Small Molecules Modulate Chromatin Accessibility to Promote NEUROG2-Mediated Fibroblast-to-Neuron Reprogramming

Abstract

Pro-neural transcription factors and small molecules can induce the reprogramming of fibroblasts into functional neurons; however, the immediate-early molecular events that catalyze this conversion have not been well defined. We previously demonstrated that neurogenin 2 (NEUROG2), forskolin (F), and dorsomorphin (D) can reprogram fibroblasts into functional neurons with high efficiency. Here, we used this model to define the genetic and epigenetic events that initiate an acquisition of neuronal identity. We demonstrate that NEUROG2 is a pioneer factor, FD enhances chromatin accessibility and H3K27 acetylation, and synergistic transcription activated by these factors is essential to successful reprogramming. CREB1 promotes neuron survival and acts with NEUROG2 to upregulate SOX4, which co-activates NEUROD1 and NEUROD4. In addition, SOX4 targets SWI/SNF subunits and SOX4 knockdown results in extensive loss of open chromatin and abolishes reprogramming. Applying these insights, adult human glioblastoma cell and skin fibroblast reprogramming can be improved using SOX4 or chromatin-modifying chemicals.

Keywords: ATAC-seq; CREB1; ChIP-seq; NEUROG2; RNA-seq; SOX4; SWI/SNF; reprogramming; small molecules; transdifferentiation.

Graphical abstract

Figure 1

Small Molecules Enhance NEUROG2 Chromatin Occupancy and Activate Synergistic Transcription (A) Stepwise timeline for fibroblast-to-neuron reprogramming with ATAC-seq, ChIP, ChIP-seq, and RNA-seq experiments annotated. DPI, days post infection; DPT, days post treatment with FD. (B) Diagram representing the effects of FD exposure on NEUROG2 peak score for binding events detected at 0.5, 1, and 2 DPT. The change in peak intensity is represented as a ratio of the peak score in the presence versus absence of FD with the Venn diagram depicting the number of NFD-unique events at 0.5, 1, and 2 DPT (biological replicates, n = 3 for each time point and condition). (C) Hierarchical clustering and heatmap of changes in transcription detected by RNA-seq relative to control fibroblasts. FD, fibroblasts exposed to FD; N, NEUROG2-transduced fibroblasts; NFD, NEUROG2-transduced fibroblasts exposed to FD (biological replicates, n = 3 for each time point and condition). (D) Pie diagrams representing the respective number of genes induced by NEUROG2, FD, and NEUROG2-FD synergistic action at 2 DPT. Synergy represents genes only induced in the NFD condition. (E) Time-course representation of the change in transcript expression intensity for genes regulated by FD, NEUROG2, or NFD synergy. (F) Sunburst diagram depicting the upregulated and downregulated genes at each time point, the intensity of expression change at 2 DPT, and corresponding NEUROG2 ChIP-seq annotation data. Each segment represents one collection of time points. For example, the section with pink, red, and deep-red segments represents genes with significant differential expression at all time points, while the section with only a pink segment represents genes differentially expressed only at 0.5 DPT. The gray outer gradient represents the expression change observed for the genes in each section. NEUROG2 binding events are annotated with a blue line segment.

