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. 2020 Mar 15;459(2):161-180.
doi: 10.1016/j.ydbio.2019.12.003. Epub 2019 Dec 17.

Combinatorial action of NF-Y and TALE at embryonic enhancers defines distinct gene expression programs during zygotic genome activation in zebrafish

William Stanney 3rd  1 , Franck Ladam  1 , Ian J Donaldson  2 , Teagan J Parsons  3 , René Maehr  3 , Nicoletta Bobola  2 , Charles G Sagerström  4
Affiliations

Combinatorial action of NF-Y and TALE at embryonic enhancers defines distinct gene expression programs during zygotic genome activation in zebrafish

William Stanney 3rd et al. Dev Biol. .

Abstract

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF-Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF-Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF-Y or TALE defines genes in specific developmental processes, such that NF-Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF-Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF-Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA.

Keywords: Embryogenesis; Enhancer; Maternal; Nucleosome; Transcription; Zygotic.

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Figures

Figure 1:
Figure 1:. Disruption of NF-Y or TALE function affects anterior embryonic development.
Zebrafish embryos were left uninjected (A, E, F, N, O) or injected with either control mRNA (GFP; B, G, H, P, Q), mRNA encoding a TALE dominant negative construct (PBCAB; C, I, J, R, S) or mRNA encoding an NF-Y dominant negative construct (NF-YA DN; D, K, L, T-W) at the 1–2 cell stage and raised to 24hpf (N-W), 28hpf (A-D) or 5dpf (E-L). Embryos were either left untreated (A-D), stained with alcian blue (E-L) or processed for detection of pax2 (at the mid/hindbrain boundary), krox20 (in rhombomeres 3 and 5) and hoxd4 (in the spinal cord) transcripts by in situ hybridization (N-W). White arrows highlight differences in eye morphology (A-L), black arrows highlight differences in head cartilage formation (E-L) and orange arrows indicate differences in rhombomere 3 krox20 expression (N-U). Tables summarize effects of TALE or NF-Y disruption on head cartilage formation (M), eye formation (X) and gene expression (Y). Panels V and W show representative images of embryos scored as having gross abnormalities in panel X. Embryos are shown in lateral (A-D, F, H, J, L, N, P, R, T, V) or dorsal (E, G, I, K, O, Q, S, U, W) views.
Figure 2:
Figure 2:. NF-Y and TALE TFs have both shared and independent transcriptional targets.
(A) Schematic of RNA-seq experiments. (B-C) Scatterplots of gene expression in PBCAB vs GFP-injected (B) and NF-YA DN vs GFP-injected (C) zebrafish embryos (expression presented as log2 of average TPM for multiple replicates; see methods). Expression of genes highlighted in orange is significantly different at 12hpf (padj≤0.01; Wald test in DESeq2). (D) Number of genes differentially expressed in PBCAB (left) or NF-YA DN (right) relative to GFP-injected embryos (p-adj ≤ 0.01; fold-change ≥ 1.5). (E) Breakdown of downregulated (left) and upregulated (right) genes exclusive or common to each experimental condition. (F, H, J) DAVID analyses showing the 25 most significant GO terms (EASE Score) associated with genes downregulated by PBCAB (F), NF-YA DN (H), and common to both (J). Blue bars correspond to transcription-related, green to embryogenesis-related, orange to homeodomain-related, yellow to cilia-related, and gray to other ontologies. (G, I, K) Selected genes downregulated by PBCAB (G), NF-YA DN (I), or both (K). Color coding is the same as in (F, H, J).
Figure 3:
Figure 3:. Identification of NF-Y and/or TALE-dependent genes in zebrafish.
(A) Read counts for the RNA-seq analysis. (B, C) Histograms, scatter plots, and Spearman’s rank correlation coefficient comparing each biological replicate of NF-YA DN with GFP (B) or PBCAB with GFP (C). (D) Venn diagram showing upregulated genes (p-adj ≤ 0.01; FC ≥ 1.5) in embryos injected with PBCAB or NF-YA DN. (E-I) GO terms associated with genes upregulated (p-adj ≤ 0.01, FC ≥ 1.5) by PBCAB (E), upregulated by NF-YA DN (F), upregulated by both PBCAB and NF-YA DN (G), downregulated exclusively by PBCAB (H) or downregulated exclusively by NF-YA DN (I). In E-I, blue bars correspond to transcription-related, green to embryogenesis-related, orange to homeodomain-related, yellow to cilia-related, and gray bars to other ontologies.
Figure 4:
Figure 4:. NF-Y and TALE occupy genomic elements associated with developmental and transcriptional control genes at ZGA
