David G. Skalnik

CpG-binding Protein (CXXC Finger Protein 1) Is a Component of the Mammalian Set1 Histone H3-Lys4 Methyltransferase Complex, the Analogue of the Yeast Set1/COMPASS Complex

CpG-binding protein (CXXC finger protein 1 (CFP1)) binds to DNA containing unmethylated CpG motifs and is required for mammalian embryogenesis, normal cytosine methylation, and cellular differentiation. Studies were performed to identify proteins that interact with CFP1 to gain insight into the molecular function of this protein. Immunoprecipitation and mass spectrometry reveal that human CFP1 associates with a ∼450-kDa complex that contains the mammalian homologues of six of the seven components of the Set1/COMPASS complex, the sole histone H3-Lys4 methyltransferase in yeast. In vitro assays demonstrate that the human Set1/CFP1 complex is a histone methyltransferase that produces mono-, di-, and trimethylated histone H3 at Lys4. Confocal microscopy reveals that CFP1 and Set1 co-localize to nuclear speckles associated with euchromatin. A Set1 complex of reduced mass persists in murine embryonic stem cells lacking CFP1. These cells carry elevated levels of methylated histone H3-Lys4 and reduced levels of methylated histone H3-Lys9. Together with the previous finding of reduced levels of cytosine methylation, these data indicate that cells lacking CFP1 contain reduced levels of heterochromatin. Furthermore, ES cells lacking CFP1 exhibit a 4-fold excess of histone H3-Lys4 methylation following induction of differentiation, indicating that CFP1 restricts the activity of the Set1 histone methyltransferase complex. These results reveal a mammalian counterpart to the yeast Set1/COMPASS complex. The presence of CFP1 in this complex implicates this protein as a critical epigenetic regulator of histone modification in addition to cytosine methylation and reveals one mechanism by which this protein intersects with the epigenetic machinery.

Cloning of a Mammalian Transcriptional Activator That Binds Unmethylated CpG Motifs and Shares a CXXC Domain with DNA Methyltransferase, Human Trithorax, and Methyl-CpG Binding Domain Protein 1

ABSTRACT Ligand screening was utilized to isolate a human cDNA that encodes a novel CpG binding protein, human CpG binding protein (hCGBP). This factor contains three cysteine-rich domains, two of which exhibit homology to the plant homeodomain finger domain. A third cysteine-rich domain conforms to the CXXC motif identified in DNA methyltransferase, human trithorax, and methyl-CpG binding domain protein 1. A fragment of hCGBP that contains the CXXC domain binds to an oligonucleotide probe containing a single CpG site, and this complex is disrupted by distinct oligonucleotide competitors that also contain a CpG motif(s). However, hCGBP fails to bind oligonucleotides in which the CpG motif is either mutated or methylated, and it does not bind to single-stranded DNA or RNA probes. Furthermore, the introduction of a CpG dinucleotide into an unrelated oligonucleotide sequence is sufficient to produce a binding site for hCGBP. Native hCGBP is detected as an 88-kDa protein by Western analysis and is ubiquitously expressed. The DNA-binding activity of native hCGBP is apparent in electrophoretic mobility shift assays, and hCGBP trans-activates promoters that contain CpG motifs but not promoters in which the CpG is ablated. These data indicate that hCGBP is a transcriptional activator that recognizes unmethylated CpG dinucleotides, suggesting a role in modulating the expression of genes located within CpG islands.

Identification and Characterization of the Human Set1B Histone H3-Lys4 Methyltransferase Complex

We previously identified a mammalian Set1A complex analogous to the yeast Set1/COMPASS histone H3-Lys4 methyltransferase complex (Lee, J.-H., and Skalnik, D. G. (2005) J. Biol. Chem. 280, 41725–41731). Data base analysis indicates that human Set1A protein shares 39% identity with an uncharacterized SET domain protein, KIAA1076, hereafter denoted Set1B. Immunoprecipitation and mass spectrometry reveal that Set1B associates with a ∼450 kDa complex that contains all five non-catalytic components of the Set1A complex, including CFP1, Rbbp5, Ash2, Wdr5, and Wdr82. These data reveal two human protein complexes that differ only in the identity of the catalytic histone methyltransferase. In vitro assays demonstrate that the Set1B complex is a histone methyltransferase that produces trimethylated histone H3 at Lys4. Both Set1A and Set1B are widely expressed. Inducible expression of the carboxyl terminus of either Set1A or Set1B decreases steady-state levels of both endogenous Set1A and Set1B protein, but does not alter the expression of the non-catalytic components of the Set1 complexes. A 123-amino acid fragment upstream of the Set1A SET domain is necessary for interaction with CFP1, Ash2, Rbbp5, and Wdr5. This protein domain is also required to mediate feedback inhibition of Set1A and Set1B expression, which is a consequence of reduced Set1A and Set1B stability when not associated with the methyltransferase complex. Confocal microscopy reveals that Set1A and Set1B each localize to a largely non-overlapping set of euchromatic nuclear speckles, suggesting that Set1A and Set1B each bind to a unique set of target genes and thus make non-redundant contributions to the epigenetic control of chromatin structure and gene expression.

