Detection and Quantitation of Acetylated Histones on Replicating DNA Using In Situ Proximity Ligation Assay and Click-It Chemistry

Lazarchuk, Pavlo; Roy, Sunetra; Schlacher, Katharina; Sidorova, Julia M.

doi:10.1007/978-1-4939-9434-2_3

Cited by 12 publications

(22 citation statements)

References 28 publications

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“…EdU was clicked to biotin and recognized by antibiotin antibody ( Fig. 2A), as we described previously (29). The specificity of the PLA was confirmed by performing HDAC1-EdU and HDAC2-EdU PLAs in hdac1-and hdac2-null clones, respectively ( Fig.…”

Section: Resultsmentioning

confidence: 76%

“…2K and L) was dramatically higher in hdac2-null clones than in controls. To distinguish between S-phase and non-S-phase cells, we also introduced a two-azide approach whereby in some Click-It reactions, biotin-azide was spiked with Alexa Fluor 488 (A488)-azide at molar ratios of 1:40 to 1:50 (A488 to biotin), as we documented previously (29). PLA foci were scored separately in A488-positive (EdU-positive [EdU ϩ ], replicating) versus A488-negative (EdU-negative [EdU Ϫ ], nonreplicating) cells to confirm that the HDAC1-EdU PLA signal was enriched in replicating cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Given the increased abundance of the compensating HDACs in nascent chromatin, we next measured the levels of their major target on replicating DNA: newly incorporated hyperacetylated histone H4. We used a PLA to detect the presence of H4K12ac on EdU-labeled, newly replicated DNA as we described previously (29). The specificity of the H4K12ac PLA signal for replicating cells was confirmed previously (29) by clicking EdU to a mixture of biotin-and Alexa Fluor 488-azides at molar ratios of 1:10 to 1:40 (A488 to biotin) and scoring the number of PLA foci and the PLA signal MFI per nucleus in A488-positive (replicating) versus A488-negative (nonreplicating) cells.…”

Section: Figmentioning

confidence: 99%

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Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

Lazarchuk

Hernandez-Villanueva

Pavlova

et al. 2020

Molecular and Cellular Biology

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Newly synthesized histone H4 that is incorporated into chromatin during DNA replication is acetylated on lysines 5 and 12. Histone deacetylase 1 (HDAC1) and HDAC2 are responsible for reducing H4 acetylation as chromatin matures. Using CRISPR-Cas9-generated hdac1- or hdac2-null fibroblasts, we determined that HDAC1 and HDAC2 do not fully compensate for each other in removing de novo acetyls on H4 in vivo. Proteomics of nascent chromatin and proximity ligation assays with newly replicated DNA revealed the binding of ATAD2, a bromodomain-containing posttranslational modification (PTM) reader that recognizes acetylated H4. ATAD2 is a transcription facilitator overexpressed in several cancers and in the simian virus 40 (SV40)-transformed human fibroblast model cell line used in this study. The recruitment of ATAD2 to nascent chromatin was increased in hdac2 cells over the wild type, and ATAD2 depletion reduced the levels of nascent chromatin-associated, acetylated H4 in wild-type and hdac2 cells. We propose that overexpressed ATAD2 shifts the balance of H4 acetylation by protecting this mark from removal and that HDAC2 but not HDAC1 can effectively compete with ATAD2 for the target acetyls. ATAD2 depletion also reduced global RNA synthesis and nascent DNA-associated RNA. A moderate dependence on ATAD2 for replication fork progression was noted only for hdac2 cells overexpressing the protein.

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Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

Lazarchuk

Hernandez-Villanueva

Pavlova

et al. 2020

Molecular and Cellular Biology

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“…Proximity ligation assay-based chromatin assembly assays (CAAs) have recently been developed and serve as a powerful method for analyzing protein dynamics on newly replicated DNA (60)(61)(62)(63). The proximity ligation technique determines whether two molecules reside close to each other in the cell by employing two species-specific secondary antibodies that are fused to oligonucleotides.…”

Section: Resultsmentioning

confidence: 99%

“…After several PBS washes, cells were incubated with 1N HCl for 10 minutes, washed with PBS until pH neutralizes, and blocked with 5% BSA for 1 hour at room temperature. BSA was removed with PBS washes and primary antibodies Images were acquired using MetaMorph version 7.8.10 and quantification was completed using ImageJ version 1.52t according to a previously described protocol (63).…”

Section: Methodsmentioning

confidence: 99%

Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

Garcia

Lovejoy

NAGARAJAN

et al. 2020

Preprint

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ABSTRACTThe replisome functions in a dynamic environment that is at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the action of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (Hat1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12 that accompanies replication-coupled chromatin assembly. Analysis of the role of Hat1 in replication-coupled chromatin assembly demonstrates that Hat1 also physically associates with chromatin near sites of DNA replication. The association of Hat1 with newly replicated DNA is transient but can be stabilized by replication fork stalling. The association of Hat1 with nascent chromatin may be functionally relevant as loss of Hat1 results in a decrease in replication fork progression and an increase in replication fork stalling. In addition, in the absence of Hat1, stalled replication forks are unstable and newly synthesized DNA becomes susceptible to Mre11-dependent degradation. These results suggest that Hat1 links replication fork function to the proper processing and assembly of newly synthesized histones.

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The Chromatin Interaction System

Kreuz

David

Medina

et al. 2022

Protein Interactions

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Detection and Quantitation of Acetylated Histones on Replicating DNA Using In Situ Proximity Ligation Assay and Click-It Chemistry

Cited by 12 publications

References 28 publications

Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

The Chromatin Interaction System

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