HBL1 Is a Human Long Noncoding RNA that Modulates Cardiomyocyte Development from Pluripotent Stem Cells by Counteracting MIR1  Juli Liu, Yang Li, Bo Lin, Yi Sheng, Lei Yang  Developmental Cell  Volume 42, Issue 4, Pages 333-348.e5 (August 2017) DOI: 10.1016/j.devcel.2017.07.023 Copyright © 2017 Elsevier Inc. Terms and Conditions

Developmental Cell 2017 42, 333-348. e5DOI: (10. 1016/j. devcel. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Identification of Lineage-Specific lncRNAs during Cardiomyocyte Differentiation from hESCs (A) Scheme of differentiation and enrichment of cardiovascular cells from human RUES2 ESCs. (B) Expression profiles of the lineage-specific marker genes (left) and lncRNA genes (right) quantitated by the normalized FPKM (fragments per kilobase of exon per million reads). (C) Scheme for the construction of pFEFW-E2-crimsom lentiviral vector for lncRNA knockdown (upper). Human S3 iPSC cells with knockdown of lncRNA TCONS_00028911 were cultured on feeders (lower). Scale bars, 200 μm. (D) Detection of lncRNA TCONS_00028911 expression by qRT-PCR. (E) Cardiomyocyte differentiation protocol using embryoid body (EB) method (upper) and images of EBs under differentiation from S34 hiPSCs (lower). Scale bars, 600 μm. (F) Percentage of CTNT+ CMs after 20 days of EB differentiation detected by flow cytometry. (G) Statistical analysis of percentage of CTNT+ cells in day-20 EBs. (H) Cardiomyocyte differentiation protocol using a monolayer differentiation method. (I) Statistical analysis of percentage of CTNT+ cells using monolayer differentiation protocol. Experiments were performed in triplicate. All error bars denote mean ± SD. n = 3. ∗p < 0.05 (Student's t test). See also Figure S1. Developmental Cell 2017 42, 333-348.e5DOI: (10.1016/j.devcel.2017.07.023) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Identification of lncRNA HBL1 (A) HBL1 transcription in human ESCs and sequence conservation analysis from the UCSC genome browser. (B) Subcellular localization of HBL1 in hiPSCs. (C) Detection of HBL1 expression in hiPSCs with Northern blotting. (D) Detection of 5′ and 3′ terminals of HBL1 using 5′ RACE and 3′ RACE. (E) Detection of absolute copy numbers of HBL1 transcripts in hiPSCs using four sets of primers for absolute qRT-PCR. Primers 1 detect T1, T2, T3, and T4 expressions. Primers 2 detect T2, T3, and T4 expressions. Primers 3 detect T3 expression. Primers 4 detect T3 and T4 expressions. (F) According to standard curve of absolute qRT-PCR, expression levels of four HBL1 transcripts are T4 > T1 ≫ T3 > T2. See also Figure S2. Developmental Cell 2017 42, 333-348.e5DOI: (10.1016/j.devcel.2017.07.023) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 HBL1 Overexpression Represses Cardiomyocyte Differentiation (A) Scheme for the construction of pHAGE-inducible lentiviral vector for HBL1 overexpression. (B) qRT-PCR detection of HBL1 expression in the HBL1-overexpressing hiPSCs. (C) Measurement of CTNT+ CMs differentiated from wild-type control and HBL1-overexpressing iPSCs by flow cytometry. (D) Statistical analysis of percentage of CTNT+ CMs from (C). (E) qRT-PCR detection of cardiac marker genes in wild-type and HBL1-overexpressing iPSC-derived CMs. All error bars denote mean ± SD. n = 3. ∗p < 0.05. All data comparisons use a one-way ANOVA (multiple groups). Developmental Cell 2017 42, 333-348.e5DOI: (10.1016/j.devcel.2017.07.023) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 HBL1 Knockout Promotes Cardiomyocyte Differentiation (A) Scheme for knockout of HBL1 using the CRISPR/Cas9 technology. Dual gRNAs were designed to completely knock out HBL1 from human genome. Two sets of PCR primers were used to detect HBL1 knockout. (B) PCR verification of HBL1 knockout in individual hiPSC clones using different primer sets. (C) Genome sequence after knockout of HBL1 in hiPSC clones. Dashed lines indicate deletion of HBL1 sequence. (D) HBL1 expression in wild-type (WT) and HBL1 null iPSCs by qRT-PCR. (E) Northern blot detection of HBL1 RNA level. (F) Representative FACS results of CM ratios after differentiation of WT and HBL1 null iPSCs. (G) Statistical