Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons

Abstract

Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.

Figure 1. Global transcriptional responses to the BAM reprogramming factors

(A) Schematic representation of the overall experimental design of this study (B) Hierarchical clustering and heatmap of genome-wide expression analysis during the iN cell reprogramming process by RNA-seq across indicated time points (n=2 biological replicates except n=1 for MEF+Brn2 and MEF+Myt1l). The day 13 and day 22 samples were FACS-purified for TauEGFP-positive cells. Shown are those 2,522 genes that changed expression at least two-fold at any time point. This emphasizes the global changes between MEFs and mature iN cells. Compare to Figure 7A showing a subset of the same data emphasizing the short term transcriptional effect. Fold change is represented in logarithmic scale normalized to the mean expression value of a gene across all samples. Right: names of selected genes significantly induced and repressed upon reprogramming. (C) Top 5 most significant Gene ontology (GO) terms enriched in the group of induced and repressed genes. See also Figure S1.

Figure 2. Genome-wide maps of BAM factors occupancy reveal Ascl1 dominant targeting capacity

(A) Heat maps of normalized tag densities in log2 scale representing genome-wide occupancy profile for Ascl1, Brn2, and Myt1l in MEFs 48h after induction of the BAM factors. For each bound site, the signal is displayed within a 4 kb window centered around the peak summit. Peaks are sorted based on intensity. The signal at the corresponding genomic regions of the transcription factor binding is displayed across the other two data sets. Note little enrichment of Brn2 and Myt1l at Ascl1 bound sites; however, strong enrichment for Ascl1 in Brn2 sites and some in Myt1l. (B) Left: Average signal intensity of Ascl1, Brn2, Myt1l ChIP-seq peaks at respective target sites represented as the average of log2 normalized reads. Right: Ascl1, Brn2, and Myt1l high confidence peaks genomic classification from ChIPseq in BAM-infected MEFs. Starting at the top (darkest shade) and moving in clockwise orientation with color gradient. (C) BAM factors occupancy profile at the Zfp238 locus revealing strong binding of Ascl1 but not Brn2 or Myt1l. The y-axis represents the total number of mapped reads. The genomic scale is in kilobases (kb). See also Figure S2.

Figure 3. Ascl1 binds similar genomic sites in fibroblasts and neural progenitor cells independent of Brn2 and Myt1l

(A) Heat maps of normalized tag densities representing genome-wide occupancy profile for Ascl1 across cell samples: MEFs infected with Ascl1, 48h post induction (MEFs+Ascl1), MEFs infected with the BAM factors, 48h post induction (MEFs+BAM), and NPCs infected with Ascl1, 18h post induction (NPCs). For each bound site, the signal is displayed within a 4 kb window centered around the peaks. (B) The E-box motif is significantly enriched at Ascl1 target sites across data sets (p-value 5.9^e-1353). (C) Average signal intensity of Ascl1 high confidence peaks across the three cell types as defined in (A), represented as the average of normalized reads in log2 scale. (D) Representative tracks comparing Ascl1 occupancy at two neuronal target genes across the three cell types. (E) Ascl1 ChIP-qPCR at selected neuronal target genes in MEFs 48h after induction of Ascl1 alone or in combination with the indicated transcription factors. Target gene binding by Ascl1 is not greatly influenced by presence of Brn2 or Myt1l. Error bars = standard deviation of the ΔC_T. See also Figure S3.

Figure 4. Brn2 is mislocalized during the early stages of the reprogramming

(A) Heat maps of normalized tag densities representing genome-wide occupancy profile for Brn2 ChIP-seq in MEFs+Brn2 alone, MEFs+BAM, and NPCs. For each bound site, the signal is displayed within a 4 kb window centered around the summits of binding sites. Bottom panel illustrates the most significant motif enriched at Brn2 binding sites in MEFs+BAM (left, an Ascl1 motif, p=3.3e⁻⁵²⁴) and in NPCs (right, a Brn2 motif, p=3.9e⁻⁴⁷²). No significant motif was enriched in the MEFs+Brn2 data set. (B) Venn diagram representing Brn2 high confidence peak overlap between targets in MEFs+Brn2, MEFs+BAM, and NPCs. (C) Representative tracks illustrating Brn2 occupancy profile in MEFs+BAM and NPCs, and Ascl1 occupancy in MEFs+BAM. Top, middle, and bottom panel depict examples corresponding to 67.5%, 5.5%, and 27% of the cases observed for Brn2 occupancy between data sets respectively. (D) 22.2% of Brn2 sites in MEFs+BAM co-localize with Ascl1, while only 9.9% of Brn2 sites in NPCs co-localize with Ascl1, which is a significant difference (p < 0.0001 t-test). Only 0.7% of Brn2 peaks in MEFs+Brn2 data set overlap with Ascl1 target sites in MEFs+BAM. Peak overlap was determined by at least 1 base pair overlap between high confidence peaks. (E) Chromatin co-immunoprecipitation (co-IP) of Brn2 in MEFs infected with Brn2 (left) or Brn2+Ascl1 (middle). Chromatin co-IP of Ascl1 in MEFs infected with Brn2+Ascl1 cells (right). IPs were probed with antibodies against Brn2 (upper row) or Ascl1 (bottom row). Both Ascl1 and Brn2 protein is detectable when chromatin is precipitated with Brn2 or Ascl1 antibodies, respectively demonstrating co-occupancy of the 2 factors on chromatin. (F) Brn2 contributes to neuronal maturation. Representative Tuj1 staining 11 days after dox-induction in the VA1 cell line (containing dox-inducible Ascl1) infected with Brn2 at different stages. From left to right shows: 1) Negative control (infected with GFP to control for possible virus toxicity effects), 2) positive control infected with Brn2 one day before dox induction, 3–4) infected with Brn2 2 days and 5 days respectively after dox induction. Brn2 contributes to iN maturation even up to 5 days after initiation of iN reprogramming. See also Figure S4.

