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Article: Elserafy, M., Abugable, A.A. orcid.org/0000-0003-1275-3328, Atteya, R. et al. (1 more author) (2018) Rad5, HLTF, and SHPRH: A fresh view of an old story. Trends in Genetics, 34 (8). pp. 574-577. ISSN 0168-9525 https://doi.org/10.1016/j.tig.2018.04.006

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[email protected] https://eprints.whiterose.ac.uk/ impact of mismatches on the recombina- The presence of dispersed Alu elements chromosomal double strand breaks. Methods Mol. Biol. 920, 379–391 fi tion ef ciency. Their reporter was inte- throughout the genome [3], and other 9. Sugawara, N. et al. (2004) Heteroduplex rejection during grated in the genome in such a way that repetitive DNA sequences, provides end- single-strand annealing requires Sgs1 helicase and mis- match repair proteins Msh2 and Msh6 but not Pms1. Proc. they could use one Crispr/Cas9 guide RNA less opportunities for RMDs that disrupt Natl. Acad. Sci. U. S. A. 101, 9315–9320 to cut near the upstream homology region genome function. The relatively efficient and a second guide RNA to cut varying excision of tens of kilobases of DNA at distances (16 bp to 28.4 kb) from the DSBs means that many copies of Alu downstream homology region. may be exposed in single-strand regions, Forum leading to many possible rearrangements. Using their assay [7], the Stark lab showed The human genome could not survive the Rad5, HLTF, and that the frequency of repeat-mediated resulting instability if it did not have strong SHPRH: A Fresh View deletions (RMDs) increased when the regulators inhibiting these RMDs. DSB is close to the repetitive elements of an Old Story and declines rapidly with distance, fol- There are a number of situations in which Menattallah Elserafy,1,2,3 lowed by a very gradual decline up to at someoftheDSBrepairpathways are either Arwa A. Abugable,2,3 least 28.4 kb. At the longer distances, the suppressed or defective in humans. Can- Reham Atteya,2 and RMD was much more dependent on cer is the most obvious situation in which 1,2, ,@ Rad52 then at shorter distances. The role some of these genes, such as msh2, Sherif F. El-Khamisy * of Rad52 in SSA is to help anneal homolo- brca1,andbrca2 are commonly defective. gous or homologous sequences to form Thus, in various cancers one might expect Not only have helicase-like tran- heteroduplexes [1], whereas NHEJ and alt- tosee increases intheabundanceofRMDs scription factor (HLTF) and SNF2 NHEJ pathways are Rad52-independent. and possibly other changes in the nature of histone-linker PHD-finger RING-fin- They also showed that even a modest level repeat-mediated genomic rearrange- ger helicase (SHPRH) proved to be of mismatch between the repetitive ments that might lead to more genetic important players in post-replication sequences (1–3%) significantly inhibited instability, which in turn helps the cancer repair like their yeast counterpart, RMD at all distances. This inhibition by evolve to a more malignant form. Rad5, but they are also involved in mismatches between repetitive elements 1 multiple biological functions and are was largely dependent on Msh2, which Tulane Cancer Center, Tulane University, New Orleans, LA 70122, USA associated with several human dis- would be expected for a process mediated orders. We provide here an updated by heteroduplex rejection [9]. *Correspondence: [email protected] (P. Deininger). view of their functions, associated https://doi.org/10.1016/j.tig.2018.05.003 diseases, and potential therapeutic This new study tested the dependence of References approaches. RMD on various proteins involved in DSB 1. Ceccaldi, R. et al. (2016) Repair pathway choices and conse- repair. For example, loss of KU70 and quences at the double-strand break. Trends Cell Biol. 26, 52–64 XRCC4 (proteins involved in c-NHEJ) 2. Symington, L.S. and Gautier, J. (2011) Double-strand break RAD5, a Gene All[134_TD$DIFF] Budding Yeast end resection and repair pathway choice. Annu. Rev. Genet. increased RMD, while loss of CtIP and 45, 247–271 Laboratories Should Know about BRCA1 (which are involved in the resec- 3. Lander, E.S. et al. (2018) Initial sequencing and analysis of Yeast has served as a prominent model the human genome. Nature 409, 860–921 tion pathway) was found to decrease organism in DNA repair research for dec- 4. Carvalho, C.M. and Lupski, J.R. (2016) Mechanisms under- RMD. This indicates that there may be lying structural variant formation in genomic disorders. Nat. ades [1]. Rad5 is a RING-finger-containing competition between the nonresection Rev. Genet. 17, 224–238 ubiquitin ligase and helicase that[135_TD$DIFF] was 5. Belancio, V.P. et al. (2008) Mammalian non-LTR retrotrans- and resection pathways (Figure 1). How- posons: for better or worse, in sickness and in health. discovered in budding yeast and was ever, the authors found that direct regu- Genome Res. 18, 343–358 found to play a role in template switching lation by heteroduplex rejection (Msh2- 6. Morales, M.E. et al. (2015) The contribution of alu elements (TS) and translesion synthesis (TLS) post- to mutagenic DNA double-strand break repair. PLoS Genet. mediated) is a stronger inhibitor of RMD 11, e1005016 replication repair (PRR) mechanisms. TS than competition with c-NHEJ. These 7. Mendez-Dorantes, C. et al. (2018) Repeat- utilizes a clever mechanismusing thenewly mediated deletions can be induced by a data indicate that there could be multiple chromosomal break far from a repeat, but multiple synthesized sister DNA strand as a tem- pathways that suppress RMD between pathways suppress such rearrangements. Genes Dev. plate to bypass lesions, while TLSswitches 32, 524–536 the high concentration of repetitive replicative polymerases to low-fidelity pol- 8. Gunn, A. and Stark, J.M. (2012) I-SceI-based assays to sequences throughout the genome. examine distinct repair outcomes of mammalian ymerases to overcome lesions [2].

