ATRX promotes maintenance of herpes simplex virus heterochromatin during chromatin stress
- Harvard Medical School, United States
Figures
Figure 1 with 3 supplements
HSV genomes colocalize with host restriction factors upon nuclear entry.
HFFs were infected with HSV-EdC at an MOI of 5 and fixed at times post infection as indicated. Host proteins were detected by immunocyctochemistry, and HSV genomes were detected with click chemistry and a streptavidin-fluorophore probe. (A) Representative confocal image showing colocalization of ATRX and IFI16 with vDNA as indicated by the white arrows. (B) Percentage of cells with at least 1 HSV-EdC focus within the nucleus as determined by foci detection software. n ≥ 270 total cell population per time point from 15 to 30 mpi derived from 3 independent experiments. Percentage of vDNA foci colocalized with (C) ATRX (blue) or IFI16 (black) and (D) ATRX (blue) or PML (black) from 15 to 30 mpi in 5 min intervals as determined by foci detection and colocalization software (see Materials and methods). n ≥ 150 total cell population per time point from 15 to 30 mpi derived from 3 independent experiments. (E) Percentage of vDNA foci colocalized with ATRX (blue) or IFI16 (black) from 30 to 120 mpi in 10 min intervals as determined by foci detection and colocalization software. n ≥ 65 total cell population per time point from 30 to 120 mpi derived from 3 independent experiments. (F) Frequency distribution of distances from the center of a vDNA focus to the center of the nearest neighbor ATRX focus within a 60 pixel radius of the vDNA focus (blue) vs a distribution of distances from vDNA to randomly generated points within the nucleus equal to the number of ATRX foci (see Figure 1—figure supplement 1A). Distance values were binned in increments of 1 pixel with the center of the first bin set to 0.5 pixels. Kolmogorov-Smirnov test was used to compare distributions and generate a p value. 0 = same underlying distribution, 1 = d distributions are significantly different (see Materials and methods). n = 183 vDNA foci. (G) Frequency distribution of distances (as in F) from the center of a vDNA focus to the center of an IFI16 focus vs vDNA to random points. n = 51 vDNA foci. All data are reported ± standard error of the mean.
Figure 1—figure supplement 1
Foci and colocalization detection.
(A) Screen shot from the frequency distribution script. For each reference channel focus (green) that is detected in a nuclear area (center), the script records the distance between the reference focus and its nearest neighbor (nn) non-reference focus (red). The script also generates random x,y positions within the nuclear mask to simulate the center of a random sample object (blue) and records the distance from the reference channel foci to random sample nn. The script generates a number of random sample points equal to the number of non-reference foci detected within that nuclear area. The script then generates a report of distances from each reference focus to its nearest neighbor in the non-random sample set and the random sample set within a defined radius from the reference focus. (B) Sample image showing ATRX (magenta) and IFI16 (red) colocalization with vDNA (green). White arrows indicate areas of colocalization.
Figure 1—figure supplement 2
EdC labeling of KOS HSV.
(A) HFF cells infected with HSV-EdC at an MOI of 5 and fixed at 2 hpi. vDNA is shown in green and viral protein ICP4 in red. (B) HFF cells infected with HSV-EdC at an MOI of 5 in the absence (-) or presence (+) of viral DNA synthesis inhibitor PAA and fixed at 4 and 6 hpi. vDNA is shown in green and viral protein ICP8 in red. (C) Immunoblot comparing ICP4 and ICP8 from HSV and HSV-EdC at 4, 8, and 12 hpi. (D) HFF cells infected with HSV-EdC at an MOI of 5 in the absence (-) or presence (+) heparin at 1 hpi. HSV-EdC genomes in green.
Figure 1—figure supplement 3
HSV DNA colocalization with ATRX, IFI16, and PML.