Figure 5. Ascl1, but not Brn2 and Myt1l, acts as a pioneer factor in embryonic fibroblasts

(A). Average FAIRE-seq signal of uninfected MEFs at Ascl1 and Brn2 target sites in MEFs infected with the BAM factors. For each target site, the signal is displayed +/− 250bp from the peak summit. While Brn2 targets are mostly nucleosome-free, the Ascl1 targets are predominantly nucleosome-bound in MEFs. (B) Left: Average enrichment of individual histone marks in MEFs based on Brn2 target sites in MEFs after infection with the BAM factors. Right: Heat maps of normalized tag densities representing MEFs chromatin marks at the same Brn2 targets sites. The signal is displayed within an 8 kb window centered around the binding sites. (C) Left: Average enrichment of individual histone marks in MEFs based on Myt1l target sites in MEFs infected with BAM. Right: Heat maps of tag densities representing indicated histone marks at Myt1l targets sites. The signal is displayed within an 8 kb window centered around the binding sites. (D) Average FAIRE-seq signal of uninfected MEFs at the genomic sites where Ascl1 (blue) or Brn2 (purple) is bound in NPCs. Dotted line represents the FAIRE-seq signal at random sites. See also Figure S5.

Figure 6. A trivalent chromatin state is characteristic and predictive of Ascl1 chromatin accessibility between different cell types

(A) Average enrichment of individual histone marks in uninfected MEFs at the sites where Ascl1 (left) or Brn2 (right) is bound in NPCs. For each binding site, the signal is displayed +/− 750bp from peak summit. (B) MEFs chromatin states based on ChromHMM analysis. Heat map of chromatin states enrichment within −2/+2 kb from Ascl1 binding site in NPCs. Arrow highlights most differentially enriched state between Ascl1 and Brn2 binding sites specified as state #5 (trivalent chromatin state = H3K4me1, H3K27acetyl, and H3K9me3). (C) Average enrichment of trivalent chromatin state at predicted Ascl1 bound sites across cell types based on Ascl1 binding in NPCs (white columns). In black columns are represented the average enrichment for trivalent chromatin state at Ascl1 bound sites from Ascl1 ChIP-seq in MEFs, NHDF, and NHEK overexpressing cells. NHDF= normal human dermal fibroblasts; NHEK= normal human epidermal keratinocytes. Error bars = SEM. (D) Venn diagram representing Ascl1 ChIP-seq peak overlap between targets in NHDF and NHEK cells. Bottom: Motif enriched in most confident thousand peaks. (E) Left: Ascl1 ChIP-qPCR at predicted Ascl1 target genes in NHDF and NHEK cells overexpressing Ascl1 reveals substantially better binding in fibroblasts than keratinocytes. Right: Validation of equivalent pull-down efficiency of Ascl1 in NHEK and NHDF cells by ChIP followed by immunoblotting against Ascl1. Error bars = standard deviation of the ΔC_T (F) Average enrichment of trivalent chromatin state at predicted Ascl1 bound sites based on Ascl1 binding in NPCs across Encode cell types with chromatin data publicly available. In orange is the highest enrichment scores predicted for Ascl1-mediated reprogramming in human skeletal muscle myoblasts. In green is one of the lowest enrichment scores predicted for Ascl1-mediated reprogramming in human osteoblasts. Error bars = SEM. (G) iN cell conversion efficiency expressed as ratio of neuronal, MAP2⁺ cells/ seeded cells in skeletal muscle myoblasts and osteoblasts infected with Ascl1, Brn2, Myt1l and NeuroD1. Measurements were carried out 20 days after induction of transgenes. A t-test was performed to compare reprogramming efficiencies of the two cell types (Error bars = SEM, n = 3 biological replicates, **p < 0.01). (H) Characterization of Ascl1-mediated reprogramming upon global loss of H3K9me3 by overexpression of wild type (WT) or catalytically mutant (H189A) JmjD2d. Left: Tuj1 staining of MEFs expressing WT or H189A JmjD2d after Ascl1 induction for 11 days. Right: Fraction of Tuj1 positive neurons in WT relative to mutant (H189A) JmjD2d overexpressing MEFs. Bottom: Detection of H3K9me3 levels by immunoblot. Error bars = SEM, n = 3 biological replicates. See also Figure S6.

