Developmental Cell Article

UTX, a H3-Lysine 27 , Acts as a Critical Switch to Activate the Cardiac Developmental Program

Seunghee Lee,1,2 Jae W. Lee,1 and Soo-Kyung Lee1,2,* 1Pediatric Neuroscience Research Program, Pape´ Family Pediatric Research Institute, Department of Pediatrics 2Vollum Institute Oregon Health and Science University, Portland, OR 97239, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2011.11.009

SUMMARY enhancers remain unclear. Addressing these questions would resolve a fundamental issue of how poised developmental The removal of histone H3 lysine27 (H3K27) trimethy- exit the transcriptionally purgatory state to achieve tissue- lation mark is important for the robust induction of specific expression during organogenesis. many cell type-specific genes during differentiation. Recent studies have identified UTX (also known as KDM6A), Here we show that UTX, a H3K27 demethylase, UTY and JMJD3 (also known as KDM6B) as H3K27 demethy- acts as a critical switch to promote a cardiac-specific lases belonging to a family of JmjC-domain-containing gene program. UTX-deficient ESCs failed to develop (Agger et al., 2007; De Santa et al., 2007; Hong et al., 2007; Jep- sen et al., 2007; Lan et al., 2007; Lee et al., 2007). UTX and heart-like rhythmic contractions under a cardiac JMJD3 displayed specific catalytic activity in demethylating differentiation condition. UTX-deficient mice show H3K27me3/me2, whereas UTY was inactive under the same severe defects in heart development and embryonic assay conditions (Hong et al., 2007; Lan et al., 2007). Interest- lethality. We found that UTX is recruited to cardiac- ingly, UTX and UTY constitute a large complex that contains specific enhancers by associating with core cardiac multiple subunits including H3K4 methyltransferase MLL3 or transcription factors and demethylates H3K27 resi- MLL4 (Cho et al., 2007; Goo et al., 2003; Issaeva et al., 2007; dues in cardiac genes. In addition, UTX facilitates Lee et al., 2007, 2006a). It is unclear whether JMJD3 forms the recruitment of Brg1 to the cardiac-specific a steady state complex like UTX. The UTX gene is encoded on enhancers. Together, our data reveal key roles for the X , but escapes inactivation UTX in a timely transition from poised to active chro- (Greenfield et al., 1998). The UTY gene, the male counterpart matin in cardiac genes during heart development and of UTX, is localized on the Y chromosome. Unlike UTX and UTY, the JMJD3 gene is encoded in an autosome. Notably, a fundamental mechanism by which a H3K27 deme- UTX is required for Hox gene activation and posterior develop- thylase triggers tissue-specific chromatin changes. ment in zebrafish embryos (Agger et al., 2007; Lan et al., 2007; Lee et al., 2007), suggesting roles of UTX in development. However, the function of H3K27 during cell fate INTRODUCTION determination and organogenesis remains unknown. To gain insight into the developmental roles of UTX, we In embryonic stem cells (ESCs), many developmental genes generated UTX-deficient ESCs and mice, and found that UTX exhibit ‘‘poised status’’ marked by bivalent trimethylation of is essential for heart development. Our study also uncovered both histone H3-lysine 27 (H3K27me3) and H3-lysine 4 the molecular basis underlying the action of UTX in inducing (H3K4me3) (Bernstein et al., 2006; Pan et al., 2007; Rada-Iglesias cardiac genes during heart development. Overall, our results et al., 2011; Zhao et al., 2007). Given that the H3K27me3 mark is provide important insights into how the histone modifying important to repress many developmental genes in self-renewing orchestrate the establishment of transcriptionally ESCs (Azuara et al., 2006; Bernstein et al., 2006; Boyer et al., active chromatin in appropriate lineage-specific genes during 2006; Lee et al., 2006b; Pan et al., 2007; Zhao et al., 2007), the organogenesis. removal of H3K27me3 in a cohort of tissue-specific genes is likely an important step in organogenesis. Despite recent progress in understanding the changes of histone post-translational modifi- RESULTS cations during ESC differentiation, how histone modifying enzymes make specific changes in a battery of lineage-specific UTX Is Highly Expressed in the Developing Embryos genes on the right time and space during development are To understand the role of UTX in embryo development, we poorly understood. In particular, the identity of the histone generated mice bearing UTXF-loxP allele, in which b-galactosi- H3K27 demethylases that control cardiac and dase gene was inserted into the UTX locus and exon 3 of UTX the mechanisms by which they are recruited to cardiac-specific was flanked by loxP sequences (Figure 1A). To examine the

Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. 25 Developmental Cell UTX in Heart Development

A

B C

DEF

Figure 1. UTX Expression Pattern in the Developing Embryos (A) Schematic representation of three mutant alleles of UTX. (B–F) Whole-mount X-gal staining in E8.5 (B and C) and E11.5 (D–F) UTXF-loxP/+ embryos. X-gal stained areas in UTXF-loxP/+ embryos depict the embryonic tissues expressing UTX. ant nt, anterior region of the neural tube; bc, bulbus cordis; br, brachial arches; ctx, cortex; f.l., forelimb; he, heart; h.l., hindlimb; is, isthmus; la, left atrium; nt, neural tube; pv, primitive ventricle; ra, right atrium; sm, somites; sp, spinal cord; v, ventricle; vz, ventricular zone of the neural tube. See also Figure S1. expression pattern of UTX in embryos, we performed X-gal stain- ESCs, in which exon 3 of UTX is deleted, by inducing a Cre-medi- ing in UTXF-loxP/+ embryos, in which b-galactosidase expression ated recombination in UTXF-loxP/y ESCs (Figure 1A). Neither UTX is controlled by UTX regulatory elements, and found that UTX is mRNA nor UTX expression was detected in UTXF-D/y broadly expressed but is enriched in several tissues. At E8.5, ESCs (Figure 2A, data not shown), establishing that UTXF-D/y UTX was highly expressed in developing heart, anterior region ESCs are UTX null. UTXF-loxP/y ESCs showed reduced levels of of the neural tube, ventricular zone of the caudal neural tube, UTX mRNA and protein (Figure 2A, data not shown), indicating neural crest cells, and somites (Figures 1B and 1C). At E11.5, that UTXF-loxP is a UTX hypomorphic allele. UTX was enriched in heart, myotome of somites, limb buds, UTX null ESCs were indistinguishable from wild-type ESCs in brachial arches, isthmus, cortex, and eyes (Figure 1D). Cross- cell proliferation and survival when cultured with leukemia inhib- sections of X-gal stained embryos revealed a high level of UTX itory factor (LIF), as monitored by MTT assay and western blot- expression in cardiomyocytes (Figures 1C, 1E, and 1F). Consis- ting assays with a-cleaved Caspase3 antibody (Figures 2B and tently, UTX transcripts are highly expressed in cardiomyocytes 2C). Alkaline phosphatase staining revealed that, like wild-type of E10.5 hearts, as shown by in situ hybridization (Figure S1 ESCs, UTX null ESCs self-renew in the presence of LIF, and available online). UTY is also expressed in E10.5 hearts (Fig- readily differentiate upon withdrawal of LIF and addition of reti- ure S1). The expression pattern of UTX suggests possible roles noic acid (RA), which trigger ESC differentiation (Figure 2D). of UTX in organogenesis of several tissues including heart. Consistently, Oct4, a transcription factor essential for self- renewal of ESCs, was downregulated and the expression of UTX Is Required for ESCs to Differentiate to a Cardiac markers for the three germ layers were induced in UTX null Lineage ESCs under an ESC differentiation condition (Figures S2A– To address the role of UTX during developmental transitions S2D). These results indicate that UTX is not required for self- from ESCs to a cardiac cell lineage, we used a well-established renewal, proliferation, survival, and differentiation of ESCs. protocol to differentiate ESCs to cardiac cell types, which closely Wild-type and UTX null ESCs were subjected to a well-estab- mimic in vivo development and provide cells at developmental lished protocol for differentiating ESCs into a cardiac lineage transitions (Boheler et al., 2002). We first generated UTXF-D/y (Figure S2E). The expression of UTX and UTY was nearly

26 Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. Developmental Cell UTX in Heart Development

