RNF1, and RNF2, the catalytic RING domain subunit) is less clear although it is generally accepted that PRC1-induced gene silencing primarily involves monoubiquitination of histone H2A at the K119 residue, the only known substrate of the complex.2 Interestingly, more recent studies showed that PRC1 can induce gene silencing independent of histone ubiquitination in the context of Drosophila or mouse embryo development.4,5 These studies suggested that the enzymatic activity of PRC1 may not be essential in gene repression, at least in the early developmental stages. Some studies also suggested that PRC1 induces a compact chromatin state that may also contribute to gene silencing.2 An interesting twist in Polycomb biology is a series of findings that BMI1 and several other Polycomb factors are rapidly recruited to DNA lesions (e.g., double-strand breaks, UV-induced lesions), and that BMI1 function is required for the DNA damage response and maintenance of genome integrity (reviewed in ref. 66). BMI1 is thought to induce the ubiquitination of histone H2A (and H2AX) CONTACT Younghoon Kee © 2016 Taylor & Francis Group, LLC
[email protected]
at lesions, but whether this is the primary mechanism responsible for transcriptional repression at the damaged sites is not clear. Our recent work unexpectedly showed that chromatin recruitment of UBR5 E3 ligase is largely dependent on BMI1 and 2 other PRC1 components, RNF1 and RNF2.7 We found that, similar to BMI1, ablation of UBR5 leads to derepression of transcription at damaged sites, suggesting that UBR5 may be a downstream effector of BMI1 in mediating transcription repression. This phenotype of UBR5depleted cells was rescued by wild-type UBR5 but not by a catalytically inactive mutant, suggesting that UBR5 must ubiquitinate a substrate to repress transcription. Further analysis revealed that UBR5 interacts with the histone chaperone complex FACT, which is a heterodimer of SPT16 and SSRP1. Interestingly, a previous report found that SPT16 is localized to UV lesions and is required for the dynamic exchange of histone H2A and H2B molecules and for RNA Pol II to resume transcription.8 Therefore, an intriguing hypothesis is that UBR5 acts to temporarily suppress FACT activity, an event controlled by BMI1 and PRC1, to allow repair to occur until transcription resumes. How might UBR5 suppress FACT activity? We found that the SPT16 foci at the lesions are enlarged in BMI1- and UBR5-depleted cells, suggesting that BMI1 and UBR5 may suppress the local concentration of FACT at the lesions. We found that UBR5 ubiquitinates SPT16, and postulate that this modification may account for transcription repression. In summary, this finding revealed an unexpected link among FACT activity, UBR5, and BMI1 in the context of controlling Pol II progression through damaged chromatin (Fig. 1). Clearly, there are still many unanswered questions: (1) Does recruitment of UBR5 to chromatin require the H2AK119-Ub mark induced by PRC1? UBR5 has a ubiquitinassociated (UBA) domain, and it is possible that this is involved in interaction with the ubiquitin mark on histones. Although our data did not indicate that this is the case, further thorough investigation is warranted. (2) Is SPT16 ubiquitination indeed inhibitory to FACT activity, as well as to the nucleosomal dynamics necessary for transcriptional elongation? Identification of the specific ubiquitination sites may be
e1244513-2
Y. KEE
Figure 1. Various models for Polycomb repressive complex 1–induced transcription repression at damaged chromatin. (A). PRC1 containing BMI1 induces ubiquitination at H2A (K119-Ub), which leads to repression of Pol II progression near the damaged regions (the catalytic subunit RNF2 is not shown). There may be a reader of the ubiquitinated histone that mediates RNA Pol II inhibition. ZRF1 was identified as a reader of ubiquitinated H2A to positively regulate the expression of developmental genes, but whether it is also involved in the DNA damage response is unknown. (B). PRC1-induced repression of RNA Pol II elongation involves UBR5 and FACT histone chaperone activity. Whether UBR5 recruitment to the damaged sites requires ubiquitinated H2A is not known.
