Supplemental Information. Pioneer Factor NeuroD1 Rearranges. Transcriptional and Epigenetic Profiles. to Execute Microglia-Neuron Conversion

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1 Neuron, Volume 101 Supplemental Information Pioneer Factor NeuroD1 Rearranges Transcriptional and Epigenetic Profiles to Execute Microglia-Neuron Conversion Taito Matsuda, Takashi Irie, Shutaro Katsurabayashi, Yoshinori Hayashi, Tatsuya Nagai, Nobuhiko Hamazaki, Aliya Mari D. Adefuin, Fumihito Miura, Takashi Ito, Hiroshi Kimura, Katsuhiko Shirahige, Tadayuki Takeda, Katsunori Iwasaki, Takuya Imamura, and Kinichi Nakashima

2 Supplemental Information Pioneer factor NeuroD1 rearranges transcriptional and epigenetic profiles to execute microglia-neuron conversion Taito Matsuda, Takashi Irie, Shutaro Katsurabayashi, Yoshinori Hayashi, Tatsuya Nagai, Nobuhiko Hamazaki, Aliya Mari D Adefuin, Fumihito Miura, Takashi Ito, Hiroshi Kimura, Katsuhiko Shirahige, Tadayuki Takeda, Katsunori Iwasaki, Takuya Imamura and Kinichi Nakashima Titles for supplemental tables Table S1. Expression of genes associated with epigenetic enzyme and chromatin remodeling related to Figure 4. Table S2. List of candidate genes and literatures related to Figure 4. Table S3. List of candidate genes and literatures related to Figure 5. Table S4. List of primers related to Figure 5, S2 and S5. 1

3 Figure S1. ND1 efficiently converts microglia to in cells, Related to Figure 1. (A) Representative images of staining for Iba1, biii-tubulin, CD11b, GFAP, CD68, Olig2, Nestin and Hoechst in primary cultured microglia. Scale bar, 50 µm. (B) Quantification of the indicated marker-positive cells in (A) (n = 5 biological replicates). (C) Experimental scheme for candidate gene screening. (D) Quantification of biii-tubulinand GFP-positive cells in microglia transduced with candidate genes; Ascl1 (A), Brn2 (B), 2

4 Myt1l (M), mir-124, Zic1 (Z), Olig2 (O), Sox2 (S), shmbd3 (shmbd3), Neurog2 (Neurog2), and ND1 (ND1) (n = 6 biological replicates). *P < by ANOVA with Tukey post-hoc tests compared with control. (E) Representative images of staining for biii-tubulin, GFP and Hoechst in ND1-transduced microglia. Scale bar, 50 µm. (F) qrt PCR analysis of exogenous FLAG-tagged ND1 expression, which almost disappeared by 7 dpt (n = 3 biological replicates). (G) Representative images and quantification of staining for biii-tubulin, Map2ab, Iba1, active-caspase3 (accaspase3) and Hoechst in ND1-transduced microglia at 7 dpt. Scale bar, 50 µm (n = 5 biological replicates). Yellow and white arrowheads indicate Iba1-positive cells and Map2ab-positive/ biii-tubulinnegative cells, respectively. (H) Quantification of the number of indicated markerpositive cells in (G). Ratios in (G) were multiplied by the total number of cells in a dish to give estimates of the total number of indicated marker-positive cells. (I) Representative images of staining for Map2ab and Hoechst in ND1-transduced microglia stimulated with or without LPS. Scale bar, 50 µm. (J) Quantification of the indicated marker-positive cells in (I) (n = 5 biological replicates). 3

5 Figure S2. ND1 directly converts microglia to in cells, Related to Figure 1. (A) Representative images of staining for GFP, GFAP, Map2, biii-tubulin, Iba1 and Hoechst in astrocytes, neurons and the microglial cell line BV2, all of which are infected with lentivirus encoding TRE-controlled EGFP together with lentivirus encoding rtta under the control of the Iba1 promoter. EGFP signals were observed only in BV2 cells. (B) Representative images of staining for biii-tubulin, DCX and Map2ab in microglia 7 days after transduction with ND1 under the control of the Iba1 promoter. Scale bar, 50 µm. (C) qrt-pcr analysis of ND1 in microglia at 2 days after ND1 transduction under 4