Figure 2. UTX Is Required for the Differenti- ation of ESCs into a Cardiac Lineage (A) RT-PCR using ESCs of various genotypes as shown above. UTX expression was not detected in UTX null (UTXF-D/y) ESCs. cycl. A, cyclophilin A. (B) MTT assays to monitor proliferation of ESCs. (C) Western blot to detect the levels of activated Caspase3 in self-renewing ESCs (day 0) and cardiac differentiated ESCs (day 10). (D) Alkaline phosphatase staining of ESC colonies under self-renewal condition (+LIF) and differenti- ation condition incubated with RA in the absence of LIF (+RA/LIF). UTX null ESCs self-renew and readily differentiate when stimulated with RA, like wild-type ESCs. (E, F, and I–L) Quantitative RT-PCR to monitor mRNA levels during cardiac differentiation of wild- type ESCs (UTX+/y, black dotted lines) and UTX null ESCs (UTXF-D/y, gray lines). x axis, differenti- ation days; y axis, relative mRNA levels. UTX null ESCs did not induce cardiac-specific genes during cardiac differentiation. The mRNA level in wild-type ESCs at day 0 was calculated as 1. (G) Quantification of cEBs that show heart-like spontaneous contraction among all EBs. UTX null ESCs failed to form beating cEBs. (H) TUNEL assays in cardiac differentiated EBs (cEBs, day 10). UTX null cEBs do not show more cell death than wild-type cEBs. Error bars repre- sent the standard deviation in all graphs (B, E–G, and I–L). See also Figure S2.

wild-type EBs during cardiac differentia- tion, as shown by detection of activated Caspase3 and TUNEL staining (Figures 2C and 2H), indicating that a failed forma- tion of contracting EBs from UTX null ESCs was not due to excessive cell death. Next, we examined the induction of cardiac-specific genes during cardio- myocyte differentiation in wild-type and UTX null ESCs. Atrial natriuretic factor (ANF), the ventricular isoform of the myosin regulatory light chain (MLC2v), a-cardiac actin (a-CA), cardiac muscle myosin heavy chain 6 (Myh6), and cardiac-specific troponin T2 were mark- edly induced in wild-type ESCs during doubled during cardiac differentiation in ESCs (Figures 2E and differentiation (Figures 2I–2L, data not shown). However, under 2F). Notably, the expression of UTY as well as UTX was abol- the same conditions, these cardiac-specific genes were not up- ished in UTX null ESCs (Figures 2E and 2F), suggesting that regulated in UTX null ESCs, consistent with the morphological UTY expression in male ESCs is dependent on UTX. We then defects in developing contraction (Figures 2G and 2I–2L, data monitored the differentiation of ESCs into cardiac lineage- not shown). Together, these data establish that UTX is required committed embryoid bodies (cEBs) that show heart beating- for cardiac lineage differentiation of ESCs and robust induction like rhythmic contractions, a unique functional property of of the cardiac genes during cardiac differentiation. pulsating cardiac muscle cells. Almost 100% of EBs from wild- type ESCs exhibited prominent beating (Figure 2G). In the UTX Null Mice Are Defective in Cardiac Development parallel condition, however, EBs derived from UTX null ESCs To address the role of UTX in vivo, we created mice with UTXD failed to develop spontaneous contraction (Figure 2G). Only allele by crossing the UTXF-loxP/+ mice with Rosa-Flp and EIIa- 26% of UTXF-LoxP/y EBs displayed spontaneous beating (Fig- Cre mice, which constitutively express Flp and Cre recombinase ure 2G). UTX null EBs did not undergo more cell death than respectively, in succession (Figure 1A) (Farley et al., 2000; Lakso

Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. 27 Developmental Cell UTX in Heart Development

Figure 3. UTX Is Required for Early Heart Development (A–F) Images of E9 littermate wild-type embryo (UTX+/y,A,A0, B, and E) and UTX null embryo (UTXD/D,C,C0, D, and F), and H&E staining of E9 embryonic hearts (E and F). At E9, UTX null embryonic hearts displayed no overt looping, whereas wild-type embryos underwent cardiac looping morphogenesis, adopting a spiral shape consisting of cardiac chambers. UTX null embryos also show neural tube closure defects (red arrow in C). (G and H) Immunohistochemical analyses with a proliferation marker Ki67, TUNEL labeling, and myocardiac lineage markers Nkx2.5 and Isl1. ht, heart tube. (I–Z) Whole-mount in situ hybridization analyses of E9 embryos with probes of various cardiac genes, including Nkx2.5 (I–L), Tbx5 (M–P), ANF (Q–T), and MLC2v (U–Z). Lateral view from left side of the embryos or ventral view are shown as indicated. Transverse sections of MLC2v-hybridized embryos corresponding to the dotted lines in (W) and (X) are shown in (Y) and (Z), respectively. In E9 UTX null embryos, expression of ANF and MLC2v was markedly downregulated compared to control embryos, and the separate chamber formation did not occur. lv, left ventricle; nt, neural tube; rv, right ventricle; v, ventricle. (A–D and I–Z) Relative position of embryos was marked by V (ventral), D (dorsal), right (R), and left (L) along with arrows. See also Figure S3 and Figure S4. et al., 1996). UTXD/D female and UTXD/y male mice did not peared to develop normally (Figures 3A–3F, data not shown). express detectable levels of UTX transcripts (Figure S3A). UTY At E9, UTXD/+ and UTX+/y sibling embryos displayed cardiac is expressed in both wild-type UTX+/y and UTX null UTXD/y looping morphogenesis, which marks chamber formation and male embryos at E8.5 (Figure S3B), suggesting that UTY expres- results in a physical separation between the primitive atrial and sion in developing male embryos is independent on UTX, unlike ventricular regions, and completed neural tube closure in the male ESCs. UTY expression was not detected in female anterior region (Figures 3A and 3B). In contrast, UTXD/D embry- embryos, as expected (Figure S3B). UTXD/D female mice, which onic hearts largely retained a tubular heart structure and their do not have the UTY gene, did not survive past E10.5, whereas neural tubes remained open in the anterior portion (Figures 3C UTXD/+ heterozygote female mice were viable and fertile. and 3D; Figure S3C). We further examined the gross structure UTXD/y male mice displayed embryonic lethality and severe of hearts by performing hematoxylin and eosin staining on the developmental retardation, but some UTXD/y mice that escaped transverse sections of E9 embryos. As predicted, hearts of embryonic lethality lived into adulthood. Thus, UTX is required UTXD/+ and UTX+/y embryos were highly looped and consisted for the survival of female embryos, and UTY partially compen- of separate chambers, whereas UTXD/D embryos were largely sates for the loss of UTX in males. linear throughout the heart tube and lacked chamber develop- UTXD/D female embryos exhibited severe cardiac malforma- ment (Figures 3E and 3F). In addition, the myocardium of UTX tion and neural tube closure defects, while their allantois ap- null embryos appeared thinner than that of littermate control

28 Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. Developmental Cell UTX in Heart Development

