The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming

Abstract

Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency, little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs, human pluripotency-associated transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2, HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells, followed by whole-transcriptome analysis, identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate.

Figure 1

Molecular single-cell gene expression and functional analyses of HPAT2, HPAT3 and HPAT5 during human embryo development. (a) Overview of single-cell gene expression analysis for human blastocysts. A total of 24 blastocysts were pooled and run on four C1 chips. (b–d) Single-cell gene expression analyses. RT, reverse transcribe. (b) Three HPATs had significantly higher expressed in ICM than in trophectoderm (TROPH). (c,d) ICM (c) and trophectoderm (d) markers are also shown. Box plots are shown for each group. The whiskers are the minimum and maximum data points. The bottom and top of each box are the first and third quartiles, respectively. n = 46 single trophoectoderm cells and n = 67 single ICM cells. (e) Immunohistochemistry and RNA FISH for OCT4 (green) and lincRNAs (red), respectively, in human blastocysts. Sections are counterstained with DAPI (blue). ICMs are circled by dotted white lines. Entire human or mouse blastocysts are circled by dotted yellow lines in the merged images. lincRNA signal was specific to the ICM. Speckles (red) are nonspecific and are observed in all human blastocysts. Mouse blastocysts initiated hatching when fixed. Images are representative (n = 9 human blastocysts for HPAT3, n = 11 human blastocysts for HPAT5 and n = 3 mouse blastocysts; n = 2 independent experiments). Scale bar, 100 μm. (f) Blastomeres with reduced expression of HPAT2, HPAT3 and HPAT5 during human embryo development did not contribute to ICM. The presence of ICM was validated with OCT4 and SOX2 staining. ICMs are circled by yellow dashed lines (n = 3 blastocysts). RRX, Rhodamine Red-X; siHPAT2/3/5, combination of siRNAs targeting HPAT2, HPAT3 and HPAT5. Scale bar, 100 μm.

Figure 2

Single-cell expression analysis of HPAT transcripts during nuclear reprogramming. (a) Dynamics in single-cell expression of HPAT transcripts during nuclear reprogramming shown as box plots. HPATs are grouped according to activation pattern (with two examples representing each group). (b) Bicluster analysis identifies five biclusters, P1–P5, and correlates HPAT2, HPAT3 and HPAT5 (gene names in orange) with core pluripotency markers (method by Lazzeroni and Owen). (c) Correlation analysis across single cells identifies positively and negatively correlated gene pairs (see supplementary table 3 for additional details). (d) Bayesian network analysis predicts a central role for HPAT2, HPAT3 and HPAT5 (orange circles) within the core regulatory network (yellow circles). The hierarchical view predicts that SALL4 (red circle) triggers a cascade of key pluripotency gene activation. Arrow thickness and circle size increase with the confidence level of interactions in the calculated network. Only pluripotency genes and lincRNAs were included. Data in a–d represent n = 578 single cells.

Figure 3

Functional analyses of HPAT2, HPAT3 and HPAT5 during nuclear reprogramming. (a) Experimental scheme of functional analysis of HPAT2, HPAT3 and HPAT5 (HPAT2/3/5) during iPSC reprogramming. AP, alkaline phosphatase. (b) Immunostaining of TRA-1-60 during reprogramming with HPAT2, HPAT3 and HPAT5 in combination. Representative images are shown (n = 8). KD, knockdown. Scale bar, 100 μm. (c) Calculated percentage of TRA-1-60–positive cells at different points during reprogramming (n = 8). Data are represented as means + s.e.m. (d) Representative images of colony size appearance at day 12 during reprogramming (n = 8). Scale bar, 100 μm. (e) Alkaline phosphatase staining at day 12. Shown are the wells of a six-well plate from one experiment (n = 2 independent experiments). (f) iPSC colony number counted on the basis of alkaline phosphatase staining (n = 3). Data are represented as means + s.e.m. (g) Cell number relative to control cells during reprogramming (n = 3). Data are represented as means + s.e.m. NS, not significant. (h) Alkaline phosphatase staining at day 12 during reprogramming (n = 2 for each condition). (i) Percentage of TRA-1-60–positive cells at day 12 of single knockdown of HPAT2, HPAT3 or HPAT5. siGlo was used as a control. Data are represented as means + s.e.m. (j) Reprogramming with POU5F1 and HPAT2, HPAT3 and HPAT5. POU5F1 only is used as a control. (k) Epigenetic and gene expression analysis of HPAT2, HPAT3 and HPAT5. NANOG mRNA was transfected into BJ fibroblasts treated or not with 5-Aza-2′-deoxycytidine (5-Aza) with gene expression measurement at 48 h (n = 6). Data are represented as means + s.e.m.

Figure 4

HPAT5 binds directly to let-7. (a–c) HPAT5-OE hESCs suppress differentiation mediated by siRNA to POU5F1 and bFGF removal. (a) Outline of the protocol. (b,c) The expression of key pluripotency markers decreases with delay in comparison to the mCherry-OE control line. P values are calculated for the comparison of the mCherry-OE and HPAT5-OE lines on the same days (c), with this evidence supported by morphological changes (after day 3) (b) (n = 2 independent experiments). Scale bar, 50 μm. (d) Two predicted let-7 binding positions in HPAT5 identified by RegRNA 2.0 (miRanda). (e,f) Target validation using luciferase reporters in HEK293 cells. The relative luciferase activity (shown as fold change relative to empty vector) was assayed 48 h after cotransfection of cells with the indicated miRNAs or control (scrambled miRNA) together with wild-type reporter (e) or the let-7 miRNAs together with wild-type or mutant reporter (f). (g) The point mutations (in red) introduced into two mutant let-7 mimics and two mutant HPAT5 reporters for compensatory analysis. WT, wild type. (h) Analysis of the effects of the point mutations using luciferase reporters in HEK293 cells. Relative luciferase activity (shown as fold change relative to empty vector) of wild-type or mutant reporters was assayed 48 h after cotransfection of cells with the indicated miRNAs or scrambled miRNA. Representative results from n = 2 (c,e,f,h) independent experiments; n = 3 samples; data are shown with s.e.m.

Figure 5

HPAT5 regulates let-7 in hESCs during differentiation. (a) Morphological changes 48 h after let-7 overexpression in HPAT5-WT and HPAT5-KO lines. (b) Table listing findings from enrichment analysis with cWords (supplementary table 8). NA, not applicable. (c) HPAT5 regulates let-7 activity. Expression levels of mature let-7a and let-7d in undifferentiated hESCs (H1) transiently transfected for 48 h with wild-type (WT) or mutant HPAT5 or with knockdown with siHPAT5. Empty vector or scrambled miRNA was used as a negative control. RNA levels (ln) and P values are shown relative to negative controls. n = 3 samples; data are shown with s.e.m. (d) RIP-qPCR showing interaction between AGO2 and HPAT5 but not GAPDH in hESCs transfected with let-7. n = 3 samples; data are shown with s.e.m.