(A) FIJI generated 3D volume projections of ATRX (blue) and IFI16 (red) colocalization with vDNA (green). Red lines define the bounding box of the X, Y, Z area being projected. (B) ATRX (magenta) and PML (red) colocalization with vDNA (green) at one hpi with HSV-EdC at an MOI of 5. (C) The number of viral DNA per infected nucleus from 30–120 mpi after infection with HSV-EdC at an MOI of 5. n ≥ 36 infected nuclei per time point. (D) Percentage of nuclei that are positive for HSV vDNA plus ATRX staining (ATRX+) or vDNA plus ICP0 staining (ICP0+) from 60–150 mpi. n ≥ 33 infected nuclei per time point. (E) HFFs infected with HSV-EdC at an MOI of 5. Cells were fixed and stained for HSV DNA, ATRX, and ICP0 at 90 mpi.
Figure 2 with 1 supplement
ATRX and IFI16 independently localize to viral DNA.
HFFs were treated with siRNAs targeting ATRX (siATRX), IFI16 (siIFI16), DAXX (siDAXX), or non-targeting (siNT). 72 hr post siRNA treatment, cells were infected with HSV-EdC at an MOI of 5. (A) Colocalization of ATRX (magenta) and IFI16 (red) with vDNA (green) in HSV-EdC infected HFFs treated with either siNT or siIFI16 at one hpi. (B) Colocalization of ATRX and IFI16 with vDNA in HSV-EdC infected HFFs treated with siATRX at 30 mpi. (C) Immunoblot detection of ATRX and IFI16 in lysates from HFFs treated with siRNA against either ATRX or IFI16 72 hr post treatment. GAPDH was used as a loading control. Percentage of vDNA that colocalizes with (D) ATRX or (E) IFI16 in cells treated with siNT, siATRX, or siIFI16 30 mpi as determined by foci detection and colocalization software. n ≥ 120 total cell population derived from two independent experiments, one-way ANOVA. All data are reported ± standard error of the mean. (F) Colocalization of ATRX and IFI16 with vDNA in HSV-EdC infected HFFs treated with siDAXX at 30 mpi.
Figure 2—figure supplement 1
siRNA depletion of ATRX and DAXX in HFF cells.
(A) Immunoblot detection of ATRX and DAXX from HFF whole cell lysates 72 h after treatment with transfection reagent only (RNAiMax only), siNT, siATRX, siDAXX, or siATRX + siDAXX (siA+D).
Figure 3
ATRX restricts HSV gene expression from input and progeny viral DNA.
(A) HFFs were infected with HSV 7134 at an MOI of 3, and infected cells were fixed and harvested 30, 60, and 240 min post infection. ChIP-qCPR and HSV specific primers were used to detect chromatin enrichment of ATRX at ICP27 (blue) and ICP8 (black) gene promoters. Two-tailed t-tests were used to compare ATRX enrichment at viral gene promoters compared to GAPDH. (B) HFFs were treated with siNT or siATRX and infected with HSV 7134 at an MOI of 5 in the absence (left panels) or presence (right panels) of PAA. Relative viral transcripts for (B) ICP27, (C) ICP8, or (D) gB were quantified by qPCR at 0, 2, 4, 6, and 8 hpi. Viral mRNA levels were normalized to cellular 18S transcripts. Results were analyzed by two-way ANOVA. All data for Figure 3 are reported as the average of 3 independent experiments ± standard error of the mean; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
Figure 4 with 1 supplement
ATRX and DAXX cooperatively restrict HSV gene expression via an IFI16-independent pathway.