embryos (Figures 3E and 3F). At E10.5, UTXD/D embryos were and serum response factor (SRF), a MADS box factor that markedly growth retarded as compared to wild-type littermates, functions as a strong activator to induce cardiac-specific gene and were completely reabsorbed by E11.5 (data not shown). transcription during development (Miano et al., 2004; Niu et al., These results indicate that UTX is needed for early heart devel- 2005; Parlakian et al., 2004; Shore and Sharrocks, 1995). We opment and neural tube closure. performed coimmunoprecipitation (CoIP) assays in HEK293T cells transfected with Myc-tagged UTX and HA-tagged SRF, Impaired Cardiac Differentiation in UTX Null Embryos and found that UTX associates with SRF in cells (Figure 4A). To understand the basis for the cardiac defects in E9 UTX null Furthermore, we detected the interaction between endogenous embryos, we analyzed cell proliferation and survival in E8.5 UTX and SRF by CoIP assays in the developing heart tissues embryos using Ki67 and TUNEL staining and found little differ- (Figure 4B; Figure S5A). Neither UTX nor SRF associated with ence between UTX null and UTXD/+ heterozygote embryonic Ezh2, a histone H3K27 methyltransferase, in the hearts (Fig- hearts (Figure 3G). Next, we monitored expression levels of ure S5A), confirming the specificity of interaction between UTX core cardiac transcription factors using quantitative RT-PCR and SRF (Figure S5A). with E8.5 littermate whole embryos. The expression of the four To test whether UTX affects the transcriptional activity of SRF, core cardiac factors, Nkx2.5, Tbx5, GATA4, and SRF, was com- we performed cell-based reporter assays using luciferase parable among embryos of various genotypes at E8.5 (Figures reporters linked to synthetic serum response elements (SREs) S3D–S3F). Immunohistochemical analyses revealed that both or enhancer elements of ANF, SM22,ora-CA gene, each of Nkx2.5+Isl1 and Nkx2.5+Isl1+ myocardial lineage cells are which contains essential SRF binding sites (Chen and Schwartz, specified in E8.5 UTX null embryos (Figure 3H). UTX is expressed 1996; Li et al., 1997; Small and Krieg, 2004; Strobeck et al., in both myocardial lineages (Figure S4). Together, these results 2001). UTX enhanced SRF-mediated transcriptional activation suggest that proliferation, survival, and initial fate specification in all SRF-responsive reporters in a dose-dependent manner, are not overtly disturbed in myocardial lineage cells of UTX null while UTX alone had little effect without SRF (Figures 4C and embryos at E8.5. 4D; Figures S5B and S5D). Conversely, shRNA-mediated knock- Next, we examined expression of Nkx2.5 and Tbx5 in E9 UTX ing down of endogenous UTX strongly suppressed the SRF- null and control littermate embryos using whole-mount in situ mediated activation, which was rescued by expression of hybridization analyses. Nkx2.5 and Tbx5 were highly expressed shRNA-insensitive form of UTX (Figures 4E and 4F; Figures in the sinoatrial structures and heart tube of UTXD/D embryos, but S5C and S5E). These data demonstrate that UTX is important the overall levels were reduced compared to those in UTXD/+ for the SRF-mediated transcriptional activation. littermates (Figures 3I–3P), suggesting that UTX plays a role in Next, we tested whether UTX cooperates with other core maintaining high levels of Nkx2.5 and Tbx5 in the developing cardiac transcription factors. CoIP assays demonstrated that heart. To monitor the expression of heart-specific genes that UTX associates with Tbx5, Nkx2.5, and GATA4 in HEK293T cells are regulated by the core cardiac transcription factors, we per- (Figures 4G–4I). Likewise, CoIP assays in E12.5 hearts revealed formed whole mount in situ hybridization analyses in littermate the interaction of UTX with endogenous Tbx5, Nkx2.5, and embryos. ANF, an atrial natriuretic peptide, is a marker of the GATA4 (Figure 4J). In contrast, UTX did not interact with Pax6 differentiating chamber myocardium. SRF, Tbx5, Nkx2.5, and homeodomain transcription factor in HEK293T cells (Figure S5F), GATA4, alone or in combination, bind to their cognate sites in confirming the specificity of association between UTX and core the ANF enhancer and activate its transcription (Bruneau et al., cardiac transcription factors. 2001; Charron and Nemer, 1999; Durocher and Nemer, 1998; The proximal enhancer of ANF bears the critical response Garg et al., 2003; Hiroi et al., 2001; McBride and Nemer, 2001; elements for Tbx5, Nkx2.5, and GATA4 in addition to SRF (Bru- Small and Krieg, 2003, 2004; Tanaka et al., 1999; Thuerauf neau, 2002; Bruneau et al., 2001; Charron and Nemer, 1999; et al., 1994). In addition, Nkx2.5 and Tbx5 also activate expres- Durocher and Nemer, 1998; Garg et al., 2003; Hiroi et al., sion of the ventricle-specific contractile protein MLC2v (Bruneau 2001; McBride and Nemer, 2001; Small and Krieg, 2003, 2004; et al., 2001; Lyons et al., 1995; Tanaka et al., 1999). Whole-mount Tanaka et al., 1999; Thuerauf et al., 1994). Consistent with the in situ hybridization revealed that ANF expression was greatly observed interactions between UTX and the cardiac transcrip- reduced in UTXD/D embryonic heart (Figures 3Q–3T). In addition, tion factors, UTX expression further potentiated Tbx5, Nkx2.5 MLC2v was also substantially decreased and the expression and GATA4-dependent activation of the ANF enhancer, while pattern of MLC2v was severely altered in UTXD/D embryos UTX-shRNA inhibited this activation (Figures 4K and 4L; Figures (Figures 3U–3Z). Together, our data establish that UTX plays S5G–S5J). Interestingly, we found that Tbx5 and SRF synergize a crucial role in maintaining high levels of Nxk2.5 and Tbx5 in activating the ANF enhancer (Figures 4M and 4N). UTX expression and marked upregulation of heart-specific genes strengthened this combinatorial action of Tbx5 and SRF, and during embryonic development. UTX-shRNA suppressed this action (Figures 4M and 4N). Simi- larly, UTX also supported the cooperative actions of Nkx2.5 UTX Functions as a Coactivator of the Core Cardiac and GATA4 in activating the ANF enhancer, and UTX-shRNA Transcription Factors inhibited this action (Figures S5K and S5L). Combined with the Given that UTX null mice exhibit severely arrested heart develop- phenotypic analyses of UTX null hearts, our results suggest ment and downregulation of ANF and MLC2v, target genes of that UTX potentiates the induction of heart-specific genes, at core cardiac transcription factors, we asked whether UTX func- least in part, by functioning as an essential common coactivator tions as a coactivator of any of the cardiac transcription factors. for the core cardiac transcription factors, SRF, Tbx5, Nkx2.5, To test this idea, we first examined the association between UTX and GATA4.

Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. 29 Developmental Cell UTX in Heart Development

A BC D E

F GHI J

KL MN

Figure 4. UTX Functions as a Coactivator of the Cardiac Transcription Factors SRF, Nkx2.5, Tbx5, and GATA4 (A and G–I) CoIP assays in HEK293T cells transfected with Myc-UTX and the cardiac transcription factors. UTX associates with SRF, Nkx2.5, Tbx5, and GATA4 in cells. (B and J) CoIP assays using a-UTX antibody for western blotting assays in dissected E12.5 embryonic hearts. The antibodies used for immunopurification were marked on the top. (C–F and K–N) Luciferase reporter assays using SRE:luciferase (C and E) and ANF:luciferase (D, F, and K–N) reporters with vectors indicated below each graph. The transcriptional activity of SRF, Tbx5, and Nkx2.5, alone or in combination, was enhanced by UTX, and inhibited by UTX-shRNA (sh-UTX). The basal luciferase activity for each reporter was calculated as 1 in y axis. + and ++ represent relative dosage of plasmids used for transfection. Error bars represent the standard deviation in all graphs. *p < 0.05; **p < 0.01 in two-tailed Student’s t test. See also Figure S5.

UTX Is Recruited to Cardiac-Specific Enhancers (Figure 5C). These data underline a heart-specific recruitment of Our results predict that UTX is recruited to cardiac-specific endogenous UTX to the cardiac enhancers, which are coregu- enhancers to control cardiac gene expression in the developing lated by core cardiac transcription factors, in embryos. heart. To test this prediction, we monitored the binding of endog- To test whether UTX binding to the cardiac enhancers is enous UTX to cardiac enhancers using chromatin immunopre- developmentally regulated, we performed ChIP assays in self- cipitation (ChIP) assays with an a-UTX antibody in dissected renewing pluripotent ESCs and differentiated cEBs that display hearts, limbs and brains from E12.5 mouse embryos. UTX was spontaneous heart-like beating. As expected, the expression strongly recruited to the ANF enhancer that contains the key of Tbx5, Nkx2.5, SRF, and GATA4 was induced as ESCs differ- responsive elements for SRF, Tbx5, Nkx2.5, and GATA4 in entiate to cEBs (Figures S6A–S6D). Interestingly, UTX was re- embryonic hears, whereas its binding to the ANF enhancer in cruited to the ANF enhancer in cEBs that express cardiac embryonic limbs and brains was much weaker (Figures 5A and transcription factors, while it did not bind to the enhancer in 5B). Similarly, UTX bound to the cardiac-specific enhancer of self-renewing ESCs (Figure 5D). Likewise, ESC differentiation a Brg1-associated factor Baf60c, which was co-occupied by into cEBs induced UTX to bind to the Baf60c enhancer (Fig- SRF, Nkx2.5, and GATA4 (He et al., 2011), in hearts, but UTX ure 5E). These data suggest that the recruitment of UTX to the did not bind to the Baf60c enhancer efficiently in limbs and brains cardiac-specific genes is facilitated during the developmental

30 Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. Developmental Cell UTX in Heart Development

A BC D

EF G

Figure 5. UTX Is Recruited to the Cardiac Genes in a Cardiac Cell-Type Specific Manner and Triggers the Demethylation of H3K27 Resides (A) Schematic representation of the ANF gene. The arrows indicate two sets of primers detecting ANF-enhancer (ANF-enh) and ANF-exon1 (ANF-ex1). The specific response elements for the cardiac transcription factors are also indicated. (B–E) ChIP assays with a-UTX antibody in dissected tissues from E12.5 embryos (B and C) or ESCs (D and E). (D and E) Wild-type ESCs (UTX+/y, black bars) and UTX null ESCs (UTXF-D/y, gray bars) were cultured under self-renewing ESC condition (E) or cardiac differentiation condition (C). UTX is efficiently recruited to the ANF-enhancer and Baf60c-enhancer only in hearts or cEBs, but UTX binding to the cardiac enhancers is very weak in limbs, brains, and self-renewing ESCs. (F and G) ChIP assays with a-H3K27me3 in P19 cells treated with serum shock and transfected with sh-UTX and UTX expression vectors as indicated below each graph. UTX-wt, wild-type UTX; UTX-mt, catalytically inactive UTX. (B–G) The quantitative PCR levels in IgG samples were calculated as 1. Error bars represent the standard deviation in all graphs. See also Figure S6. transition of ESCs into the cardiac lineage, likely due to upregu- phenotype (Figures 5F and 5G). The expression levels of UTX lation of the cardiac transcription factors and UTX and their wild-type and mutant were comparable (data not shown). These subsequent association. Together, these data show that the data demonstrate that UTX enzymatic activity is needed for the occupancy of UTX to the cardiac lineage genes is controlled in activated SRF to remove a repressive mark H3K27me3 in the a developmental stage- and tissue-specific manner. cardiac genes.

