Báo cáo khoa học: CXXC finger protein 1 restricts the Setd1A histone H3K4 methyltransferase complex to euchromatin doc

CXXC finger protein 1 restricts the Setd1A histone H3K4 methyltransferase complex to euchromatin Courtney M. Tate, Jeong-Heon Lee and David G. Skalnik Herman B. Wells Center for Pediatric Research, Section of Pediatric Hematology ⁄ Oncology, Departments of Pediatrics and Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA Introduction DNA in eukaryotic cells is complexed with histones and other proteins in the form of chromatin. The core histone tails are subject to a variety of covalent modifications, including acetylation, phosphorylation, methylation, ubiquitination, sumoylation, and ADP-ribosylation [1,2]. Histone methylation plays critical roles in gene expression, epigenetic regulation, and disease [3]. Histone methylation is catalyzed by a family of histone methyltransferase (HMT) enzymes, many of which are characterized by an evolutionarily conserved catalytic SET [Su(var)3–9, Enhancer of Zeste, Trithorax] domain [4]. A major function of the SET domain-containing proteins is to modulate gene activity [5]. Lys residues of histones can be monomethylated, dimethylated, Keywords chromatin; epigenetics; histone methylation; subnuclear targeting Correspondence D. Skalnik, Cancer Research Building, 1044 West Walnut Street, Indianapolis, IN 46202, USA Fax: +1 317 278 9298 Tel: +1 317 274 8977 E-mail: dskalnik@iupui.edu (Received 21 September 2009, revised 28 October 2009, accepted 4 November 2009) doi:10.1111/j.1742-4658.2009.07475.x CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is a compo- nent of the euchromatic Setd1A histone H3K4 methyltransferase complex, and is a critical regulator of histone methylation, cytosine methylation, cel- lular differentiation, and vertebrate development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1 ) ⁄ ) ) are viable but show increased levels of global histone H3K4 methylation, suggesting that Cfp1 functions to inhibit or restrict the activity of the Setd1A histone H3K4 methyltransferase com- plex. The studies reported here reveal that ES cells lacking Cfp1 contain decreased levels of Setd1A and show subnuclear mislocalization of both Setd1A and trimethylation of histone H3K4 with regions of heterochroma- tin. Remarkably, structure–function studies reveal that expression of either the N-terminal fragment of Cfp1 (amino acids 1–367) or the C-terminal fragment of Cfp1 (amino acids 361–656) is sufficient to restore appropriate levels of Setd1A in CXXC1 ) ⁄ ) ES cells. Furthermore, functional analysis of various Cfp1 point mutations reveals that retention of either Cfp1 DNA-binding activity or association with the Setd1 histone H3K4 methyl- transferase complex is required to restore normal Setd1A levels. In con- trast, expression of full-length Cfp1 in CXXC1 ) ⁄ ) ES cells is required to restrict Setd1A and histone H3K4 trimethylation to euchromatin, indicat- ing that both Cfp1 DNA-binding activity and interaction with the Setd1A complex are required for appropriate genomic targeting of the Setd1A complex. These studies illustrate the complexity of Cfp1 function, and identify Cfp1 as a regulator of Setd1A genomic targeting. Abbreviations CTD, C-terminal repeat domain; DAPI, 4¢,6-diaminidino-2-phenylindone; Dnmt1, DNA methyltransferase 1; ES, embryonic stem; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H3K4me3, trimethylated histone H3K4; HMT, histone methyltransferase; PHD, plant homeodomain; RNAP, RNA polymerase; Ser5-P CTD, C-terminal repeat domain phosphorylated at Ser5; SID, Set1 interaction domain. 210 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS or trimethylated, and the functional relevance of these modifications depends on the position. For example, dimethylated and trimethylated histone H3K4 is found associated with promoters and 5¢-regions of active genes [6], whereas dimethylated and trimethylated his- tone H3K9 is present at transcriptionally inactive chro- matin sites [7–9]. Yeast express a single H3K4 HMT, Set1, which associates with a complex known as COM- PASS (Complex Proteins Associated with Set1) [10] and is required for telomeric and rDNA silencing [11,12]. In contrast, mammalian cells contain numerous HMTs that show