6 the control of the human CD68 promoter (n = 4 biological replicates). *P < 0.05 by Wilcoxon rank sum test. (D) Representative images of staining for biii-tubulin and DCX in microglia at 7 days after ND1 transduction under the control of the human CD68 promoter. Scale bar, 50 µm. (E) Experimental scheme for assessing the direct conversion of microglia to in cells. (F) Representative images of staining for EdU, DCX and Hoechst in ND1-transduced microglia at 5 dpt. Scale bar, 50 µm. (G) Quantification of DCXpositive or -negative cells with or without EdU signal in ND1-transduced or control microglia at 5 dpt (n = 3 biological replicates). (H) qrt-pcr analyses of the indicated genes in ND1-transduced microglia at 1 dpt (n = 4 biological replicates). *P < 0.05 by Wilcoxon rank sum test; n.s., not significant. (I) Representative images of staining for tdtomato, GAD67 and VGLUT1 in ND1-transduced microglia at 21 dpt. Scale bar, 20 µm. (J) Quantification of GAD67- or VGLUT1-positive cells cultured as in (G) (n = 3). 5

7 Figure S3. ND1-converted in cells exhibit neuronal activity, Related to Figure 1. (A) Experimental scheme for assessing functions of in cells. (B) Representative images of staining for tdtomato and Map2ab in ND1-transduced microglia co-cultured with cortical neurons. Scale bar, 50 µm. (C) Quantification of indicated marker-positive cells 6

8 in (B) (n = 3 biological replicates). (D) Representative images of staining for tdtomato, NeuN and Map2 in ND1-transduced microglia co-cultured with cortical neurons. Scale bar, 20 µm. (E) Representative images of staining for tdtomato, PSD95 and VGLUT1 in ND1-transduced microglia co-cultured with cortical neurons. The images on the right are enlargements of the dashed box (lower left). Yellow arrowheads (lower left) indicate PSD95- and VGLUT1-positive puncta. Scale bars, 10 µm (left) and 5 µm (right). (F) Fluo-4AM calcium (Ca 2+ ) imaging of an in cell (white arrow) in response to NMDA stimulation. Color ranges from blue (low concentration of Ca 2+ ) to green, yellow, red and finally white (high [Ca 2+ ]). Scale bar, 20 µm. (G) Quantification of fluorescence intensity in in cells and cortical neurons before and after NMDA stimulation. Changes in fluorescence emission intensity (F) are expressed as Ft/F0, where F0 is the basal intensity obtained at the start of the experiment. Each gray line indicates the average Ft/F0 of five cells in four different experiments. Red lines show the average of all Ft/F0 values. (H) Bar graph showing the fluorescence ratio of microglia, in cells and cortical neurons. Fluorescence ratio is calculated by subtraction of minimum fluorescence intensity from maximum fluorescence intensity (n = 20 cells). **P < 0.01 by ANOVA with Tukey posthoc tests; n.s., not significant. (I) Representative traces of spontaneous firing activity of action potentials in in cell co-cultured with cortical neurons. (J) Representative traces of action potentials evoked by the depolarizing current steps in in cells co-cultured with cortical neurons in the absence or presence of 1 µm TTX under the current-clamp condition. Insets indicate a configuration of step-pulses elicited from the patch pipette (cumulative step stimulation from the resting potential with 30 pa for 100 ms duration). (K) Representative traces of miniature postsynaptic currents (mpscs) recorded from in cells co-cultured with cortical neurons in the presence of 1 µm tetrodotoxin (TTX) under the voltage-clamp condition. (L) Bar graph showing the membrane capacitance of cortical neurons (n = 23 cells) and in cells (n = 25 cells). Not significant (n.s.) by Student s t-test. (M) Bar graph showing the resting membrane potential of cortical neurons (n = 22 cells) and in cells (n = 24 cells). Not significant (n.s.) by Student s t-test. (N) Bar graph showing the input resistance of cortical neurons (n = 23 cells) and in cells (n = 25 cells). Not significant (n.s.) by Student s t-test. (O) Bar graph showing the frequency of mpscs recorded from cortical neurons and in cells in the presence of 1 µm TTX under the voltage-clamp condition (n = 18 cells, respectively). Not significant (n.s.) by Student s t-test. (P) Bar graph showing the amplitude of mpscs of cortical neurons 7