574 Trends in Genetics, August 2018, Vol. 34, No. 8 The wild-type W303 yeast strain used by to predict prognosis and survival. More- Vpr and DCAF1 [9]. Mutations in this motif numerous laboratories harbors a RAD5 over, in cervical, thyroid, and head and or the addition of a zinc-chelating agent, mutation (rad5-535). Some researchers neck cancers, alternative splicing of HLTF TPEN N,N,N,N[136_TD$DIFF] -tetrakis-(2-pyridylmethyl) have started to use a strain in which the mRNA increases the expression of trun- ethylenediamine, was found to disrupt mutation has been corrected. Conse- cated versions lacking functional DNA the Vpr–CRL4 (DCAF1) interaction and quently, conflicting results have been repair domains [8]. Heterozygosity in the therefore their function. Nevertheless, reported in the literature by laboratories long arm of chromosome 6, where the TPEN did not affect the assembly of the using the W303 rad5-535 versus W303 SHPRH gene resides, has been reported cellular CRL4 E3 complex. This opens the RAD5 strains [3]. While it is important to in malignant melanoma, ovarian, and cer- door to developing novel antiviral thera- raise the awareness of yeast researchers vical cancers. Furthermore, SHPRH was pies against[137_TD$DIFF] HIV-1 in which the interaction to the importance of RAD5 genotype in found to be truncated or to contain mis- between Vpr and CRL4/DCAF1 is selec- yeast, it is equally important to highlight sense mutations in tumor cell lines, which tively disrupted [9] (Figure 1B). how the mammalian Rad5 orthologs, is consistent with a tumor-suppressor such as HLTF and SHPRH, play multiple function [2]. HLTF and Inflammatory Bowel Disease important roles in mammalian cells. (IBD) Interestingly, HLTF was found to be one of Gene expression analysis of regenerating HLTF and SHPRH; Rad5 Human the genetic determinants conferring sensi- gene (REG) family members has shown that Orthologs tivity or resistance to lysosomal autophagy the genes encoding REG-Ia (REG1A), REG- HLTF is considered to be the closest inhibitors such as hydroxychloroquine Ib (REG1B), and REG-IV (REG4)areover- ortholog to yeast Rad5; it shows higher (HCQ) in several cancer cell lines [8]. HLTF expressed in IBD. Upregulation of REG sequence conservation to Rad5 than to expression was found to be low in HCQ- genesisbeneficial on the one hand because SHPRH. HLTF contains the conserved sensitive cell lines and high in resistant cell it induces the proliferation of intestinal epi- HIRAN domain that is responsible for lines. HLTF protected against HCQ- thelial cells, protecting them from damage 30-single-stranded (ss)DNA binding [4], induced cell death because it is essential caused by the immune system, but on the and is able to complement UV sensitivity for repairing the resulting reactive oxygen other hand upregulation increases therisk of in the Drad5 yeast strain. It also pos- species (ROS)-induced DNA damage [8]. developing colon cancer [10]. sesses double-stranded (ds)DNA translo- Because lysosome inhibitors are currently case activity which is utilized to reverse in clinical trials for efficacy as single or com- A recent study investigating the link stalled replication forks [5]. Similarly to bined anticancer agents, HLTF expression between REG genes and IBD reported yeast Rad5, both HLTF and SHPRH are profiling of patients may be helpful to deter- that REG1A, REG1B, and REG4 are over- E3 ubiquitin ligases and have been shown mine the response [8] (Figure 1B). We sug- expressed in the colon of Crohn’s disease to polyubiquitinate PCNA, in cooperation gest designing HLTF inhibitors for patients, and that REG4 is overexpressed with Ubc13–MMS2, in response to DNA anticancer therapy because combining in the colon of ulcerative colitis patients damage, thereby directing PRR towards themwithautophagyinhibitorscouldprove [10]. IL-22 was found to induce REG1B the TS repair pathway [6,7]. The homol- to be synergistic. Moreover, one could overexpression through activating the ogy between Rad5, HLTF, and SHPRH is question whether cancers with downregu- REG1B promoter region which contains depicted in Figure 1A [2]. lated SHPRH expression would also a binding site for HLTF. Knockdown of respond to autophagy inhibitors. Similarly, HLTF was found to decrease the expres- it is tempting to test whether inhibiting sion of IL-22-induced REG Ib [10].To HLTF and SHPRH in Disease SHPRH would also synergize with autoph- prevent the development of colon cancer Cancer agy inhibitors (Figure 1B). after intestinal epithelium recovery, the A large body of evidence indicates that study suggested that anti-IL-22 and HLTF is a tumor-suppressor gene [2]. HLTF and HIV-1 anti-REG proteins could be administered Methylation and silencing of HLTF have Upon HIV-1 infection of target cells, viral to patients. We also propose that devel- been documented in several cancers protein R (Vpr) downregulates HLTF [9]. oping inhibitors that prevent the interac- such as colon, gastric, esophageal, uter- Vpr is known to recruit CRL4 (DCAF1) E3 tion between HLTF and the REG1B ine, lung, hepatocellular, and bladder ligase to mediate HLTF degradation, and promoter could be useful to reduce the cancer [2,8]. HLTF promoter hypermethy- thus facilitates viral replication. A highly expression of REG-1b after colon regen- lation in the blood of cancer patients has conserved zinc-binding motif (HHCH) in eration, and thus reduce the risk of colon been investigated as a tumor biomarker Vpr is required for the interaction between cancer (Figure 1B).