specificity for histone H3K4, including Setd1A, Setd1B, Mll1, Mll2, Mll3 ⁄ Halr, Mll4 ⁄ Alr, Ash1L, Smyd1, Smyd2, Smyd3, and Set7 ⁄ 9, which are present as distinct multiprotein complexes and play critical roles in gene expression and development [4,13–16]. The molecular mechanisms that control the targeting and activity of HMT complexes are not well under- stood. Methylation at histone H3K4 correlates with transcriptional activation and is directly coupled to the transcription process [17]. In yeast and mammals, Set1 and Setd1A localize to the 5¢-end of actively tran- scribed genes and interact with the RNA polymerase (RNAP) II C-terminal domain (CTD) phosphorylated at Ser5 (Ser5-P CTD), a repeat marker associated with transcription initiation [18–20]. In yeast, Paf1C interac- tion with RNAP II is required for recruitment of the Set1–COMPASS H3K4 HMT complex to actively transcribed genes [19]. In mammals, Setd1A is tethered to RNAP II by Wdr82, an integral component of the Setd1A complex [18]. Wdr82 associates with the RNA recognition motif within Setd1A, and directly recog- nizes Ser5-P CTD of RNAP II [18]. In mammals, Mll1 interacts with RNAP II containing Ser5-P CTD and mediates histone H3K4 methylation at a subset of transcriptionally active genes [21]. In addition, menin, a component of the Mll2 H3K4 HMT complex, associ- ates with RNAP II containing Ser5-P CTD [22]. In yeast and mammals, the Setd2 H3K36 HMT primarily associates with the elongating hyperphosphorylated form of RNAP II [23,24]. Therefore, histone methyla- tion mediated by HMTs is involved in regulating both transcription initiation and elongation. Although generally widely expressed, mammalian H3K4 HMTs have nonredundant functions. For exam- ple, Mll2 is important for expression of the HOXB gene cluster, but not the HOXA cluster [13], whereas HOXA9 and HOXC8 are exclusive Mll1 targets [22,25]. The HMTs Ash1L and Mll1 occupy the 5¢-regions of active genes, and their localization is nearly indistinguishable, which suggests redundancy of function [14]. However, in vivo depletion of either enzyme results in diminished methylation of histone H3K4 at active HOXA genes [14]. In addition, loss of a single member of the H3K4 HMT family can lead to disease or death [26,27]. MLL1 is frequently the target of chromosomal translocations involved in acute lymphoid and myeloid leukemias [28–31]. In addition, genetic disruption of murine MLL1 or MLL2 leads to embryonic lethality [13,32]. In addition, Smyd3 expression is upregulated in colorectal and hepatocellular carcinomas, and its H3K4 HMT activity activates oncogenes and other genes associated with the cell cycle, whereas depletion of Smyd3 by small interfering RNA treatment leads to suppression of cell growth [27]. With the exception of the enzymatic Setd1 compo- nent, the subunit composition of the mammalian Setd1A and Setd1B HMTase complexes are identical [16], each containing CXXC finger protein 1 (Cfp1), Rbbp5, Wdr5, Ash2, and Wdr82 [15,16]. Setd1A and Setd1B mRNA are ubiquitously expressed in murine tissues, and Setd1A and Setd1B do not show differen- tial cell type expression [16]. However, confocal immu- nofluorescence reveals that endogenous Setd1A and Setd1B show largely nonoverlapping subnuclear locali- zation [16]. This suggests that Setd1A and Setd1B are targeted to unique sets of genomic sites, and that each has unique functions in the regulation of chromatin structure and gene expression. Consequently, it is likely that the nonredundant function of each H3K4 HMT is a result of distinct target gene specificity [16]. Cfp1 is a critical epigenetic regulator of both cytosine methylation and histone methylation, and interacts with both the maintenance DNA methyltrans- ferase [DNA methyltransferase 1 (Dnmt1)] [33] and with the Setd1A H3K4 HMT complex [15]. Cfp1 localizes nearly exclusively to euchromatic nuclear speckles, and associates with the nuclear matrix [34]. Cfp1 contains two Cys-rich plant homeodomains (PHDs); a PHD is a Cys-rich CXXC DNA-binding domain that shows specificity for unmethylated CpG dinucleotides, an acidic domain, a basic domain, a coiled-coil domain, and a Cys-rich Set1 interaction domain (SID), which is required for interaction with the Setd1A and Setd1B H3K4 HMT complexes [33,35,36]. Disruption of murine CXXC1 results in embryonic lethality shortly after implantation [37]. Murine embry- onic stem (ES) cell lines lacking Cfp1 (CXXC1 ) ⁄ ) ) are viable but show a variety of defects, including an increased population doubling time due to increased apoptosis, a  70% decrease in global cytosine methyl- ation, decreased Dnmt1 protein expression and main- tenance DNA methyltransferase activity, and an inability to achieve in vitro differentiation [38]. In C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 211 addition, CXXC1 ) ⁄ ) ES cells express elevated levels of histone H3K4 dimethylation and trimethylation, and reduced levels of histone H3K9 dimethylation [15]. Consequently, Cfp1 plays an important role in the reg- ulation of cytosine methylation, histone methylation, and cellular differentiation. The purpose of this study was to obtain insights into the molecular mechanisms regulating the activity and targeting of the Setd1A H3K4 HMT complex. The results reported here reveal that CXXC1 ) ⁄ ) ES cells contain reduced levels of Setd1A and show mislocal- ization of both Setd1A protein and trimethylated histone H3K4 (H3K4me3) to areas of heterochro- matin. Surprisingly, expression in CXXC1 ) ⁄ ) ES cells of either the amino half of Cfp1 (amino acids 1–367) or carboxyl half of Cfp1 (amino acids 361–656) is sufficient to restore appropriate levels of Setd1A. However, full-length Cfp1 is required to restrict the subnuclear localization of both Setd1A and H3K4me3 to euchromatin. Results ES cells lacking Cfp1 contain decreased levels of Setd1A Exogenous expression of Setd1A fragments in HEK293 cells competes with endogenous Setd1A binding with the Setd1A H3K4 HMT complex, resulting in decreased stability of endogenous Setd1A [16]. To examine whether loss of Cfp1 has a similar effect, western blot analysis was performed to determine protein levels of Setd1A complex components in wild-type ES cells (CXXC1 + ⁄ + ), ES cells heterozygous for the disrupted CXXC1 allele (CXXC1 + ⁄ ) ), ES cells lacking Cfp1 (CXXC1 ) ⁄ ) ), CXXC1 ) ⁄ ) ES cells transfected with a full-length Cfp1 expression vector (Rescue), and CXXC1 ) ⁄ ) ES cells cells carrying the empty expression vector (Vector). A significant decrease ( 50%) in the level of Setd1A was observed in CXXC1 ) ⁄ ) ES cells (Fig. 1A). Appropriate levels of Setd1A were restored upon introduction of a Cfp1 expression vector (Rescue), but not in ES cells carrying the empty expression vector (Vector). CXXC1 + ⁄ ) ES cells express approximately 50% as much Cfp1 as CXXC1 + ⁄ + ES cells [38], and show a slight decrease in Setd1A levels. In contrast, no difference in protein levels was observed for the other Setd1A HMT complex components (Rbbp5, Wdr5, Wdr82, and Ash2) in CXXC1 ) ⁄ ) ES cells (Fig. 1A). Previous work demonstrated that Cfp1 functions as a transcriptional activator in cotransfection assays [34,36]. Thus, further studies were performed to exam- ine whether reduced Setd1A levels in ES cells lacking Cfp1 are due to reduced transcription of the cognate gene. Surprisingly, quantitative real-time PCR analysis demonstrated that Setd1A mRNA levels were elevated four-fold to five-fold in CXXC1 ) ⁄ ) ES cells as com- pared with CXXC1 + ⁄ + and CXXC1 + ⁄ ) ES cells, and are restored to wild-type levels in rescued ES cells but not in CXXC1 ) ⁄ ) ES cells carrying the empty expres- sion vector (Fig. 1B). Therefore, the decreased levels of Setd1A observed in CXXC1 ) ⁄ ) ES cells is not explained by reduced transcription of SETD1A. Previous work by our laboratory demonstrated that disruption of the interaction between endogenous Setd1A and other components of the intact HMT complex led to a reduction of Setd1A levels as a conse- quence of a reduced Setd1A half-life [16]. Additional studies were therefore performed to assess the role of protein stability in Setd1A levels in CXXC1 ) ⁄ ) ES cells. These experiments revealed that treatment of CXXC1 ) ⁄ ) ES cells with the proteosome inhibitor MG132 led to an elevation of Setd1A levels to near wild-type levels (Fig. 1C). Cfp1 is required to restrict Setd1A and H3K4me3 to euchromatin The molecular mechanisms regulating HMT activity and genomic targeting remain largely unknown. Previ- ous studies revealed the