9 and in cells (n = 18 cells, respectively). Not significant (n.s.) by Student s t-test. (Q) Representative images of staining for FLAG and Hoechst in microglia transduced with lentivirus encoding FLAG-tagged ND1. (R) Quantification of FLAG-positive cells in (A) (n = 3 biological replicates). (S) PCA of NSC-specific genes (n = 850) of each sample; Microglia day 2 (M day 2), Microglia day 7 (M day 7), Microglia+ND1 day 2 (M+ND1 day 2), Microglia+ND1 day 7 (M+ND1 day 7), neurons, SCR029 cells (NS/PC), primary cultured NS/PCs (prins/pc), oligodendrocytes and astrocytes. 8

10 Figure S4. Binding of ND1 to bivalent domains induces subsequent gene expression, Related to Figure 4. (A) Alignment data of H3K27me3, H3K4me3, ND1 and input near the Meis2, Scrt1 and Brn2 loci in microglia (M) and ND1-transduced microglia at 7 dpt (in). Black lines at the bottom show the MACS-annotated regions for ND1 binding. (B) ChIP-qPCR analyses of H3K4me3 and H3K27me3 enrichment in the indicated bivalent genes and negative control genes in microglia (n = 5 biological replicates). *P < 0.05 and **P < 0.01 by ANOVA with Tukey post-hoc tests compared with negative control (Zfp384). (C) ChIPqPCR analyses of ND1 enrichment in the indicated bivalent genes and negative control genes. Anti-FLAG ChIP was carried out using primary cultured microglia expressing FLAG-tagged ND1 (n = 5 biological replicates). **P < 0.01 by ANOVA with Tukey posthoc tests compared with negative control (Zfp384). (D) Box plots showing the gene expression levels of up-regulated (n = 764) and unchanged genes at the indicated time 9

11 points (n = 5735). *P < by Wilcoxon rank sum test. (E) Enrichment profiles of ATAC-seq signal at ND1-bound sites located around up-regulated (red) and unchanged (blue) genes (± 4 kb). (F) Functional annotation of unchanged genes (n = 5735). The top five GO terms are displayed. (G) Bar graph showing the ND1 expression level during conversion. *q value < 0.05 by a program in Cufflinks. 10

12 Figure S5. A bivalent chromatin state predicts permissiveness for ND1-mediated reprogramming among different cell types, Related to Figure 4. (A) Representative images of staining for GFAP and Hoechst in primary cultured astrocytes. Scale bar, 50 µm. (B) Representative images of staining for GFP and Map2ab in ND1-transduced astrocytes at 7 dpt. Scale bar, 50 µm. (C) Quantification of indicated 11

13 marker-positive cells in (B) (n = 3 biological replicates). (D) Representative images of staining for Olig2 and Hoechst in oligodendrocytes. Scale bar, 50 µm. (E) Representative images of staining for APC and Hoechst in oligodendrocytes. Scale bar, 50 µm. (F) Representative image of staining for APC and MBP in oligodendrocytes. This is a magnified view of the yellow dashed box in (E). Scale bar, 50 µm. (G) Representative images of staining for GFP, Map2ab and Hoechst in ND1-transduced oligodendrocytes at 7 dpt. Scale bar, 50 µm. (H) Representative images of staining for GFP and NeuN in ND1-transduced oligodendrocytes at 7 dpt. Scale bar, 50 µm. (I) Quantification of indicated marker-positive cells in (G) (n = 4 biological replicates). (J) ChIP-qPCR analyses of H3K4me3 and H3K27me3 enrichment in the indicated bivalent genes and negative control genes in astrocytes (n = 5 biological replicates). *P < 0.05 by ANOVA with Tukey post-hoc tests compared with negative control (Zfp384). (K) ChIP-qPCR analyses of H3K4me3 and H3K27me3 enrichment in the indicated bivalent genes and negative control genes in oligodendrocytes (n = 5 biological replicates). *P < 0.05 by ANOVA with Tukey post-hoc tests compared with negative control (Zfp384 or GAPDH). (L) Scatter plots of genes associated with functions in epigenetic modification and chromatin remodeling (n = 171) from control and ND1-transduced microglia. Up- (red) and down-regulated (blue) differentially expressed genes are highlighted. (M) Bar graph showing the expression levels of Kdm6b (Jmjd3) in control and ND1-transduced microglia (n = 3 biological replicates). *q value < 0.05 by a program in Cufflinks. (N) Alignment data of ND1 locus in ND1-transduced microglia at 2 dpt. Black lines at the bottom show the MACS-annotated regions for ND1 binding. (O) Representative images of staining for Map2ab and Hoechst in microglia transduced with ND1 alone or together with Brn2 at 7 dpt. Scale bar, 50 µm. (P) Quantification of indicated marker-positive cells in (O) (n = 4 biological replicates). (Q) Representative images of staining for Map2ab and GFP in microglia transduced with ND1 alone or together with shrna for Brn2 at 7 dpt. Scale bar, 50 µm. (R) Quantification of indicated marker-positive cells in (R) (n = 4 biological replicates). *P < 0.05 by ANOVA with Tukey post-hoc tests. (S) qrt-pcr analysis of the expression of Brn2 in neurons. Each shrna significantly reduces Brn2 expression (n = 4 biological replicates). *P < 0.05 by ANOVA with Tukey post-hoc tests. 12

