DOI: 10.5061/dryad.6m905qgdg
title: Histone H1.2 dependent translocation of poly (ADP-ribose) initiates parthanatos

description: Toxic cellular insults activate the nuclear protein poly (ADP-ribose)
 (PAR) polymerase-1 (PARP-1) to initiate parthanatos, a regulated cell
 death program. PAR acts as a death signal by translocating from the
 nucleus to the cytosol, where it activates the next steps in the
 parthanatic cell death cascade.  How PAR translocates from the
 nucleus to the cytosol is not known. Here, we show that PARylation and PAR
 binding to histone H1.2 enables it to act as a carrier, transporting PAR
 out of the nucleus to the cytosol. Knocking down the expression of histone
 H1.2 via CRISPR/Cas9 and knockout of histone H1.2 reduces the
 translocation of PAR to the cytosol after treatment of human cortical
 neurons with N-methyl-D-aspartate (NMDA) or following oxygen-glucose
 deprivation (OGD). The PAR-dependent E3 ubiquitin ligase, Iduna (RNF146)
 ubiquitinates PARylated H1.2. Overexpression of Iduna reduces the
 expression levels of cytosolic histone H1.2, preventing the translocation
 of PAR following NMDA or OGD exposure, similar to inhibition of PAR
 formation by the PARP inhibitor, DPQ.  Whereas, the catalytically
 null variant Iduna C60A, or the PAR binding mutant Iduna Y156A and R157A
 (YRAA) was ineffective in ubiquitinating histone H1.2 and preventing the
 reduction in cytosolic histone H1.2 levels and PAR translocation from the
 nucleus to the cytosol. Histone H1.2 heterozygote and homozygote knockout
 mice exhibited reduced infarct volume 24 hrs post middle cerebral artery
 occlusion (MCAO) and showed better recovery in motor deficits than
 wildtype littermates at day 3 and/or day 7 post MCAO. 
 Collectively, these findings reveal histone H1.2 as the key carrier of PAR
 out of the nucleus to the cytosol where it participates in the next step
 of the parthanatic cell death cascade.   
description: # Histone H1.2 dependent translocation of poly (ADP-ribose) initiates
 parthanatos Dataset DOI:
 [10.5061/dryad.6m905qgdg](https://doi.org/10.5061/dryad.6m905qgdg) ##
 Description of the data and file structure In this study, we discovered
 how a molecule called poly(ADP-ribose) (PAR), which signals cells to die
 under toxic conditions, moves from the nucleus to the cytosol to trigger
 cell death. This form of cell death, known as parthanatos, is activated
 when the enzyme PARP-1 in the nucleus produces PAR. However, how PAR exits
 the nucleus was previously unknown. We found that a nuclear protein,
 histone H1.2, binds to and carries PAR out of the nucleus. When histone
 H1.2 was reduced or deleted in human neurons, PAR could no longer move to
 the cytosol after exposure to harmful stimuli such as NMDA or
 oxygen-glucose deprivation. We also showed that another protein, Iduna
 (RNF146), tags histone H1.2 with ubiquitin to control its movement and the
 release of PAR. In mice, deleting histone H1.2 protected the brain from
 damage after stroke and improved recovery. Together, these findings reveal
 histone H1.2 as a key carrier that transports PAR from the nucleus to the
 cytosol, uncovering a crucial step in the parthanatos cell death pathway.