paradoxical finding that ES cells lacking the Cfp1 component of the Setd1A H3K4 HMT complex have increased levels of histone H3K4 methylation. These findings suggest that Cfp1 may inhibit or restrict the activity of the Setd1A HMT complex. To examine this issue further, subnuclear localization of Setd1A relative to 4¢,6-diaminidino- 2-phenylindone (DAPI) staining was examined by confocal immunofluorescence. DAPI is a fluorescent DNA stain that preferentially binds to the condensed structure of pericentromeric heterochromatin [39]. Quantification of colocalization revealed that Setd1A showed only a slight ( 4%) overlap with DAPI- bright heterochromatin in wild-type ES cells. However, a significant (four-fold to five-fold) increase in colocal- ization of Setd1A with DAPI-bright heterochromatin was observed in CXXC1 ) ⁄ ) ES cells (Fig. 2A). Rescue of appropriate restriction of Setd1A to euchromatin was observed in CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1 (1–656), but not in cells carrying the empty expression vector (Fig. 2A). The subnuclear localization of H3K4me3, a product of Setd1A HMT activity, was similarly analyzed by confocal immunofluorescence. Consistent with the findings of Setd1A mislocalization in CXXC1 ) ⁄ ) ES cells, quantification of overlap between H3K4me3 and Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al. 212 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS DAPI-bright heterochromatin indicated that H3K4me3 showed only a slight overlap with DAPI-bright heteo- chromatin in wild-type ES cells. However, a significant (five-fold to six-fold) increase in colocalization of H3K4me3 with DAPI-bright heterochromatin regions was observed in CXXC1 ) ⁄ ) ES cells (Fig. 2B). Rescue of appropriate subnuclear localization of H3K4me3 was observed in CXXC1 ) ⁄ ) ES cells expressing full- length Cfp1 (1–656), but not in cells carrying the empty expression vector (Fig. 2B). These results demonstrate that ES cells lacking Cfp1 show partial mislocalization of both Setd1A and H3K4me3 to DAPI-bright regions of heterochromatin, and reveal that Cfp1 restricts the Setd1A H3K4 HMT complex to euchromatin. Retention of either Cfp1 DNA-binding activity or association with the Setd1A HMT complex is required to restore appropriate levels of Setd1A The defects in Setd1A level and localization observed in CXXC1 ) ⁄ ) ES cells were corrected upon introduc- tion of a full-length Cfp1 expression vector (Figs 1 and 2), thus providing a convenient method for assessment of the structure–function relationships of Cfp1. Vari- ous cDNA expression constructs encoding FLAG- tagged Cfp1 truncations and mutations were stably expressed in CXXC1 ) ⁄ ) ES cells to identify the functional domains of Cfp1 that are necessary and suf- ficient to restore normal levels of Setd1A (Fig. 3A). Isolated ES cell lines were screened for protein Fig. 1. ES cells lacking Cfp1 contain decreased levels of Setd1A. (A) Whole cell protein extracts were isolated from the ES cell lines CXXC1 + ⁄ + , CXXC1 + ⁄ ) , CXXC1 ) ⁄ ) , and CXXC1 ) ⁄ ) , expressing full-length Cfp1 (Rescue), and CXXC1 ) ⁄ ) , carrying the empty expression vector (Vector). Extracts were subjected to western blot analysis, using antisera directed against the Setd1A HMT complex components Setd1A, Cfp1, Ash2, Rbbp5, Wdr5, and Wdr82. The graph presents the relative level of Setd1A normalized to b-actin expression from at least three independent experiments, and error bars indicate standard error. Asterisks denote statistically significant (P < 0.05) differences as compared with CXXC1 + ⁄ + ES cells. (B) Quantitative RT-PCR was performed to assess Setd1A mRNA levels in the indicated ES cell lines. The graph presents Setd1A transcript levels relative to those for GAPDH from three independent experiments, and error bars indicate standard error. Asterisks denote statistically significant differences (P < 0.05) as compared with CXXC1 + ⁄ + ES cells. (C) Western blot analysis was performed as described in (A) to assess Setd1A levels in CXXC1 ) ⁄ ) ES cells following treatment with 5 lM MG132 for 6 h. C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 213 expression by western blot analysis, using an antibody against Cfp1. CXXC1 + ⁄ ) ES cells express  50% as much Cfp1 as CXXC1 + ⁄ + ES cells, but show normal levels of cytosine methylation and histone methylation, and are able to differentiate in vitro [38]. Consequently, clones were selected for analysis that have at least 50% of the level of Cfp1 observed in CXXC1 + ⁄ + ES cells [44]. Expression of a C-terminal deletion fragment of Cfp1 that lacks PHD2 (amino acids 1–481), or an N-terminal deletion fragment that lacks PHD1, the CXXC domain and the acidic domain (amino acids 302–656), leads to restoration of normal levels of Setd1A, indicating that none of these Cfp1 domains are necessary for this rescue activity (Fig. 3B). Surpris- ingly, expression of either the amino half of Cfp1 (amino acids 1–367, containing PHD1, and the CXXC, acidic and basic domains) or the carboxyl half of Cfp1 (amino acids 361–656, containing the coiled-coil domain, SID, and PHD2) is sufficient to restore appropriate levels of Setd1A, indicating that Cfp1 contains redundant functional domains that support Setd1A levels, and that no single Cfp1 domain is essential for this function (Fig. 3B). The N-terminal fragment of Cfp1 (amino acids 1–367) contains the CXXC DNA-binding domain, and the C-terminal Cfp1 fragment (amino acids 361–656) contains the SID [33]. Previous work determined that mutation of a conserved Cys residue (C169A) within the CXXC domain ablates Cfp1 DNA-binding activity [35], and mutation of a conserved Cys residue within the SID (C375A) ablates the interaction of Cfp1 with Fig. 2. Cfp1 is required to restrict Setd1A and H3K4me3 to euchromatin. (A) The sub- nuclear distribution of endogenous Setd1A was determined in CXXC1 + ⁄ + , CXXC1 ) ⁄ ) and CXXC1 ) ⁄ ) ES cells expressing full- length Cfp1 (amino acids 1–656) or carrying the empty expression vector, using rabbit antibody against Setd1A and FITC-conju- gated bovine anti-rabbit IgG as secondary antibody. Nuclei were counterstained with DAPI and observed by confocal microscopy. Colocalization is indicated by yellow in the merged and colocalization image. The num- bers inside the colocalization image indicate the percentage colocalized signal for the presented nucleus. The numbers outside of the image summarize the average percent- age overlap of Setd1A with DAPI-bright het- erochromatin and standard error for at least 30 nuclei. Asterisks denote a statistically significant difference (P < 0.05) as com- pared with CXXC1 + ⁄ + ES cells. (B) Subnu- clear distribution of endogenous H3K4me3 was detected in the indicated ES cell lines, using rabbit antibody against H3K4me3 and FITC-conjugated bovine anti-rabbit IgG as secondary antibody, as described above. The asterisks denote a statistically signifi- cant difference (P < 0.05) as compared with CXXC1 + ⁄ + ES cells. Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al. 214 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS Fig. 3. Cfp1 DNA-binding activity or associa- tion with the Setd1A complex is required for appropriate levels of Setd1A. (A) Schematic representation of full-length Cfp1 (amino acids 1–656) and Cfp1 truncations and mutations that were stably expressed in CXXC1 ) ⁄ ) ES cells. The filled circle at the N-terminus of Cfp1 represents the FLAG epitope, and NLS represents a nuclear locali- zation signal. Mutations that ablate DNA- binding activity (C169A) or interaction with Setd1A (C375A) are indicated by ‘X’. (B) Western blot analysis was performed on whole cell extracts collected from CXXC1 + ⁄ + , CXXC1 ) ⁄ ) and CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1 (amino acids 1–656) or the indicated Cfp1 muta- tions (or carrying the empty expression vec- tor), using antisera directed against Setd1A [16]. The level of b-actin serves as a loading control. The graph represents relative Setd1A levels normalized to b-actin from at least three independent experiments, and error bars indicate standard error. Asterisks denote statistically significant (P < 0.05) differences as compared with CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1 (amino acids 1–656). C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 215 the Setd1A HMT complex [33]. Additional studies were performed to assess the functional significance of these Cfp1 properties for the ability to restore normal levels of Setd1A. CXXC1 ) ⁄ ) ES cells expressing