UTX Mediates Demethylation of H3K27 Residues UTX Facilitates the Recruitment of Brg1 to in Cardiac-Specific Genes Cardiac-Specific Genes The recruitment of UTX to the cardiac genes led us to ask The ATP-dependent chromatin remodeling Swi/Snf complex whether UTX functions as a H3K27 demethylase that re- containing Brg1, an ATPase subunit, plays crucial roles in heart moves a repressive transcription mark H3K27me3 in cardiac development and potentiates the transcription activity of core genes upon the activation and/or expression of the cardiac tran- cardiac transcription factors (Hang et al., 2010; Lickert et al., scription factors. To this end, we monitored the H3K27me3 2004). Interestingly, it was reported that H3K27 demethylases levels in the cardiac enhancers with or without serum shock, mediate a functional interaction between a T-box transcription which promotes the transcriptional activity of SRF, in P19 mouse factor T-bet and the Brg1-complex (Miller et al., 2010). Our find- embryonic cells. The H3K27me3 levels in the ANF and Baf60c ings that UTX functions as a coactivator of cardiac transcription enhancers were markedly suppressed when SRF was activated factors, along with the known roles of Brg1 in heart development, by serum shock (Figures 5F and 5G). This SRF-mediated deme- raise the question of whether UTX also contributes to heart- thylation of H3K27 in the cardiac enhancers was blocked by specific gene expression by recruiting the Brg1-containing Swi/ shRNA-mediated knockdown of endogenous UTX (Figures 5F Snf chromatin remodeling complex to the cardiac genes. We and 5G), suggesting that UTX is a demethylase that eliminates found that UTX associates with Brg1 in HEK293T cells (Figure 6A). the H3K27me3 mark. Further supporting this idea, re-expression We then tested whether UTX facilitates the interaction between of UTX wild-type repressed the elevated H3K27me3 levels in the Tbx5 and Brg1 using CoIP assays in HEK293 cells transfected cardiac enhancers in UTX knockdown conditions, while a catalyt- HA-tagged Tbx5 with or without UTX. Expression of UTX greatly ically inactive UTX mutant failed to rescue the UTX knockdown promoted the interaction between Tbx5 and Brg1 (Figure 6B).

Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. 31 Developmental Cell UTX in Heart Development

AB A C

CD B

Figure 7. Role of UTX in Establishing Active Chromatin in Cardiac- Specific Genes and Model for Action of the UTX-Complex During Developmental Transition of ESCs to Cardiac Cells (A and B) ChIP assays with a-H3K27me3 (A) and a-H3K4me3 (B) antibodies in self-renewing ESCs (E) and cEBs (C) using wild-type ESCs (UTX+/y, black bars) D Figure 6. The Demethylase Activity-Independent Function of UTX in and UTX null ESCs (UTXF- /y, gray bars). The ANF gene loses a repressive Brg1 Recruitment to the Cardiac Genes mark H3K27me3 and gains an activating mark H3K4me3 during cardiac (A) CoIP assays in HEK293T cells. UTX associates with Brg1. differentiation in wild-type ESCs, but these chromatin changes do not occur in (B) CoIP assays in HEK293T cells transfected with HA-Tbx5. UTX mediates the UTX null ESCs. The quantitative PCR levels in IgG samples were calculated as interaction between Tbx5 and Brg1. 1. Error bars represent the standard deviation. (C and D) ChIP assays with a-Brg1 in P19 cells treated with serum shock and (C) UTX acts as a critical switch to drive cardiac lineage in development. X transfected with sh-UTX and UTX expression vectors as indicated below each represents the cardiac transcription factors SRF, Tbx5, Nkx2.5, and GATA4, graph. UTX-wt, wild-type UTX; UTX-mt, catalytically inactive UTX. The quan- and XRE depicts the response elements of these cardiac transcription factors titative PCR levels in IgG samples were calculated as 1. Error bars represent that control expression of a battery of cardiac-specific genes. During cardiac the standard deviation in all graphs. differentiation, the cardiac transcription factors are induced in expression, bind to the XREs of cardiac genes, and recruit the UTX-complex, which re- moves the H3K27me3 repressive mark. In addition, UTX promotes the Next, to test whether UTX plays a role in recruiting Brg1 to the recruitment of the Brg1-complex to the cardiac genes in a demethylase heart-specific genes, we monitored the recruitment of Brg1 to activity-independent manner. UTX may also facilitate the recruitment of the ANF and Baf60c enhancers in P19 cells treated with serum histone H3K4 methyltransferases MLL3 and MLL4 that form a complex with shock. Brg1 binding to the cardiac enhancers was substantially UTX. These establish active chromatin in the cardiac genes and triggering the transcription. reduced by UTX knockdown (Figures 6C and 6D), suggesting that endogenous UTX facilitates the recruitment of Brg1 to the cardiac enhancers. Interestingly, enzymatically inactive UTX, differentiation condition (Figure 7A), suggesting that UTX is as well as wild-type UTX, enhanced Brg1-binding to the cardiac required to remove the H3K27me3 repressive mark in the ANF enhancers (Figures 6C and 6D). These data suggest a demethy- enhancer during cardiac development. These changes in the lase activity-independent function of UTX in facilitating the H3K27me3 levels were not specific to the ANF gene. UTX null recruitment of the Brg1-containing chromatin remodeling com- ESCs also failed to suppress the H3K27me3 levels efficiently plex to the cardiac-specific genes. during cardiac differentiation in the Baf60c enhancer and the enhancer of a-CA, which contains the binding sites for SRF, UTX Is Required to Trigger Transcriptionally Active Nkx2.5, and GATA4 (Chen and Schwartz, 1996; Sepulveda Chromatin in Cardiac-Specific Genes et al., 1998), while their H3K27me3 levels declined in wild-type Considering the recruitment of UTX to the cardiac enhancers cEBs (data not shown). These results suggest that UTX is impor- during cardiac lineage differentiation, we asked whether UTX is tant to eliminate the H3K27me3 mark in cardiac genes during important to shift the chromatin signature of the cardiac-specific cardiac lineage specification. genes from a poised state in self-renewing ESCs to an active Interestingly, the H3K4me3 levels in the ANF gene remained state in cEBs. The H3K27me3 levels in the ANF enhancer were unchanged and relatively low during cardiac differentiation in high in ESCs, and substantially reduced as wild-type ESCs UTX null ESCs, while the ANF-H3K4me3 levels were highly differentiated into cEBs (Figure 7A), as levels of ANF transcript induced under parallel differentiation conditions in wild-type increased (Figure 2I). In sharp contrast, in UTX null ESCs, the ESCs (Figure 7B). We also obtained the similar data with the ANF-H3K27me3 levels were not suppressed under a cardiac Baf60c enhancer (data not shown). These data indicate that in