Figure 2

NEUROG2 Acts as a Pioneer Transcription Factor and Small Molecules Promote Chromatin Accessibility at Pro-neural Genetic Elements (A) Clustered heatmaps representing open chromatin events detected at NEUROG2 binding sites (2-DPT ChIP-seq, leftmost black) in fibroblasts (MRC-5, black), NEUROG2-transduced fibroblasts (N, blue), and NFD-treated fibroblasts (NFD, rightmost red). Input-normalized tag densities are plotted in log₂ scale and centered in a 4-kb window around each peak. (B) NEUROG2 ChIP-seq (2 DPT) and ATAC-seq tracks for INSRR/NTRK1, LRRC10B/SYT7, and GADD45G genomic loci. Gray shading highlights regions of NEUROG2 pioneer activity or FD-induced chromatin remodeling. (C) Super-enhancers identified using H3K27AC and 2-DPT NEUROG2 ChIP-seq datasets with a Venn diagram depicting the distribution of super-enhancers in open versus closed chromatin. (D) Genomic distribution of ATAC-seq peaks newly detected upon NFD treatment is biased toward intergenic and intronic genetic elements. (E) Gene ontology analysis of open chromatin peaks newly detected following NFD treatment reveals an enrichment of neurogenesis- and differentiation-related classifications. Additionally the enhancer box, CRE half-site, and HMG-box motifs are diversely represented within these sites. (F) Pairwise scatter plot representing the change in expression of genes annotated to ATAC-seq open chromatin peaks newly detected upon NFD treatment. Upregulated genes (red) were enriched for pro-neuronal identity, while downregulated genes (blue) had primarily non-neural function. Black dots indicate genes without significant changes.

Figure 3

Small Molecules Activate CREB1 to Promote Neuron Survival and SOX4 Upregulation (A) Four hypothetical mechanisms that might underlie NEUROG2 and FD synergy. (B) Immunoblot of shRNA-mediated CREB1 knockdown. (C) CREB1 knockdown significantly reduces the viability of reprogrammed neurons (mean ± SD; n = 10 random image fields from biological triplicates). Relative survival represents (GFP⁺ and Hoechst 33342⁺ cells at each time point)/(total GFP⁺ and Hoechst 33342⁺ cells at 1 DPT). (D) Phospho-CREB1 (pS133) ChIP-qPCR at CREB1 targets (NR4A1, SIK1), NEUROG2 targets with FD-enhanced expression (GADD45G, SOX4), and controls (NEUROG2 target, DLL3; within gene, NR4A1 open reading frame) for NEUROG2-transduced fibroblasts (blue) and NFD-treated fibroblasts (red) at 1 DPT (mean ± SD; biological triplicates). (E) NEUROG2 occupancy around the SOX4 locus. NEUROG2 occupancy is represented as an overlay of 0.5-DPT (light), 1-DPT (medium), and 2 DPT (dark) NEUROG2 ChIP-seq traces from NEUROG2-transduced fibroblasts (blue) and NFD-treated fibroblasts (red). Purple hashes indicate CRE motif sequences. (F) Condition-dependent SOX4 expression from RNA-seq results at 2 DPT (mean values from biological triplicate RNA-seq data). (G) NEUROG2 and CREB1 ChIP from control and NFD-treated fibroblasts reveals chromatin co-occupancy in NFD-treated fibroblasts.

Figure 4

SOX4 Promotes Open Chromatin Patterns during Reprogramming (A) Immunoblot validation of shRNA-mediated SOX4 knockdown. (B) SOX4 knockdown significantly reduces neuronal conversion (mean ± SD; n = 10 random image fields from biological triplicates). Reprogramming efficiency represents (GFP⁺ and MAP2⁺ cells)/(total GFP⁺ cells). (C) Area-proportional Venn diagram depicting the total number of SOX4 binding events and the proportion shared with NEUROG2. The adjacent pie diagram represents the relative fold increase in normalized transcript number for co-bound genes in NEUROG2-transduced fibroblasts in the presence versus absence of FD from RNA-seq datasets. (D) NEUROG2 ChIP-seq track (2 DPT) and ATAC-seq tracks generated from fibroblasts (MRC-5), NEUROG2-transduced fibroblasts (N), NFD-treated fibroblasts (NFD), and NFD-treated fibroblasts co-expressing SOX4 shRNA (NFD + SOX4 shRNA) around the NEUROD1 and NEUROD4 loci. Gray shading highlights representative regions of FD-enhanced chromatin accessibility lost upon SOX4 knockdown. (E) Area-proportional Venn diagram depicting the total number of ATAC-seq events in NFD-treated fibroblasts relative to the number of events detected at the same locations upon SOX4 knockdown. The adjacent pie diagram represents the relative peak score change for shared sites (NFD peak score)/(NFD + SOX4 shRNA peak score). (F) NEUROD1 and NEUROD4 ChIP-qPCR at sites of newly open chromatin or NFD-induced genes (mean ± SD; biological triplicates).