Wdr82 is a C-terminal domain-binding protein that recruits the Setd1A Histone H3-Lys4 methyltransferase complex to transcription start sites of transcribed human genes.

ABSTRACT Histone H3-Lys4 trimethylation is associated with the transcription start site of transcribed genes, but the molecular mechanisms that control this distribution in mammals are unclear. The human Setd1A histone H3-Lys4 methyltransferase complex was found to physically associate with the RNA polymerase II large subunit. The Wdr82 component of the Setd1A complex interacts with the RNA recognition motif of Setd1A and additionally binds to the Ser5-phosphorylated C-terminal domain of RNA polymerase II, which is involved in initiation of transcription, but does not bind to an unphosphorylated or Ser2-phosphorylated C-terminal domain. Chromatin immunoprecipitation analysis revealed that Setd1A is localized near the transcription start site of expressed genes. Small interfering RNA-mediated depletion of Wdr82 leads to decreased Setd1A expression and occupancy at transcription start sites and reduced histone H3-Lys4 trimethylation at these sites. However, neither RNA polymerase II (RNAP II) occupancy nor target gene expression levels are altered following Wdr82 depletion. Hence, Wdr82 is required for the targeting of Setd1A-mediated histone H3-Lys4 trimethylation near transcription start sites via tethering to RNA polymerase II, an event that is a consequence of transcription initiation. These results suggest a model for how the mammalian RNAP II machinery is linked with histone H3-Lys4 histone methyltransferase complexes at transcriptionally active genes.

Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer

Deoxycytosine methylation within CpG islands of tumor suppressor genes plays a prominent role in the development and progression of drug-resistant ovarian cancer. Consequently, epigenetic therapies directed toward tumor suppressor demethylation/reexpression could potentially reverse malignant phenotypes and chemosensitize recalcitrant tumors. In this report, we examined the demethylating agent zebularine [1-(β-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one], in comparison with the well-known methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-dC), for its ability to inhibit ovarian cancer cell proliferation and to demethylate and induce tumor suppressor genes. Zebularine exerted significant (>5-aza-dC) antiproliferative effects against the ovarian cancer cell lines Hey, A2780, and the cisplatin-resistant A2780/CP in a dose-dependent manner (65% versus 35% inhibition at 48 hours, zebularine versus 5-aza-dC). Moreover, 48-hour treatment with 0.2 mmol/L zebularine significantly induced demethylation of the tumor suppressors ras-associated domain family 1A and human MutL homologue-1. RASSF1A gene reexpression was also observed, as was reexpression of two other tumor suppressors, ARHI and BLU, although levels differed from those induced by 5-aza-dC. Global analyses of DNA methylation revealed similar overall demethylation (2.5- to 3-fold) by 5-aza-dC and zebularine as determined by methyl acceptance assay. However, differences in demethylation of individual loci were observed as determined by differential methylation hybridization. Finally, we found that zebularine could resensitize the drug-resistant cell line A2780/CP to cisplatin, with a 16-fold reduction in the IC50 of that conventional agent. In summary, zebularine seems to be a promising clinical candidate, singly or combined with conventional regimens, for the therapy of drug-resistant ovarian cancer.

CCAAT Displacement Protein Competes with Multiple Transcriptional Activators for Binding to Four Sites in the Proximal gp91phox Promoter

CCAAT displacement protein (CDP) competes with transcriptional activating proteins for binding to each of four elements within the myeloid-specific gp91phox promoter. CDP exhibits the strongest affinity for a site centered at −110 base pairs (bp) of the promoter and progressively weaker affinities for three more distal binding sites. CDP binding to each site is down-regulated during terminal phagocytic differentiation, coincident with induction of gp91phox expression. Deletion of the high affinity CDP-binding site at −110 bp leads to inappropriate gp91phox promoter activity in HeLa, K562, and HEL cells. An overlapping binding site for the CCAAT box-binding factor CP1 is required for derepressed promoter activity in HeLa and K562 cells, but is dispensable in HEL cells, indicating that different cell types require distinct cis-elements for gp91phox promoter activity. Derepressed gp91phox promoter activity is further increased upon removal of a second CDP-binding site centered at −150 bp, revealing that CDP represses gp91phox expression via multiple cis-elements. We present a model in which restriction of gp91phox expression to mature myeloid cells involves competition between transcriptional activators and repressors for binding to multiple sites within the promoter.

