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. 2012 Nov 15;125(Pt 22):5489-501.
doi: 10.1242/jcs.110148. Epub 2012 Sep 12.

Single-cell analysis of Daxx and ATRX-dependent transcriptional repression

Alyshia Newhart  1 , Ilona U Rafalska-Metcalf, Tian Yang, Dmitri G Negorev, Susan M Janicki
Affiliations

Single-cell analysis of Daxx and ATRX-dependent transcriptional repression

Alyshia Newhart et al. J Cell Sci. .

Abstract

Histone H3.3 is a constitutively expressed H3 variant implicated in the epigenetic inheritance of chromatin structures. Recently, the PML-nuclear body (PML-NB)/Nuclear Domain 10 (ND10) proteins, Daxx and ATRX, were found to regulate replication-independent histone H3.3 chromatin assembly at telomeres and pericentric heterochromatin. As it is not completely understood how PML-NBs/ND10s regulate transcription and resistance to viral infection, we have used a CMV-promoter-regulated inducible transgene array, at which Daxx and ATRX are enriched, to delineate the mechanisms through which they regulate transcription. When integrated into HeLa cells, which express both Daxx and ATRX, the array is refractory to activation. However, transcription can be induced when ICP0, the HSV-1 E3 ubiquitin ligase required to reverse latency, is expressed. As ATRX and Daxx are depleted from the activated array in ICP0-expressing HeLa cells, this suggests that they are required to maintain a repressed chromatin environment. As histone H3.3 is strongly recruited to the ICP0-activated array but does not co-localize with the DNA, this also suggests that chromatin assembly is blocked during activation. The conclusion that the Daxx and ATRX pathway is required for transcriptional repression and chromatin assembly at this site is further supported by the finding that an array integrated into the ATRX-negative U2OS cell line can be robustly activated and that histone H3.3 is similarly recruited and unincorporated into the chromatin. Therefore, this study has important implications for understanding gene silencing, viral latency and PML-NB/ND10 function.

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Figures

Fig. 1.
Fig. 1.
The histone H3.3 chaperone, Daxx, is recruited to the transgene array in U2OS cells. (A) Diagram of the inducible transgene drawn to scale. Expression of Cherry-lac repressor allows the inactive transgene array to be visualized. Transcription is induced by the activator, Cherry-tTA-ER, in the presence of 4-hydroxytamoxifen (4-OHT). The transcribed RNA encodes CFP fused to a peroxisomal targeting signal (SKL). The RNA is visualized by YFP-MS2, which binds to the stem loops in the transcript. The 3′ end of the transcription unit is the intron 2 splicing unit of the rabbit β-globin gene. The recruitment of YFP-tagged factors to the array can be monitored by co-expression with Cherry-lac repressor or Cherry-tTA-ER. (B) Localization of HIRA-YFP (a–d) and endogenous HIRA labeled with α-HIRA antibody (e–h) in relation to Cherry- or CFP-tTA-ER, at the activated transgene array in U2OS 2-6-3 cells. YFP-Daxx is enriched at the inactive array, marked by Cherry-lac repressor (i–l), and the activated array, marked by Cherry-tTA-ER (m–p). Yellow lines in enlarged merge insets show the path through which the red, green and blue intensities were measured in the intensity profiles (d, h, l and p). Asterisks mark the start of the line. Scale bar: 5 µm; inset: 1 µm.
Fig. 2.
Fig. 2.
ATRX represses transcriptional activation of the transgene array in U2OS cells. (A) Western blot of endogenous ATRX in U2OS 2-6-3 and HeLa cells. γ-Tubulin is used as a loading control. (B) Enrichment of ATRX-YFP at the inactive array, marked by Cherry-lac repressor (a–d). Enrichment of ATRX-YFP (e–h) and ATRX(K1600R)-YFP (i–l) at the activated array, marked by Cherry-tTA-ER. Yellow lines in enlarged merge insets show the path through which the red and green intensities were measured in the intensity profiles (d, h and l). Asterisks mark the start of the line. Scale bar: 5 µm; inset: 1 µm. (C) Pixel areas of the inactive array, marked by Cherry-lac repressor (n = 16), and the Cherry-tTA-ER activated arrays in control (n = 16), ATRX-YFP (n = 82) and ATRX(K1600R)-YFP (n = 68) in U2OS 2-6-3 cells. (D) Mean intensity levels of RNA pol II (4H8 Ab staining) at the inactive (n = 16) and CFP-tTA-ER activated arrays in control (n = 12), ATRX-YFP (n = 14) and ATRX(K1600R)-YFP (n = 18) expressing U2OS 2-6-3 cells. Standard deviations, in the form of error bars, are shown in the graph. P values were calculated using unpaired t-test.
Fig. 3.
Fig. 3.
Analysis of the ATRX domains required for recruitment and repression of the transgene array in U2OS cells. (A) Diagram of ATRX showing the location of the ADD domain, the Daxx interaction domain (DID), the ATPase/helicase domain, the K1600R catalytic mutation and the amino acid composition of the YFP-tagged deletion constructs. (B) Localization of the ATRX deletion constructs at the inactive array in U2OS 2-6-3 cells marked by Cherry-lac repressor. Yellow lines in enlarged merge insets show the path through which the red and green intensities were measured in the intensity profiles (d, h and l). Asterisks mark the start of the line. Scale bar: 5 µm; inset: 1 µm. (C) Integrated intensity measurements of the accumulation of Cherry-MS2 (RNA) at the inactive array (n = 20) and CFP-tTA-ER activated arrays in control (n = 34), ATRX-YFP (n = 41), ATRX(K1600R)-YFP (n = 57), ATRX(1–1201)-YFP (n = 28), ATRX(800–1670)-YFP (n = 37) and ATRX(1189–2492)-YFP (n = 38) expressing U2OS 2-6-3 cells. Standard deviations, in the form of error bars, are shown in the graph. P values were calculated using unpaired t-test and are shown in the table.
Fig. 4.
Fig. 4.
Daxx, ATRX and PML are enriched at the CMV promoter of the transgene in HeLa and U2OS cells. (A) Immunofluorescence localization of Daxx (a–d), ATRX (e–h) and PML (i–l) at the inactive array marked YFP-lac repressor in HeLa, HI 1-1 cells. Yellow lines in enlarged merge insets show the path through which the red, green and DAPI intensities were measured in the intensity profiles (d, h and l). Asterisks mark the start of the line. Scale bar: 5 µm; inset: 1 µm. (B) Diagram of the transgene showing the location of primers used for real-time PCR in the ChIP assays. Daxx, ATRX, and PML were evaluated in chromatin lysates prepared from inactive HeLa, HI 1-1 and U2OS 2-6-3 cells. Results are the average of at least three independent experiments. Standard deviations, in the form of error bars, are presented in the graphs. P values were calculated using unpaired t-test and are shown in the tables.
Fig. 5.
Fig. 5.
Histone H3.3 is specifically incorporated into the transgene array chromatin in HeLa cells. (A) Diagram of the transgene showing the location of primers used for real-time PCR in the ChIP assay. Flag-tagged histone H3.2 and H3.3 were evaluated by native ChIP in inactive HeLa, HI 1-1 and U2OS, 2-6-3 cells. Results are the average of at least three independent experiments. Standard deviations, in the form of error bars, are presented in the graphs. P values for comparison of H3.2 and H3.3 levels using each primer pair are presented in the tables and were calculated using unpaired t-test. Western blot shows histone H3.2 and H3.3 immunoprecipitated with flag antibody and in crude lysates (CLs). γ-Tubulin is used as a loading control. (B) Enrichment of transiently expressed histone H3.2-YFP (a–d) and H3.3-YFP (e–h) at the inactive transgene array, marked by Cherry-lac repressor, in HeLa HI 1-1 cells. Yellow lines in enlarged merge insets show the path through which the red and green intensities were measured in the intensity profiles (d and h). Asterisks mark the start of the line. Scale bar: 5 µm; inset: 1 µm. (C) Intensity measurements of transiently expressed H3.2-YFP (n = 25) and H3.3-YFP (n = 23) at the inactive array, marked by Cherry-lac repressor in HeLa HI 1-1 cells. Example images are shown in B. Standard deviations, in the form of error bars, are presented in the graphs. P values were calculated using unpaired t-test.
Fig. 6.
Fig. 6.
Daxx is not required for transgene array activation in U2OS cells. (A) Quantitative RT-PCR analysis of RNA collected from U2OS (2-6-3/YFP-MS2/rtTA) cells expressing control and Daxx shRNAs. Results are the average of three independent experiments. Standard deviations, in the form of error bars, are presented in the graphs. P values were calculated using unpaired t-test. Western blot shows Daxx protein levels in knockdown cells. γ-Tubulin is used as a loading control. (B) Single-cell analysis of YFP-MS2 accumulation at the activated transcription site in U2OS (2-6-3/YFP-MS2/rtTA) cells expressing control and Daxx shRNAs. The same exposure settings were used to image Daxx levels in these cells. Scale bar: 5 µm; inset: 1 µm. (C) Single-cell time lapse imaging of YFP-Daxx at the transgene array, marked by Cherry-tTA-ER, in U2OS 2-6-3 stably expressing YFP-Daxx. Western blot shows the levels of endogenous (circle) and stably expressed YFP-Daxx (arrow) in the U2OS 2-6-3 cell line. Images were taken at 0 min and 15 and 30 min after the addition of 4-OHT. Yellow lines in enlarged merge insets show the path through which the red and green intensities were measured in the intensity profiles (d, h and l). Asterisks mark the start of the line. Scale bar:10 µm; inset: 2 µm.
Fig. 7.
Fig. 7.
ICP0 makes the transgene array in HeLa cells permissible to activation. (A) Quantitative RT-PCR analysis of RNA collected from HeLa HI 1-1 cells. Inactive and Cherry-tTA-ER expressing control cells are compared to those co-expressing control and ATRX shRNAs, ICP0 and ICP0-FxE, in which the zinc-binding motif in the RING finger domain is deleted. Results are an average of at least three independent experiments. Western blot shows ATRX protein levels in knockdown cells. γ-Tubulin is used as a loading control. (B) Recruitment of YFP-ICP0 to the activated array, marked by Cherry-tTA-ER, in HeLa HI 1-1 cells (a–d). Immunofluorescence staining of ATRX at the CFP-tTA-ER activated transgene array in control (e–h) and ICP0-expressing (i–l) HeLa HI 1-1 cells. Yellow lines in enlarged merge insets show the path through which the red, green and blue intensities were measured in the intensity profiles (d, h, and l). Asterisks mark the start of the line. Scale bar: 5 µm; inset: 1 µm. (C) Measurement of the area of the activated transgene array in HeLa HI 1-1cells, (−)ICP0 (n = 43) and (+)ICP0 (n = 38). (D) Measurement of mean intensity of Daxx [(−)ICP0, n = 25; (+)ICP0, n = 25], ATRX [(−)ICP0, n = 18; (+)ICP0, n = 21] and PML [(−)ICP0, n = 25; (+)ICP0, n = 24] at the activated array in HeLa, HI 1-1, cells. Standard deviations, in the form of error bars, are presented in the graphs. P value was calculated using unpaired t-test.
Fig. 8.
Fig. 8.
Histone H3.3 is recruited to the activated transgene array in U2OS and ICP0-expressing HeLa cells. (A) H3.3-YFP enrichment at the activated transgene array, marked by Cherry-tTA-ER, in ICP0-expressing HeLa, HI 1-1 cells (a–d) and U2OS, 2-6-3 cells (e–h). Yellow lines in enlarged merge insets show the path through which the red and green intensities were measured in the intensity profiles (d and h). Asterisks mark the start of the line. Scale bar: 5 µm; inset: 1 µm. (B) Percentage of cells in which H3.3-YFP accumulated at the activated transgene array in control, ICP0 and ICP0-FxE expressing HeLa, HI 1-1 cells and U2OS 2-6-3 cells. 100 cells were counted in three independent transfections.
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