Fig 2: Whole-Genome RNAi Screening Strategy to Identify Cell-Fate-Safeguarding Factors in C. elegans(A) Schematic representation: the ASER neuron is labeled by gcy-5::GFP. Overexpression of the Zn-Finger TF CHE-1 (che-1oe) combined with genome-wide RNAi led to discovery of genes preventing ectopic gcy-5::GFP induction in adults.(B) Representative images of control animals, GFP induction in germline (lin-53, hmg-3, isw-1, and hecd-1 RNAi), intestine (spt-16, hmg-4, emb-5, and spt-5 RNAi), epidermis (hsp-1 RNAi), or germline and gut simultaneously (cco-1). Dashed lines indicate outline of animals. Scale bars represent 20 µm.(C) Percentage of 171 genes whose knockdown allowed ectopic GFP induction in specific tissues.(D) Gene ontology (GO) categories of genes from the screen.(E and F) Proteostasis and FACT protein network based on genes identified in the screen. Network plots are based on known and predicted interactions from STRING (string-db.org) with minimal confidence score of 0.4. FACT complex proteins are highlighted in red.(G) Models of FACT subunits in human and C. elegans. Conserved protein domains according to Pfam and InterPro are indicated: Nlob, N-terminal lobe domain; M24, metallopeptidase family M24; SPT16, FACT complex subunit Spt16p/Cdc68p; Rtt, histone chaperone Rttp106-like; SSRP1,structure-specific recognition protein; HMG, high mobility group box domain.See also Table S1.

Fig 3: FACT Depletion Directly and Indirectly Primes the Transcriptome for Pluripotency(A) Human fibroblasts were used for ChIP-seq analysis or transfected with siRNAs against human FACT subunits and used for ATAC-seq and RNA-seq analysis 48 hr after knockdown without OSKM induction.(B) Correlation of SSRP1 and SUPT16H log2-ChIP versus input ratios for TSS windows classified as high for at least one of the two FACT components (see STAR Methods). Density scale reflects number of TSS windows plotted per hexbin. The Spearman correlation Rho value is given.(C) Correlation of SSRP1 and SUPT16H knockdown RNA-seq log2-fold changes for genes that were significantly changed in at least one of the conditions (see STAR Methods; FDR 0.1). Density scale reflects number of genes plotted per hexbin. The Spearman correlation Rho value is given.(D) Hexbin density scatterplot of SUPT16H knockdown normalized ATAC-seq log2-fold changes plotted against SSRP1 knockdown normalized ATAC-seq log2-fold changes for ATAC-seq peaks that were significantly differential (FDR 0.01) in at least one of the conditions. Scale shows number of ATAC-seq peaks plotted per hexbin. The Spearman correlation Rho value is given.(E) Log2 ratio of SUPT16H ChIP versus Input plotted against Fragments Per Kilobase of transcript per Million mapped reads (FPKM) expression values from control RNA-seq for genes classified as FACT high and detected in the RNA-seq. Density scatterplot scale shows number of genes plotted per hexbin.(F) Log2-fold changes in expression levels from RNA-seq after SUPT16H knockdown plotted against log2 ratio of SUPT16H ChIP versus Input for genes classified as FACT high and detected in the RNA-seq. Density scatterplot scale shows number of genes plotted per hexbin.(G) Average positional profiles and heatmaps of library- and input-normalized SUPT16H ChIP-seq signal for genes whose TSS windows classified as FACT high or FACT low (see STAR Methods).(H) Violin plots of RNA-seq log2-fold change distributions following FACT knockdown for genes classified as being FACT high or FACT low. Red jitter dots reflect genes whose expression changed significantly upon FACT knockdown (FDR < 0.1).(I) Density distributions of control or FACT-knockdown ATAC-seq fragment lengths intersecting TSS windows for genes classified as FACT high or FACT low.(J) Mean positional profiles and heatmaps of control or FACT-knockdown ATAC-seq signal for genes classified as FACT high or FACT low.(K) -Log10 enrichment p values of genes assigned ATAC-seq peaks changing, left, or genes showing expression changes upon FACT-knockdown, right, intersecting with clusters of genes previously reported to display specific expression profile changes during OSKM-induced reprogramming (Cacchiarelli et al., 2015). I, early somatic; II, late somatic; III, metabolic processes; IV, late embryogenesis; V, early embryogenesis; VI, pre-implantation; VII, shared soma versus pluripotency; VIII, early pluripotency; IX, late pluripotency; X, neuro-ectoderm.(L) RNA-seq fold changes upon FACT-knockdown for key genes previously implicated in reprogramming or pluripotency. Stars reflect adjusted p values.(M) Browser shot of ATAC-seq signal and library-normalized RNA-seq signal upon SSRP1 (SSRNAi) or SUPT16H RNAi (SURNAi), and FACT ChIP-seq signals for the PRRX1 gene. ATAC-seq peaks and RNA-seq genes called as significantly changed are noted below the respective signal tracks.(N) De novo-generated motifs from ATAC-seq peaks differentially changing upon FACT-knockdown and TF family-representatives matching the given motif.(O and P) Browser shots as described in (M) for the (O) CEBPB and (P) SALL4 genes.(Q) Zoomed-in browser shot of upstream enhancer-candidate for SALL4. Shown are control and FACT-knockdown signals for ATAC-seq and publicly available CEBPB ChIP-seq in human embryonic stem cells (ESCs). Two CEBPB biding sites were found within the 387-bp accessible region by matching the consensus RTTKCAYMAY and allowing one mismatch.(R) Model for how FACT depletion primes for reprogramming: direct changes in gene expression for TFs such as CEBPB lead to promoter distal, indirect chromatin accessibility changes at genomic loci of key regulators such as SALL4.See also Figure S6 and Tables S5 and S6.