(A) Relative viral transcripts for ICP27, ICP8, and gB detected by qPCR in whole cell lysates collected from ATRX-KO or Control cells treated with siNT, siDAXX, or siATRX for 72 hr then infected with HSV 7134 at an MOI of 5. Lysates were collected at 4 and 8 hpi. Results were analyzed by two-way ANOVA. (B) Viral yields from ATRX-KO and Control cells treated with siRNAs against non-targeting, ATRX, DAXX, or ATRX +DAXX (siD +A) and infected with HSV 7134 at an MOI of 0.1. Viral lysates were collected at 24 hpi and titrated on U2OS cells. Yields were normalized to (PFU/mL)/1 × 105 cells. Results were analyzed by two-way ANOVA. (C) Viral yields from ATRX-KO and Control cells treated with siRNAs against non-targeting, ATRX, IFI16, or ATRX + IFI16 (siA + I) and infected with HSV 7134 at an MOI of 0.1. Viral lysates were collected at 48 hpi and titrated on U2OS cells. Yields were normalized to (PFU/mL)/1 × 105 cells. Results were analyzed by two-way ANOVA. All data for Figure 4 are reported as the average of 3 independent experiments ± standard error of the mean; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
Figure 4—figure supplement 1
CRISPR-mediated knockout of ATRX alleviates viral restriction.
(A) Immunoblot of immortalized fibroblasts (TERT-HF) transduced with lentivirus expressing Cas9 alone (LenitCRISPR V2 control), or Cas9 and co-expressing sgRNA against ATRX (TERT-ATRX-1 and TERT-ATRX-2). LenitCRISPR V2 control was used as the Control cell line in this study. TERT-ATRX-1 was used as the ATRX-KO cell line in this study. (B) Viral yields from Control and ATRX-KO cells infected with HSV 7134 at an MOI of 0.1 for 48 hr. Viral lysates were collected and titrated on U2OS cells. Yields were normalized to (PFU/mL)/1 × 105 cells. Results analyzed by two-tailed t-test. (C) Relative viral transcripts for ICP27, ICP8, and gB detected by qPCR in whole cell lysates collected from ATRX-KO or Control cells treated with siNT, siDAXX, or siATRX for 72 hr then infected with HSV 7134 at an MOI of 5. Lysates were collected at 4 and 8 hpi. Results were analyzed by two-way ANOVA. (D) Control and ATRX-KO cells treated with siNT, siDAXX, siATRX, or siATRX + siDAXX (siA + D). (E) Viral yields from ATRX-KO and Control cells treated with siRNAs against non-targeting, ATRX, DAXX, or ATRX + DAXX (siD + A) and infected with HSV 7134 at an MOI of 3. Results were analyzed by two-way ANOVA. Panels B, C, and E are reported as the average of 3 independent experiments ± standard error of the mean; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
Figure 5 with 1 supplement
ATRX depletion enhances ICP0-null HSV DNA Replication and removal of heterochromatin.
ATRX-KO or Control cells were infected with HSV 7134 at an MOI of 3. Infected cells were fixed and harvested 2, 4, and 8 hpi. ChIP-qCPR and HSV specific primers were used to detect chromatin enrichment of (A) H3, (B) H3.3, (C) H3K9me3, and (D) H3K27me3 at the viral gene promoter for ICP27. Results are reported as the percent of input immunoprecipitated by each antibody. Two-tailed t-tests were used to compare results from ATRX-KO versus Control cells for each antibody and each time point. (E) H3K9me3 enrichment per H3 for the ICP27 promoter. (F) Chromatin input for ICP8 relative to input GAPDH to determine relative viral genome copy numbers. All data for Figure 5 are reported as the average of 4 independent experiments ± standard error of the mean; p < 0.05 (*), p < 0.01 (**).
Figure 5—figure supplement 1
ATRX depletion enhances removal of heterochromatin from the ICP8 promoter.