 Files are labeled by figure and include imaging files [.jpg, .tif and .czi
 (image data file format developed by ZEISS),  data sets in excel and
 graphs in GraphPad Prism (.pzf and .pzfx). All files included in this
 submission are raw data files generated from experiments performed for
 this manuscript. The following abbreviations are retained as published,
 with clarified and expanded definitions for better reader intuition: ####
 Chemicals and Ischemia model NMDA or N+: N-methyl-D-aspartate (a common
 excitatory neurotransmitter receptor) OGD or O+: Oxygen-Glucose
 Deprivation (an *in vitro* model of ischemia) MCAO: Middle Cerebral Artery
 Occlusion (an *in vivo* model of ischemic stroke) PAR: Poly(ADP-ribose) (a
 key signaling polymer in the DNA damage response) DPQ: PARP1 inhibitor
 (3,4-Dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone)
 PDD00017273: PARG inhibitor (a specific inhibitor of Poly(ADP-ribose)
 glycohydrolase) XAV-939: Tankyrase Inhibitor (a specific inhibitor of
 Tankyrase, a PARP family member)  #### Genetic Status and Tools WT: Wild
 Type (the normal, non-mutated genetic state) Het: Heterogeneous (used here
 likely for Heterozygous, having two different alleles for a gene) sg or
 sgRNA: Single Guide RNA for CRISPR-Cas9 targeting KD: Knock Down (reduced
 expression of a gene/protein) KO: Knockout (complete removal of a
 gene/protein function) OE: Over Expression (artificially increased
 expression of a gene/protein) sgRNAres: Guide RNA insensitive/resistant (a
 genetic construct or cell line not targeted by a specific sgRNA) Cas9:
 Cas9 only treatment (used to denote a condition where the Cas9 enzyme is
 present without an active sgRNA) #### Specific Protein Constructs and
 Mutants Iduna-GFP: RNF146/Iduna wildtype protein fused with Green
 Fluorescent Protein C60A-GFP: RNF146/Iduna C60A mutant (Cysteine to
 Alanine substitution at position 60) fused with Green Fluorescent Protein
 YRAA-GFP: RNF146/Iduna YRAA mutant (substitutions at a Tyr−Arg−Ala−Ala
 motif) fused with Green Fluorescent Protein K213A: H1.2 K213A mutant
 (Lysine to Alanine substitution at position 213 of Histone H1.2) C1:
 Truncated form of H1.2 mutant (amino acids 1 through 202) C2: Truncated
 form of H1.2 mutant (amino acids 1 through 190) ### Assays and
 Instrumentation MSD: Electrochemiluminescent system developed by Meso
 Scale Diagnostics IVUA: In Vitro Ubiquitination Assay ### File structure
 The archived files are organized into a hierarchical structure to ensure
 easy access and verification of the data presented in the manuscript. Main
 Figure folder contains all files corresponding to Figures 1 through 6.
 Supplementary Figure folder contains all files corresponding to Figures S1
 through S13. Individual data files are stored within designated subfolders
 under these major categories. The full path to each archived file is
 explicitly listed in the separated file named
 Jing_et_al_Nat_Comms_data_path.xlsx ## Files and variables ### File:
 Jing_et_al_Nat_Comms_data.zip #### Description:  Main figure 1\. Figure 1:
 Histone H1.2 physically interacts with PAR and translocates from the
 nucleus to the cytosol and mitochondria in response to NMDA or OGD
 stimuli. The data illustrate the stimulus-induced redistribution of H1.2
 and its association with PAR signaling. Fig1A: Immunoblot showing the
 interaction of H1.2 with PAR in human cortical neurons after NMDA
 stimulation (Immunoprecipitation with Anti-PAR antibody). Fig1B:
 Immunoblot showing the interaction of PAR with H1.2 in human cortical
 neurons after OGD stimulation (Immunoprecipitation with Anti-H1.2
 antibody). Fig1C: Representative immunofluorescence images showing the
 co-translocation of H1.2 and PAR from nucleus to cytosol following NMDA
 challenge, with or without DPQ/XAV-939 treatment. Fig1D: Quantification of
 the Nuclear-to-Cytosolic translocation ratio of H1.2 and PAR following
 NMDA stimulation (Data from Fig. 1C). Fig1E: Representative
 immunofluorescence images showing the co-translocation of H1.2 and PAR
 from nucleus to cytosol following OGD challenge, with or without
 DPQ/XAV-939 treatment. Fig1F: Quantification of the Nuclear-to-Cytosolic
 translocation ratio of H1.2 and PAR following OGD stimulation (Data from
 Fig. 1E). Fig1G: Immunoblot of subcellular fractionation showing the
 time-dependent shift of H1.2 and PAR after NMDA challenge, with or without
 DPQ treatment. Fig1H: Quantification of the post-nuclear fraction
 (Cytosol/Mitochondria) shift of H1.2 and PAR after NMDA stimulation (Ratio
 to GAPDH, data from Fig. 1G). Fig1I: Immunoblot of subcellular
 fractionation showing the time-dependent shift of H1.2 and PAR after OGD
 challenge, with or without DPQ treatment. Fig1J: Quantification of the
 post-nuclear fraction (Cytosol/Mitochondria) shift of H1.2 and PAR after
 OGD stimulation (Ratio to GAPDH, data from Fig. 1I). 2\. Figure
 2: Deletion of H1.2 reduces PAR/H1.2 translocation from the nucleus and
 protects human neurons from NMDA- and OGD-induced cell death. The data
 support a functional role for H1.2 in PAR-dependent neuronal injury.