necessary to answer these questions. (3) How does the role of UBR5 in transcriptional repression relate to a previously known role of UBR5 in suppressing RNF168-mediated ubiquitin signaling? Knockdown of UBR5 (and another E3 ligase, TRIP12) increases the 53BP1 and ubiquitin foci at the damage sites, which in turn negatively affects RNA synthesis.9 While we could not convincingly reconcile the seemingly contradictory effects of this study and our results, there are several notable differences in the experimental conditions. For example, our experiments were performed under UV-damage conditions during which overall RNA synthesis is suppressed, whereas the study by Gudjonson et al. monitored spontaneous 53BP1 foci spots when UBR5 was depleted. It is possible that in the UBR5-depleted cells 53BP1 and SPT16 exhibit opposing activity between DSB signaling and transcription, with SPT16 exerting dominating effects at the UV lesions. There is a clear need for further investigation to explain these differences. (4) Do ATM, ATR, or DNA-PK kinases, which are all known to participate in the transcriptional repression albeit under different contexts, crosstalk with UBR5 in transcriptional
repression, possibly through BMI1? UBR5 was shown to ubiquitinate the ATM-associated protein ATMIN, an event that leads to activation of the ATM signaling.10 Therefore, it is conceivable that a feedback loop may exist between UBR5 and ATM in transcriptional silencing. In summary, future studies will hopefully provide better insight into the role of BMI1 and histone ubiquitination in transcriptional silencing at damaged chromatin and their other putative non-canonical activity in supporting the DNA damage response and genomic integrity.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References 1. Svejstrup JQ. The interface between transcription and mechanisms maintaining genome integrity. Trends Biochem Sci 2010; 35:333-8; PMID:20194025; http://dx.doi.org/10.1016/j.tibs.2010.02.001
MOLECULAR & CELLULAR ONCOLOGY
2. Sparmann A, van Lohuizen M. Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer 2006; 6:846-56; PMID:17060944; http://dx.doi.org/10.1038/nrc1991 3. Di Croce L, Helin K. Transcriptional regulation by Polycomb group proteins. Nat Struct Mol Biol 2013; 20:1147-55; PMID:24096405; http://dx.doi.org/10.1038/nsmb.2669 4. Pengelly AR, Kalb R, Finkl K, Muller J. Transcriptional repression by PRC1 in the absence of H2A monoubiquitylation. Genes Dev 2015; 29:1487-92; PMID:26178786; http://dx.doi.org/10.1101/gad.265439.115 5. Illingworth RS, Moffat M, Mann AR, Read D, Hunter CJ, Pradeepa MM, Adams IR, Bickmore WA. The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development. Genes Dev 2015; 29:1897902; PMID:26385961; http://dx.doi.org/10.1101/gad.268151.115 6. Vissers JH, van Lohuizen M, Citterio E. The emerging role of Polycomb repressors in the response to DNA damage. J Cell Sci 2012; 125:3939-48; PMID:23104738; http://dx.doi.org/10.1242/jcs.107375 7. Sanchez A, De Vivo A, Uprety N, Kim J, Stevens SM, Jr, Kee Y. BMI1UBR5 axis regulates transcriptional repression at damaged chromatin.
e1244513-3
Proc Natl Acad Sci U S A 2016; 113:11243-8; PMID:27647897; http:// dx.doi.org/10.1073/pnas.1610735113 8. Dinant C, Ampatziadis-Michailidis G, Lans H, Tresini M, Lagarou A, Grosbart M, Theil AF, van Cappellen WA, Kimura H, Bartek J, et al. Enhanced chromatin dynamics by FACT promotes transcriptional restart after UV-induced DNA damage. Mol Cell 2013; 51:469-79; PMID:23973375; http://dx.doi.org/ 10.1016/j.molcel.2013.08.007 9. Gudjonsson T, Altmeyer M, Savic V, Toledo L, Dinant C, Grofte M, Bartkova J, Poulsen M, Oka Y, Bekker-Jensen S, et al. TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes. Cell 2012; 150:697-709; PMID:22884692; http://dx.doi. org/10.1016/j.cell.2012.06.039 10. Zhang T, Cronshaw J, Kanu N, Snijders AP, Behrens A. UBR5mediated ubiquitination of ATMIN is required for ionizing radiation-induced ATM signaling and function. Proc Natl Acad Sci U S A 2014; 111:12091-6; PMID:25092319; http://dx.doi.org/10.1073/ pnas.1400230111