Fig 4: FACT Depletion Increases Efficiency of Reprogramming Human Fibroblasts(A) Reprogramming with human secondary fibroblasts carrying a doxycycline (DOX)-inducible OCT4/SOX2/KLF4/c-MYC (OSKM) cassette (Cacchiarelli et al., 2015) that were transfected with siRNAs against human SSRP1 or SUPTH16 before DOX induction.(B) Alkaline phosphatase (AP) staining of stem cell colonies 21 days after DOX treatment. Control experiment is scrambled siRNA (see STAR Methods).(C) Quantification of iPSC colonies (6 replicates) based on SSEA4 immunostaining for each knockdown condition. Paired Student’s t test was used for statistical comparison, *p < 0.05, *** p < 0.001. Error bars represent SD.(D) Histological characterization of teratomas derived from grafting SSRP1 or SUPT16H depletion-derived iPSCs in mice. Teratomas reached 1.5 cm2 in size after 51–70 days and showed tissues of all three germ layers (ectoderm, endoderm, mesoderm). Scale bars represent 50 µm.(E) Forced expression of Ascl1, Brn2, and Mytl1 in human fibroblasts (Normal Human Dermal Fibroblats = NHDF cells) and SSRP1 or SUPT16H depletion enhances reprogramming of fibroblasts to neurons. One-way ANOVA test was used for statistical comparison, ** p < 0.01. Error bars represent SEM.See also Figure S5.

Fig 5: DTC domain required for substrate ubiquitination.(A) Immunoblots of Myc-trap pull-downs of whole-cell lysates from HEK293 cells expressing Myc-DTX2 WWE variants or EV (-) in the presence and absence of ABT-888 using anti-SPT16, anti–histone H3.1, anti–Myc-tag, or anti-actin as indicated. (B) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by DTX2 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and EV (-) or full-length Myc-DTX2 variants: WT (wild type) or WDM (WWE domain mutant); cell lysates and Ni2+–pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. (C) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by 2RD in the presence and absence of ABT-888 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and Myc-2RD or EV. The cell lysates and Ni2+–pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. (D) Immunoblot of in vitro ADP-ribosylation of 2RD and histone H3.1 by PARP1. Biotinylated NAD+ was used as a source of ADP-ribose and immunoblotted with NeutrAvidin protein. (E) Immunoblots of in vitro ubiquitination of biotinylated ADP-ribosylated H3.1 tail-GFP (ADPrn-H3.1 tail-GFP) or H3.1 tail-GFP by 2RD. Biotinylated NAD+ was used as a source of ADP-ribose. Top: Anti-Ub (red) and NeutrAvidin protein. Bottom: Anti-Ub (red) and anti-GFP (green). Asterisk likely denotes a truncated H3.1 tail-GFP fragment (see fig. S2C). (F) Immunoblot of in vitro ubiquitination of biotinylated ADP-ribosylated His-HaPARP (ADPrn-His-HaPARP) or His-HaPARP by 2RD performed in the presence of E1, UbcH5B, and Ub. Biotinylated NAD+ was used as a source of ADP-ribose. Ni2+–pull-down products were immunoblotted with anti-His, anti-Ub, or NeutrAvidin protein as indicated to detect Ubn-ADPrn-His-HaPARP products.