Acetylated Histone H3K56 Interacts with Oct4 to Promote Mouse Embryonic Stem Cell Pluripotency

Acetylated histone H3K56 interacts with Oct4 to promote mouse embryonic stem cell pluripotency

Yuliang Tan, Yong Xue, Chunying Song, and Michael Grunstein1

Department of Biological Chemistry and the Molecular Biology Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095

Contributed by Michael Grunstein, May 29, 2013 (sent for review April 29, 2013) The presence of acetylated histone H3K56 (H3K56ac) in human ES E14Tg2a ESCs. Approximately 85.5 million reads uniquely mapped cells (ESCs) correlates positively with the binding of Nanog, Sox2, to the mouse genome and 92,549 H3K56ac significantly enriched and Oct4 (NSO) transcription factors at their target gene pro- regions were identified. We then wished to determine whether moters. However, the function of H3K56ac there has been unclear. H3K56ac presence is associated with the key transcriptional fac- We now report that Oct4 interacts with H3K56ac in mouse ESC tors genome-wide. By comparing the correlation of H3K56ac and nuclear extracts and that perturbing H3K56 acetylation decreases known transcription factors using our H3K56ac chromatin im- Oct4–H3 binding. This interaction is likely to be direct because it munoprecipitation sequencing (ChIP-Seq) results and published can be recapitulated in vitro in an H3K56ac-dependent manner data (4), we found using Cistrome (19) that H3K56ac correlates and is functionally important because H3K56ac combines with most positively with the presence of Oct4 and Sox2 and much less NSO factors in chromatin immunoprecipitation sequencing to mark so with Nanog (Fig. 1A;Pearson’s correlation values in SI Appendix, the regions associated with pluripotency better than NSO alone. Table S1). This is consistent with the frequent association of Oct4 Moreover, reducing H3K56ac by short hairpin Asf1a decreases ex- and Sox2 at their regulatory regions (4, 20) and the less abun- pression of pluripotency-related markers and increases expression dant interaction of these factors with Nanog (21, 22). Therefore, of differentiation-related ones. Therefore, our data suggest that mouse H3K56ac correlates best genome wide with the presence H3K56ac plays a central role in binding to Oct4 to promote the of Oct4 and Sox2. pluripotency of ESCs. H3K56ac Interaction with Oct4 in Mouse E14Tg2a Cells and in Vitro. It GENETICS he core transcriptional network, which contains transcription is known that despite their functional association on chromatin, Tfactors, chromatin regulators, and signaling proteins, helps Oct4 and Sox2 do not necessarily coimmunoprecipitate (23–25). determine self-renewal and pluripotency in ES cells (ESCs) and Therefore, we wished to determine by coimmunoprecipitation cell fate upon their differentiation (1, 2). This network is defined (co-IP) whether Oct4 or Sox2 separately interact with H3K56ac. in part through the action of the key transcriptional regulators We found that, unlike Sox2, Oct4 coimmunoprecipitates strongly Nanog, Sox2, and Oct4 (NSO) that recognize defined DNA se- with H3K56ac in E14Tg2a cell nuclear extracts (Fig. 1B). We quence motifs at target genes of the core transcriptional net- also noticed the trace interaction between Nanog and H3K56ac, work. There, the key regulators are involved in the activation of an interaction we have not pursued. We conclude that of the key pluripotency-related genes and repression of differentiation re- transcription factors, Oct4 interacts most with H3K56ac in lated ones (3–5). Consequently, changes in the expression levels nuclear extracts. of the key factors perturb this transcriptional circuitry, leading to We next asked whether the acetylation of H3K56 enhances the the loss of pluripotency (6). Of the key factors, Oct4, a homeo- interaction between histone H3 peptides and Oct4 by peptide pull- domain transcription factor of the POU family, is unique (7). down assays (26). Biotinylated H3 peptides containing acetylated Although ectopic expression of four factors (Oct4, Sox2, Klf4, K56 (or acetylated K9 used as a control) were used to pull down and c-Myc) together can generate induced pluripotent stem cells Oct4 in nuclear extracts of E14Tg2a cells. We found that acety- from somatic cells (8), only Oct4 is essential and sufficient in lation of K56 in peptides improves the efficiency of Oct4 binding reprogramming adult neural stem cells to pluripotency (9). compared with unmodified peptides and control peptides con- Our previous work using Agilent microarrays found that his- taining acetylated K9 (Fig. 1C). We further confirmed the im- tone H3K56ac is associated with upstream regulatory DNA portance of H3K56 in Oct4 binding using substitutions of histone sequences of the core transcriptional network in human ESCs H3K56. Plasmids that express wild type (WT) histone H3.1 with (10). H3K56ac is conserved from yeast to mammals and is very a C-terminal