Figure 5

Small Molecules Enhance H3K27 Acetylation at NEUROG2- and SOX4-Targeted Genetic Elements (A and B) Distribution of H3K27 acetylation (H3K27AC) ChIP-seq reads from untreated fibroblasts (black), NEUROG2-transduced fibroblasts (blue), and NFD-treated fibroblasts (red) centered around (A) SOX4 ChIP-seq peaks and (B) NEUROG2 and SOX4 co-bound peaks within a 4-kb window. (C and D) ChIP-seq tracks for SOX4, NEUROG2, H3K27 acetylation, and H3K27 trimethylation generated from fibroblasts (MRC-5), NEUROG2-transduced fibroblasts (N), and NFD-treated fibroblasts (NFD) around the (C) DLL3 and (D) SMARCC2 loci.

Figure 6

Increased Chromatin Accessibility Promotes Human Adult Fibroblast and Glioblastoma Cell Reprogramming (A) Expression of SOX4 RNA and protein in adult fibroblast lines relative to fetal MRC-5 fibroblasts (mean ± SD; biological quadruple samples for qPCR). (B) Percentage of rounded adult fibroblasts expressing TUBB3 when treated with NFD, FGF2, and mouse Sox4 (red) or human SOX4 (gray) (mean ± SD; n = 10 random image fields from biological triplicates). (C) Examples of progressive neuronal conversion using adult fibroblasts treated with reprogramming factors. Fibroblasts adopt a rounded morphology (5–10 DPT), outgrow low-complexity neurites (10–20 DPT), and infrequently adopt complex neuronal morphology (≥21 DPT). Scale bars, 20 μm. (D) SOX4 ChIP-qPCR time course demonstrating SOX4 chromatin occupancy at 1, 2, and 4 DPT in adult fibroblasts treated with reprogramming factors (mean ± SD; biological triplicates for each ChIP). (E) Screen for chromatin-modifying chemicals and SWI/SNF factors that promote neuronal reprogramming. The presence (+) or absence (−) of morphological change, soma rounding, neurite outgrowth, and TUBB3 expression at 8 DPT was evaluated. (F) Exposure to FK228 for 20–24 hr induces rapid neuron-like morphology. Arrows indicate cells with advanced morphology, while partially converted cells at the bottom left are still undergoing morphological restructuring. (G) BCL2L1 significantly enhances the survival of FK228 treated cells, enables the adoption of complex morphology, and promotes expression of neuronal markers such as TUBB3, MAP2, and RBFOX3. Scale bars, 50 μm. (H) Percentage of adult AG05811 fibroblasts overexpressing NEUROG2 and BCL2L1 that reprogram into TUBB3⁺ neurons at 10 DPT. Each time point represents FK228 exposure during the 24-hr window from 2–3, 3–4, or 4–5 days after initial lentiviral transduction (mean ± SD; n = 10 random image fields from biological triplicates for two virus conditions at each time point). (I) Percentage of reprogrammed U-251 glioblastoma cells expressing TUBB3 (red) and MAP2 (gray) when treated with NFD or NFD with SOX4. MAP2 and TUBB3 immunocytochemical staining of neurons generated at 10 DPT (mean ± SD; n > 10 independent experiments with biological triplicates for each experiment). Scale bar, 50 μm.

Figure 7

Simplified Molecular Mechanisms Underlying Fibroblast-to-Neuron Reprogramming NEUROG2, a pioneer transcription factor, targets thousands of pro-neural genetic elements. Small molecules simultaneously activate signaling cascades that promote NEUROG2 and CREB1 co-transcription, induce SOX4 expression, enhance H3K27 acetylation, and promote SOX4-dependent chromatin remodeling. NEUROG2 and SOX4 synergize to enhance the expression of diverse pro-neural transcription factors and drive neuron identity in diverse fibroblast and glioblastoma cell types.