ATRX-KO or Control cells were infected with HSV 7134 at an MOI of 3. Infected cells were fixed and harvested 2, 4, and 8 hpi. ChIP-qCPR and HSV specific primers were used to detect chromatin enrichment of (A) H3, (B) H3.3, (C) H3K9me3, and (D) H3K27me3 at the viral gene promoter for ICP8. Results are reported as the percent of input immunoprecipitated by each antibody. Two-tailed t-tests were used to compare results from ATRX-KO versus Control cells for each antibody and each time point. (E) H3K9me3 enrichment per H3 for the ICP8 promoter. ChIP-PCR experiments in Figure 5—figure supplement 1 are reported as the average of 4 independent experiments ± standard error of the mean; p < 0.05 (*), p < 0.01 (**). (F) Relative viral transcripts for ICP27, ICP8, and gB detected by qPCR in whole cell lysates collected from ATRX-KO or Control cells that were infected with HSV 7134R at an MOI of 5. Lysates were collected at 0, 2, 4, 6, and 8 hpi. Viral mRNA was normalized to cellular 18S transcripts.
Figure 6 with 2 supplements
ATRX promotes maintenance of heterochromatin on input viral genomes.
ATRX-KO or Control cells were infected with HSV 7134 at an MOI of 3. Infected cells were fixed and harvested 8 hpi. ChIP-qCPR and HSV specific primers were used to detect chromatin enrichment of at the viral gene promoters in the absence (Figure 6—figure supplement 1A–D) or presence of PAA. Enrichment of (A) H3 and (B) H3K9me3 at the ICP27 gene promoter. Enrichment of (C) H3 and (D) H3K9me3 at the ICP8 gene promoter. Results reported as Relative IP (the percent of input immunoprecipitated by each antibody normalized to the 4 hr control sample - set to 1.0 for each replicate). (E) Chromatin input for ICP8 relative to input GAPDH to determine relative viral genome copy numbers in the absence or presence of PAA. All data for Figure 6 are reported as the average of 4 independent experiments ± standard error of the mean; two-tailed t-test, p < 0.05 (*), p < 0.01 (**).
Figure 6—figure supplement 1
Untreated controls for ChIP.
ATRX-KO or Control cells were infected with HSV 7134 at an MOI of 3. Infected cells were fixed and harvested 8 hpi. ChIP-qCPR and HSV specific primers were used to detect chromatin enrichment of at the viral gene promoters in the absence (this figure) or presence of PAA (Figure 6). Enrichment of (A) H3 and (B) H3K9me3 at the ICP27 gene promoter. Enrichment of (C) H3 and (D) H3K9me3 at the ICP8 gene promoter. Results are reported as ‘Relative IP’ (the percent of input immunoprecipitated by each antibody normalized to the 4 hr control sample set to 1.0 for each replicate). All data for Figure 6 are reported as the average of 4 independent experiments ± standard error of the mean; two-tailed t-test, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
Figure 6—figure supplement 2
ATRX promotes maintenance of H3K27me3 and H3.3 on input viral genomes.
ATRX-KO or Control cells were infected with HSV 7134 at an MOI of 3. Infected cells were fixed and harvested 8 hpi. ChIP-qCPR and HSV specific primers were used to detect chromatin enrichment of at the viral gene promoters in the absence (solid lines) or presence (dotted lines) of PAA. Enrichment of (A) H3.3 and (B) H3K27me3 at the ICP27 gene promoter. Enrichment of (C) H3.3 and (D) H3K27me3 at the ICP8 gene promoter. Results are reported as ‘Relative IP’ (the percent of input immunoprecipitated by each antibody normalized to the 4 hr control sample set to 1.0 for each replicate). All data for Figure 6 are reported as the average of 4 independent experiments ± standard error of the mean; two-tailed t-test, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
Figure 7 with 3 supplements
ATRX promotes heterochromatin maintenance during challenges to chromatin stability.