Flag tag and similar constructs bearing substitutions ∼ abundant in yeast, marking 30% of total histone H3. Yeast at K56 (H3.1K56A, H3.1K56Q, and H3.1K56R) or at K9 H3K56ac is involved in gene transcription (11), replication- (H3.1K9A) were transfected into E14Tg2a cells. We then immu- independent histone assembly (12), DNA repair (13), silencing noprecipitated H3.1-Flag and examined the immunoprecipitate at heterochromatin (14), transcription elongation (15), and life for Oct4 presence using Western blots. Although H3.1-Flag and span regulation (16). However, its role in mammalian cells where Oct4 co-IP are not decreased by substitution of H3.1K9A, sub- H3K56ac marks less than 1% of total H3 (10, 17, 18) is less clear stitution of H3.1K56 by A, Q, or R decreased Oct4 co-IP con- and is the focus of this article. Remarkably, we found that siderably (Fig. 1D). Collectively, our data indicate that H3K56ac is H3K56ac interacts with Oct4 both in vivo in mouse ESCs and required for the interaction between histone H3 and Oct4 in vivo. directly in vitro. This interaction is in addition to that provided by the Oct4 recognition element in DNA. Our data indicate that H3K56ac has a central function in ESCs, where it binds the key Author contributions: Y.T. and M.G. designed research; Y.T., Y.X., and C.S. performed transcription factor Oct4 to promote pluripotency. research; Y.T. and M.G. analyzed data; and Y.T. and M.G. wrote the paper. fl Results The authors declare no con ict of interest. Data deposition: The primary sequencing data reported in this paper have been depos- H3K56ac Presence Is Correlated with That of Key Transcriptional ited in the Gene Expression Omnibus database (GEO), www.ncbi.nlm.nih.gov/geo (acces- Regulators in Mouse ESCs. To determine whether H3K56ac is as- sion no. GSE47387). sociated with the pluripotency network in mouse ESCs, we used 1To whom correspondence should be addressed. E-mail: [email protected]. massively parallel ChIP-DNA sequencing using Illumina HiSeq- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2000 to examine the genome-wide distribution of H3K56ac in 1073/pnas.1309914110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1309914110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Fig. 1. H3K56ac interaction with Oct4 in mouse Sox2 Nuclear Extracts E14Tg2a cells and in vitro. (A) Correlation heat map: A D the degree of target cooccupancy of factors in the Oct4 mouse genome is shown. Pearson’s correlation val- H3K56ac ues were calculated by Cistrome (19). Red indicates H3.1-wt H3.1K9AH3.1K56AH3.1K56QH3.1K56R Oct4 more frequent colocalization of each pair of factors Nanog as shown using Cluster 3.0 (47). (B) Co-IP shows the Flag interactions between IgG, Nanog, Sox2, and Oct4 Smad1 with H3K56ac in nuclear extracts of E14Tg2a cells. A CTCF total of 2 μgofα-IgG, α-Nanog, α-Sox2, and α-Oct4 antibodies was used for each co-IP and immuno- pPolII blotted with antibody against H3K56ac. Ten percent E14Tg2a cell nuclear extracts was loaded as input control. (C) In vivo peptide pull-down to 1.34 probe the interaction between peptides containing 0.67 0.00 Recombinant Oct4 H3K56ac and Oct4 in mouse E14Tg2a nuclear Oct4 Sox2 pPolII -0.67 CTCF E Nanog extracts. Biotinylated histone peptides that are ei- -1.34 Smad1 ther unmodified or modified at K9 or K56 were H3K56ac immobilized on Streptavidin beads. Experimental Peptides: details are described in SI Appendix, Methods. Ten K9-unmod.K9ac K56-unmod.K56ac percent cell nuclear extracts was loaded as input Oct4 control and probed using Western blots and anti- Nuclear Extracts body against Oct4. (D) Flag-IP to show the effect of B 20% Input mutations in K9 to A (Ala) or K56 to A (Ala), Q (Gln), and R (Arg) on the interactions between histone H3.1 and Oct4 in mouse ESCs. Histone H3.1 with C- 10% InputIgG IPNanogSox2 IP IPOct4 IP terminal Flag tag was transfected into E14Tg2a cells H3K56ac for 48 h, α-Flag antibody was used for immunoprecipitation, and the precipitate was then immunoblotted with indicated antibodies. (E) In vitro Nucleosomes peptide pull-down to probe the interaction between K56ac containing peptide and recombinant C Nuclear Extracts F Oct4 protein. A total of 100 ng of recombinant Oct4 protein was used for each pull-down experiment. H3-unmod.H3-K9ac H3-K56ac K9ac peptide and unmodified K9 and K56 contain- Peptides: Oct4 ing peptides were used as controls. Twenty percent K9-unmod.K9ac K56-unmod.K56ac H3K56ac recombinant input Oct4 protein was loaded as input control and probed with α-Oct4 antibody. (F) Oct4 H3K9ac Mononucleosome immunoprecipitation using recom- 10% Input H3 binant Oct4, histone H3–unmodified nucleosomes, or H3K9ac- or H3K56ac-modified nucleosomes. Mononucleosomes were assembled by salt dilution using histone octamers and biotin-labeled DNA as described in SI Appendix, Methods. Proteins were pulled down with streptavidin beads and immunoblotted with indicated antibody.