14 Figure S6. Lyl1 and Mafb safeguard immune gene expression, Related to Figure 5. (A) qrt-pcr analyses showing down-regulation of ND1-silenced immune genes (Cebpa, Irf8, Ccl3, Pu.1, p65 and Ccl4) by Lyl1 and Mafb knockdown. Bar graphs indicate the expression of these immune genes in microglia at 4 days after single or combinatorial shrna transduction of Lyl1 and Mafb (n = 4 biological replicates). (B) Venn diagrams showing the overlap of microglia-specific genes (green, n = 1244) with genes significantly decreased in ND1-transduced microglia at 2 dpt (blue, n = 200) and 7 dpt (magenta, n = 4048) (fold change 0.5, q value < 0.05 by a program in Cufflinks). The top three GO terms enriched in overlapped genes are displayed (right side). 13

15 Figure S7. ND1 reprograms microglia into functional in cells in the adult mouse striatum, Related to Figure 7. (A) Experimental scheme for microglial ablation in the brain. (B) Representative images of staining for Iba1, S100b, NeuN and Hoechst in the adult mouse striatum with or without PLX5622 (PLX) treatment at 2 weeks after injection of CD68 promoter-driven ND1-P2A-EGFP lentivirus. Scale bar, 20 µm. (C) Representative images of staining for 14

16 EGFP, DCX and Hoechst in the adult mouse striatum with or without PLX treatment at 2 weeks after injection of CD68 promoter-driven ND1-P2A-EGFP lentivirus. Scale bar, 20 µm. (D) Quantification of DCX-positive cells in (C) (n = 3 biological replicates). (E) Representative images of staining for YFP, DCX and Hoechst in the adult striatum at 2 weeks after injection of CD68 promoter-driven ND1-P2A-Cre lentivirus into Rosa-YFP reporter mice. Scale bar, 20 µm. (F) Quantification of DCX-positive cells in (E) (n = 3 biological replicates). (G) Representative images of recorded EGFP-positive cells labeled with biotin at 4 weeks after injection of CD68-ND1-P2A-EGFP lentivirus together with CD68-EGFP lentivirus. The right image is a magnified view of the white dashed box in the left panel. Scale bar, 20 µm (left) and 10 µm (right). (H) Representative traces of spontaneous firing activity of action potentials by the depolarizing current steps in an in cell in the striatum under the current-clamp condition at 4 weeks after injection of CD68- ND1-P2A-EGFP lentivirus together with CD68-EGFP lentivirus. The inset indicates the configuration of step-pulses elicited from the patch pipette (cumulative step stimulation from the resting potential with 10 pa for 500 ms duration). (I) Bar graph showing the resting membrane potential of in cells in the striatum (n = 8 cells from 2 animals). (J) Representative traces of spontaneous excitatory postsynaptic currents (sepscs) recorded from in cells in the striatum under the voltage-clamp condition. (K) Bar graph showing the frequency (left) and amplitude (right) of sepscs of in cells in the striatum (n = 8 cells from 2 animals). (L) Representative traces of spontaneous inhibitory postsynaptic currents (sipscs) recorded from in cells in the striatum. Bar graph showing the frequency (left) and amplitude (right) of sipscs of in cells in the striatum (n = 8 cells from 2 animals). (M) Bar graph showing the frequency (left) and amplitude (right) of sipscs of in cells in the striatum (n = 6 cells from 2 animals). 15