(A) ATRX-KO and Control cells were infected with HSV d109 at an MOI of 3 and fixed and harvested at 4 and 8 hpi. ChIP-qPCR was used to detect H3 and H3K9me3 enrichment at the ICP8 promoter. (B) ATRX-KO and Control cells were treated with flavopiridol (flavopiridol; 1 µM) from 1 hr prior to infection until time of harvest. Treated and untreated (Figure 7—figure supplement 1A–B) cells were infected with HSV 7134 at MOI three and fixed at 4 and 8 hpi. ChIP-qPCR was used to detect H3 and H3K9me3 enrichment at the ICP8 promoter. (C) ATRX-KO and Control cells were treated with α-amanitin (2 µg/mL) from 16 hr prior to infection until time of harvest. Treated and untreated (Figure 7—figure supplement 1C–D) cells were infected with HSV 7134 at MOI three and fixed at 4 and 8 hpi. ChIP-qPCR was used to detect H3 and H3K9me3 enrichment at the ICP8 promoter. (D) ATRX-KO and Control cells were infected with HSV 7134 at a MOI of 3 and treated with ActD (5 µg/mL) at 4 hpi. Samples were fixed and collected a 4 and 8 hpi. Chromatin enrichment is reported as percent input immunoprecipitated by antibodies specific for H3 and H3K9me3 as detected by qPCR using specific primers for the promoter of ICP8. All data for Figure 7 are reported as the percent of input immunoprecipitated by each antibody and are the average of 3 independent experiments ± standard error of the mean; two-tailed t-test, p < 0.05 (*).
Figure 7—figure supplement 1
Untreated controls for flavopiridol and α-amanitin ChIPs.
ATRX-KO and Control cells, not treated with flavopiridol, were infected with HSV 7134 at MOI three and fixed at 4 and 8 hpi as controls for Figure 7B. ChIP-qPCR was used to detect (A) H3 and (B) H3K9me3 enrichment at the ICP8 promoter. ATRX-KO and Control cells, not treated with α-amanitin, were infected with HSV 7134 at MOI three and fixed at 4 and 8 hpi as controls for Figure 7C. ChIP-qPCR was used to detect (C) H3 and (D) H3K9me3 enrichment at the ICP8 promoter. All data for Figure 7 are reported as the percent of input immunoprecipitated by each antibody and are the average of 3 independent experiments ± standard error of the mean; two-tailed t-test, p < 0.05 (*).
Figure 7—figure supplement 2
ATRX promotes heterochromatin maintenance at the ICP27 promoter during chromatin stress.
(A) ATRX-KO and Control cells were treated with flavopiridol (flavopiridol; 1 µM) from 1 hr prior to infection until time of harvest. Treated cells were infected with HSV 7134 at MOI three and fixed at 4 and 8 hpi. ChIP-qPCR was used to detect H3 and H3K9me3 enrichment at the ICP27 promoter. (B) ATRX-KO and Control cells were treated with α-amanitin (2 µg/mL) from 16 hr prior to infection until time of harvest. Treated cells were infected with HSV 7134 at MOI three and fixed at 4 and 8 hpi. ChIP-qPCR was used to detect H3 and H3K9me3 enrichment at the ICP27 promoter. (C) ATRX-KO and Control cells were infected with HSV 7134 at an MOI of 3 and treated with ActD (5 µg/mL) at 4 hpi. Samples were fixed and collected a 4 and 8 hpi. Chromatin enrichment is reported as percent input immunoprecipitated by antibodies specific for H3 and H3K9me3 as detected by qPCR using specific primers for the promoter of ICP27. (D) Immunoblot showing inhibition of ICP8 production in Control and ATRX-KO cells infected with HSV d109. (E) Immunoblot showing inhibition of ICP8 production in Control and ATRX-KO cells when treated with flavopiridol. (F) Immunoblot showing inhibition of ICP8 production in Control and ATRX-KO cells when treated with α-amanitin. All data for Figure 7 are reported as the percent of input immunoprecipitated by each antibody and are the average of 3 independent experiments ± standard error of the mean; two-tailed t-test, p < 0.05 (*).
Figure 7—figure supplement 3
Relative HSV genomes during drug treatment.