To determine whether there is a possible direct interaction and clustered them based on the combinatorial binding of NSO between H3K56ac and Oct4, purified recombinant Oct4 protein and H3K56ac. Four major clusters were identified that represent was incubated with H3 peptides that were unacetylated (K9- the enrichment of one or more of the NSO proteins and unmod, K56-unmod) or acetylated (K9ac, K56ac) for peptide pull H3K56ac (Fig. 2A). Interestingly, gene ontology (GO) anal- down. We found that recombinant Oct4 protein binds best in ysis using Genomic Regions Enrichment of Annotations Tool vitro to the H3 peptide in which K56 is acetylated (Fig. 1E). We (GREAT) (31) showed that the genes of cluster 4 containing then asked whether a recombinant nucleosome in which the only NSO at Oct4 peak centers but not H3K56ac were not enriched in posttranslational modification is the acetylation of H3 at K56 any functional categories. In contrast, the copresence of NSO promotes binding to recombinant Oct4 in vitro. Nucleosomes in and H3K56ac (in cluster 1) caused strong functional enrichment which only H3K56 or H3K9 is acetylated were prepared as de- for genes involved in early embryogenesis and pluripotency (Fig. scribed (27, 28). Using the in vitro binding assay, we found that 2B and SI Appendix, Table S2). For example, Oct4, Klf4, Nanog, acetylation of H3K56 promotes the binding of Oct4 to nucleo- and Nodal proteins are all associated with pluripotency (4) and somes much more strongly than to the unmodified nucleosomes are members of cluster 1. The copresence of H3K56ac and NSO or to nucleosomes containing H3K9ac (Fig. 1F). Thus, acetylation proteins at these cluster 1 upstream regulatory sequences is of H3K56 not only decreases nucleosome stability (29), increases shown in SI Appendix, Fig. S1.Wefind in a similar manner that unwrapping of nucleosomal DNA ends, and may affect histone- the regions marked by Oct4 and Sox2 alone or Oct4 and Nanog histone interactions (27, 30), but H3K56ac may also free H3K56 alone (in the relevant portions of cluster 2) are enriched in sufficiently from local nucleosomal constraints to enable H3K56ac pluripotency associated functions only when they are also recognition by Oct4. Taken together, our data indicate that marked by the copresence of H3K56ac (Fig. 2C and D and SI H3K56ac interacts with Oct4 in vivo in mouse ESCs and directly Appendix, Tables S3 and S4). We conclude that H3K56ac links in vitro. genes marked by NSO proteins to pluripotency in mouse ESCs.

