Supplemental Figure 1 A

Transcription

1 Supplemental Figure A prebleach postbleach 2 min 6 min 3 min mh2a.-gfp mh2a.2-gfp mh2a2-gfp GFP-H2A..9 Relative Intensity mh2a. GFP n=8.4 mh2a.2 GFP n=4.3 mh2a2 GFP n=2.2 GFP H2A n=24. GFP n= Time (min) Supplemental Figure. FRAP analysis of canonical H2A and mh2a isoforms. (A) Representative images from long-term FRAP time series of HeLa cells transiently transfected with GFP-H2A or mh2a-gfp isoforms. Squares of 5 µm x 5 µm within the nucleus were photobleached and fluorescence recovery followed for at least 5 hours. Prebleach, postbleach, and 2min, 6min, 3min postbleach are shown. () Quantitative FRAP evaluation. mh2a isoforms (orange and yellow) display a decreased recovery compared to canonical H2A (blue). mh2a2 (red) displays decreased recovery compared with canonical H2A and mh2a isoforms. In contrast, GFP alone shows full recovery within less than a minute (green).

2 Supplemental Figure 2 A Imp9 ATRX Nuclear Import and Export Factors Protein H2A peptide count macroh2a peptide count C Ncln NAP SET H2A-GFP m.2-gfp Importin Importin Importin Importin KapA 48 4 Imp Npm KapA KapA KapA KapA KapA D GFP H2A-GFP Input GFP mh2a.2-gfp IP H2A-GFP mh2a.2-gfp Kap 4 4 Exportin Exportin 5 3 Exportin 7 6 Transportin 4 7!Imp9 mh2a.2-gfp GFP-H2A GFP!GFP Supplemental Figure 2. H2A and mh2a.2 associate with nuclear-cytoplasmic shuttling factors and chromatin-associated factors in chromatin-free extracts. (A) Proteins interacting with either GFP-H2A or mh2a.2-gfp in chromatin-free extracts were resolved on 4-2% NuPAGE and silver stained. Each lane was cut into ten slices and MS analyzed. Chromatin-associated factors identified by MS are presented alongside the gel (see Fig. D for peptide counts). lue characters = H2A or mh2a.2-specific factors; black characters = chromatin-associated factors which interact with both H2A and mh2a.2; green = GFP histones. () Table of nuclear-cytoplasmic shuttling factors that interact with H2A or mh2a.2 in chromatin free extracts. These data were collected from MS analysis of the gel presented in (A) and Supplemental Table. (C) Chromatin-free IP and silver staining of proteins interacting with GFP-H2A or mh2a.2-gfp resolved on 4-2% NuPAGE. Unique bands were excised and MS analyzed. Arrow indicates Imp9; molecular weight marker shown on right. (D) GFP, GFP-H2A or mh2a.2-gfp IPs were resolved on 2% PAGE and I d for Imp9 (upper panel) or GFP (lower panel).

3 A Protein H2A Peptide counts mh2a.2 Peptide counts C Supplemental Figure 3 NAP SET 2 9 Nucleolin 79 8 Nucleophosmin 7 42 ATRX 9 Importin Input IP αgfp αatrx αh EEGTSSSEK LIETTANMNSSYVK SVLADIKKAHLALEEDLNSEFRAMDAVNKEKNTK RQTQSESSNYDSELEKEIK TSQEGSSDDAERKQERETFSSAEGTVDK NGAYGREK RNTKEIQSGSSSSDAEESSEDNK AVIVKEKK LTVSDGESGEEKKTK EEEEEEEEEEEEEEEDENDDSKSPGKGRKKIR RPQERSYMLQRWQEDGGVMIIGYEMYRNLAQGRNVK NEASAVSKAMNSIRSRRR FLAQGTMEDKIYDR RDTPMLPK ENMNLSEAQVQALALSRQASQELDVKRR (6-292) ADD (ATRX-DNMT3- DNMT3L) (56-595) HPαinteracting domain (89-326) DID Daxx-interacting domain (44-47) Acidic Patch ( ) ATPase Domain Supplemental Figure 3. ATRX interacts with mh2a in chromatin-free extracts. (A) Peptide counts of chaperones identified by MS for H2A and mh2a.2 (See corresponding gel in Supplemental Fig. 2A). () ATRX peptides identified by MS analysis in mh2a.2 IPs from chromatin-free extracts. Peptides presented next to a schematic of ATRX protein with its various domains shown. Nineteen peptides in total were identified (5 are presented here as 4 peptides were overlapping). (C) Is of GFP and ATRX from chromatin-free extracts by independent chromatin fractionation method (Mendez and Stillman 2).

