Figure 2. H2A.Z.1 and H2A.Z.2 regulate both distinct and overlapping sets of genes.
RNA-Seq data was analysed for differential gene expression in samples transfected with either H2A.Z.1 siRNA or H2A.Z.2 siRNA versus the control siRNA sample. (A) Venn diagram showing the intersection between genes differentially expressed upon H2A.Z.1 and H2A.Z.2 inhibition. The p value indicated below the diagram indicates the significance of the intersection calculated using the Chi square test considering all expressed genes. The numbers in brackets indicate the expected number of genes considering the total number of expressed genes if intersection was random. (B) Same as in A, except that the intersections indicate genes that are up-regulated or down-regulated in each sample and those that are regulated in an opposite way. Note that the intersection between genes up-regulated upon H2A.Z.1 depletion and down regulated upon H2A.Z.2 depletion is not highly significant. (C) U2OS cells expressing endogenously Flag-tagged H2A.Z.1 or H2A.Z.2 were subjected to ChIP-Seq experiments using anti-Flag antibodies. Metadata showing ChIP-Seq signals around TSS were computed for the five classes of genes (Unch: unchanged upon H2A.Z.1 or H2A.Z.2 depletion) defined from RNA-Seq data obtained in U2OS upon H2A.Z.1 or H2A.Z.2 depletion (see Figure 2—figure supplement 3). A representative experiment is shown. A replicate is shown in Fig. Figure 2—figure supplement 5A.
Figure 2—figure supplement 1. Validation of RNA Seq results.
WI38 cells were transfected with the indicated siRNAs. 72 hr later, total RNA was prepared and further purified to be subjected to RNA-Seq or analysed by RT-qPCR. RNA-Seq signals were averaged for the indicated genes and calculated relative to one for the control siRNA sample. RT-qPCR data for the indicated genes was standardised using GAPDH mRNA levels and calculated relative to one for cells transfected with the control siRNA. The data for the two samples sent to RNA-Seq are shown.
Figure 2—figure supplement 1—source data 1. Source data of the histogrammes representing the validation of RNA-seq effects by RT-qPCR on Figure 2—figure supplement 1.
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Figure 2—figure supplement 2. Effect of a second siRNA H2A.Z.2 (siZ2#).
WI38 cells were transfected using a control siRNA or a second independent siRNA against H2A.Z.2 (siZ2#). 72 hr later, total RNAs was prepared. The amount of the indicated mRNA was quantified by RT-qPCR, and was standardised using GAPDH mRNA levels and calculated relative to one in cells transfected using the control siRNA. The mean and SDOM from three independent experiments are shown.
Figure 2—figure supplement 2—source data 1. Source Data for the histogrammes representing the effect of a second siRNA H2A.Z.2 (siH2A.Z.2#) on Figure 2—figure supplement 2.
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Figure 2—figure supplement 3. RNA-seq analysis after H2A.Z.1 and H2A.Z.2 depletion in U2OS cells.
(A) U2OS cells were transfected with the indicated siRNAs. 72 hr later, total RNA was prepared. The amount of H2A.Z.1 and H2A.Z.2 mRNA was quantified by RT-qPCR, standardised using GAPDH mRNA levels and calculated relative to one for cells transfected with the control siRNA. The mean and SDOM from three independent experiments are shown. (B) H2A.Z.1 and H2A.Z.2 regulate specific and common genes in U2OS cells. RNA Seq data from U2OS cells were analysed for differential gene expression in samples transfected by either the H2A.Z.1 siRNA or the H2A.Z.2 siRNA versus the control siRNA sample. Venn diagram showing the intersection between the genes differentially expressed upon H2A.Z.1 and H2A.Z.2 inhibition, between genes up-regulated in the two samples, down-regulated in the two samples or regulated in an opposite way in the two samples. Note that, as for WI38, the intersection between genes upregulated upon H2A.Z.1 depletion and down-regulated upon H2A.Z.2 depletion is not highly significant. The numbers in brackets indicate the expected number of genes considering the total number of expressed genes if intersection was random. The p-value was calculated using the Chi-square test considering all expressed genes. (C) H2A.Z.1 and H2A.Z.2 regulate different genes in WI38 and U2OS cells. Venn diagram showing the intersection between lists of differentially expressed genes from RNA Seq data from WI38 and U2OS cells. Note that despite significant overlap, gene lists were mostly different. The numbers in brackets indicate the expected number of genes considering the total number of expressed genes if intersection was random. The p-value was calculated using the Chi-square test considering all expressed genes.
Figure 2—figure supplement 3—source data 1. Source data of the histogramme representing the depletion of H2A.Z.1 and H2A.Z.2 in response to siRNAs in U2OS cells in Figure 2—figure supplement 3.
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Figure 2—figure supplement 4. Profiles of tagged H2A.
Z.1 and H2A.Z.2 ChIP-Seq data at the CDKN1A/p21 and GAPDH loci. The two replicates of ChIP-Seq experiments using Flag antibodies from U2OS cells expressing either genome edited H2A.Z.1 or H2A.Z.2 were visualized on IGB (Integrated Genome Browser). The tracks show the normalized number of aligned reads of ChIP-seq datasets at the CDKN1A/p21 (A) and GAPDH (B) genes. RefSeq genes (hg38) are also shown with their DNA strands in brackets (if several transcript variants exist, only the 1st transcript variant is shown for simplicity).
Figure 2—figure supplement 5. Analysis of H2A.Z.1 or H2A.Z.2 presence around TSS and enhancers.
(A) Replicates of ChIP-Seq experiments using Flag antibodies from U2OS cells expressing either genome edited H2A.Z.1 (left) or H2A.Z.2 (right) analysed as in Figure 2C. (B) Box plots representing the ratio of the mean amount of H2A.Z isoforms ChIP-Seq signals around all TSS (59,553 TSSs), at all U2OS enhancers (obtained through enhanceratlas: http://www.enhanceratlas.org/, 14,764 enhancers) and at 1000 random genomic sequences of 1000 bases. (C) Metadata showing the mean of H2A.Z.1 and H2A.Z.2 ChIP Seq signals on the 10 kB region encompassing all TSSs and all enhancers. For enhancers, the ‘0’ position corresponds to the centre of enhancers defined in enhanceratlas. (D) TSSs and enhancers were sorted in five classes according to the total levels of H2A.Z. The box plots show the ratio of the mean amount of H2A.Z isoforms ChIP-Seq signals around TSS (24,695/22,263 TSSs), and U2OS enhancers (1,707/1,459 enhancers) falling into the highest class (4 < ln (H2A.Z.1 + H2A.Z.2 ChIP-Seq signals)<5). (E) Metadata showing the mean of H2A.Z.1 and H2A.Z.2 ChIP Seq signals on the 10 kB region encompassing the TSS and enhancer populations defined in D). For enhancers, the ‘0’ position corresponds to the centre of enhancers defined in enhanceratlas.