32 Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. Developmental Cell UTX in Heart Development

the absence of UTX, cardiac-specific genes fail to undergo T-bet in T helper cells (Miller et al., 2010). Thus, H3K27 demethy- removal of H3K27me3 and deposition of H3K4me3, thus main- lases may act to bridge the Brg1-containing Swi/Snf complex taining the repressive chromatin in cardiac genes (Figure 7C). and developmental transcription factors in multiple cell types. This failure of establishment of active chromatin in cardiac genes It is worth noting that Baf60c, a heart-specific component likely contributes to various defects in UTX null embryonic of the Brg1-complex, mediates the association between Brg1 hearts. and multiple cardiac transcription factors, such as Tbx5, Nkx2.5, and GATA4, and potentiates cardiac gene expression DISCUSSION (Lickert et al., 2004). In the future, it will be interesting to investi- gate the relationship between UTX and Baf60c in the Brg1- Cardiac lineage decisions and the subsequent morphogenesis dependent transcriptional activation of cardiac genes. of the early developing heart are orchestrated by a set of tran- Our studies revealed that loss of UTX disrupts the coordination scription factors, including SRF, Tbx5, Nkx2.5, and GATA4, of H3K4 and H3K27 methylations on poised cardiac genes which support each other’s expression and act in combination during cardiac differentiation. These observations suggest that to control the overlapping target genes (Bruneau, 2002; Garg H3K27 demethylation is intricately linked to H3K4 methylation et al., 2003; Hiroi et al., 2001; McBride and Nemer, 2001; Olson, and that UTX is essential in orchestrating the chromatin signa- 2006; Searcy et al., 1998; Sepulveda et al., 1998; Small and ture of the cardiac genes from a poised to active state in the Krieg, 2004; Tanaka et al., 1999). In spite of crucial roles of developing heart. UTX may be important to bring the H3K4 meth- histone methylation in gene regulation and development (Bhau- yltransferases MLL3/4 to the cardiac genes during development mik et al., 2007; Cloos et al., 2008), the identity of enzymes that (Figure 7C), given they form a steady-state complex with MLL3 control histone methylation levels for cardiac lineage-specific or MLL4 (Cho et al., 2007; Goo et al., 2003; Issaeva et al., genes was unclear. We found that UTX is recruited to the cardiac 2007; Lee et al., 2007, 2006a). Investigating a role of UTX in re- genes by associating with core cardiac transcription factors cruiting MLL3/4 methyltransferases to cardiac genes will help Nkx2.5, Tbx5, GATA4, and SRF, thereby activating heart- understand how histone codes are tightly coordinated in cell specific genes in cardiac lineage cells, but not in other cell types lineage-specific genes during organogenesis. that do not express the cardiac transcription factors (Figure 7C). Altogether, our findings establish UTX as a central player in Considering broad expression of UTX in multiple tissues, the heart development and point to an intriguing possibility that association of UTX with the cardiac transcription factors likely cardiac-specific gene and signaling networks converge on plays an important role for specifically targeting UTX to cardiac UTX to trigger a wide range of cardiac gene transcription during genes only in cells committed to a cardiac lineage. The adjacent development. Our findings may contribute to development of locations of SRF, Tbx5, Nkx2.5, and GATA4 binding sites in the novel strategies to efficiently produce cardiomyocytes from enhancer regions of many developmentally regulated cardiac stem cells for therapeutic purpose. genes (Bruneau, 2002; He et al., 2011; McBride and Nemer, The removal of the transcriptionally repressive chromatin mark 2001; Small and Krieg, 2004) are likely crucial to recruit UTX H3K27me3 is likely important for the gene expression for more efficiently to cardiac genes, as UTX associates with multiple lineages. Relatively broad expression of UTX suggests multiple transcription factors that co-occupy the enhancers. its possible role beyond cardiac development. Indeed, UTX Our data support two mechanisms by which UTX activates null embryos exhibit neural tube closure impairment as well as transcription of cardiac genes after UTX is recruited to the genes cardiac defects, consistent with an exencephaly phenotype of via association with cardiac transcription factors. First, UTX, mutant mice with a UTX gene trap allele (Cox et al., 2010). Inter- a histone H3K27 demethylase, eliminates the H3K27me3 repres- estingly, our data show that UTX null ESCs are not defective sive mark in heart-specific genes during cardiac differentiation to in cellular differentiation in general. Under a differentiation allow transcriptional activation. UTX knockdown blocks the acti- condition with RA, UTX null ESCs stop self-renewal and readily vated SRF from reducing the H3K27me3 levels in the cardiac differentiate. In addition, UTX null embryos develop until E9.5. genes, which was rescued by UTX wild-type but not by enzymat- We also found that UTX null ESCs differentiate into neurons, ically inactive UTX. Consistently, in the absence of UTX, cardiac similarly to wild-type ESCs (data not shown). It is possible that genes are still marked by the H3K27me3 even under a cardiac UTX is dispensable for differentiation of certain cell lineages differentiation condition. These data suggest that the enzymatic and/or that JMJD3, another H3K27 demethylase, functions activity of UTX is crucial in establishing a transcriptionally active redundantly with UTX. Analyses of JMJD3-deficient mice and chromatin in cardiac genes. Second, UTX associates with Brg1 mutant mice lacking both JMJD3 and UTX may be employed and facilitates the recruitment of the Brg1-containing ATPase- to address specific and redundant functions of JMJD3 and dependent Swi/Snf chromatin-remodeling complex to the UTX in future. cardiac genes. Interestingly, this function of UTX appears inde- A single allele mutation in UTX gene in males is sufficient to pendent of its demethylase activity. Consistently, a catalytically inactivate UTX gene as it is located on the X chromosome. In light inactive UTX mutant enhanced the activity of the cardiac tran- of this, it is noteworthy that somatic mutations of UTX were found scription factors in luciferase assays and partially restored the in various human cancers (van Haaften et al., 2009). UTXD/y male transcription of some UTX target genes inhibited by UTX knock- mice displayed a wide range of phenotypes from embryonic down (data not shown). Our data indicate that UTX strongly lethality with severe cardiac malformation to tumor formation in promotes the association between Brg1 and Tbx5. JMJD3, adults, while UTXD/+ heterozygote female mice were viable and another histone H3K27 demethylase, has also been shown to fertile. UTXD/D female mice invariably show embryonic lethality. mediate the association between Brg1 and a T-box factor These analyses of UTX-deficient mice suggest that UTY

Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. 33 Developmental Cell UTX in Heart Development

compensates for the loss of UTX but only partially. Given MTT Cell Proliferation Assays previous studies that UTY lacks the H3K27 demethylase enzy- A total of 500 cells/well were seeded on MEF feeder cells in 96-well plate in matic activity in vitro (Hong et al., 2007; Lan et al., 2007), propagation medium. Ten microliters of MTT (3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide, 5 mg/ml, Sigma) solution was added to each compensation of UTX deficiency by UTY might be attributed to well containing cells and incubated at 37 C. Six hours later, medium was its demethylase activity-independent functions such as recruit- removed and 200 ml of dimethyl sulfoxide (DMSO) was added and absorbance ment of Brg1 to the developmental transcription factors. was measured at 550 nm using spectrophotometer. However, it is still possible that UTY exhibits demethylase activity under certain in vivo contexts. Further investigation into TUNEL Assays the redundant and unique actions of UTX and UTY will provide To monitor cell death, differentiated cEBs and E8.5 embryos were analyzed important insights into not only the developmental roles of with In situ cell death detection kit, TMR red (Roche). Briefly, tissues were fixed in 4% paraformaldehyde for 20 min at room temperature, washed with PBS histone demethylases but also their anti-tumorigenic functions. twice, permeabilized (0.1% Triton X-100 in 0.1% sodium citrate), washed Our UTX-deficient mice will serve as crucial tools for future with PBS twice and TUNEL reaction mixture was added and incubated for investigation. 1hrat37C.