full- length Cfp1 that lacks DNA-binding activity (amino acids 1–656, C169A) or interaction with the Setd1A H3K4 HMT complex (amino acids 1–656, C375A) contain normal levels of Setd1A. This was expected, given that expression of either half of Cfp1 is sufficient to restore normal Setd1A levels. However, ablation of DNA-binding activity within the N-terminal fragment of Cfp1 (amino acids 1–367, C169A), or disruption of Setd1A interaction with the C-terminal Cfp1 fragment (amino acids 361–656, C375A), results in the loss of Setd1A rescue activity (Fig. 3B). Finally, rescue activ- ity was lost upon introduction of both point mutations into full-length Cfp1 (amino acids 1–656, C169A ⁄ C375A). These data indicate that retention of either Cfp1 DNA-binding activity or interaction with the Setd1A H3K4 HMT complex is required to restore appropriate Setd1A levels in CXXC1 ) ⁄ ) ES cells. Full-length Cfp1 is required to restrict Setd1A and H3K4me3 to euchromatin CXXC1 ) ⁄ ) ES cells expressing various Cfp1 trunca- tions and mutations were analyzed by confocal immu- nofluorescence to determine the functional domains of Cfp1 required to restrict the subnuclear localization of Setd1A and H3K4me3 to euchromatin. The vast majority of Setd1A and H3K4me3 was localized to DAPI-dim euchromatic regions in CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1 (amino acids 1–656) (Figs 4 and 5). In contrast to the pattern of Cfp1 res- cue activity seen for Setd1A levels, however, expres- sion of the N-terminal (amino acids 1–481 or 1–367) or C-terminal (amino acids 302–656 or 361–656) frag- ments of Cfp1 in CXXC1 ) ⁄ ) ES cells is not sufficient to exclude Setd1A and H3K4me3 from DAPI-bright heterochromatin (Figs 4 and 5). In addition, CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1 that lacks DNA-binding activity (amino acids 1–656, C169A) or fails to interact with the Setd1A H3K4 HMT complex (amino acids 1–656, C375A) also fail to restrict Setd1A and H3K4me3 to euchromatin (Figs 4 and 5). As expected, ablation of the DNA- binding activity within the N-terminal fragment of Cfp1 (amino acids 1–367, C169A), disruption of the Setd1A interaction with the C-terminal fragment of Cfp1 (amino acids 361–656, C375A) or introduction of both mutations within full-length Cfp1 (1-656 C169A, C375A) also results in a failure to exclude Setd1A and H3K4me3 from DAPI-bright heterochro- matin (Figs 4 and 5). Therefore, full-length Cfp1 is required to restrict Setd1A and H3K4me3 localization to euchromatin, and Cfp1 DNA-binding activity and interaction with the Setd1A H3K4 HMT complex are both required for proper restriction of Setd1A and H3K4me3 to euchromatin. Discussion The results of the studies reported here reveal that ES cells lacking the epigenetic regulator Cfp1 contain decreased levels of the histone H3K4 methyltransferase Setd1A. Yeast cells lacking Spp1, the Cfp1 homolog, also express reduced amounts of Set1 [40], and Spp1 is thought to stabilize Set1 [40]. Furthermore, expression of Cfp1-interacting Setd1A fragments in human cells disrupts the association of endogenous Setd1A with the intact HMT complex, resulting in reduced Setd1A levels as a consequence of reduced Setd1A half-life [16]. Thus, the reduced levels of Setd1A observed in ES cells lacking Cfp1 may be due to decreased Setd1A stability. The observed increase of Setd1A level in CXXC1 ) ⁄ ) ES cells following treatment with the prote- osome inhibitor MG132 supports this hypothesis. In contrast, the levels of the other components of the Setd1A complex (Ash2, Rbbp5, Wdr5, and Wdr82) are not altered in CXXC1 ) ⁄ ) ES cells, which may be due to their association with additional H3K4 HMT com- plexes (Setd1B, Mll1, Mll2, and Mll3) [16,18,22,28, 41–43]. Despite reduced Setd1A levels, CXXC1 ) ⁄ ) ES cells express an approximately five-fold increased level of Setd1A mRNA, suggesting that these cells increase transcription of the SETD1A gene to compensate for reduced levels of Setd1A. Expression of either an N-terminal fragment (amino acids 1-367) or C-terminal fragment (amino acids 361–656) of Cfp1 is sufficient to restore normal levels of Setd1A in CXXC1 ) ⁄ ) ES cells. These results are consistent with previous findings that expression in CXXC1 ) ⁄ ) ES cells of either Cfp1(1–367) or