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

Jeong-Heon Lee,David G Skalnik

doi:10.1074/jbc.m508312200

Abstract

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 approximately 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.

Highlights

CpG-binding protein exhibits a unique DNA binding specificity for unmethylated CpG motifs and acts as a transcriptional activator [1]
CFP1 contains a cysteinerich CXXC DNA-binding domain [1, 3], which is present in several other proteins, including DNA methyltransferase 1 (Dnmt1) [4], the major maintenance DNA methyltransferase; human trithorax (HRX), a histone H3-Lys4 methyltransferase encoded by a gene frequently involved in chromosomal translocations in leukemia [5,6,7,8,9,10]; methyl-binding domain protein 1, which binds to methylated CpG dinucleotides [11, 12]; leukemia-associated protein LCX [13]; and MLL-2, a histone H3-Lys4 methyltransferase, which is often amplified in solid tumors [14, 15]
Previous work demonstrated that CFP1 is essential for early mammalian embryogenesis, embryonic stem (ES) cell differentiation, and normal cytosine methylation patterns [21, 25]

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—Human embryonic kidney cells (HEK-293) were cultured and transfected as described [20], and transfected cells were selected in 200 ␮g/ml hygromycin B. Purification of a CFP1 Complex—HEK-293 cells expressing FLAGCFP1 were incubated in hypotonic buffer for 10 min on ice and homogenized, and nuclei were harvested by centrifugation. Ten milliliters of extraction buffer (10 mM PIPES (pH 7.0), 300 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, supplemented with the protease inhibitors leupeptin, aprotinin, pepstatin (1 ␮g/ml each), and 1 mM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100) was added and incubated for 10 min on ice. The pellets were extensively Dounce homogenized on ice. The solubilized nuclear fraction was separated by centrifugation and used for immunoprecipitation. FLAG-IgG-agarose slurry (Sigma) was added, incubated for 3 h, and washed five times with 12 ml of extraction buffer. Bound proteins were eluted twice with 0.5 ml of extraction buffer containing 250 ␮g/ml FLAG peptide. Protein-A-agarose beads were washed four times with extraction buffer containing 300 mM NaCl. Proteins were eluted with SDS sample buffer and analyzed by Western blotting. Cells were mounted with 10 ␮l of Fluoromount G (Southern Biotechnology Associates) and scanned with a Zeiss LSM 510 laser scanning confocal microscope

RESULTS

DISCUSSION

Mammalian the Yeast