Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including 3'-end cleavage and polyadenylation [1][2][3][4][5][6][7][8] . Despite the characterization of a large number of proteins that are required for the cleavage reaction, the identity of the endoribonuclease is not known 4,9,10 . Recent analyses suggested that the 73 kD subunit of cleavage and polyadenylation specificity factor (CPSF-73) may be the endonuclease for this and related reactions [10][11][12][13][14][15] , although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 Å resolution, complexed with zinc ions and a sulfate that may mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1p) at 2.5 Å resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-β-lactamase domain and a novel β-CASP domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses endoribonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3'-end processing endonuclease. Keywordspolyadenylation; metallo-β-lactamase; pre-mRNA processing; Artemis; V(D)J recombination; double-strand break repair CPSF-73 belongs to the metallo-β-lactamase superfamily of zinc-dependent hydrolases 11,12 . Canonical metallo-β-lactamases contain five signature sequence motifs-Asp (motif 1), His-X-His-X-Asp-His (motif 2), His (motif 3), Asp (motif 4) and His (motif 5), most of which are ligands to the two zinc ions in their active site. Sequence conservation between CPSF-73 and the canonical metallo-β-lactamases is limited to these signature motifs. While the first four motifs can be identified in the N-terminal segment of CPSF-73 (Supplemental Fig. 1a, Supplemental Table 1), the fifth motif was uncertain, with three candidates, A (Asp or Glu), B (His), and C (His) (Supplemental Fig. 1a), in the so-called β-CASP motif 12 . Motif B was proposed to be equivalent to motif 5 in the canonical metallo-β-lactamases. Another subunit of CPSF, CPSF-100, shares sequence conservation (Supplemental Fig. 1b) Fig. 1a) with CPSF-73 but lacks the putative Zn 2+ binding residues.To understand the roles of CPSF-73 and CPSF-100 in pre-mRNA 3'-end processing, we determined the structures of human CPSF-73 (residues 1-460), and yeast CPSF-100 (residues 1-720) (the crystallographic data are summarized in Supplemental Table 2). The two structures obtained for CPSF-73 were crystallized in the absence or presence of 0.5 mM zinc (although both structures contained zinc atoms; see below). We discovered serendipitously that in situ proteolysis by a fungal protease is crucial for the crystallization of yeast CPSF-100 16 .The structure of CPSF-73 can be divided into two domains (Fig. 1a). The N-terminal residues (amino acids 1-208) form a domain similar to the structure of canonical me...

We have examined changes in the chromatin landscape during muscle differentiation by mapping the genome-wide location of ten key histone marks and transcription factors in mouse myoblasts and terminally differentiated myotubes, providing an exceptionally rich dataset that has enabled discovery of key epigenetic changes underlying myogenesis. Using this compendium, we focused on a well-known repressive mark, histone H3 lysine 27 trimethylation, and identified novel regulatory elements flanking the myogenin gene that function as a key differentiation-dependent switch during myogenesis. Next, we examined the role of Polycomb-mediated H3K27 methylation in gene repression by systematically ablating components of both PRC1 and PRC2 complexes. Surprisingly, we found mechanistic differences between transient and permanent repression of muscle differentiation and lineage commitment genes and observed that the loss of PRC1 and PRC2 components produced opposing differentiation defects. These phenotypes illustrate striking differences as compared to embryonic stem cell differentiation and suggest that PRC1 and PRC2 do not operate sequentially in muscle cells. Our studies of PRC1 occupancy also suggested a "fail-safe" mechanism, whereby PRC1/Bmi1 concentrates at genes specifying nonmuscle lineages, helping to retain H3K27me3 in the face of declining Ezh2-mediated methyltransferase activity in differentiated cells.chip-Seq | chromatin modifications | muscle development | transcriptional regulation R egulation of the transcriptome through dynamic changes in chromatin plays an important role in lineage commitment and differentiation. Multiple