Cfp1(361–656) is sufficient to rescue defects in ES cell plating efficiency, cytosine methylation, and in vitro differentiation [44]. Interestingly, Cfp1(1–367) fails to interact with the Setd1A complex [33], but still restores appropriate levels of Setd1A, indicating that a physical interaction of Cfp1 with the Setd1A complex is not required for appropriate levels of Setd1A. In addi- tion, analysis of point mutations within the CXXC domain (C169A) or SID (C375A) reveals that reten- tion of either Cfp1 DNA-binding activity or interac- tion with the Setd1A H3K4 HMT complex is necessary to restore normal levels of Setd1A in CXXC1 ) ⁄ ) ES cells. Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al. 216 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS ES cells that lack Cfp1 show increased levels of histone H3K4 dimethylation and trimethylation [15], despite expressing decreased levels of Setd1A, suggest- ing that Cfp1 restricts the activity of the Setd1A H3K4 HMT complex. Consistent with this model, confocal immunofluorescence reveals that both Setd1A and H3K4me3 are partially mislocalized to DAPI-bright regions of heterochromatin in CXXC1 ) ⁄ ) ES cells. In contrast to the pattern of Cfp1 rescue activity observed for Setd1A levels, expression of full-length Cfp1 in CXXC1 ) ⁄ ) ES cells is required to properly restrict subnuclear localization Fig. 4. Full-length Cfp1 is required to restrict Setd1A to euchromatin. The subnu- clear distribution of endogenous Setd1A was detected in CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1 (amino acids 1–656) or the indicated Cfp1 trunca- tions and mutations, using rabbit antibody against Setd1A and FITC-conjugated bovine anti-rabbit IgG as secondary antibody, as described for Fig. 2. Asterisks denote a statistically significant difference (P < 0.05) as compared with CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1 (amino acids 1–656). C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 217 of Setd1A and H3K4me3 to euchromatin. These studies further indicate that Cfp1 DNA-binding acti- vity and interaction with the Setd1A H3K4 HMT complex are both required for proper subnuclear localization of Setd1A. The requirement for an intact Cfp1 CXXC domain for proper genomic localization may indicate that Cfp1 DNA-binding activity restricts the Setd1A H3K4 HMT complex to euchromatin by binding to unmethylated CpG dinucleotides in euchromatin. Individual CXXC1 ) ⁄ ) ES cell nuclei show a range (5–30%) of colocalization between Setd1A and H3K4me3 with DAPI-bright heterochromatin, and 20–30% mislocalization of Setd1A and H3K4me3 is observed in 35–40% of CXXC1 ) ⁄ ) ES cell nuclei. It is possible that cell-to-cell variation in the degree of Fig. 5. Full-length Cfp1 is required to restrict H3K4me3 to euchromatin. The subnuclear distribution of H3K4me3 was detected in CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1 (amino acids 1–656) or the indicated Cfp1 truncations and mutations, using rabbit antibody against H3K4me3 and FITC-conjugated bovine anti-rabbit IgG as secondary antibody, as described for Fig. 2. Asterisks denote statistically significant differences (P < 0.05) as compared with CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1 (amino acids 1–656). Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al. 218 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS colocalization may be cell cycle-dependent. However, significant mislocalization of Setd1A and H3K4me3 is never observed in wild-type ES cells or in rescued CXXC1 ) ⁄ ) ES cells expressing full-length Cfp1. The persistence of DAPI-bright staining colocalizing with H3K4me3 indicates that deposition of this euchroma- tin epigenetic mark is insufficient to induce general chromatin remodeling in these heterochromatin regions. Little is known regarding the relative contributions of each mammalian histone H3K4 HMT complex. However, Cfp1 has been shown to be an integral com- ponent of only the Setd1A and Setd1B HMT com- plexes [15,16]. The localization of Setd1B in the absence of Cfp1 has not been determined, but the find- ing that the extent of Setd1A mislocalization is similar to that of H3K4me3 localization suggests that the Setd1 HMT complexes are responsible for the bulk of histone H3K4 