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Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt signaling by Daam1/2

ABSTRACT: Purification of RNF43 interactors was performed with the following 4 different constructs: (i) empty vector (EV) as a control for background contaminants/non-specific protein binding; (ii) full-length RNF43 protein (RNF43 FL); (iii) deleted version of RNF43 harboring only the PA-TA domain (RNF43 Δ1); and (iv) deleted version of RNF43 harboring only the PA-TA-RING domain (RNF43 Δ3). HEK293T cells were transfected with pcDNA3-FLAG-HA- RNF43 (FL, Δ1 or Δ3).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Bon-Kyoung Koo

PROVIDER: PXD043151 | JPOST Repository | Thu Oct 19 00:00:00 BST 2023

REPOSITORIES: jPOST

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Project description:In order to identify epigenetic protein complexes recruited to histone PTMs in the deadly human malaria parasite Plasmodium falciparum, we set out to perform histone peptide pulldowns with various histone peptides. Biotinylated peptides corresponding to P. falciparum histone variant H2A.Z, H2B.Z and H3.3, canonical P. falciparum histone H4 or human histone H4 tail sequences (differing on position 21 from the P. falciparum peptide) bearing multiple histone post-translational modifications (acetylations and/or methylations) as well as the unmodified control peptides were coupled to beads and incubated with native nuclear extract obtained from mixed-stage asexual cultures. Proteins preferentially binding to the modified histone peptide were identified by quantitative proteomics using di-methyl labelling. This yielded 6 out of 7 P. falciparum bromo-domain proteins preferentially binding to the acetylated histone peptides, as well as many putative bromo-domain protein interactors. In addition, a PHD-domain containing protein was found to be recruiting a PfSAGA-like complex to H3K4 di- and tri-methylated peptides. 7 reader-domain containing proteins (BDP1, BDP2, BDP4, TAF1, GCN5, PHD1, PHD2) and 5 putative interactors (PF3D7_0306100, PF3D7_1124300, PF3D7_1128000, PF3D7_1225200, PF3D7_1451200) were endogenously tagged by GFP or HA and used in quantitative GFP-/HA-IP-MS/MS experiments to further characterize epigenetic reader-complex composition. As negative control, GFP- and HA-IPs were performed on native nuclear extract not encoding tagged protein. Most HPP experiment employed a 3-label setup, while few only relied on “light” and “heavy”-label sample pools (labels and conditions listed below). GFP- and HA-IP experiments were using a 2-label “light” and “heavy” di-methyl setup only. For all experiments technical replicates were performed using a label-swap approach (called F (forward) and R (reverse)) and independent biological replicates were performed for each. For GFP- and HA-IP forward reactions are set-up as: GFP-/HA-beads = “heavy” di-methyl label, control-beads = “light” di-methyl label. Reverse reactions are set-up as: GFP-/HA-beads = “light” di-methyl label, control-beads = “heavy” di-methyl label. For GFP-/HA-IP raw files the bait and tag used for fishing are included in the file name.

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Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt signaling by Daam1/2

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