EXPERIMENTAL PROCEDURES Immunohistochemistry and In Situ Hybridization Assays For immunohistochemistry and in situ hybridization analyses, the embryos Generation of UTX Null ESCs were harvested at indicated stage, fixed in 4% paraformaldehyde, embedded m UTXF-loxP ESCs were obtained from the European Conditional Mouse in OCT, and cryosectioned at 12–18 m. Immunohistochemistry was per- Mutagenesis Program (EUCOMM). UTXF-loxP ESCs and their wild-type coun- formed as described (Lee et al., 2009) using the following antibodies and a a a terparts were maintained on top of irradiated mouse embryonic fibroblast reagents: -Ki67 (Novocastra), -Nkx2.5 (H-114, Santa Cruz), -Isl1 (DSHB), a (MEF) feeder cells in propagation medium consisting of Knockout-DMEM and -UTX (a gift from K. Helin) antibodies and 4,6-diamidino-2-phenylindole (Dulbecco’s modified Eagle’s medium), 10% fetal bovine serum, 2 mM (DAPI) (Sigma). L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM b-mercaptoethanol, For in situ hybridization analysis, embryos were processed with digoxigenin- and leukemia inhibitory factor (LIF; 1,000 units/ml, Chemicon). To generate labeled Nkx2.5, Tbx5, MLC2v (gifts from J. Martin), ANF (gift from B. Bruneau), UTXF-D/y ESCs that are deficient in exon 3 of the UTX gene, 2000 UTXF-loxP UTX and UTY riboprobes, as described previously (Wilkinson and Nieto, 1993). ESCs were plated in 35 mm dish with MEF feeder cells for 24 hr and trans- cDNA for mouse UTX and UTY were cloned to pBluescript vector to generate fected with pOG231-Cre plasmid (Addgene) using Lipofectamine 2000 riboprobes. (Invitrogen). Three days later, single colonies were picked, further amplified, For H&E staining, transverse sections of E9 embryos were stained with and verified for the loss of UTX expression by western blot and RT-PCR hematoxylin and eosin according to standard techniques. analyses. PCR was used to determine the genotype of ESCs with the primers 50-GTTGTGGTTTGTCCAAACTCA-30 and 50-CGAGTGATTGGTCTAATTT RNA Extraction and Quantitative RT-PCR GG-30. The amplified product from the UTXF-D allele is 209 bp, whereas that Total RNAs were extracted using the Trizol (Invitrogen) and reverse-tran- from the UTXF-loxP allele is 996 bp. Additional set of primers was also used scribed using the SuperScript III First-Strand Synthesis System (Invitrogen). to determine the UTXF-loxP allele, which produces 264 bp PCR product; The levels of mRNA were determined using quantitative RT-PCR (Mx3000P, 50-GTTGTGGTTTGTCCAAACTCA-30 and 50-CTGTAATCTCTGCTGATACAA-30. Stratagene). The levels of mRNA were normalized against the levels of cyclo- philin A. The primers used were as follows: ANF, forward 50-GAAAAGGCAG TCGATTCTGC-30, reverse 50-CAGAGTGGGAGAGGCAAGAC-30; MLC2v, Generation of UTX-Deficient Mice forward 50-ACAGAGACGGCTTCATCGAC-30, reverse 50-TGAATGCGTTGA UTXF-loxP ESCs were introduced into mouse blastocysts. Chimeric mice were GAATGGTC-30; Myh6, forward 50-CACTCAATGAGACGGTGGTG-30, reverse mated with wild-type C57BL6 mice. UTXF-loxP/+ heterozygous F1 mice were 50-GTGGGTGGTCTTCAGGTTTG-30; SRF, forward 50-CCTTCAGCAAGAGG mated to Rosa-Flp mice to delete the sequences between FRT sites (Farley AAGACG-30, reverse 50-GAGAGTCTGGCGAGTTGAGG-30; Nkx2.5, forward et al., 2000). To delete exon 3 of UTX constitutively, EIIa-Cre mice (Lakso 50-CTTGAACACCGTGCAGAGTC-30, reverse 50-GGTGGGTGTGAAATCTGAG et al., 1996) were crossed with mice bearing UTX-floxed allele. Genotyping G-30; UTX, forward 50-CTGAAGGGAAAGTGGAGTCTG-30, reverse 50-TCGAC was performed by PCR using primers 50-TCTGGTATAGATTTTGGCTAA-30 ATAAAGCACCTCCTG-30; UTY, forward 50- ATAGTGTCCAGACAGCTTCA-30, and 50-CGAGTGATTGGTCTAATTTGG-30. PCR reaction results in 932 bp reverse 50-GAGGTAGGAATACGTAAGAA-30; Tbx5, forward 50- GTGGCTGAA product from wild-type allele and 286 bp product from UTX null allele. All GTTCCACGAAG-30, reverse 50-GGCTCTGCTTTGCCAGTTAC-30; a-CA, animal procedures were conducted in accordance with the Guidelines for forward 50-TGCTTCTGGAAGAACCACAG-30, reverse 50-TCACGGACAATTTC the Care and Use of Laboratory Animals and were approved by the Institutional ACGTTC-30; GATA4, forward 50-CCCCTCATTAAGCCTCAGC-30, reverse Animal Care and Use Committee at Oregon Health and Science University. 50-ATTCAGGTTCTTGGGCTTCC-30; cyclophilin A, forward 50-GTCTCCTTCG AGCTGTTTGC-30, reverse 50-GATGCCAGGACCTGTATGCT-30. Differentiation ESCs into a Cardiac Lineage Cardiac differentiation of ESCs was performed as previously described (Boh- Whole-Mount X-Gal Staining eler et al., 2002). Briefly, 500 ESCs in 20 ml of differentiation medium (ESC Embryos were harvested, fixed in 4% paraformaldehyde for 10 min at 4C, and growth medium without LIF) were placed on the lids of 150 mm Petri dishes washed in rinse solution (100 mM sodium phosphate, pH7.5, 2 mM MgCl2, and for 2 days. The aggregated ESCs, now termed embryoid bodies (EBs), were 0.02% NP-40) three times for 20 min each at 4C. Then, embryos were stained floated in bacteriological low attach dishes for 5 days and medium was with X-gal staining solution (1 mg/ml X-gal, 5 mM potassium ferricyanide and changed every other day. At day 7, EBs were plated onto tissue culture dishes 5 mM potassium ferrocyanide in rinse solution) at 37C overnight. Embryos coated with 0.1% gelatin. Spontaneous beating areas could be detected as were postfixed in 4% paraformaldehyde for 1 hr at room temperature and early as day 8 (day 0 means the day when hanging drops were seeded). washed in rinse buffer. To make the embryos transparent, embryos were incu- bated in serial dilution of glycerol (25%, 50%, 80% in PBS) for at least 5 hr in AP Staining each step. To measure AP activity, 3,000 cells/well were seeded on MEF feeder cells in 24-well plate in propagation medium. For ESC differentiation, 1 mM of retinoic DNA Constructs acid (RA) was added in the absence of LIF. At day 4, cells were fixed in 4% ANF:Luc, Flag-Nkx2.5, Flag-GATA4, HA-Tbx5 (gifts from B. Bruneau), Myc- paraformaldehyde/PBS and AP activity was measured using alkaline phos- UTX (a gift from K. Gei), shRNA-human UTX (a gift from K. Helin), SM22:Luc phatase detection kit (Chemicon #SCR004). (a gift from J. Epstein), aCA:Luc (a gift from M. Parmacek), SRE:luc, and

34 Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. Developmental Cell UTX in Heart Development

HA-SRF vectors were described previously (Agger et al., 2007; Bruneau et al., ACKNOWLEDGMENTS 2001; Chen et al., 2002; Hong et al., 2007; Lee et al., 2000). Mouse Pax6 full- length was cloned to pcDNA3HA vector. shRNA-mouse UTX target sequence We are grateful to Juhee Kim and Seongkyung Seo for their excellent technical is AAGTTAGCAGTGGAACGTTAT. support; J. Christian and D. Goldman for advice on embryo analyses; R. Goodman, G. Mandel, S. Impey, K. Thiebes, N. Vo, L. Cambronne, J. Oyer, and D. Goldman for critically reading the manuscript; B. Bruneau, Luciferase Reporter Assays J. Martin, K. Helin, K. Gei, J. Epstein, and M. Parmacek for various reagents. HEK293T cells were cultured in DMEM media supplemented with 10% fetal This research was supported by grants from NIH/NINDS (R01 NS054941), bovine serum. Cells were plated in 48-well plate and incubated for 24 hr, March of Dimes Foundation, and Christopher and Dana Reeve Foundation and transient transfections were performed using Superfect (QIAGEN). An (to S.-K.L.) and NIH/NIDDK (R01 DK064678) (to J.W.L.). actin-b-galactosidase plasmid was cotransfected for normalization of trans- fection efficiency, and empty vectors were used to equalize the total Received: August 6, 2011 amount of DNA. Cells were harvested 48 hr after transfection. Cell extracts Revised: October 26, 2011 were assayed for luciferase activity and the values were normalized with Accepted: November 21, 2011 b-galactosidase activity. All transfections were repeated independently at Published online: December 22, 2011 least three times. Data are represented as the mean of triplicate values ob- tained from representative experiments. Error bars represent standard deviation. REFERENCES

CoIP and Western Blotting Assays Agger, K., Cloos, P.A., Christensen, J., Pasini, D., Rose, S., Rappsilber, J., HEK293T cells were seeded on 10 cm tissue cultures dishes and expression Issaeva, I., Canaani, E., Salcini, A.E., and Helin, K. (2007). UTX and JMJD3 vectors were transfected as described above. Forty eight hours later, cells are histone H3K27 demethylases involved in HOX gene regulation and were harvested and lysed in IP buffer (20 mM Tris-HCl, pH 8.0, 0.5% NP-40, development. Nature 449, 731–734.