Trends in Genetics, August 2018, Vol. 34, No. 8 575 (A) HIRAN SNF2 RING Rad5 1169 aa

HIRAN SNF2 RING HLTF 1009 aa

SNF2 H15 PHD RING SHPRH 1683 aa

(B) IBD GH4C1 cells

Regulaピon of clock ? -controlled genes

Inhibitors

? Brain development

SHPRH HLTF Collagen biogenesis Cancer in heart on ピ Downregulaピon/ truncaピon Degrada CRL4 ? + (DCAF1) HIV-Vpr HIV

Lysosomal autophagy inhibitor Zinc-chelaピng agent

Cancer cell death Prevenピng HLTF degradaピon

Figure 1. A Fresh View of HLTF and SHPRH. (A) Homology between Rad5, HLTF, and SHPRH. Similarly to Rad5, HLTF contains the conserved HIRAN domain. All three proteins contain the RING finger domain embedded between the SNF2 helicase motifs. SHPRH also contains a H15 linker histone H1/H5 domain and a PHD- finger domain. Panel adapted, with permission, from [2]. (B) Biological functions and associated diseases for HLTF and SHPRH, and potential therapeutic approaches. HLTF plays a role in the regulation of clock-controlled genes in GH4C1 cells, in mouse embryonic and postnatal brain development, and in collagen biogenesis in the heart. In addition, downregulation or truncation of HLTF and SHPRH have been implicated in different cancers. Cancer cells with low HLTF expression were found to be sensitive to lysosomal autophagy inhibitors.[131_TD$DIFF] HLTF is also involved in REG1B overexpression, increasing the risk of colon cancer in IBD patients. Zinc-chelating agent prevents HLTF degradation by HIV-1. We propose testing the effect of the lysosomal autophagy inhibitors on cancers with low SHPRH levels. We also propose designing HLTF and SHPRH inhibitors to be tested in combination with[132_TD$DIFF] lysosomal autophagy inhibitors. Abbreviations: aa, amino acids; IBD, in[133_TD$DIFF] flammatory bowl disease.

576 Trends in Genetics, August 2018, Vol. 34, No. 8 HLTF in Biological Functions addition, HLTF regulates the expression of *Correspondence: s.el-khamisy@sheffield.ac.uk (S.F. El-Khamisy). HLTF in the Regulation of Clock- the cohesion and condensin complexes, https://doi.org/10.1016/j.tig.2018.04.006 Controlled Genes (CCGs) as well as of cell-cycle regulators, during