 Fig2A: Representative immunofluorescence images demonstrating H1.2 and PAR
 translocation in human neurons after NMDA challenge, comparing GFP
 control, Cas9 control, H1.2 knockdown (sgRNA #1/#2), and sgRNA-resistant
 H1.2 rescue. Fig2B: Quantification of the Nuclear-to-Cytosolic
 translocation ratio of H1.2 and PAR under various genetic conditions
 following NMDA stimulation (Data from Fig. 2A). Fig2C: Representative
 immunofluorescence images demonstrating H1.2 and PAR translocation in
 human neurons after OGD challenge, comparing GFP control, Cas9 control,
 H1.2 knockdown (sgRNA #1/#2), and sgRNA-resistant H1.2 rescue. Fig2D:
 Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 and
 PAR under various genetic conditions following OGD stimulation (Data from
 Fig. 2C). Fig2E: Representative images of PI/Hoechst staining used to
 assess neuronal cell death 24 hours after NMDA or OGD insults in control
 (Cas9 only) and H1.2 knockdown cells. Fig2F: Quantification of the
 percentage of neuronal cell death following NMDA insult in control and
 H1.2 knockdown cells (Assessed via PI/Hoechst staining). Fig2G:
 Quantification of the percentage of neuronal cell death following OGD
 insult in control and H1.2 knockdown cells (Assessed via PI/Hoechst
 staining). 3\. Figure 3: Inhibiting nuclear export with Leptomycin B (LMB)
 diminishes PAR/H1.2 translocation following NMDA or OGD and lowers
 neuronal death. The results indicate that nuclear export contributes to
 H1.2-mediated injury signaling. Fig3A: Representative immunofluorescence
 images showing the co-translocation of H1.2 and PAR following NMDA
 challenge under various conditions, including Leptomycin B (LMB) and
 specific inhibitors. Fig3B: Quantification of the Nuclear-to-Cytosolic
 translocation ratio of H1.2 and PAR after NMDA stimulation and various
 drug treatments (Data from Fig. 3A). Fig3C: Representative
 immunofluorescence images showing the co-translocation of H1.2 and PAR
 following OGD challenge under various conditions, including LMB and
 specific inhibitors. Fig3D: Quantification of the Nuclear-to-Cytosolic
 translocation ratio of H1.2 and PAR after OGD stimulation and various drug
 treatments (Data from Fig. 3C). Fig3E: Quantification of the percentage of
 neuronal cell death following NMDA insult, showing the protective effect
 of LMB (Assessed via PI/Hoechst staining). Fig3F: Quantification of the
 percentage of neuronal cell death following OGD insult, showing the
 protective effect of LMB (Assessed via PI/Hoechst staining). Fig3G:
 Immunoblot of Cytosol (Cyto) and Nuclear (Nu) fractions showing H1.2 and
 PAR separation following NMDA challenge with or without LMB. Fig3H:
 Immunoblot of Cytosol (Cyto) and Nuclear (Nu) fractions showing H1.2 and
 PAR separation following OGD challenge with or without LMB, along with the
 corresponding quantification. 4\. Figure 4: Overexpression of Iduna, but
 not its mutant forms, reduces PAR and H1.2 translocation and protects
 neurons from NMDA- and OGD-induced injury. The data highlight Iduna’s role
 as a protective regulator of PAR-dependent cell death pathways. Fig4A:
 Representative immunofluorescence images of neurons overexpressing
 Iduna-GFP (Wild-Type RNF146) following NMDA challenge, with or without DPQ
 treatment. Fig4B: Representative immunofluorescence images of neurons
 overexpressing C60A-GFP (Iduna Mutant) following NMDA challenge, with or
 without DPQ treatment. Fig4C: Representative immunofluorescence images of
 neurons overexpressing YRAA-GFP (Iduna Mutant) following NMDA challenge,
 with or without DPQ treatment. Fig4D: Quantification of the
 Nuclear-to-Cytosolic translocation ratio of PAR from Fig 4A-4C after NMDA
 challenge, comparing Wild-Type Iduna and its mutants. Fig4E:
 Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2
 from Fig 4A-4C after NMDA challenge, comparing Wild-Type Iduna and its
 mutants. Fig4F: Quantification of neuronal Cell Death (PI/Hoechst ratio)
 after NMDA challenge in neurons overexpressing Iduna-GFP, C60A-GFP, or
 YRAA-GFP. Fig4G: Representative immunofluorescence images of neurons
 overexpressing Iduna-GFP following OGD challenge, with or without DPQ
 treatment (Same experimental setup as Fig 4A). Fig4H: Representative
 immunofluorescence images of neurons overexpressing C60A-GFP following OGD
 challenge, with or without DPQ treatment (Same experimental setup as Fig
 4B). Fig4I: Representative immunofluorescence images of neurons
 overexpressing YRAA-GFP following OGD challenge, with or without DPQ
 treatment (Same experimental setup as Fig 4C). Fig4J: Quantification of
 the Nuclear-to-Cytosolic translocation ratio of PAR after OGD challenge,
 comparing Wild-Type Iduna and its mutants (Same experimental setup as Fig
 4D). Fig4K: Quantification of the Nuclear-to-Cytosolic translocation ratio
 of H1.2 after OGD challenge, comparing Wild-Type Iduna and its mutants
 (Same experimental setup as Fig 4E). Fig4L: Quantification of neuronal
 Cell Death (PI/Hoechst ratio) after OGD challenge in neurons
 overexpressing Iduna-GFP, C60A-GFP, or YRAA-GFP (Same experimental setup
 as Fig 4F). 5\. Figure 5: The carboxyl-terminal region of H1.2 is required
 for PAR/H1.2 translocation and neuronal death triggered by NMDA and OGD.
 The data define specific H1.2 domains essential for Parthanatos signaling.
 Fig5A: Schematic illustrating the structure of human Histone H1.2 and the
 locations of its key variants: the K213A point mutation and the two
 carboxyl-tail truncated forms (C1 and C2). Fig5B: Representative
 immunofluorescence images showing PAR and H1.2 translocation following
 NMDA challenge in neurons rescued with V5-tagged H1.2 (WT, K213A, C1, or
 C2) constructs. Fig5C: Quantification of the Nuclear-to-Cytosolic
 translocation ratio of PAR and H1.2 following NMDA stimulation, comparing
 the effects of H1.2 WT and mutant overexpression (Data from Fig5B). Fig5D:
 Representative immunofluorescence images showing PAR and H1.2
 translocation following OGD challenge in neurons rescued with V5-tagged
 H1.2 (WT, K213A, C1, or C2) constructs. Fig5E: Quantification of the
 Nuclear-to-Cytosolic translocation ratio of PAR and H1.2 following OGD
 stimulation, comparing the effects of H1.2 WT and mutant overexpression
 (Data from Fig5D). Fig5F: Representative images of PI/Hoechst staining
 used to assess neuronal cell death after NMDA or OGD insults in neurons
 overexpressing H1.2 WT or its K213A, C1, or C2 mutants. Fig5G:
 Quantification of the percentage of neuronal cell death following NMDA
 insult, comparing the effects of overexpressing H1.2 WT versus its K213A,
 C1, and C2 mutants. Fig5H: Quantification of the percentage of neuronal
 cell death following OGD insult, comparing the effects of overexpressing
 H1.2 WT versus its K213A, C1, and C2 mutants. 6\. Figure 6: Partial or
 complete deletion of H1.2 reduces neuronal death in mouse cortical
 cultures exposed to NMDA or OGD and mitigates injury in an MCAO stroke
 model. The data indicate that H1.2 contributes to ischemic and excitotoxic
 neurodegeneration *in vivo*. Fig6A: Representative images of TTC-stained
 wild-type (WT) and H1.2−/− mouse brain slices 24 hours after a 1-hour
 transient MCAO (Middle Cerebral Artery Occlusion). Fig6B: Pooled data
 showing the total brain infarct volume percentage in the whole hemisphere
 24 hours post-MCAO, comparing WT, H1.2+/−, and H1.2−/− mice. Fig6C: Pooled
 data showing the total brain infarct volume percentage in the striatum 24
 hours post-MCAO, comparing WT, H1.2+/−, and H1.2−/− mice. Fig6D: Pooled