analysis of percentage of CTNT+ CMs from (F). (H and I) Representative FACS results of KDR and PDGFRα staining of day-6 dissociated EBs (H), with statistical analysis shown in (I). All error bars denote mean ± SD. n = 3. ∗p < 0.05. All data comparisons use a one-way ANOVA (multiple groups). See also Figures S3 and S4. Developmental Cell 2017 42, 333-348.e5DOI: (10.1016/j.devcel.2017.07.023) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 HBL1 Interacts Directly with MIR1 and Regulates Cardiomyocyte Differentiation (A) Locations and sequences of two putative hsa-miR-1(MIR1) binding sites in HBL1. Mutations in the binding sites disrupted MIR1 binding capability. (B) Detection of luciferase activities in 293T cells after transfection with pmiR-GLO vectors containing WT HBL1 and mutated HBL1 sequences. GJA1 was used as a positive control for the MIR1 binding assessment. (C) Disruption of two MIR1 binding sites in HBL1 one by one using CRISPR/Cas9 technology. (D) Detection of MIR1 binding site (BS) genomic editing by SURVEYOR assay. (E) HBL1 expression in S3 iPSCs with MIR1 binding site disruption. (F) Detecting ratios of CTNT+ CMs differentiated from S3 iPSCs by flow cytometry. (G) Statistical analysis of percentage of CTNT+ CMs from (F). All error bars denote mean ± SD. n = 3. ∗p < 0.05; N.S, not significant. All data comparisons use a one-way ANOVA (multiple groups) or Student's t test (two groups). See also Figure S5. Developmental Cell 2017 42, 333-348.e5DOI: (10.1016/j.devcel.2017.07.023) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 HBL1 and MIR1 Form an RNA-Induced Silencing Complex (A) Scheme of pull-down assays to verify physical interactions between HBL1 and MIR1. (B) RNA immunoprecipitation in hiPSCs using an anti-AGO2 antibody. An anti-IgG antibody was used as control. (C) RNA pull-down in hiPSCs using 5′ biotin-labeled HBL1 DNA probes. qRT-PCR was used to detect enrichments of HBL1 and MIR1 by these probes. (D) RNA pull-down in hiPSCs using 5′ biotin-labeled MIR1 DNA probes. qRT-PCR was used to detect enrichment of HBL1 by this probe. (E) Examination of HBL1 and MIR1 expressions in hiPSCs with overexpression of MIR1. (F and G) Detecting ratios of CTNT+ CMs by flow cytometry. (H) qRT-PCR detection of expression of cardiac markers in hiPSC derived-CMs. All error bars denote mean ± SD. n = 3. ∗p < 0.05. All data comparisons use a one-way ANOVA (multiple groups) or Student's t test (two groups). See also Figure S6. Developmental Cell 2017 42, 333-348.e5DOI: (10.1016/j.devcel.2017.07.023) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 SOX2 Activates HBL1 Transcription by Binding to Its Promoter (A) A putative SOX2 binding site on HBL1 promoter. (B) qRT-PCR examinations of SOX2 and HBL1 expressions during cardiomyocyte differentiation from hiPSCs using the EB method. (C) Luciferase activities of WT HBL1 promoter in 293T cells when co-transfected with SOX2 overexpression vector or SOX2-shRNA vector. (D) ChIP-PCR detection of SOX2 binding on WT HBL1 promoter in hiPSCs. A set of specific primers flanking putative SOX2 binding site were used to detect whether SOX2 binds to the putative binding site. IgG antibody is used as a negative control. Anti-polymerase II antibody is used as a positive control. (E) Quantitative results of SOX2 binding to HBL1 promoter by ChIP-qPCR. (F) Luciferase activities of WT and mutated promoters in 293T cells when co-transfected with SOX2 overexpression vector. (G) SOX2 protein expression level detected by Western blotting. (H and I) qRT-PCR examination of HBL1 (H) and MIR1 (I) expression in hiPSCs after SOX2 was knocked down. (J and K) Flow-cytometry analysis of CTNT+ cell ratios after CM differentiation from hiPSCs with SOX2 knockdown. (L) qRT-PCR detection of cardiac marker genes in control hiPSCs versus SOX2 knockdown hiPSCs after CM differentiation. All error bars denote mean ± SD. n = 3. ∗p < 0.05. All data comparisons between groups were analyzed using Student's t test (two groups). See also Figure S7. Developmental Cell 2017 42, 333-348.e5DOI: (10.1016/j.devcel.2017.07.023) Copyright © 2017 Elsevier Inc. Terms and Conditions