(A-B) Representative UCSC Genome Browser tracks for NF-YA, Pbx4 and Prep1 ChIP-seq analyses at 3.5hpf. (C, F, I, L) Venn diagrams showing the overlap (at least 1bp shared between 200bp fragments centered on peaks) of two Pbx4 ChIP-seq replicates (C), the overlap of Pbx4 and Prep1 ChIP-seq peaks (F), the overlap of two NF-YA ChIP-seq replicates (I) and the overlap of TALE and NF-Y ChIP-seq peaks (L). (D, G, J, M) The top sequence motif returned by MEME for Pbx4-occupied sites (D), Pbx4/Prep1 co-occupied sites (G), NF-YA occupied sites (J) and TALE/NF-YA co-occupied sites (M). (E, H, K, N) The top 25 gene ontology (GO) terms returned by the GREAT analysis tool for genes associated with Pbx4-occupied sites (E), Pbx4/Prep1 co-occupied sites (H), NF-YA occupied sites (K) and TALE/NF-YA co-occupied sites (N). (O) Chart showing percent of ChIP-seq peaks found within 5kB or 30kB of a promoter. (P, Q) Top sequence motif returned by MEME for peaks bound by TALE, but not NF-YA (P) and peaks bound by NF-YA, but not TALE (Q). Only peaks with a 10-fold or greater enrichment over input (FE≥10) were considered for the analyses in C-Q.
Figure 5:
Figure 5:. Identification of genomic binding sites for NF-Y and Pbx4 in 3.5hpf zebrafish.
(A) Table showing data for Pbx4 and NF-YA ChIP-seq biological replicates with our previous Prep1 ChIP-seq data REF included as reference. (B) Table showing number of peaks that overlap (at least 1bp shared between 200bp fragments centered on peaks) between Prep1, Pbx4 and NF-YA ChIP-seq data sets. Only peaks with a 10-fold or greater enrichment over input were considered. (C) Table showing extent of overlap of Pbx4 peaks with Prep1 peaks and TALE peaks with NF-YA peaks at three different cutoffs (FE≥4, FE≥10 and top 10% of peaks).
Figure 6:
Figure 6:. Combinatorial function of NF-Y and TALE defines distinct gene expression programs
(A) Table showing correlation between NF-Y and/or TALE-dependent genes and binding by the corresponding TF at a nearby site (ChIP peaks enriched by 4-fold or greater over input were considered). (B) Graphical breakdown of NF-Y and/or TALE occupancy near NF-Y and/or TALE-dependent genes. (C-F) Top 25 GO terms returned by DAVID for TALE-dependent genes associated with TALE peaks (C), NF-Y dependent genes associated with NF-YA peaks (D), TALE/NF-Y dependent genes associated with both NF-YA and TALE peaks (E), and TALE/NF-Y dependent genes associated with overlapping TALE and NF-YA peaks (F). Blue bars correspond to transcription-related, green to embryogenesis-related, orange to homeodomain-related, yellow to cilia-related, and gray bars to other ontologies.
Figure 7:
Figure 7:. Genomic elements occupied by NF-Y and TALE act as enhancers in vivo.
(A-F) Average histone mark signals at genomic regions containing only TALE peaks (dark blue), only NF-YA peaks (light blue), or NF-YA/TALE peaks (yellow) for H3K27ac at 4.3hpf (A) and 9hpf (B), H3K4me1 at 4.3hpf (C) and 9hpf (D), H3K4me3 at 4.3hpf (E) and 9hpf (F). (G, I, K, M, O) UCSC Genome Browser tracks showing NF-YA, Pbx4, and Prep1 ChIP-seq data for the tcf3a (G), tle3a (I), dachb (K), fgf8a (M) and yap1 (O) loci. The diagrams above the tracks show the putative enhancer region in green, DECA motifs in orange and CCAAT boxes in blue. (H, J, L, N, P) GFP expression in 24hpf F1 tcf3a:E1b-GFP (H), tle3a:E1b-GFP (J), dachb:E1b-GFP (L), fgf8a:E1b-GFP (N) and yap1:E1b-GFP (P) transgenic embryos resulting from crosses between male founders and wild type females.
Figure 8:
Figure 8:. Characterization of NF-Y/TALE-regulated enhancers in zebrafish.
(A-C) GFP expression in F1 embryos from yap1:E1b-GFP founder #11 (A), founder #5 (B) and representative image of a 24hpf GFP-negative embryo (C). (D-F) UCSC Genome Browser tracks showing NF-YA, Pbx4, and Prep1 ChIP-seq data for the pax5 (D), her6 (E) and prdm14 (F) loci. The diagrams above the tracks show the putative enhancer region in green, DECA motifs in orange and CCAAT boxes in blue. (G) Table summarizing information about each enhancer element. Note that embryos that inherited the transgene from a female founder were GFP positive already at fertilization, indicating that these enhancer elements are active in the female germline. For this reason, all images in figures 7 and 8 are of embryos that inherited the transgene from a male founder.
Figure 9:
Figure 9:. Disruption of TALE and NF-Y function reduces enhancer activity.
(A) Schematic showing workflow for dominant negative disruption of tcf3a:E1b-GFP. (B-D) Representative images showing no GFP (B), weak GFP (C), and strong GFP (D) of dominant negative-injected embryos. (E) Distribution of GFP expression in uninjected embryos and embryos injected with PBCAB, NF-YA DN or control RNA. (F) RT-qPCR-based detection of GFP expression in embryos injected with PBCAB, NF-YA DN or control RNA. Data are shown as mean +/− SEM. Statistical test: unpaired t-test. (G-N) Representative examples of RFP (G, K, I, M) and GFP (H, L, J, N) signal in tcf3a-WT:sv40 (G, H), tcf3a-mut:sv40 (I, J), tle3a-WT:sv40 (K, L) and tle3a-mut:sv40 (M, N) embryos at 32hpf. Insets in panels L, J, N show higher magnification of GFP expression in lens. Note that embryo in panels G, H is at a later stage than embryos in panels I-N. (O) Table quantifying results from experiment in panels G-N.
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