 data showing the total brain infarct volume percentage in the cortex 24
 hours post-MCAO, comparing WT, H1.2+/−, and H1.2−/− mice. Fig6E:
 Quantification of the coronal section infarct volume percentage in the
 whole hemisphere 24 hours post-MCAO across different genotypes. Fig6F:
 Quantification of the coronal section infarct volume percentage in the
 striatum 24 hours post-MCAO across different genotypes. Fig6G:
 Quantification of the coronal section infarct volume percentage in the
 cortex 24 hours post-MCAO across different genotypes. Fig6H:
 Representative images of TTC-stained WT and H1.2−/− mouse brain slices 7
 days after a 1-hour transient MCAO. Fig6I: Pooled data showing the total
 brain infarct volume percentage in the whole hemisphere 7 days post-MCAO,
 comparing WT, H1.2+/−, and H1.2−/− mice. Fig6J: Pooled data showing the
 total brain infarct volume percentage in the striatum 7 days post-MCAO,
 comparing WT, H1.2+/−, and H1.2−/− mice. Fig6K: Pooled data showing the
 total brain infarct volume percentage in the cortex 7 days post-MCAO,
 comparing WT, H1.2+/−, and H1.2−/− mice. Fig6L: Quantification of the
 coronal section infarct volume percentage in the whole hemisphere 7 days
 post-MCAO across different genotypes. Fig6M: Quantification of the coronal
 section infarct volume percentage in the striatum 7 days post-MCAO across
 different genotypes. Fig6N: Quantification of the coronal section infarct
 volume percentage in the cortex 7 days post-MCAO across different
 genotypes. Fig6O: Pooled data from the Cylinder Test, assessing
 sensorimotor function in the three genotypes at the 7-day endpoint
 post-MCAO. Fig6P: Pooled data from the Treadmill Test, assessing motor
 endurance in the three genotypes at the 7-day endpoint post-MCAO. Fig6Q:
 Pooled data from Spontaneous Activity monitoring, assessing general motor
 function in the three genotypes at the 7-day endpoint post-MCAO. 7\.
 Figure 7: Schematic illustration of roles of H1.2 in Parthanatos ###
 Supplementary figure 1\. Figure S1: Histone H1.2 directly binds PAR and
 can be ribosylated by PARP-1 *in vitro*. These data establish H1.2 as a
 PAR-interacting and substrate of PARP-1. FigS1A: Electromobility gelshift
 assay testing the PAR binding capacity of recombinant H1.2. Increasing
 amounts of H1.2 were incubated with [32P]-PAR and resolved by PAGE; both
 bound and free [32P]-PAR signals were visualized via autoradiography.
 FigS1B: *In vitro* ribosylation assay testing the ribosylation of H1.2 by
 PARP1. Ribosylated products of PARP1 (auto-ribosylation control), GST
 (negative control), and H1.2 were separated by SDS-PAGE and visualized by
 autoradiography to demonstrate that H1.2 can be modified by PARP1. 2\.
 Figure S2: NMDA and OGD treatments trigger nuclear translocation of
 Parthanatos markers AIF and MIF in cortical neurons. Representative
 photomicrographs showing nuclear translocation of the parthanatos markers
 AIF and MIF induced by NMDA or OGD treatment. 3\. Figure S3: Experimental
 validation of the immunoprecipitation conditions used in the study. The
 data confirm specificity and efficiency of the IP assays applied
 throughout the work. FigS3A: Immunoblots validating the H1.2-PAR
 interaction by showing the dependence on PAR polymer integrity. Lysates
 from NMDA/OGD challenged neurons were subjected to Immunoprecipitation
 (IP) with anti-PAR or anti-H1.2, followed by treatment with active DNase
 (to remove DNA) or PARG (to cleave PAR polymer) to confirm the specificity
 of the binding. FigS3B: Immunoblots validating the H1.2-PAR interaction
 using inhibitors. Lysates from NMDA/OGD challenged neurons treated with
 inhibitors for PARP1, Tankyrase, or PARG were subjected to IP (anti-PAR or
 anti-H1.2) to confirm that the observed interaction is enzyme-dependent.