H3K56ac Links NSO Factors to Pluripotency. To investigate the Relationship Among H3K56ac, Oct4, and Oct4-Binding Motifs. We spatial and functional relationships between NSO proteins and then probed the extent to which Oct4 presence in cluster 1 cor- H3K56ac genome wide, we used ChIP-Seq to select and center relates with H3K56ac in addition to its association with Oct4- all Oct4 peaks in the mouse genome including regions ±5kb binding motifs. To do so, we divided cluster 1 regions into three from the Oct4 peak centers. We then plotted the relative en- groups based on the enrichment of H3K56ac (Fig. 3A). We found richment of Nanog, Sox2, and H3K56ac at the Oct4 peak regions that the enrichment of Oct4 at Oct4 peak centers (Fig. 3B)is

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1309914110 Tan et al. Downloaded by guest on October 1, 2021 Oct4 Nanog Sox2 H3K56ac H3K56ac/Oct4/Sox2/Nanog A B TS4_compacted morula 1 TS4_inner cell mass Theiler_stage_4 TS4_embryo TS4_second polar body TS4_zona pellucida 2 Theiler_stage_5 MGI expression detected TS5_inner cell mass TS4_extraembryonic TS5_embryo TS3_zona pellucida 3 Theiler_stage_3 TS3_second polar body +4 TS3_4-8 cell stage 0 4

Z score TS3_4-cell stage 4 4 -4 - --55 0 ++55 -55 0 ++55 -55 0 ++55 -55 0 +5+5 0 35 70 Relative distance to Oct4 peak center (kb) -Log10P

CDH3K56ac/Oct4/Sox2 H3K56ac/Oct4/Nanog TS5_embryo TS4_inner cell mass TS5_inner cell mass TS3_4-cell stage TS21_nasal cavity TS4_second polar body TS21_nasal cavity; TS4_zona pellucida TS28_olfactory TS4_compacted morula TS28_nasal cavity TS5_embryo MGI expression detected TS12_embryo; ectoderm TS25_smooth muscle MGI expression detected TS12_embryo; ectoderm; TS8_ectoplacental cone GENETICS TS28_trigeminal V TS20_midbrain; lateral TS17_latero-nasal TS11_primitive streak TS14_unsegmented TS12_head mesenchyme; TS14_2nd arch TS20_midbrain; lateral TS23_urinary bladder TS8_polar trophectoderm TS23_sublingual gland TS18_diencephalon TS19_tail; mesenchyme; TS24_oviduct 0 35 70 0 35 70 -Log10P -Log10P

Fig. 2. H3K56ac links NSO factors to mark pluripotency. (A) The distribution of Oct4, Nanog, Sox2, and H3K56ac at the Oct4-binding regions are shown as heat maps. Purple indicates that the factor is enriched at those regions. All regions were grouped into four clusters based on combinatorial presence of Oct4, Nanog, Sox2, and H3K56ac. (B–D) Functional annotation of H3K56ac/Oct4/Sox2/Nanog–enriched elements in cluster 1 (B), H3K56ac/Oct4/Sox2 (C), and H3K56ac/Oct4/Nanog–enriched (D) elements in cluster 2 in mouse ESCs were performed using GREAT (31). Mouse Genome Informatics (expression detected) that indicates the information on tissue and developmental stage–speciﬁc expression in mouse is used as GO category. The x axes values (in logarithmic scale) correspond to the binomial raw P values.

