Volume 24, Issue 2, Pages 261-275 (February 2016) SET1A Cooperates With CUDR to Promote Liver Cancer Growth and Hepatocyte-like Stem Cell Malignant Transformation Epigenetically  Tianming Li, Qidi Zheng, Jiahui An, Mengying Wu, Haiyan Li, Xin Gui, Hu Pu, Dongdong Lu  Molecular Therapy  Volume 24, Issue 2, Pages 261-275 (February 2016) DOI: 10.1038/mt.2015.208 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 The expression analysis of CUDR, SET1A, pRB1, RB1, H3K4me1/2/3 and the interplay among histone H3, SETA1, and pRB in human ES-derived hepatocyte-like stem cells and human liver cancer tissues. (a) RT-PCR for CUDR and western blotting for anti-SET1A, anti-pRB1, anti-RB1, anti-H3K4me1, anti-H3K4me2, and anti-H3K4me3 in human ES-derived hepatocyte-like stem cells at 0 day, 0.5 day, 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, and 14 days, respectively. β-Actin was used as an internal control. (b) Co-immunoprecipitation (IP) with anti-SET1A or anti-pRB followed by western blotting with anti-pRB1 or anti-histone H3 in derived hepatocyte-like stem cells at 0 day,0.5 day, 1 day, 2 days, 4 days, 6 days,8 days,10 days,12days,14 days, respectively. IgG IP was considered as negative control. Input refers to western blotting with anti-SET1A or anti-histone H3. (c) RT-PCR for CUDR and western blotting with anti-SET1A, anti-pRB, anti-RB, anti-H3K4me1, anti-H3K4me2, anti-H3K4me3 in human liver cancer tissue (C) and its paracancerous liver tissues (P), respectively. β-Actin was used as an internal control. Molecular Therapy 2016 24, 261-275DOI: (10.1038/mt.2015.208) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 SET1A cooperates with CUDR to accelerate liver cancer cells HepG2 growth in vitro and in vivo. (a) Western blotting with anti-SET1A, anti-pRB, anti-RB, and RT-PCR for CUDR in HepG2 cell lines stably transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-SET1A, pCMV6-AC-GFP-SET1A plus pCMV6-A-GFP-CUDR, pCMV6-AC-GFP-SET1A plus pGFP-V-RS-CUDR, pGFP-V-RS, pGFP-V-RS-SET1A, pGFP-V-RS-SET1A plus pCMV6-A-GFP-CUDR, pGFP-V-RS-SET1A plus pGFP-V-RS-CUDR, respectively. β-Actin was used as an internal control. (b) Cells growth analysis with CCK8 assay. The pCMV6-AC-GFP control group is denoted by solid line; the pCMV6-AC-GFP-SET1A group is denoted by square dotted line; the pCMV6-AC-GFP-SET1A plus pCMV6-AC-GFP-CUDR group is denoted by dashes separated by one dots; the pCMV6-AC-GFP-SET1A plus pGFP-V-RS-CUDR group is denoted by round dot line; the pGFP-V-RS-AC-GFP group is denoted by dashes line; the pGFP-V-RS-SET1A group is denoted by long dash dots; the pGFP-V-RS-SET1A plus pCMV6-AC-GFP-CUDR group is denoted by long dash; the pGFP-V-RS-SET1A plus pGFP-V-RS-CUDR group is denoted by dashes separated by two dots. Each value was presented as mean ± sSEM. *P < 0.05; **P < 0.01. (c) Cells plate colony formation assay. Each value was presented as mean ± SEM. **P < 0.01. (d) (A) The mice were stratified and the tumors were recovered. The photography of xenograft tumors in the eight groups (indicated in left). (B) The wet weight of each xenograft tumor was determined for each mouse. Each value was presented as mean ± SEM. *P < 0.05;**P < 0.01. (e) Western blotting with anti-PCNA in xenograft tumors. β-Actin was used as an internal control. PCNA, proliferating cell nuclear antigen. Molecular Therapy 2016 24, 261-275DOI: (10.1038/mt.2015.208) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 SET1A incorporates CUDR to accelerate human ES-derived hepatocyte-like stem cells malignant transformation. (a) Western blotting analysis with anti-SET1A, anti-pRB, anti-RB, and RT-PCR for CUDR in MEL-2–derived hepatocyte-like stem cells stably transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-SET1A, pCMV6-AC-GFP-SET1A plus pCMV6-A-GFP-CUDR, pCMV6-AC-GFP-SET1A plus pGFP-V-RS-CUDR, pGFP-V-RS-AC-GFP, pGFP-V-RS-SET1A, pGFP-V-RS—SET1A plus pCMV6-A-GFP-CUDR, and pGFP-V-RS-SET1A plus pGFP-V-RS-CUDR, respectively. β-Actin has been considered as an internal control. (b) Cell growth analysis with CCK8 assay. The pCMV6-AC-GFP control group is denoted by solid line; the pCMV6-AC-GFP-SET1A group is denoted by square dot line; the pCMV6-AC-GFP-SET1A plus pCMV6-AC-GFP-CUDR group is denoted by dash line; the pCMV6-AC-GFP-SET1A plus pGFP-V-RS-CUDR group is denoted by long dash line; the pGFP-V-RS-AC-GFP group is denoted by round line; the pGFP-V-RS-SET1A group is denoted by long dash dot line; the pGFP-V-RS-SET1A plus pCMV6-AC-GFP-CUDR group is denoted by dashes separated by two dots; the pGFP-V-RS-SET1A plus pGFP-V-RS-CUDR group is denoted by dashes separated by one dots. Each value was presented as mean ± SEM. *P < 0.05; **P < 0.01. (c) Cells soft-agar colony formation assay. Each value was presented as mean ± SEM. *P < 0.05; **P < 0.01. (d) (A) The mice were stratified and the tumors were recovered. The photography of xenograft tumors in the eight groups (indicated in left). (B) The wet weight of each tumor was determined for each mouse. Each value was presented as mean ± SEM. *P < 0.05; **P < 0.01. (e) A portion of each tumor was fixed in 4% paraformaldehyde and embedded in paraffin for histological hematoxylin–eosin staining (original magnification: ×100). Molecular Therapy 2016 24, 261-275DOI: (10.1038/mt.2015.208) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 CUDR enhances the phosphorylation of RB1 (pRB1), and the interplay between and SET1A and pRB1 dependent on C-myc. (a) RT-PCR for CUDR, RB1 and C-myc and western blotting with anti-RB, anti-pRB, anti-C-myc in CUDR excessive or depleted HepG2 cell lines. β-Actin has been considered as an internal control. (b) RNA immunoprecipitation (RIP) with anti-C-myc followed by RT-PCR with CUDR mRNA primers in CUDR excessive or depleted HepG2 cell lines. IgG RIP is negative control. CUDR mRNA is input. (c) Co-immunoprecipitation (Co-IP) with anti-C-myc followed by western blotting with anti-SET1A in CUDR overexpression or knockdown HepG2 cell lines. IgG IP as negative control. Western blotting with anti-C-myc as input. (d) RIP with anti-SET1A followed by RT-PCR with CUDR mRNA primers in CUDR overexpression or knockdown HepG2 cell lines. IgG RIP is negative control. CUDR mRNA is input. (e) RIP with anti-SET1A followed by RT-PCR with CUDR mRNA primers in CUDR excessive or CUDR excessive plus C-myc depleted HepG2 cell lines. IgG RIP is negative control. CUDR mRNA is input. (f) Western blotting analysis for pRB in CUDR excessive or CUDR excessive plus C-myc depleted HepG2 cell lines. β-Actin was used as an internal control. (g) RIP with anti-pRB followed by RT-PCR with CUDR mRNA primers in CUDR excessive or CUDR excessive plus C-myc depleted HepG2 cell lines. IgG RIP is negative control. CUDR