Research ArticleGENETICS

Primate-restricted KRAB zinc finger proteins and target retrotransposons control gene expression in human neurons

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Science Advances  28 Aug 2020:
Vol. 6, no. 35, eaba3200
DOI: 10.1126/sciadv.aba3200
  • Fig. 1 Genomic characterization of ZNF417/587.

    (A) Expression from a PGK-GFP cassette cloned downstream of KAP1-restricted (R) or nonrestricted (NR) HERVK PBS sequences in control (Ctrl) or ZNF417/587 KD hESC (average and SD values of duplicates). unT, untransduced. (B) LoF mutations for ZNF417/587 with numbers of most frequent alleles among >140,000 individuals (in red homozygous LoF mutations). Dark and light purple boxes indicate intact and degenerated ZFs. (C) ZNF417/587 expression in brain development and substructures according to BrainSpan Atlas of the Developing Human Brain. pcw, post-conception weeks; FTS, forebrain fetal transient structures; D, diencephalon; My, myelencephalon; Me, metencephalon; M1C_S1C, primary motor sensory cortex; PCx, parietal cortex; TCx, temporal neocortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; URL, upper rhombic lip; CB, cerebellum; CBC, cerebellar cortex. (D) Percentage of integrants from indicated TE families and subfamilies overlapping with ZNF417 and ZNF587 peaks as obtained by ChIP-seq triplicates in hESC. Only families with at least five bound integrants are shown. (***P < 0.001 and **P < 0.01, hypergeometric test). (E) Top: PBS consensus sequences of bound LTR/ERVs and SVAs compared with PBSLys1.2 and R- and NR-HERVK14C sequences. Bottom: Predicted binding motifs according to Rsat.

  • Fig. 2 Impact of ZNF417/587 depletion in pluripotent stem cells.

    (A) Dot plots of RNA-seq from ZNF417/587 KD quadruplicates versus control hESC triplicates, outlining DE TEs and genes (fold change > 2, FDR < 0.05). TEs with predicted ZNF417 (yellow), ZNF587 (green), or both (pink) binding motifs (left) or bound by KAP1 in hESC (middle) or genes with a TSS closer than 100 kb from a ZNF binding site (right) are highlighted. (B) Bar plots depicting loss of H3K9me3 or gain in H3K4me1 or H3K27ac at indicated loci as obtained by independent ChIP-seq duplicates in hESC. Top: ZNF417/587-bound versus ZNF417/587-unbound TEs; middle: TSS of coding genes close to <20 kb versus distant from a KZFP peak; bottom: TSS of coding DE genes versus all genes (P values, hypergeometric test). Right: Fold change (Fc) in expression in KD versus wild-type (WT) hESC of loci illustrated on the left (P values, Wilcoxon test). ns, not significant.

  • Fig. 3 ZNF417/587-mediated repression of TEeRS and neuron-specific genes.

    (A) Expression at indicated stages of human development using single-cell (left) or in naïve versus primed hESC (right) RNA-seq data (P values, Wilcoxon test) of TEs found or not found to be bound by ZNF417/587 in ESC. (B) IGV (Integrative Genomics Viewer) screenshots of independent RNA-seq replicates from control (Ctrl) and KZFP KD hESC with boxed ZNF417/587 peaks at HERVK integrants upstream of AADAT (top) and PRODH (bottom). EREs, Endogenous Retroelement. (C) Spatial representation of ZNF417, HERVK, and PRODH expression in early prenatal and childhood brains, using RNA-seqs from the Brain Span Atlas of the Developing Human Brain. Prenatal brain is depicted as anatomically adult for consistency. (D) Repression assay in control or KD hESC using LVs carrying upstream of a GFP the genomic region encompassing the PRODH promoter and either the WT HERVK LTR5hs and PBS sequences (HERVK/PRODH), the sequence mutated in the two SOX2 binding sites (ΔSOX2 HERVK/PRODH), or the PBS sequence mutated into an R PBS binding site (HERVK.R/PRODH). The previously described PGK-GFP LV with the strong KZFP recruiting 39-bp R.PBS sequence was used in parallel as a positive control of silencing (PBS.R/PGK). Data have been collected from independent duplicates of KD and control cells, at day 6 after transduction with equal amounts of LVs. Average and SD of the duplicates are shown. The WT PBS sequence as found on the WT HERVK upstream of the PRODH promoter (PBSPRODH) and the mutated one (PBSR) is shown.

  • Fig. 4 Impact of ZNF417/587 on neuronal differentiation, function, and homeostasis.

    (A) Number of DE elements in indicated cells. (B) Expression in KD (left) or HERVK-silenced (right) versus control iN of genes classified with Allen Brain Atlas Gene Ontologies (GOs) compared to random genes (P values, Wilcoxon test). (C) Examples of DE genes related to GABAergic pathway. (D) HERVK with (top) RNA-seqs of control versus KD and (middle) control versus HERVK-CRISPRi iN, and (bottom) KZFPs ChIP-seqs in hESC. (E) Western blot of NCCIT cell lysates probed with anti-ENV or anti-actin antibodies. (F) Expression of ISG (with fold change > 5 in IFN-treated normal tissues or cells according to the Interferome database) in KD versus control iN. In red, DE ISG (fold change > 2, FDR < 0.05). Venn diagram indicates number of DE genes stimulated by each IFN type. (G) Fold change expression of antiviral ISG in indicated conditions versus control iN (ND, not detected). (H) Forty-three–day organoids, with size quantification underneath (P values, Mann Whitney U test). (I) PAX6 immunostaining and RT-qPCR quantification (P values, two-tailed t test), and (J) fold change expression of neuronal function–related genes and (K) ISG in 20-day organoids. Independent duplicates (A to G) or triplicates (D, J, and K) were used. Error bars represent the SD (***FDR < 0.001, **FDR < 0.01, and *FDR < 0.05).

Supplementary Materials

  • Supplementary Materials

    Primate-restricted KRAB zinc finger proteins and target retrotransposons control gene expression in human neurons

    Priscilla Turelli, Christopher Playfoot, Dephine Grun, Charlène Raclot, Julien Pontis, Alexandre Coudray, Christian Thorball, Julien Duc, Eugenia V. Pankevich, Bart Deplancke, Volker Busskamp, Didier Trono

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