highest in group I that also contains the highest level of H3K56ac. related regions. To further test this possibility, we cloned a single In contrast, group III contains lower enrichment of both Oct4 DNA–binding site (ATTTG-N5-CAAAT) (32), 16 bp from H3K56ac and Oct4. Interestingly, the low presence of H3K56ac the 3′ end of the 601 nucleosome positioning sequence (Fig. 4A) in group III is not accompanied by lower enrichment of Oct4- (33). This construct was assembled with histone octamers using salt binding motifs [calculated by Cistrome (19)]. Instead, group III dilution as described (34). Only histone H3K56 was acetylated in contains a high level of Oct4-binding motifs and a lower but the assembled nucleosome. We then performed gel shift experi- signiﬁcant level of Oct4 at the Oct4 peak centers. This argues for ments with recombinant Oct4 protein (Origene TP311998) as de- the use of Oct4 motifs in binding a lower level of Oct4 when scribed (28, 35) to probe the interaction of Oct4 protein to its DNA H3K56ac is absent. A similar relationship between H3K56ac and recognition element with and without an adjacent nucleosome Oct4 presence is seen when comparing the four clusters of Fig. acetylated at H3K56. We found that recombinant Oct4 protein 2A. As shown in SI Appendix,Fig.S2, the greatest enrichment of slows the mobility in the native gel of the construct containing the H3K56ac (in clusters 1 and 2) is accompanied by the highest levels H3K56 unmodiﬁed nucleosome (II) and shifts it to a new position of Oct4. In contrast, the lowest levels of H3K56ac in clusters 3 and (III). In contrast, mobility of the construct containing both the Oct4 4 are accompanied by the lowest levels of Oct4. Oct4-binding DNA–binding site and the H3K56ac nucleosome is slowed further motif presence is similar in all four clusters. Furthermore, when (to position IV). We conclude that acetylation of H3K56 promotes we analyzed the functions associated with each of the three groups increased Oct4 binding to the construct containing a nucleosome within cluster 1, we found that regions highly enriched for adjacent to the Oct4-binding site (Fig. 4B). H3K56ac in group I are most associated with pluripotency (SI Appendix,Fig.S3A and Table S5), whereas the lower enrichment Decreased H3K56ac Promotes the Loss of Pluripotency of Mouse ESCs. of H3K56ac in groups II and III is more associated with differ- Finally, we wished to determine whether H3K56ac promotes entiation (SI Appendix,Fig.S3B and Tables S6 and S7). pluripotency in mouse ESCs by examining the effect of reduced These data are consistent with a role for H3K56ac in recruiting H3K56ac level on the expression of markers for pluripotency and or stabilizing Oct4 in vivo in addition to the interaction between differentiation of ESCs. The histone chaperone Asf1 is required Oct4 DNA–binding motifs and Oct4 protein at pluripotency- for the acetylation of H3K56, whereas the effect of Asf1 deletion

Tan et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 A Group I Group II Group III 25 25 20 15 20 0 5 10 15 0 5 10 15 20 25 0510 Average Profile of H3K56ac -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 Relative distance to Relative distance to Relative distance to Oct4 peak centers (kb) Oct4 peak centers (kb) Oct4 peak centers (kb)

B Group I Group II Group III 345 2345 12 Average Profile of Oct4 01 0 012345 -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 Relative distance to Relative distance to Relative distance to Oct4 peak centers (kb) Oct4 peak centers (kb) Oct4 peak centers (kb)

C Group I Group II Group III 0.5 0.2 0.3 0.4 0.5 0.2 0.3 0.4 Average Profile 0.1 0.1 0.1 0.2 0.3 0.4 0.5 of Oct4 binding motifs

-4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 Relative distance to Relative distance to Relative distance to Oct4 peak centers (kb) Oct4 peak centers (kb) Oct4 peak centers (kb)

Fig. 3. Relationship between H3K56ac, Oct4, and Oct4-binding motifs. Cluster 1 regions (from Fig. 2A) are divided into ﬁve groups based on the enrichment of H3K56ac surrounding regions ±5 kb from the Oct4 peak centers. Group I contains 20% of Oct4 regions with highest enrichment of H3K56ac; group II contains 20% of Oct4 regions in the middle (40–60%) with moderate enrichment of H3K56ac; and group III contains 20% of Oct4 regions with lowest enrichment of H3K56ac. (A) H3K56ac, (B) Oct4 and (C) Oct4-binding motifs (MA0142 in JASPAR) (48). Signal distribution relative to the center of group I, II, and III regions was determined by Cis-regulatory Element Annotation System (CEAS)-sitepro (49). The x axis represents the distance to the center of Oct4 peaks in mouse ESCs. The y axis represents the signals normalized by ChIP-Seq tag count numbers.