trimethylation. This conclusion is con- sistent with a recent report that small interfering RNA-mediated depletion of Setd1A and Setd1B leads to a dramatic global reduction in histone H3K4 trime- thylation [45]. The full-length Cfp1 that is required to restrict subnuclear localization of Setd1A and H3K4me3 to euchromatin contains two PHDs. PHDs are thought to be involved in chromatin-mediated transcriptional control [46], and can serve as binding modules for unmodified and methylated histone H3K4 and methy- lated histone H3K36 [17,47–51]. For example, PHD1 of Spp1, the yeast homolog of Cfp1, binds dimethylat- ed and trimethylated histone H3K4 [51]. In addition, the PHD finger of the tumor suppressor Ing2 directly associates with H3K4me3, and this interaction is criti- cal for proper occupancy of the Ing2–HDAC1 complex at target promoters during the DNA damage response and active transcriptional repression [48]. Therefore, the PHDs of Cfp1 may be important for binding mod- ified histone H3K4 and targeting the Cfp1–Setd1A complex to specific genomic sites. The mechanisms responsible for appropriate subnu- clear localization of histone H3K4 HMTs are complex, and involve gene-specific recruitment by DNA-binding factors. For example, the insulator DNA-binding pro- tein Boris recruits Setd1A to the MYC and BRCA1 genes [52]; NF-E2 recruits Mll2 to the b-globin locus [53]; the Ap2d transcription factor recruits Ash2L and Mll2 to the HOXC8 locus [54]; and the paired-box transcription factor Pax7 recruits Mll2 to the MYF5 gene [55]. In addition, several integral components of the mammalian Set1-like histone H3K4 HMT complexes have been implicated in genomic targeting. Wdr5, which is common to each member of the mammalian Set1-like HMT complex family, has been reported to bind directly to histone H3 [56–59]. In addition, the Wdr82 component of the Setd1A and Setd1B HMT complexes binds to RNAP II containing Ser5-phos- phorylated CTD, thus recruiting these complexes to sites of transciption initiation [18]. Furthermore, the compositions of the Setd1A and Setd1B HMT com- plexes are identical, except for the identity of the enzy- matic (Setd1) component [15,16], but confocal microscopy reveals that these complexes show a nearly nonoverlapping euchromatic subnuclear localization [16]. This finding strongly suggests that these closely related complexes regulate distinct sets of target genes, and that this specificity is mediated by each Setd1 pro- tein, presumably through interactions with distinct tar- geting effector molecules. The data reported here reveal that Cfp1 plays a novel role in restricting the subnuclear localization of Setd1A and H3K4me3 to euchromatin, thus identifying Cfp1 as another critical regulator of histone H3K4 HMT genomic targeting. Experimental procedures Cell culture Generation of murine CXXC1 ) ⁄ ) ES cell lines was as previ- ously described [38]. ES cells were cultured on 0.1% gela- tin-coated tissue culture dishes in high-glucose DMEM (Gibco BRL, Life Technologies, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (Gibco BRL), 100 unitsÆmL )1 penicillin ⁄ streptomycin (Invitrogen, Carls- bad, CA, USA), 2 mml-glutamine (Invitrogen), 1% nones- sential amino acids (Invitrogen), 0.2% leukemia inhibitory factor-conditioned medium, 100 nm b-mercaptoethanol, 0.025% Hepes (pH 7.5) (Invitrogen), and 1% Hank’s balanced salt solution (Invitrogen). Plasmid construction and transfection of ES cells Murine Cfp1 cDNA [38,60] was subcloned into pcDNA3.1 ⁄ Zeo (Invitrogen). The Cfp1 expression vector or the empty expression vector was electroporated into CXXC1 ) ⁄ ) ES cells as previously described [38]. Single amino acid substitutions within Cfp1 were performed using the QuikChange II site-directed mutagenesis kit (Strata- gene, La Jolla, CA, USA) according to the manufacturer’s protocol, as previously described [33,35]. For structure– function studies, cDNA constructs encoding full-length FLAG epitope-tagged human Cfp1 (amino acids 1–656) and various Cfp1 truncations and ⁄ or mutations were subcloned into the pcDNA3.1 ⁄ Hygro mammalian expres- sion vector (Invitrogen). The N-terminal bipartite nuclear localization signal of Cfp1 (amino acids 109–121) was C. M. Tate et al. 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