Figure 7. Zfp238 is a critical Ascl1 target and iN cell reprogramming mediator

(A) Left: Heat map representing RNA-seq data in MEFs 48h after infection with the indicated factors. Shown are 790 genes that changed expression at least two-fold in any given condition. Fold change of genes expression is represented in logarithmic scale. Of the 790 genes, 607 are up-regulated in MEFs+Ascl1 vs. MEFs and 183 are down-regulated. 143 of the 790 genes classify as Ascl1 targets in MEFs+BAM of which 133 are up- and 10 are down-regulated. Right: Ascl1, Brn2 or Myt1l targets within these 790 genes. Genes were identified as targets based on binding peaks within −10 to +2 kb from the TSS of genes. Data are represented as mean +/− SEM. (B) Quantification of average changes in gene expression of Ascl1 target genes in MEFs 48h after BAM induction, which were identified in (A) and are depicted across various time points of reprogramming. (13d, and 22d samples were TauEGFP-sorted). Y-axis represents the average expression (normalized reads in log2 scale) for the identified RefSeq genes. Data are represented as mean +/− SEM. (C) Left: Quantification of average change in gene expression of Brn2 target genes based on overlapping peaks between MEFs and NPCs, depicted across time points (as in (B) and neuronal cell populations. Right: Quantification of average change in gene expression of Brn2 target genes based on MEF-specific peaks, depicted across time points and neuronal cell populations. Y-axis represents the average expression (normalized reads in log2 scale) for the identified RefSeq genes. (D) Left: Expression level heat map (log2 based) of the BAM factors and twelve selected transcription factors induced during reprogramming. Right: schematic summarizing screen strategy and results for iN cell formation upon expression of the candidate factors alone, in combination with Myt1l, or in combination of Zfp238 and Myt1l. (E) Immunofluorescent detection of Tuj1 (red) and DAPI (blue) of iN cells derived from MEFs after infection with Ascl1+Myt1l or Zfp238+Myt1l. (F) Quantification of reprogramming efficiency by measuring number of cells with neuronal morphology and positive for MAP2 and Tuj1. Shown are averages of average numbers of labeled cells per 10x visual field in at least 10 randomly picked fields (Error bars = SEM, n = 3 biological replicates, t-test ** p < 0.01, *** p < 0.001). (G) Immunofluorescent detection of Tuj1 (red) and DAPI (blue) iN cells derived from MEFs after infection with Zfp238+Myt1l and Rfx1 (left), Lmo2 (middle), or Tcfl5 (right). (H) Average number of Tuj1+ cells and total neurite length of MEFs expressing Zfp238+Myt1L (Z+M) plus the indicated factor. Also shown are MEFs infected with Ascl1 (A) and Ascl1+Myt1l (A+M). A t-test was performed compared to the GFP control (Error bars = SEM, n=5 visual fields, **P<0.01, ***P<0.001). (I–J). Electrophysiological characterization of iN cells derived from MEFs by overexpressing either Ascl1 (black) or Zfp238 (red) in combination with Myt1l. (I) Cells patched in current-clamp mode. Action potential (AP)-generation pattern induced by current pulses of step-wise increasing amplitudes (top panel: stimulation protocol). Example traces from cells generating multiple (middle panels) or single (bottom panels) APs in either condition. Pie-charts indicate fraction of cells firing single (green), multiple (blue) or no (yellow) action potentials (n = 18 cells / 3 batches). (J) Cumulative plots (Cum freq) representing intrinsic properties (lines) of iN cells capable of firing action potentials in Ascl1+Myt1l (n = 18 cells / 3 batches) and Zfp+Myt1l (n = 14 cells / 3 batches) conditions with their corresponding averages ± SE (filled circles with error bars). No significant differences found in AP threshold (AP_threshold, p > 0.1) (i) or AP height (AP_height, p > 0.2) (ii). (iii) However, the number of APs fired (AP #) plotted with respect to current pulses (I) of increasing amplitude was significantly different between the 2 conditions (asterisks, p < 0.02). (iv) No significant differences found in resting membrane-potential (V_rest, p > 0.05). Cumulative plots with average values ± SE show significant difference (asterisks) in capacitance (P > 0.005) (C_m,v) and membrane resistance (p > 0.002) (R_m, vi) measured at holding potential −70 mV. See also Figure S7.