on other histone acetylation sites is relatively minor as determined and do not coimmunoprecipitate (23–25), and we find that by Western blots and MS (18, 36, 37). Using Western blots, we H3K56ac coimmunoprecipitates only with Oct4 in nuclear found that knockdown of Asf1a with shAsf1a in mouse E14Tg2a extracts. We cannot exclude the possibility that other histone cells decreases H3K56ac significantly but has very little effect on acetylation sites also interact with Oct4. For example, H3K27 and other acetylation sites in histone H3 (Fig. 5A). We also used H3K56 are both acetylated by the histone acetyltransferase p300 quantitative PCR to examine the expression of pluripotency and H3K27ac is also associated with NSO cooccupancy (40). related markers after shAsf1a transfection. We observed the However, although the interaction between Oct4 and H3K56ac is down-regulation of NSO pluripotency related markers and the up- specific in that H3K9ac, which marks enhancers, is not required regulation of the marker genes for endoderm, ectoderm, and me- for Oct4 binding (Fig. 1), we do not know whether H3K27ac soderm 10 d after transfection using the protocol described (38, 39) binds to Oct4 or to other regulators such as Sox2 and Nanog. (Fig. 5B). Thus, our results argue that decreasing H3K56ac through Nevertheless, the interaction between Oct4 and H3K56ac is loss of Asf1a results in the dissolution of the pluripotency net- unique. Previous studies have demonstrated that H3K56ac dis- work to initiate the expression of differentiation-related markers places nucleosomal DNA in vitro to allow DNA “breathing” (27) of mouse ESCs, indicating that H3K56ac helps maintain the and access of a LexA DNA-binding protein to nucleosomal DNA pluripotency of ESCs. (41, 42). Surprisingly, our data argue not for an interaction between Oct4 and nucleosomal DNA, but for a direct interaction Discussion between Oct4 and nucleosomal H3K56ac. We have shown previously that H3K56ac is enriched at NSO How H3K56 becomes acetylated in a localized manner to bind target gene promoters in human ESCs (10). However, the data Oct4 is unclear. Localized H3K56ac may result from the pres- presented here support a special role for H3K56ac in recognizing ence of the histone acetyltransferase p300 that colocalizes often Oct4 in mouse ESCs. Oct4 and Sox2 are associated with each at enhancers with NSO proteins (4, 40), resulting in acetylation of other at their targets in the core transcriptional network (4), and H3K56 in a bimodal manner surrounding p300 (SI Appendix,Fig. we find that H3K56ac presence correlates with that of Oct4 and S4). Thus, histone chaperone Asf1a and p300 may collaborate to Sox2. However, Oct4 and Sox2 recognize different DNA motifs acetylate H3K56 on histone H3 (17), which might subsequently

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1309914110 Tan et al. Downloaded by guest on October 1, 2021 A Nucleosomal DNA Template Oct4 binding site

601 nucleosome positioning sequence 16bp DNA linker B

++------+ ++ K56ac modified nucleosome --+++- - -- Unmodified nucleosome

00 100 300 0 100 300 Recombinant Oct4 (ng)

IV Nucleosome-Oct4 Complex III

I GENETICS

Fig. 4. Native gel mobility analysis for Oct4 binding to H3K56ac-modiﬁed nucleosomes adjacent to Oct4-binding motif. (A) DNA template for nucleosome assembly containing 601 nucleosome positioning sequence adjacent to a 16-bp spacer and a single Oct4-binding site (prepared by PCR from pGEM3Z601R plasmids) (33, 34). (B) Approximately 25 ng of nucleosomes were incubated with indicated recombinant Oct4 protein for 2 h at room temperature in each reaction. Position I, unassembled nucleosomal DNA template; II, assembled nucleosomal DNA template; III, assembled nucleosomal DNA template with Oct4 molecules recruited to the adjacent Oct4-binding site; IV, assembled nucleosomal DNA template containing H3K56ac with Oct4-recruited to H3K56ac histones and to Oct4-bindingsite.

replace unacetylated H3K56 in chromatin (12). This may tar- ation (1, 2). This helps explain why the clustering of NSO protein get H3K56ac to sites surrounding Oct4-binding sites to enable presence at Oct4 peak regions is not associated by GO analysis H3K56ac–Oct4 interactions. preferentially with pluripotency. In contrast, when H3K56ac is It is known that NSO proteins target not only genes of the core clustered with NSO, those regions are strongly enriched function- transcriptional network but also many genes related to differenti- ally for pluripotency (Fig. 2; cluster 1) and regions in this cluster with the highest enrichment of H3K56ac were shown to be most enriched for Oct4 (Fig. 3) and most associated with pluripotency (SI Appendix,Fig.S3). In support, shAsf1a, which is expected to A B ﬁ shGFPshAsf1a decrease H3K56ac, speci cally (Fig. 5) decreased the levels of NSO shGFP H3K9ac 4 mRNAs pluripotency related markers while promoting the pres- shAsf1a H3K14ac ence of differentiation related ones. Collectively, these data argue 3 H ession