Reduced genomic cytosine methylation and defective cellular differentiation in embryonic stem cells lacking CpG binding protein

ABSTRACT Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is essential for early murine development. Embryonic stem cell lines deficient for CGBP were generated to further examine CGBP function. CGBP − / − cells are viable but show an increased rate of apoptosis and are unable to achieve in vitro differentiation following removal of leukemia inhibitory factor from the growth media. Instead, CGBP − / − embryonic stem cells remain undifferentiated as revealed by persistent expression of the pluripotent markers Oct4 and alkaline phosphatase. CGBP − / − cells exhibit a 60 to 80% decrease in global cytosine methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total DNA methyltransferase activity is reduced by 30 to 60% in CGBP − / − cells, and expression of the maintenance DNA methyltransferase 1 protein is similarly reduced. However, de novo DNA methyltransferase activity is normal. Nearly all aspects of the pleiotropic CGBP − / − phenotype are rescued by introduction of a CGBP expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine methylation and for cellular differentiation, consistent with the requirement for CGBP during early mammalian development.

CpG Binding Protein Is Crucial for Early Embryonic Development

ABSTRACT Epigenetic modification of DNA via CpG methylation is essential for the proper regulation of gene expression during embryonic development. Methylation of CpG motifs results in gene repression, while CpG island-containing genes are maintained in an unmethylated state and are transcriptionally active. The molecular mechanisms involved in maintaining the hypomethylation of CpG islands remain unclear. The transcriptional activator CpG binding protein (CGBP) exhibits a unique binding specificity for DNA elements that contain unmethylated CpG motifs, which makes it a potential candidate for the regulation of CpG island-containing genes. In order to assess the global function of this protein, mice lacking CGBP were generated via homologous recombination. No viable mutant mice were identified, indicating that CGBP is required for murine development. Mutant embryos were also absent between 6.5 and 12.5 days postcoitum (dpc). Approximately, one-fourth of all implantation sites at 6.5 dpc appeared empty with no intact embryos present. However, histological examination of 6.5-dpc implantation sites revealed the presence of embryo remnants, indicating that CGBP mutant embryos die very early in development. In vitro blastocyst outgrowth assays revealed that CGBP-null blastocysts are viable and capable of hatching and forming both an inner cell mass and a trophectoderm. Therefore, CGBP plays a crucial role in embryo viability and peri-implantation development.

Identification and Characterization of a Novel Human PP1 Phosphatase Complex

Mammalian Wdr82 is a regulatory component of the Setd1a and Setd1b histone H3-lysine 4 methyltransferase complexes and is implicated in the tethering of Setd1 complexes to transcriptional start sites of active genes. In the studies reported here, immunoprecipitation and mass spectrometry analyses reveal that Wdr82 additionally associates with multiple protein complexes, including an RNA polymerase II complex, four distinct histone H3-Lys4 methyltransferase complexes, protein phosphatase 1 (PP1)-associated proteins, a chaperonin-containing Tcp1 complex, and other uncharacterized proteins. Further characterization of the PP1-associated proteins identified a stable multimeric complex composed of regulatory subunits PNUTS, Tox4, and Wdr82 and a PP1 catalytic subunit (denoted as the PTW/PP1 phosphatase complex). The PTW/PP1 complex exhibits in vitro phosphatase activity in a PP1-dependent manner. Analysis of protein-protein interactions reveals that PNUTS mediates phosphatase complex formation by providing a binding platform to each component. The PNUTS and Tox4 subunits are predominantly associated with the PTW/PP1 phosphatase complex in HEK293 cells, and the integrity of this complex remains intact throughout cell cycle progression. Inducible expression of a PP1 interaction-defective form of PNUTS (W401A) or small interfering RNA-mediated depletion of PNUTS in HEK293 cells causes cell cycle arrest at mitotic exit and apoptotic cell death. PNUTS (W401A) shows normal association with chromosomes but causes defects in the process of chromosome decondensation at late telophase. These data reveal that mammalian Wdr82 functions in a variety of cellular processes and reveal a potential role of the PTW/PP1 phosphatase complex in the regulation of chromatin structure during the transition from mitosis into interphase.

CXXC finger protein 1 restricts the Setd1A histone H3K4 methyltransferase complex to euchromatin.

CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is a component of the euchromatic Setd1A histone H3K4 methyltransferase complex, and is a critical regulator of histone methylation, cytosine methylation, cellular 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 complex. 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 heterochromatin. 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 methyltransferase complex is required to restore normal Setd1A levels. In contrast, expression of full‐length Cfp1 in CXXC1−/− ES cells is required to restrict Setd1A and histone H3K4 trimethylation to euchromatin, indicating 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.