histone modifications control gene expression through recruitment of factors that alter compaction of the chromatin fiber. Transient and long-term gene silencing is enforced through trimethylation of histone H3 on lysines 9 and 27 (hereafter H3K9me3 and H3K27me3) as well as H4K20, whereas gene activation is regulated by methylation of H3K4 and acetylation of the amino-terminal tails of H3 and H4 (reviewed in refs. 1 and 2). Chromatin modifications are often asymmetrically deposited with respect to the transcription start sites (TSS) of genes. Whereas H3K27me3 is found at promoters, throughout gene bodies, and in intergenic regions, histone tail acetylation and H3K4me3 are predominantly found at promoters and the 5′ ends of genes. On the other hand, H3K36 trimethylation marks gene bodies, signifying the passage of RNA polymerase II (PolII) on actively transcribed genes. Promoter acetylation and H3K4 trimethylation are often coordinated, whereas H3K27 and H3K4 trimethylation are largely anticorrelated, except within bivalent regions poised to adopt either active or repressed states at the appropriate developmental stage (3).Previous studies have shown that the pluripotent state of embryonic stem (ES) cells is in part governed by bivalent nucleosomes, characterized by simultaneous H3K4 and H3K27 trimethylation of nucleosomes in lineage commitment genes (3, 4). During ES cell differentiation, ...

To identify the compendium of distal regulatory elements that govern myogenic differentiation, we generated chromatin state maps based on histone modifications and recruitment of factors that typify enhancers in myoblasts and myotubes. We found a striking concordance between the locations of these newly defined enhancers, MyoD1-binding events, and noncoding RNA transcripts. These enhancers recruit several sequencespecific transcription factors in a spatially constrained manner around MyoD1-binding sites. Remarkably, MyoD1-null myoblasts show a wholesale loss of recruitment of these factors as well as diminished monomethylation of H3K4 (H3K4me1) and acetylation of H3K27 (H3K27ac) and reduced recruitment of Set7, an H3K4 monomethylase. Surprisingly, we found that H3K4me1, but not H3K27ac, could be restored by re-expression of MyoD1 in MyoD1 -/-myoblasts, although re-expression of this factor in MyoD1-null myotubes restored both histone modifications. Our studies identified a role for MyoD1 in condition-specific enhancer assembly through recruitment of transcription factors and histone-modifying enzymes that shape muscle differentiation.

c Ubiquitylation of H2B on lysine 120 (H2Bub) is associated with active transcriptional elongation. H2Bub has been implicated in histone cross talk and is generally regarded to be a prerequisite for trimethylation of histone 3 lysine 4 (H3K4me3) and H3K79 in both yeast and mammalian cells. We performed a genome-wide analysis of epigenetic marks during muscle differentiation, and strikingly, we observed a near-complete loss of H2Bub in the differentiated state. We examined the basis for global loss of this mark and found that the H2B ubiquitin E3 ligase, RNF20, was depleted from chromatin in differentiated myotubes, indicating that recruitment of this protein to genes substantially decreases upon differentiation. Remarkably, during the course of myogenic differentiation, we observed retention and acquisition of H3K4 trimethylation on a large number of genes in the absence of detectable H2Bub. The Set1 H3K4 trimethylase complex was efficiently recruited to a subset of genes in myotubes in the absence of detectable H2Bub, accounting in part for H3K4 trimethylation in myotubes. Our studies suggest that H3K4me3 deposition in the absence of detectable H2Bub in myotubes is mediated via Set1 and, perhaps, MLL complexes, whose recruitment does not require H2Bub. Thus, muscle cells represent a novel setting in which to explore mechanisms that regulate histone cross talk. Modifications of histones, including lysine methylation, acetylation, and ubiquitylation, are closely associated with the control and modulation of gene transcription. Chromatin modifications are often asymmetrically deposited with respect to transcription start sites (TSS) of genes. In mammalian cells, lysine 4 trimethylation of histone H3 (H3K4me3) is associated with the TSS and 5= ends of