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 …
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 …
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 …
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) …
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) …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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) …
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) …
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 …
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 …
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…
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, …
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 …
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
-
Transparent reporting form
- https://doi.org/10.7554/eLife.40228.024