The majority of CCGs involved in circa- embryonic development of mouse brain. References dian rhythm regulation lack the specificE- Therefore,asignificantpercentageofHLTF 1. Abugable, A.A. et al. (2017) Personalised medicine: genome maintenance lessons learned from studies in yeast as a box response element required for regu- null mice die, and those that survive suffer model organism. Adv. Exp. Med. Biol. 1007, 157–178 lation. A study was conducted on the from DNA damage, increased apoptosis in 2. Unk, I. et al. (2010) Role of yeast Rad5 and its human [138_TD$DIFF] orthologs, HLTF and SHPRH in DNA damage tolerance. GH4C1 rat pituitary somatolactotroph cell brain, brain encephalomalacia, G2/M tran- DNA Repair 9, 257–267 line to investigate the mechanism of tran- sition defects, and fibrillar network disorga- 3. Elserafy, M. and El-Khamisy, S.F. (2018) Choose your scription of prolactin (Prl), a CCG that nization [12,13] (Figure 1B). yeast strain carefully: the RAD5 gene matters. Nat. Rev. Mol. Cell Biol. Published online April 3, 2018. http://dx.doi. lacks the specific E-box element. HLTF org/10.1038/s41580-018-0005-2 was found to interact with PIT-1 transcrip- Concluding Remarks 4. Hishiki, A. et al. (2015) Structure of a novel DNA-binding domain of helicase-like transcription factor (HLTF) and its tion factor at the P2 region of the Prl There is increasing interest among scientists functional implication in DNA damage tolerance. J. Biol. promoter, which is different from the spe- in discovering different roles for the Rad5 Chem. 290, 13215–13223 cific E-box that is usually bound by CCGs. human homologs. HLTF is especially inter- 5. Blastyak, A. et al. (2010) Role of double-stranded DNA translocase activity of human HLTF in replication of dam- HLTF and PIT-1 are not rhythmically esting because any associated defects aged DNA. Mol. Cell. Biol. 30, 684–693 expressed; however, HLTF interacts with would not only affect the DNA repair activity 6. Unk, I. et al. (2006) Human SHPRH is a ubiquitin ligase for Mms2–Ubc13-dependent polyubiquitylation of proliferat- NONO and SFPQ proteins which rhyth- in cells but also the expression level of mul- ing cell nuclear antigen. Proc. Natl. Acad. Sci. U. S. A. 103, mically bind to the Prl gene. Therefore, tiple genes that are potentially implicated in 18107–18112 this leads to Prl expression that is neces- different disorders. We propose that defects 7. Unk, I. et al. (2008) Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitina- sary for maintaining the normal circadian in HLTF and SHPRH could be associated tion. Proc. Natl. Acad. Sci. U. S. A. 105, 3768–3773 pattern. These findings have uncovered a with multiple disorders that have not yet 8. Piao, S. et al. (2017) ALDH1A1 and HLTF modulate the fi activity of lysosomal autophagy inhibitors in cancer cells. novel role for HLTF in the regulation of the been identi ed. They could also be utilized Autophagy 13, 2056–2071 oscillatory expression of CCGs that lack as biomarkers and exploited as potential 9. Wang, H. et al. (2017) Inhibition of Vpx-mediated SAMHD1 the specific E-box response element [11] therapeutic targets. Further work will shed and Vpr-mediated host helicase transcription factor deg- radation by selective disruption of viral CRL4 (DCAF1) E3 (Figure 1B). light on novel approaches to selectively tar- ubiquitin ligase assembly. J. Virol. 91, e00225-17 get the perturbed functions of HLTF and 10. Tsuchida, C. et al. (2017) Expression of REG family genes in human inflammatory bowel diseases and its regulation. SHPRH while retaining their important role Biochem. Biophys. Rep. 12, 198–205 HLTF in Mouse Embryonic and in unaffected tissues. 11. Guillaumond, F. et al. (2011) Chromatin remodeling as a mech- Postnatal Development anism for circadian prolactin transcription: rhythmic NONO and HLTF has been shown to play crucial roles 1Krebs Institute, Department of Molecular Biology and SFPQ recruitment to HLTF. FASEB J. 25, 2740–2756 Biotechnology, Firth Court, University of Sheffield, 12. Helmer, R.A. et al. (2013) Role of helicase-like transcription in embryonic and postnatal development Sheffield S10 2TN, UK factor (Hltf) in the G2/M transition and apoptosis in brain. of the murine heart and brain [12,13].In 2Center for Genomics, Helmy Institute for Medical PLoS One 8, e66799 mouse heart, HLTF is necessary for hyp- Sciences, Zewail City of Science and Technology, Giza 13. Helmer, R.A. et al. (2013) Helicase-like transcription factor 12578, Egypt (Hltf) regulates G2/M transition, Wt1/Gata4/Hif-1a cardiac oxia-induciblefactor 1a (HIF-1a) regulation 3These authors contributed equally to the manuscript transcription networks, and collagen biogenesis. PLoS One 8, e80461 to achieve normal collagen biogenesis. In @Twitter: @SEK2016T

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