(A) Chromatin input for ICP8 relative to input GAPDH to determine relative viral genome copy numbers in cells infected with HSV d109. (B) Chromatin input for ICP8 relative to input GAPDH to determine relative viral genome copy numbers in the absence or presence of flavopiridol. (C) Chromatin input for ICP8 relative to input GAPDH to determine relative viral genome copy numbers in the absence or presence of α-amanitin. (D) Chromatin input for ICP8 relative to input GAPDH to determine relative viral genome copy numbers in the absence or presence of ActD. (E) Chromatin input for ICP8 relative to input GAPDH to determine relative viral genome copy numbers in cells expressing a control short hairpin or short hairpin against PML.
Figure 8
PML promotes maintenance of viral chromatin.
(A) Immunoblot for PML in shControl and shPML cells. shPML and shControl cells were infected with HSV 7134 at an MOI of 3. Samples were fixed and harvested at 4 and 8 hpi. ChIP-qPCR for (B) H3 and (C) H3K9me3 at the ICP27 viral gene promoter. ChIP-qPCR for (D) H3 and (E) H3K9me3 at the ICP8 viral gene promoter. Figure 8B–E are reported as the percent of input immunoprecipitated by each antibody and are the average of 3 independent experiments ± standard error of the mean; two-tailed T test, p < 0.05 (*).
Figure 9
Model – ATRX promotes heterochromatin maintenance during challenges to chromatin stability.
(A) Incoming HSV genomes are rapidly sensed by IFI16, PML, and ATRX upon nuclear entry. (B) The de novo formation of heterochromatin on input viral genomes occurs in an ATRX-independent manner, possibly through a HIRA/ASF1A mediated pathway in conjunction with histone methyltransferases (HMTs). However, our results indicate that ATRX is required to maintain viral heterochromatin stability during destabilizing events such as transcription or replication. (C) We hypothesize that ATRX maintains viral heterochromatin integrity resulting in reduced chromatin dynamics, stabilized heterochromatin, and reduced access for transcription factors, viral replication factors, and polymerases.
Videos
Video 1
3D rendering of HSV-EdC colocalization with ATRX and IFI16 at 30 min post infection.
3D rendering from confocal imaging data captured from HFFs infected with HSV-EdC at 30 min post infection. HSV DNA is shown in green; ATRX is shown in magenta; IFI16 is shown in red; DAPI is shown in blue.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (H. sapiens) | ATRX | NA | NCBI Gene ID: 546 | |
| Strain, strain background (Herpes simplex virus 1) | KOS | Smith, 1964 | ||
| Strain, strain background (Herpes simplex virus 1) | HSV-EdC | This Paper | From HSV KOS | |
| Strain, strain background (Herpes simplex virus 1) | HSV 7134 | Cai and Schaffer, 1992, PMID: 1313909 | ||
| Strain, strain background (Herpes simplex virus 1) | HSV 7134R | Cai and Schaffer, 1992, PMID: 1313910 | ||
| Strain, strain background (Herpes simplex virus 1) | HSV d109 | Samaniego et al., 1998; PMID: 9525658 | ||
