Figure 1. Novel pioneer factor CLAMP is essential for early embryonic development.

(A) Control maternal egfp depletion (left), maternal clamp depletion (middle), and maternal zld depletion (right) syncytial blastoderm stage embryos probed using smFISH for the pair-rule patterning genes run (green) and eve (red). Embryos were co-labeled with Hoechst (blue) to visualize nuclei. Scale bar represents 10 µm. Quantification (%) of eve/run defective embryos is on the right, p-values were calculated with the Fisher’s exact test; number of embryos is in parentheses. (B) Control maternal egfp depletion (left), maternal clamp depletion (middle), and maternal zld depletion (right) syncytial blastoderm stage embryos were assessed for integrity of the developing cytoskeleton using anti-NRT antibody (green) and Hoechst (blue) to label nuclei. Scale bar represents 10 µm. Quantification (%) of NRT defective embryos is on the right, p-values were calculated with the Fisher’s exact test; number of embryos is in parentheses. (C) Electrophoretic mobility shift assay (EMSA) showing the binding of increasing amounts of CLAMP DNA-binding domain (DBD) fused to MBP to 5C2 naked DNA or 5C2 in vitro reconstituted nucleosomes (Nucs). Concentrations (µM) of CLAMP DBD increase from left to right. (D) EMSA showing the binding of increasing amounts of full-length (FL) CLAMP (fused to MBP) to 5C2 DNA or 5C2 Nucs. Concentrations (µM) of CLAMP FL increase from left to right. (E) Effect of maternal clamp RNAi on maternally deposited (orange) or zygotically transcribed (yellow) gene expression log2 (clamp-i/MTD) in 0–2 hr (left) or 2–4 hr (right). Maternal versus zygotic gene categories were as defined in Lott et al., 2011. p-values of significant expression changes between maternal and zygotic genes were calculated by Mann-Whitney U-test and noted on the plot. CLAMP, chromatin-linked adaptor for male-specific lethal (MSL) proteins; smFISH, single-molecule fluorescence in situ hybridization.

Figure 1—figure supplement 1. Novel pioneer factor CLAMP is essential for early embryonic development.

(A) Expression of early zygotic genes (even-skipped, runt, and neurotactin) in MTD and clamp-i embryos measured by RNA-seq (Rieder et al., 2017). Log₂fold change and p-value were calculated using DESeq2. (B) Expression of mRNAs (clamp and zld) in MTD, clamp-i, and zld-i embryos in 0–2 hr and 2–4 hr embryos measured by RT-qPCR. Log₂fold change was calculated using the ΔΔCt method (Rao et al., 2013) and normalized to reference gene pka. (C) Western blot of CLAMP, ZLD, and reference control Actin in MTD, clamp-i, and zld-i embryos in 0–2 hr and 2–4 hr embryos. MTD: MTD-Gal4 line. clamp-i: MTD-Gal4-clamp mRNAi line, zld-i: MTD-Gal4-zld mRNAi line. (D) Genome browser tracks for a region of the CES 5C2 locus (red bar is 500 bp) used to make in vitro reconstituted nucleosomes (Urban et al., 2017a). Locations of three CLAMP binding motifs are marked as black dots. CLAMP chromatin immunoprecipitation-sequencing (ChIP-seq) normalized sequencing reads are shown in green. MNase-seq MACC scores (dark blue) show chromatin accessibility in S2 cells in control (egfp-i), clamp, and msl2 RNAi treatment. The nucleosome (Nuc) profile is shown in purple. The dashed red rectangle highlights the genomic region (240 bp) used to reconstitute nucleosomes in vitro. (E) Effect of maternal zld RNAi (Schulz et al., 2015) on maternally deposited (orange) or zygotically transcribed (yellow) gene expression log₂ (clamp-i/MTD) in 0–2 hr (left) or 2–4 hr (right) measured by RNA-seq. Maternal versus zygotic gene categories were as defined in Lott et al., 2011. p-values of significant expression changes between maternal and zygotic genes were calculated by Mann-Whitney U-test and noted on the plot. CLAMP, chromatin-linked adaptor for male-specific lethal (MSL) proteins; MBP, maltose-binding protein.