PIP5Pase activity is essential for VSG gene silencing and switching.

A) Diagram of bloodstream-form ESs (BES, top) and metacyclic-form ESs (MES, bottom). B) RNA-seq analysis of T. brucei bloodstream forms comparing 24h exclusive expression of Mut to WT PIP5Pase. FC, fold-change. C-D) RNA-seq read coverage and FC (Mut vs WT) of silent BES7 (C, top), the active BES1 (C, middle), a silent MESs (C, bottom), and chromosome 4 subtelomere (D). Heat-map in D shows RNA-seq bins per million (BPM) reads. Gray rectangles represent genes. A 99.9% reads mapping probability to the genome (mapQ>30) retained alignments to subtelomeric regions. E) VSG-seq analysis of T. brucei bloodstream forms after temporary (24h) exclusive expression of Mut PIP5Pase, and re-expression of WT PIP5Pase (60h). B1-B3, biological replicates. The color shows normalized read counts per million. A 3’-end conserved VSG sequence was used to capture VSG mRNAs (41). F) VSG-seq analysis of isolated clones after PIP5Pase temporary knockdown (tet -, 24h) followed by its re-expression (Tet +) and cloning for 5-7 days. Clones of non-knockdown (tet +) cells were analyzed as controls. G) Western blot of V5-tagged PIP5Pase knockdown in T. brucei procyclic forms. The membrane was stripped and reprobed with anti-mitochondrial heat shock protein 70 (MtHSP70). H) Expression analysis of ES VSG genes after knockdown of PIP5Pase in procyclic forms. Data are the result of three biological replicates.

ChIP-seq analysis of RAP1-HA in T. brucei.

A) RAP1-HA binding sites on the silent BES17. Below, sequence bias of bound regions. B) RAP1-HA binding to selected regions in silent BES sequences. Each dot represents the mean of a silent BES. C) RAP1-HA enrichment to the active BES1. Reads were filtered for over 99% mapping probability (primary reads only, mapQ>20). D) Comparison of RAP1-HA binding to 70 bp in silent and active ESs. E) RAP1-HA enrichment over all MESs. F) RAP1-HA binding to subtelomere 3B of chromosome (Chr) 9 (left) or chromosome 1 core (right). Yellow fluorescent protein (YFP)-tagged KKT2 protein ChIP-seq from (27) is shown. RAP1-HA ChIP-seq in cells expressing Mut PIP5Pase is shown below. p-values (p) were calculated using Model-based Analysis of ChIP-Seq (MACS) from three biological replicates. All data show ChIP vs Input analysis. See Table S1 and Data S2 for detailed statistics. ****, p-value < 0.0001.

Binding kinetics of rRAP1 to telomeric repeats, 70 bp repeats, and phosphoinositides.

Data show the mean ± standard deviation of the mean (SDM).

rRAP1 binds to telomeric and 70 bp repeats via its Myb and MybL domain.

A) Top, RAP1 diagram shows VHP, BRCT, Myb and MybL domains. Numbers indicate residue positions; asterisk, nuclear targeting sequence. Bottom, telomeric (10 repeats), 70 bp (one repeat), or scrambled (generated from telomeric repeats) sequences used in binding assays. B) His-tagged rRAP1, rRAP11-300, rRAP301-560, and rRAP1561-855 resolved in 10% SDS/PAGE and Coomassie-stained. C) EMSA of His-tagged rRAP1 with biotinylated telomeric repeats (Tel rep), 70 repeats (70 bp rep), or scrambled sequences resolved in 6% native/PAGE and developed with Streptavidin-HRP. D) MST binding kinetics of rRAP1 with Cy5-labelled telomeric repeats or 70 bp repeats. Data shown are the mean ± SDM of four biological replicates. E-G) EMSA of rRAP1301-560 (E), rRAP1561-855 (F), or rRAP11-300 (G) with telomeric, 70 bp repeats, or scrambled sequences resolved in 6% native/PAGE. rRAP1301-560 were used as a positive control in G.

rRAP1 binds to PI(3,4,5)P3 through its N-terminus.

A) Modeling and alignment of RAP1 VHP domain. Left, RAP1 modeled structure; middle, VHP domain structure of human supervillin protein (PDB accession number 2K6N); and right, superposition of T. brucei RAP1 modeled VHP and human supervilin VHP domains. Alignment of T. brucei RAP1 VHP with human (Hs), Arabidopsis thaliana (At), and Gallus gallus (Gg) VHP domains from Villin proteins. PDB accession numbers are indicated in parenthesis. % of aa identity to T. brucei sequence are shown. B) Binding assays with rRAP1, rRAP11-300, rRAP301-560, or rRAP1561-855 and PI(3,4,5)P3-biotin. Beads, Streptavidin-beads; FT, flow-through. Proteins were resolved in 10% SDS/PAGE and Western developed with α-His mAbs. C) Binding of His-tagged rRAP11-300 to biotinylated phosphoinositides or IPs. D) Binding of rRAP11-300 to PI(3,4,5)P3-biotin in presence of unlabelled PI(3,4,5)P3 or PI(4,5)P2. For C and D, proteins were analyzed as in B. E) Binding kinetics of rRAP1 with unlabelled PI(3,4,5)P3 or PI(4,5)P2. ΔTm, change in melting temperature. Data show the mean ± SDM of three biological replicates. F) Quantification of RAP1-bound PI(3,4,5)P3 (top) or total cellular PI(3,4,5)P3 (bottom) levels in T. brucei exclusively expressing WT or Mut PIP5Pase. Data show the mean ± SDM of four biological replicates. ****, p-value < 0.0001.

PIP5Pase controls rRAP1 binding to telomeric ESs via PI(3,4,5)P3.

A) EMSA of rRAP1 with biotinylated telomeric repeats and increasing concentrations of PI(3,4,5)P3. B) EMSA of rRAP1 with biotinylated telomeric repeats (left) or 70 bp repeats (right) and 30 μM of PI(3,4,5)P3 or PI(4,5)P2. For A-B, samples were resolved in 6% native/PAGE, transferred to nylon membranes, and developed with streptavidin-HRP. C) MST binding kinetics of rRAP1 with Cy5-labelled telomeric repeats (top) or 70 bp repeats (bottom) with 30 μM of PI(3,4,5)P3 or PI(4,5)P2. Data show the mean ± SDM of four biological replicates. D-E) ChIP-seq of RAP1-HA binding to BES7 (D) or (MES_Chr11_5A) from cells that exclusively express WT or Mut PIP5Pase for 24h. RNA-seq comparing exclusive expression of Mut vs WT PIP5Pase for 24h is shown. F-G) Violin plots show RAP1-HA mean enrichment over 70 bp or telomeric repeats from all silent BESs (F) or MESs (G). Each dot represents an ES. BPM, bin per million. ChIP-seq and RNA-seq were performed in three biological replicates. ****, p<0.0001.