| Cell line (H. sapiens) | Human Foreskin Fibroblast (HFF) | American Type Culture Collection | SCRC-1041 | |
| Cell line (H. sapiens) | TERT-HF | Bresnahan and Shenk, 2000, PMID: 10875924 | via Robert Kalejta | |
| Cell line (H. sapiens) | TERT-HF Control | This paper | ||
| Cell line (H. sapiens) | TERT-HF ATRX-KO | This paper | ||
| Cell line (H. sapiens) | 293T | American Type Culture Collection | CRL-3216 | |
| Cell line (H. sapiens) | shControl | Wagenknecht et al., 2015, PMID: 26057166 | ||
| Cell line (H. sapiens) | shPML | Wagenknecht et al., 2015, PMID: 26057166 | ||
| Cell line (H. sapiens) | U2OS | American Type Culture Collection | HTB-96 | |
| Antibody | Anti-ATRX, rabbit polyclonal | Abcam | ab97508 | 1:500 IF; 1:2000 WB; 6 µg/ChIP rxn |
| Antibody | Anti-IFI16 [IFI-230], mouse monoclonal | Abcam | ab50004 | 1:2500 WB;1:500 IF |
| Antibody | Anti-PML, rabbit polyclonal | Bethyl | A301-167A | 1:2000 WB |
| Antibody | Anti-DAXX, rabbit polyclonal | Sigma | D7810 | 1:2000 WB |
| Antibody | Anti-ICP4 (58S), mouse monoclonal | Showalter et al., 1981, PMID: 6277788 | 1:2000 WB; 1:500 IF | |
| Antibody | Anti-ICP8 (383), rabbit polyclonal | Knipe et al., 1987, PMID: 3027360 | 1:2000 WB; 1:500 IF | |
| Antibody | anti-GAPDH [EPR16891], rabbit monoclonal | Abcam | ab181602 | 1:5000 WB |
| Antibody | anti- HSV 1-ICP0, mouse monoclonal | East Coast Bio | H1A207 | 1:5000–10000 IF |
| Antibody | Anti-PML [C7], mouse monoclonal | Abcam | ab96051 | 1:500 IF |
| Antibody | Anti- Histone H3 ChIP Grade, rabbit polyclonal | Abcam | ab1791 | 2.5 µg/ChIP rxn |
| Antibody | Anti-Histone H3.3, rabbit polyclonal | Millipore | 09–838 | 2.5 µg/ChIP rxn |
| Antibody | Anti-H3K9me3 ChIP Grade, rabbit polyclonal | Abcam | ab8898 | 2.5 µg/ChIP rxn |
| Antibody | Anti-Histone H3K27me3, rabbit polyclonal | Active Motif | 39156 | 2.5 µg/ChIP rxn |
| Antibody | Normal Rabbit IgG, rabbit polyclonal | Millipore | NG1893918 | 2.5 µg/ChIP rxn (6 µg/ATRX ChIP) |
| Recombinant DNA reagent | lentiCRISPR v2 | Addgene | Plasmid #52961 | |
| Sequence-based reagent | ICP8 cDNA FWD | IDT | 5'-GTCGTTACCGAGGGCTTCAA | |
| Sequence-based reagent | ICP8 cDNA REV | IDT | 5'-GTTACCTTGTCCGAGCCTCC | |
| Sequence-based reagent | ICP27 cDNA FWD | IDT | 5'-GCATCCTTCGTGTTTGTCATT | |
| Sequence-based reagent | ICP27 cDNA REV | IDT | 5'-GCATCTTCTCTCCGACCCCG | |
| Sequence-based reagent | gB cDNA FWD | IDT | 5'-TGTGTACATGTCCCCGTTTTACG | |
| Sequence-based reagent | gB cDNA REV | IDT | 5'-GCGTAGAAGCCGTCAACCT | |
| Sequence-based reagent | ICP4 cDNA FWD | IDT | 5'-GCGTCGTCGAGGTCGT | |
| Sequence-based reagent | ICP4 cDNA REV | IDT | 5'-CGCGGAGACGGAGGAG | |
| Sequence-based reagent | Q-RT 18 S F | IDT | 5'-GCCGCTAGAGGTGAAATTCTTG | |
| Sequence-based reagent | Q-RT 18 S R | IDT | 5'-CTTTCGCTCTGGTCCGTCTT | |
| Sequence-based reagent | ICP27 Promoter FWD (ChIP) | IDT | 5'-ACCCAGCCAGCGTATCCACC | |
| Sequence-based reagent | ICP27 Promoter REV (ChIP) | IDT | 5'-ACACCATAAGTACGTGGC | |
| Sequence-based reagent | ICP8 Promoter FWD (ChIP) | IDT | 5'-GAGACCGGGGTTGGGGAATGAATC | |