H3K18ac r for the biological importance of the interaction between Oct4 and PD

H3K23ac A 2 H3K56ac in regulating pluripotency. G

o Although H3K56ac is generally important for Oct4 occupancy, H3K27ac t 1

H3K56ac Expelative there are also DNA regions containing Oct4-binding motifs, at R Histone 3 0 which Oct4 binds in the absence of H3K56ac. In either case, the 2 6 6 Asf1a binding of Oct4 uses Oct4 recognition motifs, and we have found Oct4 Tbx Sox Pax Sox17 FoxA2 Nanog α-Tubulin Vcam1 that Oct4 can bind its recognition motifs to a lower, albeit sig- niﬁcant, extent in the absence of H3K56ac [Figs. 3 (group III) and 4]. These different binding mechanisms are both important func- EndodermEctodermMesoderm Pluripotency tionally. When Oct4 enrichment occurs through DNA-binding Fig. 5. Decreased H3K56ac promotes the loss of pluripotency of mouse motifs and H3K56ac, those Oct4-bound regions are preferentially ESCs. (A) Down-regulation of Asf1a by shAsf1a results in decreased associated with pluripotency (SI Appendix,Fig.S3). When Oct4 H3K56ac with little apparent effect on other sites of acetylation in histone enrichment occurs through its DNA-binding motifs only, those H3. shAsf1a treatment of mouse E14Tg2a ESCs for 48 h was followed by Oct4-bound regions are preferentially associated with differenti- Western blot with indicated antibodies. Short hairpin GFP (shGFP) was used ation. Therefore, the presence of H3K56ac delineates Oct4 func- α B as a control. Histone H3 and -Tubulin were used as loading controls. ( ) tion biologically. Mouse E14Tg2a cells were transfected with plasmids that express shAsf1a Interestingly, although our data (Figs. 1 and 4) support a direct or shGFP and were selected with puromycin from day 4 after transfection. ﬁ Prolonged decrease of H3K56ac by shAsf1a plasmids for 10 d caused the interaction between Oct4 and H3K56ac, the H3K56ac modi ca- reduction of pluripotency markers and up-regulation of differentiated tion is generally absent at the actual Oct4-binding peaks (Figs. 2A makers for endoderm, ectoderm, and mesoderm. and 3) that contain Oct4 recognition motifs and may exclude

Tan et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 histones. Instead, H3K56ac surrounds the Oct4 peak centers and Materials and Methods DNA recognition motifs in a bimodal manner at pluripotency- All experiments performed with mouse ESCs were cultured under standard associated regions (SI Appendix,Fig.S4). Therefore, we propose feeder-free conditions, and were approved by the University of California Los that the DNA recognition motifs may determine the sites of Oct4 Angeles (UCLA) Embryonic Stem Cell Research Oversight Committee. ChIP binding, whereas adjacent nucleosomes acetylated at H3K56 DNA was sequenced with Illumina Genome Analyzer HiSeq2000 at the UCLA Broad Stem Cell Research Center high-throughput sequencing facility. For stabilize Oct4 at those sites. In support, our gel shift experiment details, please refer to SI Appendix. indicates that a nucleosomal construct containing H3K56ac placed adjacent to an Oct4-binding site is likely to interact with ACKNOWLEDGMENTS. We thank members of the M.G. laboratory for com- more Oct4 than the construct that is not acetylated at H3K56 ments and discussion throughout this work; Yongxin Yu, Arnie Berk, Kathrin (Fig. 4). How this occurs in vivo in the presence of Oct4 homo- Plath, Siavash Kurdistani, and Roberto Ferrari for critique of the manuscript; – Gerald Crabtree for the gift of short hairpin GFP plasmids; Jason W. Chin for dimers, Oct4-Sox2 heterodimers (32, 43 45), and multiple adja- the plasmids used to express acetyl-histones; Michael Carey for plasmids used cent DNA recognition motifs (46) remains to be determined. to express recombinant histones; the UCLA Broad Stem Cell Research Center Nevertheless, the unique role of H3K56ac in Oct4 binding is High Throughput Sequencing core facility for sequencing services; and Roberto Ferrari for help with the analysis of ChIP-Seq data. This work was supported likely one factor that contributes to Oct4 stability and to the by National Institutes of Health Grant GM23674 and by a Broad Center of function of Oct4 as a master regulator of pluripotency. Regenerative Medicine and Stem Cell Research at UCLA grant (to M.G.).

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