 4\. Figure S4: CRISPR/Cas9-mediated H1.2 knockdown reduces PAR/H1.2
 translocation in neurons exposed to NMDA and OGD. FigS4A: Representative
 immunofluorescence images demonstrating PAR translocation in human
 cortical neurons following NMDA challenge combined with DPQ (PARP1
 inhibitor) or PDD00017273 (PARG inhibitor). Cells were stained for PAR
 (red) and DAPI (blue). FigS4B: Representative immunofluorescence images
 demonstrating PAR translocation in human cortical neurons following OGD
 challenge combined with DPQ or PDD00017273. Cells were stained for PAR
 (red) and DAPI (blue). FigS4C: Quantification of the Nuclear-to-Cytosolic
 PAR translocation ratio after NMDA stimulation and treatment with
 inhibitors (DPQ, PDD00017273). FigS4D: Quantification of the
 Nuclear-to-Cytosolic PAR translocation ratio after OGD stimulation and
 treatment with inhibitors (DPQ, PDD00017273). 5\. Figure S5: H1.2
 sgRNA-mediated knock down reduces PAR/H1.2 translocation by NMDA and OGD
 stimulation. FigS5A: Representative immunoblots validating the H1.2
 knockdown efficiency. Lysates from neurons expressing Cas9 and
 H1.2-targeting sgRNAs (#1 and #2) were probed with anti-H1.2 and
 alpha-Tubulin (loading control). Quantification of H1.2 protein levels is
 shown, normalized to alpha-Tubulin. FigS5B: Representative immunoblots of
 nuclear and cytosolic fractions from control (Cas9) and H1.2 knockdown
 (sg#1 and sg#2) neurons. The blots were probed with anti-PAR, anti-H1.2,
 and fraction markers (TBP for nucleus, GAPDH for cytosol). FigS5C:
 Quantification of the NMDA-induced release of PAR and H1.2 from the
 nucleus to the cytosol, comparing control (Cas9 only) and H1.2 knockdown
 conditions (Data from FigS5B). Release is indicated by normalization to
 the cytosolic marker GAPDH. FigS5D: Representative immunoblots of nuclear
 and cytosolic fractions from control (Cas9) and H1.2 knockdown (sg#1 and
 sg#2) neurons following OGD challenge. The blots were probed with
 anti-PAR, anti-H1.2, and fraction markers. FigS5E: Quantification of the
 OGD-induced release of PAR and H1.2 from the nucleus to the cytosol,
 comparing control (Cas9 only) and H1.2 knockdown conditions (Data from
 FigS5D). Release is indicated by normalization to the cytosolic marker
 GAPDH. 6\. Figure S6: CRISPR-mediated H1.2 knockdown does not increase
 total cellular PAR levels as confirmed by Western blot. Western blot
 validation confirming the effect of CRISPR-mediated H1.2 knockdown on
 total cellular PAR levels. Human cortical neurons were transduced with
 lentiviruses carrying either a control gRNA or the H1.2-targeting gRNA #1,
 and the total cellular PAR levels were subsequently assessed by
 normalization to the Actin. 7\. Figure S7: Quantification of PAR levels in
 neurons after NMDA or OGD using MSD assays. FigS7A: Standard curve
 analysis for the PAR MSD platform. This curve establishes the linear range
 and sensitivity of the assay using serial dilutions of PAR calibrators.
 FigS7B: Quantification of relative PAR concentration in neurons using the
 MSD system. Neurons expressing either Cas9 (control) or H1.2 sgRNA #1 were
 challenged with NMDA or OGD. PAR was captured by a coated antibody (HuPAR
 clone #19) and detected by a Sulfo-tagged antibody (HuPAR clone #25).