1 mM EDTA, 150 mM NaCl, 2 mM PMSF, 10% glycerol, 4 mM Na3VO4, Azuara, V., Perry, P., Sauer, S., Spivakov, M., Jørgensen, H.F., John, R.M.,

200 mM NaF, 20 mM Na-pyroPO4, and protease inhibitor cocktail). In these Gouti, M., Casanova, M., Warnes, G., Merkenschlager, M., and Fisher, A.G. studies, precipitations were performed with a-Myc (Abcam), a-HA (Covance) (2006). Chromatin signatures of pluripotent cell lines. Nat. Cell Biol. 8, 532–538. and a-Brg1 (Millipore) antibodies. The interactions were monitored by western Bernstein, B.E., Mikkelsen, T.S., Xie, X., Kamal, M., Huebert, D.J., Cuff, J., Fry, blotting assays using a-Flag (Sigma), a-Myc (Abcam), a-Brg1 (Millipore), and B., Meissner, A., Wernig, M., Plath, K., et al. (2006). A bivalent chromatin a-UTX (a gift from K. Helin) antibodies. structure marks key developmental genes in embryonic stem cells. Cell 125, E12.5 mouse embryonic heart tissues were lysed in IP buffer, immunopre- 315–326. cipitated with a-UTX (a gift from K. Helin), a-SRF (G-20), a-Tbx5 (H-70), a-Nkx2.5 (H-114), and a-Gata4 (H-112) antibodies from Santa Cruz, and Bhaumik, S.R., Smith, E., and Shilatifard, A. (2007). Covalent modifications of then the presence of UTX in immunopurified complex was monitored by immu- during development and disease pathogenesis. Nat. Struct. Mol. Biol. noblotting with a-UTX antibody. 14, 1008–1016. Western blotting assays with a-cleaved Caspase3 (Asp175) (Cell Signaling) Boheler, K.R., Czyz, J., Tweedie, D., Yang, H.T., Anisimov, S.V., and Wobus, antibody was used to monitor apoptotic cells in ESCs and cEBs. a-Oct34 A.M. (2002). Differentiation of pluripotent embryonic stem cells into cardio- (N-19, Santa Cruz) antibody was used to monitor the self-renewal and differen- myocytes. Circ. Res. 91, 189–201. tiation of ESCs. a-tubulin (Santa Cruz) antibody was used as loading control. Boyer, L.A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L.A., Lee, T.I., Levine, S.S., Wernig, M., Tajonar, A., Ray, M.K., et al. (2006). Polycomb ChIP Assays complexes repress developmental regulators in murine embryonic stem cells. P19 cells were transfected with shRNA against mouse UTX, knockdown insen- Nature 441, 349–353. sitive form of human UTX wild-type or catalytically inactive UTX mutant Bruneau, B.G. (2002). Transcriptional regulation of vertebrate cardiac morpho- UTX-H1146A using Lipofectamine2000 (Invitrogen). Forty eight hours later, genesis. Circ. Res. 90, 509–519. cells were washed with PBS and growth media (minimum essential medium Bruneau, B.G., Nemer, G., Schmitt, J.P., Charron, F., Robitaille, L., Caron, S., a, 7.5% bovine calf serum and 2.5% fetal bovine serum) was replaced with Conner, D.A., Gessler, M., Nemer, M., Seidman, C.E., and Seidman, J.G. MEMa supplemented with 0.5% fetal bovine serum and incubated for 17 hr. (2001). A murine model of Holt-Oram syndrome defines roles of the T-box Serum shock was applied by changing the low serum to 20% fetal bovine transcription factor Tbx5 in cardiogenesis and disease. Cell 106, 709–721. serum for 2 hr. E12.5 mouse embryonic heart, limbs, and brain were used for ChIP experiments. ChIP experiments were performed as previously Charron, F., and Nemer, M. (1999). GATA transcription factors and cardiac described (Lee and Pfaff, 2003). Briefly, cells were crosslinked with formalde- development. Semin. Cell Dev. Biol. 10, 85–91. a hyde and sonicated. Protein-DNA complexes were precipitated with -UTX (a Chen, C.Y., and Schwartz, R.J. (1996). Recruitment of the tinman homolog a a a gift from K. Helin), -Brg1 (Millipore), -H3K27me3 and -H3K4me3 (Millipore) Nkx-2.5 by serum response factor activates cardiac alpha-actin gene tran- antibodies. The purified DNA was amplified using PCR and the SYBR green kit scription. Mol. Cell. Biol. 16, 6372–6384. (Invitrogen) for quantitation. Quantitative results were normalized to the total input control. Primers used were ANF-enhancer, forward 50-TGAATCAGGTG Chen, F., Kook, H., Milewski, R., Gitler, A.D., Lu, M.M., Li, J., Nazarian, R., TGAAGCTAGCTCC-30, reverse 50-GGAGCAGAAAAGAGTCCTTGGTTA-30; Schnepp, R., Jen, K., Biben, C., et al. (2002). Hop is an unusual homeobox ANF-exon1, forward 50-GGGAGATGCTGGCAGCTAGGAGAC-30, reverse gene that modulates cardiac development. Cell 110, 713–723. 50-CTTGAAATCCATCAGATCTGTGTT-30; Baf60c-enhancer, forward 50-TTA Cho, Y.W., Hong, T., Hong, S., Guo, H., Yu, H., Kim, D., Guszczynski, T., AGCAGTCAGAAGATCTCCTTG-30, reverse 50-GGCTCTCCACCTCAATAGG Dressler, G.R., Copeland, T.D., Kalkum, M., and Ge, K. (2007). PTIP associates TTCCT-30. All experiments were repeated independently at least three times. with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase Data are represented as the mean of triplicate values obtained from represen- complex. J. Biol. Chem. 282, 20395–20406. tative experiments. Error bars represent standard deviation. Cloos, P.A., Christensen, J., Agger, K., and Helin, K. (2008). Erasing the methyl mark: histone demethylases at the center of cellular differentiation and SUPPLEMENTAL INFORMATION disease. Genes Dev. 22, 1115–1140. Cox, B.J., Vollmer, M., Tamplin, O., Lu, M., Biechele, S., Gertsenstein, M., van Supplemental Information includes six figures and can be found with this Campenhout, C., Floss, T., Ku¨ hn, R., Wurst, W., et al. (2010). Phenotypic anno- article online at doi:10.1016/j.devcel.2011.11.009. tation of the mouse X chromosome. Genome Res. 20, 1154–1164.

Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. 35 Developmental Cell UTX in Heart Development

