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 ϳ450-kDa complex that contains the mammalian homologues of six of the seven components of the Set1/COMPASS complex, the sole histone H3-Lys 4 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 Lys 4 . 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-Lys 4 and reduced levels of methylated histone H3-Lys 9 . 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-Lys 4 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.CpG-binding protein exhibits a unique DNA binding specificity for unmethylated CpG motifs and acts as a transcriptional activator (1). This factor, encoded by the CXXC1 gene, has recently been designated CXXC finger protein 1, and will hereafter be referred to as CFP1.2 Originally identified in mammals, homologues of CFP1 have been detected in Drosophila, Caenorhabditis elegans, and both Saccharomyces cerevisiae and Schizosaccharomyces pombe (1, 2). 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) (also known as ALL-1 or MLL), a histone H3-Lys 4 methyltransferase encoded by a gene frequently involved in chromosomal translocations in leukemia (5-10); methyl-binding domain protein 1, which binds to methylated CpG dinucleotides (11, 12); leukemia-associated protein LCX (13); and MLL-2, a histone H3-Lys 4 methyltransferase, which is often amplified in solid tumors (14, 15). CFP1 also contains two plant homeodomain (PHD) motifs (1, 3), which are characteristic of chromatin-associated proteins and/or regulators of gene expression (1, 16) and mediate protein/protein interactions (17-19). CFP1 is a component of the nuclear...

Trimethylation of histone H3 Lys 4 (H3K4me3) is a mark of active and poised promoters. The Set1 complex is responsible for most somatic H3K4me3 and contains the conserved subunit CxxC finger protein 1 (Cfp1), which binds to unmethylated CpGs and links H3K4me3 with CpG islands (CGIs). Here we report that Cfp1 plays unanticipated roles in organizing genome-wide H3K4me3 in embryonic stem cells. Cfp1 deficiency caused two contrasting phenotypes: drastic loss of H3K4me3 at expressed CGI-associated genes, with minimal consequences for transcription, and creation of “ectopic” H3K4me3 peaks at numerous regulatory regions. DNA binding by Cfp1 was dispensable for targeting H3K4me3 to active genes but was required to prevent ectopic H3K4me3 peaks. The presence of ectopic peaks at enhancers often coincided with increased expression of nearby genes. This suggests that CpG targeting prevents “leakage” of H3K4me3 to inappropriate chromatin compartments. Our results demonstrate that Cfp1 is a specificity factor that integrates multiple signals, including promoter CpG content and gene activity, to regulate genome-wide patterns of H3K4me3.

We previously identified a mammalian Set1A complex analogous to the yeast Set1/COMPASS histone H3-Lys4 methyltransferase complex (Lee, J. . 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.

Human CCAAT displacement protein (CDP), a putative repressor of developmentally regulated gene expression, was purified from HeLa cells by DNA binding-site affinity chromatography. cDNA encoding CDP was obtained by immunoscreening a lambda gt11 library with antibody raised against purified protein. The deduced primary amino acid sequence of CDP reveals remarkable homology to Drosophila cut with respect to the presence of a unique homeodomain and "cut repeats". As cut participates in determination of cell fate in several tissues in Drosophila, the similarity predicts a broad role for CDP in mammalian development.

Mammalian development requires commitment of cells to restricted lineages, which requires epigenetic regulation of chromatin structure. Epigenetic modifications were examined during in vitro differentiation of murine embryonic stem (ES) cells. Global histone acetylation, a euchromatin marker, declines dramatically within 1 day of differentiation induction and partially rebounds by day 2. Histone H3-Lys9 methylation, a heterochromatin marker, increases during in vitro differentiation. Conversely, the euchromatin marker H3-Lys4 methylation transiently decreases, then increases to undifferentiated levels by day 4, and decreases by day 6. Global cytosine methylation, another heterochromatin marker, increases slightly during ES cell differentiation. Chromatin structure of the Oct4 and Brachyury gene promoters is modulated in concert with their pattern of expression during ES cell differentiation. Importantly, prevention of global histone deacetylation by treatment with trichostatin A prevents ES cell differentiation. Hence, ES cells undergo functionally important global and gene-specific remodeling of chromatin structure during in vitro differentiation. genesis 38:32-38, 2004.

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.Eukaryotic gene expression can be regulated by modification of chromatin structure through the enzymatic control of covalent histone modifications. Methylation of histone H3 at lysine 4 is associated with an active chromatin structure that is permissive to transcription (24,26,42). The physiologic functions of histone H3-Lys4 methylation are largely elusive, but identification of proteins recognizing H3-Lys4 methylation marks address possible functions. For example, histone H3-Lys4 trimethylation is recognized by the plant homeodomain fingers of NURF, an ISWI-related remodeling factor, and ING2, which is associated with a histone deacetylase (39). The WD40 domain-containing Wdr5 protein recognizes histone H3-Lys4 and is found in mammalian Set1-like histone H3-Lys4 methyltransferase complexes (5, 40, 52). These findings suggest that histone H3-Lys4 methylation modulates chromatin structure by recruiting chromatin remodeling and histone-modifying activities. Recently, it was proposed that histone H3-Lys4 methylation in higher eukaryotes counters the generally repressive chromatin environment imposed by histone H3-Lys9 and H3-Lys27 methylation (2).The C-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit is comprised of 25 to 52 tandem copies of the consensus repeat heptad Y 1 S 2 P 3 T 4 S 5 P 6 S 7 and is evolutionarily conserved from yeast to humans (34). The phosphorylation state of the CTD dramatically changes as RNAP II progresses through the transcription cycle (7). RNAP II containing CTD phosphorylated at serine 5 (Ser5-P CTD) is localized near t...

Background:The WDR5 interaction (Win) motif of mixed lineage leukemia-1 (MLL1) is required for core complex assembly. Results: Win motifs from human SET1 family methyltransferases differ 70-fold in their specificity for WDR5. Conclusion: Residue differences around the conserved Win motif contribute to differences in affinity. Significance: Knowledge of WDR5 recognition of SET1 Win motifs is crucial for understanding regulation of H3K4 methylation in cells.

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-Lys 4 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. Protein phosphatase 1 (PP1)2 is a serine/threonine protein phosphatase involved in diverse cellular processes, such as transcription, replication, pre-mRNA splicing, protein synthesis, muscle contraction, carbohydrate metabolism, neuronal signaling, cell survival, and cell cycle progression (1-3). Mammals express three PP1 catalytic isoforms, PP1␣, PP1␥, and PP1␤/, which show distinct subcellular localization patterns (4, 5). PP1 catalytic isoforms do not exist freely in cells but rather associate with regulatory subunits to form distinct multimeric holoenzymes. In general, PP1 regulatory subunits function as signaling modules by regulating the enzymatic activity or targeting of catalytic subunits to specific substrates (6, 7). PP1 is involved in cell cycle progression, especially during mitosis and mitotic exit, and dysregulation of PP1 activity causes mitotic arrest or deficient cytokinesis in mammals (3,8,9). PP1 is associated with multiple mitotic structures, such as chromosomes, centrosomes, and spindles (4, 8, 10), and is implicated as a major phosphatase acting on phosphoproteins at mitotic exit (11-14). However, the specific regulatory or targeting subunits required for this activity are not well und...