 Results are expressed as fold changes, demonstrating the impact of H1.2
 knockdown on PAR accumulation following injury. 8\. Figure
 S8: shRNA-mediated H1.2 knockdown reduces PAR/H1.2 translocation and
 neuronal death following NMDA and OGD exposure. FigS8A: Representative
 immunoblots validating H1.2 knockdown efficiency using shRNA. Lysates from
 neurons expressing the empty vector (pSME) or H1.2-targeting shRNAs (#1
 and #2) were probed with anti-H1.2 and alpha-Tubulin. Quantification of
 normalized H1.2 levels is shown. FigS8B: Representative immunofluorescence
 image showing PAR/H1.2 translocation in control shRNA neurons following
 NMDA challenge 15 minutes and/or 2 hrs post challenge combined with DPQ. 
 FigS8C: Representative immunofluorescence image showing PAR/H1.2
 translocation in H1.2 knockdown (shRNA mixed) neurons following NMDA
 challenge 15 minutes and/or 2 hrs post challenge combined with DPQ.
 FigS8D: Quantification of the Nuclear-to-Cytosolic translocation ratio of
 H1.2 after NMDA stimulation, comparing control and H1.2 shRNA knockdown
 conditions. FigS8E: Quantification of the Nuclear-to-Cytosolic
 translocation ratio of PAR after NMDA stimulation, comparing control and
 H1.2 shRNA knockdown conditions. FigS8F: Representative immunofluorescence
 image showing PAR/H1.2 translocation in control shRNA neurons following
 OGD challenge 15 minutes and/or 2 hrs post challenge combined with DPQ.
 FigS8G: Representative immunofluorescence image showing PAR/H1.2
 translocation in H1.2 knockdown (shRNA mixed) neurons following OGD
 challenge 15 minutes and/or 2 hrs post challenge combined with DPQ.
 FigS8H: Quantification of the Nuclear-to-Cytosolic translocation ratio of
 H1.2 after OGD stimulation, comparing control and H1.2 shRNA knockdown
 conditions. FigS8I: Quantification of the Nuclear-to-Cytosolic
 translocation ratio of PAR after OGD stimulation, comparing control and
 H1.2 shRNA knockdown conditions. FigS8J: Quantification of neuronal Cell
 Death (PI/Hoechst ratio) 24 hours after NMDA insult, comparing control
 (pSME) and H1.2 shRNA knockdown conditions. FigS8K: Quantification of
 neuronal Cell Death (PI/Hoechst ratio) 24 hours after OGD insult,
 comparing control (pSME) and H1.2 shRNA knockdown conditions. 9\. Figure
 S9: The release of PAR polymer after MNNG treatment to isolated nuclei of
 cortical tissues from wild type and Histone H1.2 knockout mice FigS9A: PAR
 polymer release from isolated nuclei after MNNG treatment. Representative
 immunoblots show lysates and post-nuclei buffer of nuclei isolated from
 cortical tissues of Wild Type (WT), H1.2+/− (Het), and H1.2−/− (KO) mice.
 Blots were probed for PAR, TBP (nuclear marker), and GAPDH (cytosolic
 marker). FigS9B: Quantification of PAR release is shown as the PAR/GAPDH
 ratio, demonstrating the effect of H1.2 deletion on PAR nuclear efflux.
 10\. Figure S10: Representative ubiquitination blots and quantification
 for Iduna mutants. The data compare the ubiquitination status of wild-type
 Iduna with its mutant variants. FigS10A: *In vitro* ubiquitination assay
 comparing the E3 ligase activity of immunoprecipitated GFP-Iduna (WT)
 against the mutants GFP-Iduna YRAA and GFP-Iduna C60A. The target
 substrates were recombinant H1.2 or Ribosylated H1.2 (Ribo-H1.2).