4 Supplemental Figure 4 ATRX fragments Full length ATRX N-term (-84) Middle (8-67) C-term ( ) Supplemental Figure 4. Expression of ATRX deletion constructs. Fluorescent images of GFPtagged ATRX constructs transiently expressed in HEK293 cells. While the N-terminal construct expressed strongly, enrichment of mh2a signal observed in the N-terminal IP (Fig. 2C, D) suggests that this region is sufficient for binding to mh2a.

5 Supplemental Figure 5 A αatrx αatrx Amido lack Amido lack HeLa.2. HEK 293 C DAPI αatrx Merge shluc sh92 Supplemental Figure 5. shrna-mediated depletion of ATRX in 293 and HeLa.2. cells. (A) αatrx I of HEK 293 cells expressing five lentivirally introduced ATRX shrnas, and control shluc. sh9 and sh92, used for subsequent studies, are highlighted in red; Amido black of histones shown for loading. (, C) ATRX depletion in HeLa.2. cells shown by I () and immunofluorescence (C). Note presence of ATRX at telomeres.

6 Supplemental Figure 6 ATRX sh_ 92 Relative Abundance m/z Supplemental Figure 6. ATRX knockdown results in mh2a accumulation in chromatin. Comparison of an averaged full mass spectrum for the +2 charged mh2a peptide (SAKAGVIFPVGR) from sh92 ATRX knockdown (d -labeled) and shluc control (d 5 -labeled) histones extracted from chromatin.

7 Supplemental Figure 7 A HEK 293 K562 shluc sh9 Probe: telomere repeat shluc sh shrna: mh2a/igg luc 9 sh92 mh2a/input Probe: telomere repeat shrna: luc 9 92 Supplemental Figure 7. Loss of ATRX results in telomeric accumulation of mh2a. (A) One of two representative ChIP-telomere Southern blots for HEK293 cells shows the increased association of mh2a with telomeric chromatin in the absence of ATRX. Only sh9 was used as it induced the most efficient knockdown in this cell line (see Supplemental Fig. 4). () iological replicate of telomere Southern blot in K562 cell line. mh2a s presence at telomeres of K562 cells is increased in the absence of ATRX in both sh9 and sh92 lines. Densitometry quantitation presented below each graph.

8 A ATRX mh2a Supplemental Figure 8 Normalized to GAPDH shrna: luc luc 9 92 POLR3K MPG C6orf35 ITFG3 TMEM8A NME4 DECR2 CDK8 Normalized to GAPDH sh_9 sh_9 sh_9 sh_9 sh_9 sh_9 sh_9 sh_9 Supplemental Figure 8. ATRX knockdown results in reduced RNA levels of sub-telomeric chromosome 6 genes. (A) shrna-mediated knockdown of ATRX (sh9 and sh92) in K562 cells results in the loss of ATRX mrna, compared to shluc, without affecting mh2a mrna. () Knock down of ATRX results in decreased mrna levels of genes found in the α globin cluster of subtelomeric chromosome 6. Starting with the most telomere proximal, genes assayed include POLR3K, MPG, C6orf35, ITFG3, TMEM8A, NME4 and DECR2. Genes assayed are circled in blue in the UCSC browser above. CDK8 transcription (chromosome 3) is unaffected by ATRX knockdown, similar to mh2a (chromosome 5) in (A). Expression was measured relative to GAPDH and to the control shluc, whose expression values were arbitrarily set as.

9 Supplemental Figure 9 A [FU] 5 Input Luc mh2a 92 mh2a Luc [bp] sh92 shluc Input (shluc) ATRX Input (ATRX) Raw reads owtie alignments (wiggle) Alignments analyzed (MACS) Total peaks count (MACS) Total peak length (bp) (MACS) C shluc 6543 ~54% sh ~57% Supplemental Figure 9. ChIP-sequencing of mh2a in shluc and sh92 K562 cells. (A) ioanalyzer traces of ChIP (mh2a) and Input DNA from MNase digested chromatin (High sensitivity DNA chip; Agilent Technologies). Isolated mononucleosomal DNA (arrow) was size selected for ChIP-Seq library preparation. Input (red), sh92 (blue) and shluc (green). Peaks at 35bp and 38bp are internal size markers. () Raw number of reads obtained by Illumina Hi-Seq, total number of alignments (owtie: -m 2 -k 2 -n 2 -l 5), and alignments used for peak calling (MACS) for sh92, shluc and Input shown. Raw reads for the ATRX ChIP-Seq were downloaded from GEO (GSE2262). Also shown are the total number of peaks for sh92 and shluc (p value cutoff =.e-4) and total number of base pairs covered by significant peaks (MACS). (C) Overlap of significant mh2a peaks (MACS) from shluc and sh92 ChIP-seq data sets (HOMER software, Heinz et al. 2).