| Sequence-based reagent | ICP8 Promoter REV (ChIP) | IDT | 5'-CCCCGGGGGTTGTCTGTGAAGG | |
| Sequence-based reagent | GAPDH Promoter FWD (ChIP) | IDT | 5'-CAGGCGCCCAATACGACCAAAATC | |
| Sequence-based reagent | GAPDH Promoter REV (ChIP) | IDT | 5'-TTCGACAGTCAGTCAGCCGCATCTTCTT | |
| Sequence-based reagent | sgATRX 1 F | This paper | 5'-TCTACGCAACCTTGGTCGAA | |
| Sequence-based reagent | sgATRX 1 R | This paper | 5'-TTCGACCAAGGTTGCGTAGA | |
| Sequence-based reagent | sgATRX 2 F | This paper | 5'-CGAAACTAACAGCTGAACCC | |
| Sequence-based reagent | sgATRX 2 R | This paper | 5'-GGGTTCAGCTGTTAGTTTCG | |
| Sequence-based reagent | siNT | Dharmacon | ON-TARGETplus Non-targeting pool D-001810–10 | |
| Sequence-based reagent | siATRX | Dharmacon | ON-TARGETplus ATRX pool D-006524–00 | |
| Sequence-based reagent | siIFI16 | Dharmacon | ON-TARGETplus IFI16 pool D-20004–00 | |
| Sequence-based reagent | siDAXX | Dharmacon | ON-TARGETplus DAXX pool D-004420–10 | |
| Peptide, recombinant protein | Streptavidin-488 | Invitrogen | 532354 | 1:1000 |
| Peptide, recombinant protein | RNase A | Thermo Science | EN0531 | |
| Commercial assay or kit | DNase Free Kit | Invitrogen | AM1906 | |
| Commercial assay or kit | RNeasy Kit | Qiagen | 74106 | |
| Commercial assay or kit | QIAquick PCR Purification Kit | Qiagen | 28106 | |
| Commercial assay or kit | High Capacity CDNA Kit | Applied BioSys | 4368814 | |
| Software, algorithm | Foci-detection and colocalization software | This paper - Image and Data Analysis Core (IDAC) at Harvard Medical School; doi: 10.5061/dryad.95fs76f | MATLAB based package; https://datadryad.org/resource/doi:10.5061/dryad.95fs76f | |
| Chemical compound, drug | Picolyl-biotin azide | Click Chemistry Tools | product# 1167 | 10 µM final conc. |
| Chemical compound, drug | sodium ascorbate | Sigma | #a4034 | |
| Chemical compound, drug | CuSO4 | Fisher | C489 | |
| Chemical compound, drug | EdC | Sigma | T511307 | 25 mM stock |
| Chemical compound, drug | Prolong gold | Invitrogen | P36934 | |
| Chemical compound, drug | EM Formaldehyde (IF) | Thermo Science | 2908 | 16% methanol free - 2% working conc. |
| Chemical compound, drug | Formaldehyde (ChIP) | Sigma | F8775 | 1% for ChIP crosslinking |
| Chemical compound, drug | Actinomycin-D | Sigma | A1410 | 5 µg/mL final |
| Chemical compound, drug | α-Amanitin | Sigma | A2263 | 2 µg/mL final |
| Chemical compound, drug | Phosphono Acetate | Sigma | 284270 | 400 µg/mL final |
| Chemical compound, drug | Puromycin | Santa Cruz | CAS 53-79-2 | 5 µg/mL final |
| Chemical compound, drug | Roche Complete protease inhibitor | Sigma-Aldrich | 11697498001 | |
| Chemical compound, drug | Fast-Sybr sybr green (qPCR) | Applied Biosystems | 4385612 | |
| Chemical compound, drug | Lipofectamine RNAiMAX transfection reagent | Invitrogen | 56532 |
Additional files
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ATRX promotes maintenance of herpes simplex virus heterochromatin during chromatin stress
eLife 7:e40228.
https://doi.org/10.7554/eLife.40228