 Ubiquitination was detected by immunoblots using anti-His, anti-EGFP, and
 anti-GST antibodies. FigS10B: Representative immunoblots of nuclear and
 cytosolic fractions from human neurons overexpressing GFP (control),
 Iduna-GFP (WT), C60A-GFP, or YRAA-GFP following NMDA treatment. Blots were
 probed for PAR, H1.2, and fraction markers (TBP and GAPDH). FigS10C:
 Quantification of the NMDA-induced release of PAR and H1.2 from the
 nucleus, comparing WT Iduna and its mutants (Data from FigS10B). Release
 is calculated as the ratio of protein levels to the cytosolic marker
 GAPDH. FigS10D: Representative immunoblots of nuclear and cytosolic
 fractions from human neurons overexpressing GFP (control), Iduna-GFP (WT),
 C60A-GFP, or YRAA-GFP following OGD treatment. Blots were probed for PAR,
 H1.2, and fraction markers. FigS10E: Quantification of the OGD-induced
 release of PAR and H1.2 from the nucleus, comparing WT Iduna and its
 mutants (Data from FigS10D). Release is calculated as the ratio of protein
 levels to the cytosolic marker GAPDH.  11\. Figure S11: Treatment with
 MG132 in neurons overexpressing wild-type Iduna restores PAR translocation
 to the cytoplasm following NMDA or OGD stimulation. FigS11A:
 Representative immunofluorescence images showing the translocation of PAR
 and H1.2 in human cortical neurons overexpressing Iduna-GFP following NMDA
 challenge and treated with the proteasome inhibitor MG132. FigS11B:
 Representative immunofluorescence images showing the translocation of PAR
 and H1.2 in human cortical neurons overexpressing Iduna-GFP following OGD
 challenge and treated with the proteasome inhibitor MG132. FigS11C:
 Quantification of the Nuclear-to-Cytosolic translocation ratio of PAR and
 H1.2 following NMDA stimulation, under the experimental conditions
 described in FigS11A. FigS11D: Quantification of the Nuclear-to-Cytosolic
 Translocation Ratio of PAR and H1.2 following OGD stimulation, under the
 experimental conditions described in FigS11B. 12\. Figure S12: Evaluation
 of PAR binding and ribosylation of H1.2 mutants. The data identify regions
 of H1.2 required for PAR interaction and modification. FigS12A:
 Immunoblots confirming the expression of Flag-tagged H1.2 WT and its
 mutants (H213A, C1, and C2) following overexpression in human cortical
 neurons with prior H1.2 knockdown (gRNA targeting H1.2). Blots were probed
 with anti-H1.2, anti-Flag, and beta-actin (loading control). FigS12B:
 Electromobility Shift Assay (EMSA) testing the PAR binding capacity of
 recombinant H1.2 WT versus its mutants (K213A, C1, and C2). The assay
 separates bound [32P]-PAR from unbound free [32P]-PAR, which are
 visualized by autoradiography. Recombinant H3 and BSA were used as
 controls. FigS12C: Representative image of the *in vitro* ribosylation
 assay demonstrating the ability of PARP1 to ribosylate H1.2 WT versus its
 mutants (K213A, C1, and C2). The proteins used in the reaction were
 visualized by Coomassie staining, and the level of ribosylation was
 quantified by autoradiography from three independent experiments. 13\.
 Figure S13: Quantification of neuronal cell death (PI/Hoechst ratio) in
 primary cortical cultures from H1.2 wild-type and knockout mice. H1.2−/−
 neurons show resistance to NMDA (Fig. S13A) and OGD (Fig. S13B)
 challenges, demonstrating a protective effect of H1.2 deletion. FigS13A:
 Quantification of neuronal Cell Death (PI/Hoechst ratio) following NMDA
 insult in primary cortical neuron cultures isolated from H1.2+/+ (Wild
 Type) versus H1.2−/− (Knockout) mice, demonstrating resistance in knockout
 neurons. FigS13B: Quantification of neuronal Cell Death (PI/Hoechst ratio)
 following OGD insult in primary cortical neuron cultures isolated from
 H1.2+/+ versus H1.2−/− mice, demonstrating resistance in knockout neurons.
 14\. Figure S14: PI/Hoechst staining data assessing cell death in human
 cortical neurons expressing pSME or H1.2 shRNAs following NMDA (Fig. S8J)
 or OGD (Fig. S8K) treatment. 15\. Figure S15: PI/Hoechst staining data
 assessing cell death in human cortical neurons expressing Iduna-GFP,
 C60A-GFP, or YRAA-GFP following NMDA (Fig. 4F) or OGD (Fig. 4L) treatment.
 16\. Figure S16: PI/Hoechst staining data assessing cell death in human
 cortical neurons expressing Cas9/sgH1.2 and GFP-tagged H1.2 variants after
 NMDA (Fig. 5G) or OGD (Fig. 5H) treatment File:
 Jing_et_al_Nat_Comms_data_path.csv **Description:** The full path to each
 archived file is explicitly listed