A) ChIP-seq profiles across 120 MB of chromosome 1 in mouse ES cells, using antibodies as indicated. Y-axis represents the number of reads spanning a genomic position (scale on right, (baseline, maximum)). CpG islands (green) and genes (refseq (+) and refseq (−)) are shown below plots. Data for general H3 in ES cells is from ().
B) Annotations of H3 variant enriched regions. Y-axis is the number of enriched regions identified for each H3 variant, while the X-axis represents different categories of gene annotation and ES TFBS (defined in ).
C) Association of H3.3 with repetitive elements. Data are presented as fold enrichment over input. SAT = satellite repeats, (TTAGGG)n = telomeric DNA repeats. See for details of ZFN-mediated generation and characterization of ES cell lines, and for genome-wide correlation of ChIP-seq datasets.

A–F) Profiles of H3 variants, H3, H3 modifications, or RNAPII as indicated above each panel across the TSS and TES for highly active (red), medium expressing (black), or low expressing (green) CpG rich genes in ES cells. Y-axis represents the average number of tags per gene per 200 bp per 1 million mapped reads. H3 data is from (). A–E is from crosslinking ChIP-seq, while F is from native ChIP-seq.
G) H3.3 is enriched around the TSS, but not into the gene body of the H3K4me3/H3K27me3 bivalent and transcriptionally repressed Zfpm2 gene in ES cells. In G-I, hybrid indicates F1 hybrid ES background.
H) H3.3 and RNAPII Ser5p are enriched in the gene body and after the TES of the highly expressed gene Actb (beta-actin) in ES cells. Data for the RNAPII unphos 8WG16 track are from (). RNA-seq data are from ().
I) H3.3 is enriched around the TSS and into the body of the non-coding RNA gene Gas5, but less enriched in the gene body of the neighboring low expressing Zbtb37 gene in ES cells. Data is represented as in . Gene is shown to scale below plot, with start and direction or transcription indicated by arrow. Green rectangles above the gene represent CpG islands. See also .

A) H3.3, H3K4me1, and H3K36me3 are enriched in the gene body of the highly expressed pluripotency gene Esrrb in ES cells, and this enrichment is largely lost upon differentiation to NPCs.
B) The epidermal growth factor receptor gene Egfr is H3K4me3/H3K27me3 bivalent and transcriptionally repressed in ES cells. Upon differentiation to NPCs, H3.3, H3K4me1, and H3K36me3 are enriched in the Egfr gene body.
C) H3.3, H3K4me1, and H3K36me3 are enriched in the gene body of the housekeeping gene lactate dehydrogenase A Ldha in both ES cells and NPCs, and is not enriched in the neighboring Ldhc gene. The Y-axes in panels A-C are identical (indicated in the right side of panel C).
D–E) H3.3 is enriched genome-wide around ES cell TFBS. Crosslinking ChIP-seq genome-wide profiles of H3 variants as indicated around ES binding sites for Nanog, Oct4, Sox2, Smad1, and STAT3. TFBS in mouse ES cells were from a previous ChIP-seq study (), and classified as promoter (black), gene body (dark blue), or intergenic (light blue). Panel D represents data from ES cells, while panel E represents data from NPCs at identical regions. Y-axis represents number of tags per binding site per 200 bp per 1 million mapped reads. Data for H3 in ES cells is from (). In A–E, “(native)” indicates native H3.3-HA ChIP-seq, and “(replicate)” indicates biological replicate H3.3-HA ChIP-seq from F1 hybrid ES background.
F) H3.3 and H3K4me1 are enriched at an intergenic region bound by multiple transcription factors () specifically in ES cells.
G) H3.3 and H3K4me1 are enriched at an intergenic region specifically in NPCs. See also .

A) Immunoblots showing expression of H3.3-EYFP in wild-type and Hira−/− ES cells, with antibodies as indicated.
B–E) Native ChIP-seq profiles of H3 variants, in wild-type ES cells (B-D) and Hira −/− ES cells (E) across the TSS and TES for highly active (red), medium expressing (black), or low expressing (green) CpG rich genes, with data represented as in . See also .
F) H3.3 enrichment at the TSS of repressed bivalent gene Pparg is Hira-dependent.
G) H3.3 enrichment at the active, ribosomal-protein coding gene Rps19 is Hira-dependent.

A) Native ChIP-seq genome-wide profiles of H3.3 around previously described ES TFBS (), with data analyzed as in .
B–C) Localization of H3.3 in ES cells at intergenic enhancer elements near Mmp17 (B) and Foxi3 (C) is Hira-independent. See also and .

A) H3.3-EYFP, H3.2-EYFP, H3.1-EYFP, and Hira −/− H3.3-EYFP associated proteins were immunopurified from heterozygous H3.3B ES cells via the EYFP tag, resolved by SDS-PAGE, and visualized by Coomassie Blue. Proteins were identified by mass spectrometry, and those listed in red are specific to H3.3. The arrow indicating Hira points to a polypeptide in the H3.3 lane running immediately above the polypeptide Nasp (*), which is common to all purified H3 variants. A full list of identified proteins is presented in .
B) Immunoblots of immunopurified H3 variant associated proteins from ES cells with antibodies as indicated. See also .
C) Immunoblots of Atrx, Daxx and FLAG-HA-H3 from HeLa cells stably expressing H3.3-FLAG-HA (FHA) or H3.1-FHA ().
D) Oligonucleosomes purified from H3.3-FHA and H3.1-FHA HeLa cells. Micrococcal nuclease digested chromatin was incubated with M2 agarose beads in order to purify H3.3-FHA and H3.1-FHA oligonucleosomes and associated proteins. Two adjacent FLAG elution fractions (FE1 and FE2) from each H3.3-FHA and H3.1-FHA purification are shown in D–E. Oligonucleosomal DNA was purified and run on a 1% agarose gel, with fragment size indicated.
E) Immunoblots of eluate from two adjacent FLAG elution fractions (FE1 and FE2) following FLAG affinity purification from H3.3-FHA and H3.1-FHA oligonucleosomes (D).

A) Immunoblots showing expression of H3.3-EYFP in Atrxflox, Atrxnull, and Hira −/− ES cells, with antibodies as indicated.
B) ChIP-seq analysis of H3.3 enrichment at repetitive elements in Atrxflox, Atrxnull, and Hira −/− ES cells. Fold enrichment of repetitive elements in ChIP-seq data are plotted over input.
C) Immunofluorescence (IF) and telomere fluorescence in situ hybridization (FISH) of Atrxflox, Atrxnull, and Hira −/− ES cells expressing H3.3B/H3.3-EYFP. Representative confocal images of interphase ES cells demonstrate that H3.3 co-localization with telomeres is lost in Atrxnull ES cells (middle), but maintained in Hira −/− ES cells (right). Bar = 5μm.
D) ChIP of Atrx, control IgG, and H3 was performed in Atrxflox and Atrxnull ES cells, followed by slot blot with a telomere probe.
E) Immunoblots of Atrx in Atrxflox, Atrxnull, and Atrxflox ES cells 4 days after treatment with retroviral Cre. Atrxt refers to a previously identified alternative splicing product of Atrx that remains expressed in Atrxnull ES cells ().
F) Representative northern blot for TERRA (upper panel) and Gapdh (lower panel) in Atrxflox and Atrxnull ES cells in the presence and absence of Cre. To ensure that signal is from RNA, samples on the right were treated with RNase for 30 minutes as indicated.
G) Quantification of TERRA normalized to Gapdh. Error bars represent the standard deviation of three independent experiments. See also .