genes, whereas H2B monoubiquitylation at lysine 120 (H2Bub) and H3K36 trimethylation are associated with transcribed regions of genes (22). H2Bub is catalyzed by a heterodimeric E3 ubiquitin ligase complex comprised of RNF20 and RNF40 (Bre1a/b in yeast) (11,46) and the E2 ubiquitinconjugating enzyme, Rad6 (12,13,30). H2Bub has been associated with active transcription and, more specifically, with transcriptional elongation (43). Several groups have independently demonstrated that H2Bub is a prerequisite for H3K79 and H3K4 methylation in yeast through a transtail mechanism (6,8,24,37). While monomethylation of H3K4 and H3K79 was found to be H2Bub independent (7, 33), H2Bub has been shown to direct diand trimethylation of H3K4 and H3K79 through the recruitment of relevant enzymes, Set1 and Dot1, respectively, facilitating histone cross talk in both yeast and mammals (12,19,37).The PAF1 complex (Paf1C) has also been shown to play a role in the regulation of H2B ubiquitylation in both yeast and mammals via recruitment and activation of Rad6/RNF20 on transcribed regions of chromatin in a manner that is dependent on elongating RNA polymerase (24,43,46). In addition, the Bur1 kinase in yeast and CDK9 in humans were shown to promote deposition of H2Bub (15,29). Thus, H2B mono...

Eukaryotic pre-mRNA 3-end formation is catalyzed by a complex set of factors that must be intricately regulated. In this study, we have discovered a novel role for the small ubiquitin-like modifier SUMO in the regulation of mammalian 3-end processing. We identified symplekin, a factor involved in complex assembly, and CPSF-73, an endonuclease, as SUMO modification substrates. The major sites of sumoylation in symplekin and CPSF-73 were determined and found to be highly conserved across species. A sumoylation-deficient mutant was defective in rescuing cell viability in symplekin small interfering RNA (siRNA)-treated cells, supporting the importance of this modification in symplekin function. We also analyzed the involvement of sumoylation in 3-end processing by altering the sumoylation status of nuclear extracts. This was done by the addition of a SUMO protease, which we show interacts with both symplekin and CPSF-73, or by siRNAmediated depletion of ubc9, the SUMO E2-conjugating enzyme. Both treatments resulted in a marked inhibition of processing. The assembly of a functional polyadenylation complex was also impaired by the SUMO protease. Our identification of two key polyadenylation factors as SUMO targets and of the role of SUMO in enhancing the assembly and activity of the 3-end-processing complex together reveal an important function for SUMO in the processing of mRNA precursors.The poly(A) tail at the 3Ј end of nearly all eukaryotic mRNAs is essential for the stability of the transcript, for its transport into the cytoplasm, and for translation initiation. The 3Ј ends of pre-mRNAs are formed in a two-step process, with an endonucleolytic cleavage generating a 3Ј OH end followed by the synthesis of a poly(A) tail (reviewed in references 10 and 47). This apparently simple reaction requires a surprisingly complex set of factors. The multisubunit cleavage/polyadenylation specificity factor (CPSF) and cleavage stimulatory factor (CstF) define the poly(A) site by binding cooperatively to the conserved AAUAAA and GU-rich sequence elements upstream and downstream, respectively, of the cleavage site (32,43,55). Cleavage factors I and II help in the complex assembly and in the first step (7,15,58). Poly(A) polymerase (PAP) catalyzes poly(A) addition and is also, in most cases, required in vitro for the cleavage reaction (48). The C-terminal domain of the largest subunit of RNA polymerase II (CTD) participates in the 3Ј-end-processing reaction and plays a critical stimulatory role (25, 39).CPSF-73, one of the subunits of the CPSF complex, has generated considerable interest recently as evidence has accumulated that it is the endonuclease that catalyzes the cleavage reaction. This was suggested first by its identification as a member of the metallo-␤-lactamase family of Zn-dependent hydrolytic enzymes (8, 49). More conclusively, recent structural and biochemical studies carried out with purified CPSF-73 provided unequivocal evidence that CPSF-73 indeed possesses endonucleolytic activity (38).In addition to the factors...