mRNA is input. (h) RIP with anti-pRB followed by RT-PCR with CUDR mRNA primers in CUDR excessive or CUDR excessive plus C-myc depleted HepG2 cell lines. IgG RIP is negative control. CUDR mRNA is input. (i) Co-IP with anti-pRB followed by western blotting with anti-SET1A in CUDR excessive or depleted HepG2 cell lines. IgG IP as negative control. Western blotting with anti-pRB as input. Molecular Therapy 2016 24, 261-275DOI: (10.1038/mt.2015.208) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 CUDR cooperates with SET1A and pRB1 to promote tri-methylation of forth lysine of histone H3 (H3K4me3). (a) Co-immunoprecipitation (IP) with anti-SET1A or anti-pRB1 followed by western blotting with anti-histone H3 or anti-pRB in CUDR excessive or depleted HepG2. IgG IP as a negative control. Input refers to western blotting with anti-histone H3 or anti-pRB1. (b) Co-IP with anti-SET1A or anti-pRB1 followed by western blotting with anti-histone H3 or anti-pRB1 in HepG2 cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-SET1A, pCMV6-AC-GFP-SET1A plus pCMV6-A-GFP-CUDR, pCMV6-AC-GFP-SET1A plus pGFP-V-RS-CUDR, pGFP-V-RS-AC-GFP, pGFP-V-RS-SET1A, pGFP-V-RS-SET1A plus pCMV6-A-GFP-CUDR, pGFP-V-RS-SET1A plus pGFP-V-RS-CUDR, respectively. IgG IP as a negative control. Input refers to western blotting with anti-histone H3 or anti-pRB. (c) Co-IP with anti-SET1A or anti-pRB followed by western blotting with anti-histone H3 or anti-pRB1 in HepG2 cell lines transfected pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pGFP-V-RS-RB, pGFP-V-RS-RB, respectively. IgG IP as a negative control. Input refers to western blotting with anti-histone H3 or anti-pRB. (d) Western blotting with anti-H3K4me1, anti-H3K4me2, anti-H3k4me3, anti-pRB1, anti-RB1 in HepG2 cell lines transfected pCMV6-AC-GFP, pCMV6-AC-GFP-SET1A, pCMV6-AC-GFP-SET1A plus pRB1 inhibitor, pCMV6-AC-GFP-SET1A plus GFP-V-RS-CUDR, respectively. Histone H3 was used as an internal control. (e) Western blotting with anti-H3K4me3, anti-SET1A, anti-pRB1 in HepG2 cell lines transfected pCMV6-AC-GFP, pCMV6-AC-GFP-SET1A, pCMV6-AC-GFP-SET1A plus pCMV6-A-GFP-CUDR, pCMV6-AC-GFP-SET1A plus pGFP-V-RS-CUDR, pGFP-V-RS-AC-GFP, pGFP-V-RS-SET1A, pGFP-V-RS-SET1A plus pCMV6-A-GFP-CUDR, pGFP-V-RS-SET1A plus pGFP-V-RS-CUDR, respectively. Histone H3 was used as an internal control. (f) In vitro translation assay with SET1A protein, RB1 protein, and nuclear extract (CUDR overexpression/knockdown, SET1A knockdown or RB1 knockdown) (indicated in upper). Western blotting with anti-H3K4me3, anti-H3K4me2, anti-SET1A, anti-RB1, and anti-pRB1. Histone H3 was used as an internal control. Molecular Therapy 2016 24, 261-275DOI: (10.1038/mt.2015.208) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 SET1A cooperates with CUDR to boost up TRF2 activity and increase telomere length depending on H3K4me3. (a) Native chromatin immunoprecipitation (CHIP) with anti-H3K4me3 followed by PCR with TRF2 promoter primers in HepG2 cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-SET1A, pCMV6-AC-GFP-SET1A plus pCMV6-A-GFP-CUDR, pCMV6-AC-GFP-SET1A plus pGFP-V-RS-CUDR, pGFP-V-RS-AC-GFP, pGFP-V-RS-SET1A, pGFP-V-RS-SET1A plus pCMV6-A-GFP-CUDR, pGFP-V-RS-SET1A plus pGFP-V-RS-CUDR, respectively. IgG CHIP as a negative control. TRF2 promoter DNA as input. (b) TRF2 promoter luciferase activity assay. Each value was presented as