De Santa, F., Totaro, M.G., Prosperini, E., Notarbartolo, S., Testa, G., and Lee, T.I., Jenner, R.G., Boyer, L.A., Guenther, M.G., Levine, S.S., Kumar, R.M., Natoli, G. (2007). The histone H3 lysine-27 demethylase Jmjd3 links inflamma- Chevalier, B., Johnstone, S.E., Cole, M.F., Isono, K., et al. (2006b). Control of tion to inhibition of polycomb-mediated gene silencing. Cell 130, 1083–1094. developmental regulators by Polycomb in human embryonic stem cells. Cell Durocher, D., and Nemer, M. (1998). Combinatorial interactions regulating 125, 301–313. cardiac transcription. Dev. Genet. 22, 250–262. Li, L., Liu, Z., Mercer, B., Overbeek, P., and Olson, E.N. (1997). Evidence for Farley, F.W., Soriano, P., Steffen, L.S., and Dymecki, S.M. (2000). Widespread serum response factor-mediated regulatory networks governing SM22alpha recombinase expression using FLPeR (flipper) mice. Genesis 28, 106–110. transcription in smooth, skeletal, and cardiac muscle cells. Dev. Biol. 187, 311–321. Garg, V., Kathiriya, I.S., Barnes, R., Schluterman, M.K., King, I.N., Butler, C.A., Rothrock, C.R., Eapen, R.S., Hirayama-Yamada, K., Joo, K., et al. (2003). Lickert, H., Takeuchi, J.K., Von Both, I., Walls, J.R., McAuliffe, F., Adamson, GATA4 mutations cause human congenital heart defects and reveal an inter- S.L., Henkelman, R.M., Wrana, J.L., Rossant, J., and Bruneau, B.G. (2004). action with TBX5. Nature 424, 443–447. Baf60c is essential for function of BAF chromatin remodelling complexes in heart development. Nature 432, 107–112. Goo, Y.H., Sohn, Y.C., Kim, D.H., Kim, S.W., Kang, M.J., Jung, D.J., Kwak, E., Barlev, N.A., Berger, S.L., Chow, V.T., et al. (2003). Activating signal cointegra- Lyons, I., Parsons, L.M., Hartley, L., Li, R., Andrews, J.E., Robb, L., and tor 2 belongs to a novel steady-state complex that contains a subset of Harvey, R.P. (1995). Myogenic and morphogenetic defects in the heart tubes trithorax group proteins. Mol. Cell. Biol. 23, 140–149. of murine embryos lacking the homeo box gene Nkx2-5. Genes Dev. 9, 1654–1666. Greenfield, A., Carrel, L., Pennisi, D., Philippe, C., Quaderi, N., Siggers, P., Steiner, K., Tam, P.P., Monaco, A.P., Willard, H.F., and Koopman, P. (1998). McBride, K., and Nemer, M. (2001). Regulation of the ANF and BNP promoters The UTX gene escapes X inactivation in mice and humans. Hum. Mol. by GATA factors: lessons learned for cardiac transcription. Can. J. Physiol. Genet. 7, 737–742. Pharmacol. 79, 673–681. Hang, C.T., Yang, J., Han, P., Cheng, H.L., Shang, C., Ashley, E., Zhou, B., and Miano, J.M., Ramanan, N., Georger, M.A., de Mesy Bentley, K.L., Emerson, Chang, C.P. (2010). Chromatin regulation by Brg1 underlies heart muscle R.L., Balza, R.O., Jr., Xiao, Q., Weiler, H., Ginty, D.D., and Misra, R.P. development and disease. Nature 466, 62–67. (2004). Restricted inactivation of serum response factor to the cardiovascular system. Proc. Natl. Acad. Sci. USA 101, 17132–17137. He, A., Kong, S.W., Ma, Q., and Pu, W.T. (2011). Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in Miller, S.A., Mohn, S.E., and Weinmann, A.S. (2010). Jmjd3 and UTX play a de- heart. Proc. Natl. Acad. Sci. USA 108, 5632–5637. methylase-independent role in chromatin remodeling to regulate T-box family member-dependent gene expression. Mol. Cell 40, 594–605. Hiroi, Y., Kudoh, S., Monzen, K., Ikeda, Y., Yazaki, Y., Nagai, R., and Komuro, I. (2001). Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyo- Niu, Z., Yu, W., Zhang, S.X., Barron, M., Belaguli, N.S., Schneider, M.D., cyte differentiation. Nat. Genet. 28, 276–280. Parmacek, M., Nordheim, A., and Schwartz, R.J. (2005). Conditional mutagen- esis of the murine serum response factor gene blocks cardiogenesis and the Hong, S., Cho, Y.W., Yu, L.R., Yu, H., Veenstra, T.D., and Ge, K. (2007). transcription of downstream gene targets. J. Biol. Chem. 280, 32531–32538. Identification of JmjC domain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases. Proc. Natl. Acad. Sci. USA 104, 18439–18444. Olson, E.N. (2006). Gene regulatory networks in the evolution and develop- ment of the heart. Science 313, 1922–1927. Issaeva, I., Zonis, Y., Rozovskaia, T., Orlovsky, K., Croce, C.M., Nakamura, T., Mazo, A., Eisenbach, L., and Canaani, E. (2007). Knockdown of ALR (MLL2) Pan, G., Tian, S., Nie, J., Yang, C., Ruotti, V., Wei, H., Jonsdottir, G.A., Stewart, reveals ALR target genes and leads to alterations in cell adhesion and growth. R., and Thomson, J.A. (2007). Whole-genome analysis of histone H3 lysine 4 Mol. Cell. Biol. 27, 1889–1903. and lysine 27 methylation in human embryonic stem cells. Cell Stem Cell 1, 299–312. Jepsen, K., Solum, D., Zhou, T., McEvilly, R.J., Kim, H.J., Glass, C.K., Hermanson, O., and Rosenfeld, M.G. (2007). SMRT-mediated repression of Parlakian, A., Tuil, D., Hamard, G., Tavernier, G., Hentzen, D., Concordet, J.P., an H3K27 demethylase in progression from neural stem cell to neuron. Paulin, D., Li, Z., and Daegelen, D. (2004). Targeted inactivation of serum Nature 450, 415–419. response factor in the developing heart results in myocardial defects and embryonic lethality. Mol. Cell. Biol. 24, 5281–5289. Lakso, M., Pichel, J.G., Gorman, J.R., Sauer, B., Okamoto, Y., Lee, E., Alt, F.W., and Westphal, H. (1996). Efficient in vivo manipulation of mouse genomic Rada-Iglesias, A., Bajpai, R., Swigut, T., Brugmann, S.A., Flynn, R.A., and sequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93, 5860–5865. Wysocka, J. (2011). A unique chromatin signature uncovers early develop- mental enhancers in humans. Nature 470, 279–283. Lan, F., Bayliss, P.E., Rinn, J.L., Whetstine, J.R., Wang, J.K., Chen, S., Iwase, S., Alpatov, R., Issaeva, I., Canaani, E., et al. (2007). A histone H3 lysine 27 Searcy, R.D., Vincent, E.B., Liberatore, C.M., and Yutzey, K.E. (1998). A GATA- demethylase regulates animal posterior development. Nature 449, 689–694. dependent nkx-2.5 regulatory element activates early cardiac gene expression in transgenic mice. Development 125, 4461–4470. Lee, M.G., Villa, R., Trojer, P., Norman, J., Yan, K.P., Reinberg, D., Di Croce, L., and Shiekhattar, R. (2007). Demethylation of H3K27 regulates polycomb Sepulveda, J.L., Belaguli, N., Nigam, V., Chen, C.Y., Nemer, M., and Schwartz, recruitment and H2A ubiquitination. Science 318, 447–450. R.J. (1998). GATA-4 and Nkx-2.5 coactivate Nkx-2 DNA binding targets: role for regulating early cardiac gene expression. Mol. Cell. Biol. 18, 3405–3415. Lee, S., Lee, D.K., Dou, Y., Lee, J., Lee, B., Kwak, E., Kong, Y.Y., Lee, S.K., Roeder, R.G., and Lee, J.W. (2006a). Coactivator as a target gene specificity Shore, P., and Sharrocks, A.D. (1995). The MADS-box family of transcription determinant for histone H3 lysine 4 methyltransferases. Proc. Natl. Acad. factors. Eur. J. Biochem. 229, 1–13. Sci. USA 103, 15392–15397. Small, E.M., and Krieg, P.A. (2003). Transgenic analysis of the atrial natriuretic Lee, S., Lee, B., Lee, J.W., and Lee, S.K. (2009). Retinoid signaling and neuro- factor (ANF) promoter: Nkx2-5 and GATA-4 binding sites are required for atrial genin2 function are coupled for the specification of spinal motor neurons specific expression of ANF. Dev. Biol. 261, 116–131. through a chromatin modifier CBP. Neuron 62, 641–654. Small, E.M., and Krieg, P.A. (2004). Molecular regulation of cardiac chamber- Lee, S.K., and Pfaff, S.L. (2003). Synchronization of neurogenesis and motor specific gene expression. Trends Cardiovasc. Med. 14, 13–18. neuron specification by direct coupling of bHLH and homeodomain transcrip- Strobeck, M., Kim, S., Zhang, J.C., Clendenin, C., Du, K.L., and Parmacek, tion factors. Neuron 38, 731–745. M.S. (2001). Binding of serum response factor to CArG box sequences is Lee, S.K., Na, S.Y., Jung, S.Y., Choi, J.E., Jhun, B.H., Cheong, J., Meltzer, necessary but not sufficient to restrict gene expression to arterial smooth P.S., Lee, Y.C., and Lee, J.W. (2000). Activating protein-1, nuclear factor- muscle cells. J. Biol. Chem. 276, 16418–16424. kappaB, and serum response factor as novel target molecules of the Tanaka, M., Chen, Z., Bartunkova, S., Yamasaki, N., and Izumo, S. (1999). The cancer-amplified transcription coactivator ASC-2. Mol. Endocrinol. 14, cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple 915–925. genes essential for heart development. Development 126, 1269–1280.

36 Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. Developmental Cell UTX in Heart Development

Thuerauf, D.J., Hanford, D.S., and Glembotski, C.C. (1994). Regulation of rat Wilkinson, D.G., and Nieto, M.A. (1993). Detection of messenger RNA by in situ brain natriuretic peptide transcription. A potential role for GATA-related hybridization to tissue sections and whole mounts. Methods Enzymol. 225, transcription factors in myocardial cell gene expression. J. Biol. Chem. 269, 361–373. 17772–17775. van Haaften, G., Dalgliesh, G.L., Davies, H., Chen, L., Bignell, G., Greenman, Zhao, X.D., Han, X., Chew, J.L., Liu, J., Chiu, K.P., Choo, A., Orlov, Y.L., Sung, C., Edkins, S., Hardy, C., O’Meara, S., Teague, J., et al. (2009). Somatic W.K., Shahab, A., Kuznetsov, V.A., et al. (2007). Whole-genome mapping of mutations of the histone H3K27 demethylase gene UTX in human cancer. histone H3 Lys4 and 27 trimethylations reveals distinct genomic compart- Nat. Genet. 41, 521–523. ments in human embryonic stem cells. Cell Stem Cell 1, 286–298.

Developmental Cell 22, 25–37, January 17, 2012 ª2012 Elsevier Inc. 37