Figure 1.
sspt-16 is a novel ncRNA: (A) Models of FACT subunits in H. sapiens (top) and C. elegans (middle). Conserved protein domains according to Pfam (pfam.xfam.org) and InterPro (ebi.ac.uk/interpro) are indicated. The pseudogene
sspt-16 and its mother gene
spt-16 are less than 2 kb apart from each other (bottom). The
ok1829 deletion allele of
sspt-16 is indicated in red. RNA image adapted from https://commons.wikimedia.org (CC BY-SA 3.0, free to use and change). (B) After broad induction of the ASE neuron specifying TF CHE-1 in L4 animals, no ectopic expression of the ASE neuron fate reporter
gcy-5p::GFP can be detected 24 h later (top and middle left). However, in the
sspt-16(
ok1829) deletion background, ectopic
gcy-5p::GFP expression can be detected in the germline (top and middle right). Some reprogrammed germ cells show axo-dendridic like projections (bottom right, indicated by white arrows) and some also show the pan-neuronal marker
rab-3p::NLS::tagRFP (bottom left and middle, indicated by orange arrows). Scale bars = 20 µm. (C) We could verify
sspt-16 RNA transcripts through RT-PCR (top; blue primers result in 143 bp for
spt-16, while green primers result in 764 bp for
spt-16 and 289 bp for
sspt-16). All primers were designed at intron/exon borders, so that the expected sizes correspond to spliced mRNA or ncRNA, but not DNA. Additionally, we could detect
sspt-16 ncRNA transcripts through long-read (Nanopore) and short-read (Illumina) RNA-seq (bottom). (D) Using CRISPR/Cas9 gene-editing, a 3xFLAG-tag was introduced at the C-terminal end of endogenous
sspt-16 / F55A3.7 (top). SSPT-16::3xFLAG worm lysates did not show any immunofluorescent FLAG staining, while the LMN-1::2xFLAG positive control did (bottom left). N2 (negative control), HIS-71::2xFLAG (positive control) and SSPT-16::3xFLAG worm lysates were used in a western blot. A positive band for SSPT-16::3xFLAG would have been expected at 24.3 kDa, if a tagged protein was generated from the edited
sspt-16 locus. (E) We checked the
sspt-16(
ok1829) deletion background for gene expression changes of the germline FACT members
spt-16 and
hmg-3, as well as of the histone chaperone and reprogramming barrier
lin-53, by using qPCR (left) and western blots (right), but could not detect any significant difference neither on mRNA nor on protein level for the tested genes. Error bars represent SD, n.s.: not significant according to a one-way ANOVA. (F) Using the CRISPR/Cas system, we generated an additional
sspt-16 deficient strain (
bar37) by knocking in an aptamer-ribozyme sequence that depletes transcribed
sspt-16 ncRNA molecules and confirmed its functionality by qPCR using two different primer pairs which target
sspt-16 ncRNA. ***: p<0.001 according to a one-way ANOVA. (G) The phenotype penetrance (ectopic GFP expression in the germline after broad CHE-1 induction) was assessed for WT control, the Δ
sspt-16(
ok1829) deletion strain and the Δ
sspt-16(apta) aptamer-ribozyme strain. n = 147-252; 4 biological repeats. ***: p<0.001 according to a one-way ANOVA. Error bars represent SEM. (H) Germline specific ATAC-seq of N2 and Δ
sspt-16(
ok1829) strains revealed that there are basically no significant differences in chromatin accessibilities. Two loci that were identified as different (turquoise) were the
ok1829 deletion itself, as well as another undocumented deletion that is present in the original
ok1829 strain, so represent deletions on DNA level rather than differentially accessible chromatin. (I) Genomic location of
sspt-16 and
spt-16 loci showing active and repressive chromatin marks. A screenshot of JBrowse implemented in wormbase.org (WS280) is shown with publicly available tracks for repressive H3 modifications: H3K27me3 (emb/L3/adult), H3K9me2/3 (emb/L3/adult); active: H3K36me3 (emb/L3), H3K9ac (L3), H3K77ac (adult). The loci are located at the right arm of chromosome I. The red line indicates the
sspt-16 /
spt-16 location, which appears to lie within a putative chromosomal compartment border region.