10 A. TSS TES Supplemental Figure.8 Normalized Read Count (per Million reads) ,-(.%&%'()%!"#$%&%'()%!"#$%&%*#% Full High Low C mh2a_shluc ~99% mh2a_sh ~99% ATRX 5643 ~93% ATRX 553 ~92% position!"#$%&''&()$#"*+,$-(.)/!!"#$%&''&()$#"*+,$-(.)/$ 7.''$8")"$,"/!!#*"!#)"!#("!#'"!#&"!#%"!#$"!" +(!!!" +'!!!" +&!!!" +%!!!" +$!!!"!" $!!!" %!!!" &!!!" '!!!" (!!!" &,/*)-"$/($22$3456! 7&89$":5#",,"+$8")",!!#*"!#)"!#("!#'"!#&"!#%"!#$"!" +(!!!" +'!!!" +&!!!" +%!!!" +$!!!"!" $!!!" %!!!" &!!!" '!!!" (!!!" &,/*)-"$/($22$3456!!"#$%&''&()$#"*+,$-(.)/$ 7(8$"95#",,"+$:")",!!#*"!#)"!#("!#'"!#&"!#%"!#$"!" +(!!!" +'!!!" +&!!!" +%!!!" +$!!!"!" $!!!" %!!!" &!!!" '!!!" (!!!" &,/*)-"$/($22$3456!,-./%",-.2" 3456" 789:",-./%",-.2" 3456" 789:",-./%",-.2" 3456" 789:" Supplemental Figure. TSS/TES analysis and peak overlap of mh2a and ATRX. (A) Read counts (2bp window) normalized to total number of reads (counts per million reads), plotted against the distance (-5Kb, +5Kb), from the nearest annotated Transcription Start Site (TSS, left), and Transcription End Site (TES, right). Genes were grouped by expression levels to Full (all annotated genes), High and Low categories based on K562 RNA-Seq data from the ENCODE project. sh92 (black), shluc (red), and Input (green). () Same TSS analysis as in (A) also containing ATRX (blue). Scale is different than in (A). (C) Overlap of significant mh2a and ATRX peaks (MACS) in shluc and sh92 samples (Law et al. 22; Heinz et al. 2).

11 A CHR6: 7 - Supplemental Figure qpcr amp: mh2a (sh92) 2Kb Normalized alignments count (5bp window, sliding 25bp) RefSeq Transcription Enhancer Promoter SINE LINE LTR DNA SIMPLE L. COMPLEXITY mh2a (shluc) ATRX Input (shluc) C6orf35 HZ HM HA2 HA HQ < < < Significant peaks Dup >bp Percentage Input" (!" '!" &!" %!" $!" #!" mh2a ChIP" sh92" shluc"!" mh2a mock mh2a mock -27kb -5kb -4kb -kb" -kb TSS Primer position: " " Supplemental Figure. mh2a is enriched at the α globin cluster. (A) Capture of UCSC genome browser showing ~5kb region around α globin locus. ChIP seq analysis of mh2a in K562 cells. Loss of ATRX (sh92) results in redistribution of mh2a compared to control cells (shluc) as shown in Fig. 5D (shown on the X-axis genomic position in Kb; Y-axis alignment counts in 5bp window sliding 25bp. Window counts are normalized to total number of alignments and scaled by ^7 for mh2a and ^6 for ATRX). ChIP-seq data for mh2a was compared to published ATRX ChIP-seq data (Law et al. 2). Shluc Input enrichment is shown for reference. Threshold line set at 35 to facilitate visualization. Regions of significant enrichment are indicated by black bars below the respective enrichment plots. UCSC custom tracks are shown, RefSeq gene annotation, K562 Chromatin State Segmentation by HMM from ENCODE/road, Repeat elements by RepeatMasker, and Duplications of > ases of Non-RepeatMasked Sequence are shown, respectively. Primer pairs used in (native ChIP) and C (fixed ChIP) shown on top. As the HA genes are duplicated, primer pair 6 at the TSS is presented twice. () Validation by qpcr of the ChIPsequencing library indicates enrichment of mh2a at regions upstream of the HA/2 genes. (C) ChIP via standard formaldehyde cross-linking and sonication demonstrates increased mh2a levels kb upstream and at TSS of the HA/2 genes when ATRX is depleted. Mock = no antibody. One of three replicates (two biological and one technical) shown. The stronger enrichment of mh2a signal in native ChIP () compared with formaldehyde-fixed ChIP (C) is likely due to greater antibody affinity for mh2a in the native ChIP protocol. C Percentage Input !"#$%&!"'(&!"')&