mean ± SEM. **P < 0.01. (c) RT-PCR analysis for TRF2 mRNA (upper) and western blotting with anti-TRF2 (lower). β-Actin was used as an internal control. (d) CHIP with anti-TRF2 followed by PCR with telomere primers. IgG IP as a negative control. Telomere DNA as input. (e) Multiple PCR with various telomere primers in HepG2 cell lines. β-Actin was used as an internal control. (f) Real-time PCR of telomere length in HepG2 cell lines. Total DNA was used as an internal control. Molecular Therapy 2016 24, 261-275DOI: (10.1038/mt.2015.208) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 The rescued experiment of carcinogenesis effect of the CUDR combined with SET1A. (a) (A) The western blotting analysis with anti-TRF2 and anti-SET1A, and the RT-PCR for CUDR in HepG2 cells line stably transfected with pCMV6-AC-GFP, pCMV6-AC-SET1A plus pCMV6-A-GFP-CUDR, pCMV6-AC-GFP-SET1A plus pCMV6-A-GFP-CUDR plus pGFP-V-RS-TRF2. β-Actin was used as an internal control. (B) Cell growth analysis with CCK8 assay. Each value was presented as mean ± SEM. *P < 0.05; **P < 0.01. (C) Cells soft-agar colony formation assay. Each value was presented as mean ± SEM. *P < 0.05; **P < 0.01. (D) Tumorigenesis test in vivo. Upper panel: the mice were stratified and the tumors were recovered. The photography of xenograft tumor in the four groups (indicated in left). Lower panel: the wet weight of each tumor was determined for each mouse. Each value was presented as mean ± SEM. *P < 0.05; **P < 0.01. (b) (A) The western blotting analysis with anti-TRF2 and anti-SET1A, and the RT-PCR for CUDR in hepatocyte-like stem cell lines stably transfected with pcDNA3.1, pcDNA3.1-SET1A plus pCMV6-A-GFP-CUDR and pcDNA3.1-SET1A plus pCMV6-A-GFP-CUDR plus pGFP-V-RS-TRF2. β-Actin was used as an internal control. (B) Cells growth analysis with CCK8 assay. Each value was presented as mean ± SEM. *P < 0.05; **P < 0.01. (C) Cells soft-agar colony formation assay. Each value was presented as mean ± SEM. *P < 0.05; **P < 0.01. (D) Tumorigenesis test in vivo. Upper panel: the mice were stratified and the tumors were recovered. The photography of xerograft tumor in the four groups. Lower panel: the wet weight of each tumor was determined for each mouse. Each value was presented as mean ± SEM. *P < 0.05; **P < 0.01. Molecular Therapy 2016 24, 261-275DOI: (10.1038/mt.2015.208) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 A schematic representation of the mechanisms for SET1A cooperates with lncRNA CUDR to accelerate hepatocarcinogenesis and liver stem cell malignant transformation. Notably, CUDR enhances the phosphorylation of RB1, and the interplay between the SET1A and pRB1 dependent on C-myc. Intriguingly, CUDR acts as a sponge cushion that link between SET1A and pRB, producing a activated pRB–SET1A complex. Furthermore, the complex may carry methyls(me) to occupy the position of H3K4, resulting in tri-methylation of forth lysine of histone H3 (H3K4me3). Thereby, the H3K4me3 loads on the TRF2 promoter region which causes the TRF2 overexpression. Ultimately, the excessive TRF2 binds to telomere repeat DNA which prolongs the telomere length and accelerates hepatocyte-like stem cells malignant transformation and the liver cancer cells malignant growth. Molecular Therapy 2016 24, 261-275DOI: (10.1038/mt.2015.208) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions