Transcriptional activation of the interferon-β gene is correlated with a strong decrease of the association of the IFN-β locus with pericentromeric heterochromatin clusters
In murine cells, PCH is constituted of clusters of γ-satellite sequences (28
). In order to analyze the degree of association of the endogenous IFN-β locus with clusters of γ-satellite sequences with respect to IFN-β expression, we carried out two-color DNA FISH experiments using a rhodamine-labeled γ-satellite probe (red) and a biotin-labeled IFN-β probe (green) containing the IFN-β locus region from −1153 to +1660 cloned into pBR322 plasmid. Experiments were carried out in murine fibroblastic L929 cells either NI or infected with the non-pathogenic Clone13 strain (C13) of the bunyavirus RVFV, which has been described as a good inducer of IFN-β expression (23
). As a control, we used the virulent ZH548 strain (ZH) of RVFV known to be lethal for mice and to abnormally maintain the IFN-β promoter in a transcriptionally silent state (23
). For each condition, the percentage of IFN-β signals colocalizing with γ-satellite FISH signals was quantified and correlated to the induction of IFN-β mRNA synthesis that was assessed by qRT-PCR.
The images corresponding to single confocal sections of nuclei of L929 cells either NI (a–c) or 8
h after infection with the C13 (d–f) or the ZH (g–i) strain of RVFV are shown in A. The number of IFN-β signals present per cell was counted in a total of at least 137 cells from two independent experiments under NI, C13 and ZH conditions. As expected for an endogenous locus in asynchronously growing cells, the majority of the cells displayed two IFN-β FISH signals per cell (B) with only a small amount of cells displaying three or four IFN-β FISH signals corresponding to those cells that have duplicated their genome and where the corresponding sister chromatides could be individually visualized. However, in ~30% of the cells, only one IFN-β FISH signal was visible per cell. This is a situation that could result from allelic pairing, similarly to what has been observed in the case of immunoglobulin loci Igh and Igk (30
) or correspond to two alleles separated by a distance inferior to the resolution of the objective used for image acquisition (200
nm). The FISH signals obtained with the IFN-β probe were specific since no signals were obtained with the empty pBR322 plasmid under the same conditions (Supplementary Figure S1A
Figure 1. In murine fibroblasts, the silent endogenous IFN-β promoter is associated with clusters of γ-satellite sequences. Murine fibroblastic L929 cells either NI or virus-infected by non-pathogenic C13 (C13) or pathogenic ZH548 (ZH) RVFV strain (more ...)
In NI cells, the endogenous IFN-β FISH signals appeared colocalizing with clusters of γ-satellite DNA (A, a–c). Measurement of the overlapping distance of the respective fluorescence intensities through the region of colocalization translated a true colocalization event (Supplementary Figure S1B
). From a total of 256 FISH signals counted from 153 cells from two independent experiments, 49.6
1.5% of the endogenous IFN-β signals colocalized with γ-satellite clusters (C). As expected, no IFN-β mRNA was detected under NI conditions (D) confirming the silent state of the promoter in the absence of virus infection.
After infection with C13 strain (A, d–f), that activated the transcriptional capacity of the IFN-β promoter (D), we observed dissociation of the IFN-β promoter from clusters of γ-satellite sequences. From 276 FISH signals counted (from a total of 167 cells from two independent infections), only 32.97
2.6% of the endogenous IFN-β signals still colocalized with γ-satellite clusters at 8
h after infection by C13 (C). Contrary to the results obtained after infection with C13, no dissociation was observed after infection with pathogenic ZH strain (A, g–i) that maintained the IFN-β promoter in a transcriptionally silent state (D). From 242 FISH signals counted in 137 cells from two independent experiments, 54.95
3% of IFN-β signals (C) remained colocalizing with clusters of γ-satellite sequences after infection with the pathogenic ZH548 strain of RVFV.
Overall, these results showed a correlation between association with PCH clusters and the absence of transcriptional activation as well as vice versa a correlation between statistically significant virus-induced dissociation from PCH and virus-induced IFN-β transcriptional activation.
In NI cells, a maximum of ~50% of the silent endogenous IFN-β loci appeared colocalizing with γ-satellite clusters suggesting the possibility of a monallelic pericentromeric recruitment. In order to determine if the association of IFN-β locus with γ-satellite clusters was indeed monoallelic, we counted, among the population of cells displaying two IFN-β signals per cell, the percentage of cells displaying 2, 1 or 0 FISH signals colocalizing with γ-satellite clusters. As shown in E, under NI conditions, 52.3% of these cells displayed a monoallelic pericentromeric recruitment with only one out of the two FISH signal colocalizing with γ-satellite clusters, whereas under the same conditions, only 25.5% of the cells displayed biallelic pericentromeric recruitment.
In order to further confirm and by doing so enhance the relevance of monoallelic PCH recruitment as a mechanism to regulate IFN-β expression before and after virus infection, FISH experiments as those described in were carried out on macrophages that constitute a major IFN-β producing cell type. Compared to fibroblasts, macrophages displayed a much faster kinetics of virus-induced IFN-β mRNA expression (A) displaying 5 h p.i. a degree of IFN-β expression analogous to the one displayed, under the same conditions, by fibroblasts 8
h p.i. Therefore, in the case of macrophages, the percentage of association was measured 5
h p.i. instead of 8
h p.i. as in the case of fibroblasts. As it can be observed in , results obtained on macrophages 5
h p.i. were very similar to those obtained on fibroblasts 8
h p.i. indicating that monoallelic PCH recruitment of the IFN-β locus was not specific of a single cell type.
Figure 2. In murine macrophages, the silent endogenous IFN-β promoter is associated with clusters γ-satellite sequences. (A) Kinetics of induction (in log10) of IFN-β expression in murine L929 fibroblasts and RAW264.7 macrophages after infection (more ...)
The proximal upstream region of the IFN-β promoter-induced association of the silent IFN-β promoter with pericentromeric heterochromatin
In order to determine if the proximal region of the IFN-β promoter was sufficient to induce the association of the promoter with PCH, FISH experiments were carried out with the murine fibroblastic L929wt330 cell line. This cell line corresponds to a pool of 10 independent L929 clones each one containing the murine IFN-β wt330 promoter region, from −330 to +20 cloned into plasmid pBLCAT3 in front of the CAT reporter gene, randomly integrated into their genome (24
). Until now, this pool of murine IFN-β wt330 promoters has been shown to display the same properties than the endogenous murine IFN-β promoter (24
). For FISH experiments, we used here a biotin-labeled γ-satellite probe (green) and a rhodamine-labeled pBLCAT3IFN-βwt330 probe (red) specific of the integrated IFN-βwt330 promoter. During the establishment of the stably transfected L929wt330 clones, the entire plasmid was integrated into the genome of L929 cells so that during FISH experiments the entire probe hybridized with the integrated locus.
Experiments were carried out in NI cells, when the promoter is maintained in a transcriptionally silent state as well as in cells infected with two different viruses corresponding to the avian paramyxovirus NDV and the previously described Clone13 strain (C13) of RVFV, both being good inducers of IFN-β promoter activity (13
). As before, we used as a control the virulent ZH548 strain (ZH) of RVFV that maintained the promoter in a transcriptionally silent state. For each condition, the percentage of colocalizing FISH signals was quantified and correlated to the transcriptional capacity of the promoter that was determined by measuring the corresponding CAT activities.
The images corresponding to single confocal sections of nuclei of L929wt330 cells either NI (a–c) or collected at 8
h after infection with NDV (d–f), C13 (g–i) or ZH (j–l) are shown in A. The FISH signal obtained with the 5300-bp long pBLCAT3IFN-βwt330 probe was specific of the integrated promoter since no signal corresponding to the endogenous IFN-β promoter was detected with this probe in L929 cells (Supplementary Figure S2A
). As a matter of fact, the only region that the pBLCAT3IFN-βwt330 probe shares with the endogenous IFN-β locus corresponds to the −330 to +20 IFN-β promoter region. This region of 350
bp is much too short to give rise to a visible fluorescent signal compared to the 5300
bp pBLCAT3IFNβwt330 probe that hybridizes with the integrated locus. Also, no signal was obtained in L929wt330 cells with plasmid pENTR/U6 used here as a rhodamine-labeled irrelevant non-specific probe (Supplementary Figure S2B
) further confirming the specificity of the hybridization signal obtained with the pBLCAT3IFN-βwt330 probe on L929wt330 cells.
Figure 3. The −330 to +20 region of the IFN-β promoter induces association of the silent promoter with clusters of γ-satellite sequences. Murine fibroblastic L929wt330 cells either NI or virus-infected by non-pathogenic NDV or RVFV C13 (C13) (more ...)
In NI cells, the transcriptionally silent integrated IFN-βwt330 promoter appeared in its great majority colocalizing with clusters of γ-satellite DNA (A, a–c). As expected for an integrated locus, the large majority of the cells (90.4
3.85%) displayed only one FISH signal per cell (data not shown). From a total of 247 FISH signals counted from 220 cells from six independent experiments, 80.97
8.78% of IFN-βwt330 FISH signals appeared colocalizing with clusters of γ-satellite DNA (B). As expected, no CAT activity was detected in NI cells (C) confirming that in the absence of virus infection the integrated IFN-βwt330 promoter was, as the endogenous promoter, maintained in a constitutive silent state. As in the case of the endogenous promoter, measurement of the overlapping distance of the respective fluorescence intensities through the region of colocalization translated a true colocalization event (Supplementary Figure S2C
Overall, these results suggested that the proximal upstream region from −330 to +20 of the IFN-β promoter could be sufficient to drive association of the silent IFN-β with PCH clusters in NI cells. The −330 to +20 region of the promoter appeared all the more sufficient by itself to drive association with clusters of γ-satellite sequences that association occurred independently of the genomic environment of the integration site. Indeed, in the great majority of NI L929wt330 cells, the IFN-βwt330 FISH signals appeared colocalizing with PCH even though the pool of L929wt330 cells derives from 10 independent clones that have, each one, randomly integrated IFN-β promoter in the genome of L929 cells. Contrary to the endogenous promoter that displayed a monoallelic PCH association (with a maximum of ~50% of IFN-β loci associated with PCH), 80% of the integrated IFN-β loci were observed associated with PCH. This observation suggests that the presence of two alleles could be necessary to regulate monoallelic distribution with respect to PCH as described in the case of immunoglobulin loci (31
As previously observed in the case of the endogenous promoter, dissociation of the integrated IFN-βwt330 promoter from clusters of γ-satellite sequences was observed after infection with non-pathogenic viruses NDV and C13 (A, d–i) that induced the activation of the transcriptional capacity of the IFN-β promoter (C). From 91 (NDV) and 140 (C13) FISH signals counted (from a total of two NDV and three C13 independent infections), only 38.4
2% and 42.85
2.64% of the IFN-β signals still colocalized with γ-satellite clusters at 8
h after infection by NDV and C13, respectively (B), as opposed to 80.97
8.78% of colocalization observed in NI cells. Contrary to results obtained after infection with NDV and C13, no dissociation was observed after infection with pathogenic ZH strain (A, j–l) that maintained the IFN-β promoter in a transcriptionally silent state (C). From 216 FISH signals counted from 195 cells from four independent experiments, 85.18
6.45% of IFN-β signals (B) remained colocalizing with clusters of γ-satellite sequences after infection with pathogenic ZH548 strain of RVFV.
Given the stronger homogeneity of the population IFN-βwt330 FISH signals compared to endogenous IFN-β FISH signals, the subsequent experiments aiming at characterizing the mechanism regulating IFN-β promoter association with γ-satellite clusters before and after virus infection were carried out on the integrated IFN-β promoters.
Virus infection affected the IFN-β promoter region without affecting the structure or organization of γ-satellite sequences
Some viruses, such as HERPES simplex virus type 1, severely damage the structure of cellular centromeres (32
). In order to determine if the viruses used in this work affected the general structure of PCH, we analyzed here the effect of virus infection on the presence of the trimethylated form of lysine 9 of histone H3 (H3K9me3) on γ-satellite sequences as compared to IFN-β promoter as well as on the number and the radial distribution of γ-satellite clusters. Indeed, H3K9me3 is an epigenetic mark concentrated within clusters of γ-satellite sequences that plays an essential role in the general organization of PCH and that it is also commonly found associated with transcriptionally silent regions of the genome in relation with heterochromatization (33–35
As shown in A, H3K9me3 was found present over the IFN-βwt330 promoter under NI conditions, whereas, epigenetic marks predominantly associated with transcriptionally active regions such as the acetylated forms of lysine 8 of histone H4 (H4K8Ac) and of lysine 14 of histone H3 (H3K14Ac), as well as co-activator CBP and transcription factor IRF3, were absent from the silent promoter under NI conditions. The presence of H3K9me3 on the promoter region strongly diminished after NDV-induced promoter activation, contrary to the presence of H4K8Ac, H3K14Ac, CBP and IRF3 that was enhanced. The specificity of the interaction was assessed: no signal was obtained from immunoprecipitates carried out with antibody anti-C23 directed against nucleoline that was used here as an irrelevant antibody.
Figure 4. Virus infection regulates the rate of H3K9me3 present over the IFN-β promoter region without affecting γ-satellite sequences. (A and B) DNA immunoprecipitated (IP) with the indicated antibodies and inputs corresponding to non-immunoprecipitated (more ...)
The effect of virus infection upon the presence of H3K9me3 on γ-satellite sequences as compared to the integrated IFN-βwt330 promoter was analyzed in NI and C13- with respect to ZH-infected cells. As shown in B, the transcriptionally silent promoter from either NI or ZH-infected cells appeared associated with H3K9me3, whereas the presence of H3K9me3 on the transcriptionally active promoter strongly diminished in C13-infected cells as previously observed after NDV infection. This diminution was specific of the integrated IFN-βwt330 promoter with respect to γ-satellite sequences since no change was observed when the same immunoprecipitates were amplified with primers specific for γ-satellite sequences (B). In order to further confirm the lack of effect of virus infection upon PCH structures, γ-satellite cluster formation and distribution were analyzed before and after virus infection. The total number of γ-satellite clusters present per nuclei as well as the radial distribution of these clusters with respect to the edge or the center of the nucleus remained unchanged during virus infection (data not shown). Overall, these results strongly suggested that virus infection affected the degree of association of the IFN-β promoter with clusters of γ-satellite sequences without affecting the organization and structure of PCH itself.
Interestingly, ChIP experiments carried out with antibodies directed against H3K9me3 as well as against the acetylated form of lysine 9 of histone H3 (H3K9Ac) demonstrated that in NI cells both these epigenetic marks were present on the integrated IFN-βwt330 promoter associated with γ-satellite clusters, whereas as expected only the H3K9me3 mark was present over γ-satellite sequences (C). Therefore, even though the integrated IFN-βwt330 promoter colocalized with PCH clusters, it displayed a characteristic combination of epigenetic marks different from that of its proximal surrounding environment.
Virus-induced dissociation of the IFN-β promoter region from γ-satellite clusters preceded strong promoter transcriptional activation
In order to further analyze the correlation between dissociation and transcriptional activation, the percentage of association of the integrated IFN-βwt330 promoter with γ-satellite clusters was analyzed during the time course of infection with either Clone 13 or ZH 548 strains of the RVFV virus. As shown in A, the degree of association of the integrated IFN-βwt330 promoter with γ-satellite clusters observed in NI cells, corresponding to around 80% of the FISH signals, remained constant up to 5
h after C13 infection. In contrast, 6
h p.i., at the onset of strong transcriptional activity, the percentage of FISH signals showing association with γ-satellite clusters was strongly reduced to ~40%. Dissociation and transcriptional activation were not linearly correlated since the highest percentage of dissociation that was reached 6
h p.i. remained constant between 6 and 8
h p.i. in spite of the strong increase of the promoter transcriptional capacity measured between 6 and 8
h p.i. (B). Dissociation therefore appeared as a phenomenon occurring between 5 and 6
h p.i. preceding the onset of strong transcriptional activity. Results shown in A correspond to the average obtained after counting cells from at least three independent experiments. Statistical analysis using the chi-square test showed that the respective population of cells counted was homogenous for all the time points except for 6
h p.i. The stronger variability observed at this time suggested that 6
h p.i. was probably the time of transition from the state of association to the state of dissociation induced after virus infection.
Figure 5. Dissociation of the IFN-β promoter from γ-satellite clusters preceded strong virus-induced promoter transcriptional activation and was reversible. (A–C) L929 wt330 cells infected with either non-pathogenic RVFV C13 (C13) strain (more ...)
Contrary to the results obtained during infection with Clone 13, the transcriptionally silent IFN-β promoter remained associated with PCH during the complete time course of ZH infection (A and B).
Virus-induced dissociation of the IFN-β promoter from clusters of γ-satellite sequences is a reversible phenomenon
Virus-induced IFN-β transcriptional activation is known as a transient, reversible phenomenon. Reversibility is regulated by a post-infection transcriptional turn-off mechanism that is set up 10–12
h after infection that is necessary to bring the IFN-β gene back to its pre-infection silent state (13
). Therefore, if association with PCH was required for the establishment of IFN-β promoter silencing, the IFN-β promoter virus-induced dissociation from PCH was expected to be a reversible phenomenon. In order to analyze the reversibility of the dissociation of the integrated IFN-βwt330 promoter from γ-satellite clusters, we measured the percentage of IFN-βwt330 FISH signals colocalizing with γ-satellite sequences at 8, 12 and 20
h p.i. with C13 or ZH strains of RVFV with respect to NI cells. As shown in C, between 8 and 12
h p.i., the percentage of IFN-βwt330 FISH signals colocalizing with γ-satellite clusters increased from 40% to 70% reaching at 12
h p.i., a percentage of association almost identical to the one observed for the silent integrated IFN-βwt330 promoter in NI cells. All infections were carried out on a population of asynchronously growing L929wt330 cells whose cycling time is of ~20
h. As observed in C, the percentage of association observed 12
h after infection remained constant from 12 to 20
h p.i. indicating that reversibility occurred independently of cell division. Results shown here correspond to the average obtained after counting cells from at least three independent experiments. The strongest variability was observed at 12
h p.i., suggesting that this time was probably the time of transition from the state of virus-induced dissociation to the state of post-infection reassociation.
YY1 binding to the IFN-β promoter regulated the promoter's association with γ-satellite clusters
The IFN-β promoter contains two functional YY1 DNA binding sites present at positions −90 and −122 (18
) that regulate the promoter transcriptional capacity with a dual repressor/activator role (18
). In NI cells, YY1 is predominantly bound to its −90 site. The binding of YY1 to its −122 site is enhanced after NDV or C13 infection but not after ZH infection (19
). After virus infection, simultaneous binding of YY1 to both its −90 and −122 sites was shown to be essential for the recruitment of an activator complex containing histone acetyltransferase CBP.
In order to analyze the eventual role of YY1 binding to the IFN-β promoter upon the regulation of the association/dissociation rate of the promoter with/from γ-satellite clusters, we carried out double FISH experiments on previously constructed L929mut90 and L929mut122 cell lines. These cell lines were constructed as the L929wt330 cell line except that the corresponding IFN-β promoters (from −330 to +20) carry a single point mutation either on the YY1 −90 (L929mut90) or −122 (L929mut122) sites (18
The percentage of the corresponding IFN-β FISH signals associated with γ-satellite clusters was measured before and at 8
h after NDV and C13 infection. Results shown in A and B indicated that virus-induced promoter dissociation from γ-satellite clusters was strongly impaired by the introduction of either mutation mut90 or mut122. Contrary to the results obtained with the wild-type wt330 IFN-β promoter, both the mut90 and the mut122 promoter remained predominantly associated with clusters of γ-satellite sequences after NDV as well as C13 infection (A and B). The inability of promoter mut90 and mut122 to reposition away from γ-satellite clusters was correlated with the particularly weak transcriptional capacity characteristic of both these promoters (C) and (18
). Interestingly, the mut122 promoter that displayed a slightly, but statistically significant, higher degree of dissociation after infection than the mut90 promoter (A and B) also corresponded, as previously shown (18
), to the promoter displaying a slightly higher virus-induced transcriptional capacity (C), indicating once more a correlation between repositioning away from PCH and activation of the promoter transcriptional capacity.
Figure 6. Binding of transcription factor YY1 to the IFN-β promoter regulated the rate of association and dissociation of the promoter with and from γ-satellite sequences. (A) Confocal sections of murine fibroblastic cells containing the IFN-β (more ...)
In NI cells, both the mut90 and mut122 IFN-β promoters displayed a percentage of association with clusters of γ-satellite sequences identical to that determined for the wild-type wt330 promoter (A and B). This indicated that contrary to the dissociation process, either one of the two YY1 binding sites was dispensable for the establishment of IFN-β association with γ-satellite clusters. This could be the result of two different situations, either association was totally independent of YY1 binding to the IFN-β promoter or the presence of only one site was sufficient to allow association.
In order to answer this question, we carried out double FISH experiments on the L929mut122/90 cell line that was generated as previous cell lines except that the corresponding IFN-β promoter (from −330 to +20) was simultaneously mutated on both the −90 and −122 YY1 binding sites (18
). As shown in A and B, only 20% of mut122/90 promoter FISH signals (247 FISH signals counted from 200 cells from three independent experiments) were found associated with γ-satellite clusters in NI cells. As opposed to ~80% of association observed under the same conditions for the wild-type wt330 or the mut90 and mut122 promoters, indicating that binding of YY1 to the promoter was also necessary for the promoter to associate with γ-satellite clusters in NI cells.
The mut122/90 IFN-β promoter is only weakly activated after virus infection (D) and (18
). Nevertheless, in NI cells the mut122/90 promoter remained silent and did not reach the transcriptional activity displayed by this promoter after virus infection even though 80% of mut122/90 IFN-β promoter were dissociated from γ-satellite clusters (D). Therefore, dissociation from γ-satellite clusters was not by itself sufficient to allow transcriptional activation.
In order to further analyze the role of YY1 in the establishment of the association of the IFN-β locus to PCH, we used RNA interference (RNAi) to suppress YY1 in NI L929wt330 cells (A). Diminution of YY1 expression was correlated with a clear diminution of the association of the IFN-β promoter with PCH (B and C). From a minimum of 120 cells counted from two independent experiments, the percentage of association dropped from 84.7% in NI and non-treated cells to 42.7% in NI cells treated with siRNA directed against YY1 (siYY1). A slight diminution of YY1 expression was also observed under the same conditions after treatment of the cells with random control siRNAs (A). Interestingly, this slight diminution of YY1 expression was also correlated with a slight diminution of the percentage of association of the IFN-β promoter with PCH (C) but as expected, the diminution observed after transfection with control siRNAs was significantly less pronounced than the one observed after treatment with siYY1. Overall, these results confirmed the role of YY1 regulating the association of the IFN-β promoter to PCH.
Figure 7. Transcription factor YY1 regulated the association of IFN-β promoter to clusters of γ-satellite sequences in NI cells. (A) RT-qPCR analysis of the expression of the yy1 gene after transfection of NI L929wt330 cells with 50nM final (more ...)
As previously shown in D, a particular combination of opposite epigenetic marks, H3K9me3 and H3K9Ac, was detected over the integrated IFN-βwt330 promoter in NI cells. Since simultaneous binding of YY1 to both its sites present on the IFN-β promoter has been shown to regulate CBP promoter recruitment as well as the acetylation of histones positioned over the promoter region (19
), we analyzed the effect of YY1 binding upon the presence over the promoter region of H3K9me3 and H3K9Ac (). The presence of both these epigenetic marks appeared diminished on YY1-mutated promoters with mutation mut90 predominantly affecting the presence of H3K9me3 and mutation mut122 predominantly affecting the presence of H3K9Ac. Nevertheless, the ratio H3K9me3:H3K9Ac remained >1 in the case of promoters wt330, mut90 and mut122 under NI conditions when these promoters were observed associated with γ-satellite clusters, whereas the H3K9me3:H3K9Ac ratio became <1 in the case of promoters that were dissociated from γ-satellites clusters such as promoter mut122/90 or the wild-type wt330 promoter 8
h after infection with C13. It is therefore tempting to hypothesize that YY1-dependent regulation of the ratio of H3K9me3:H3K9Ac over the IFN-β promoter region could influence the degree of association of this promoter with clusters of PCH.
Figure 8. YY1 binding and virus infection regulates the rate of H3K9me3/H3K9Ac present over the integrated IFN-β promoters. (A and C) DNA immunoprecipitated (IP) with the indicated antibodies and inputs corresponding to non-immunoprecipitated genomic DNA, (more ...)