SummaryWe have used a human artificial chromosome (HAC) to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric α-satellite repeat containing one natural monomer with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability. Targeting of several tet-repressor (tetR) fusions into the centromere had no effect on kinetochore function. However, altering the chromatin state to a more open configuration with the tTA transcriptional activator or to a more closed state with the tTS transcription silencer caused missegregation and loss of the HAC. tTS binding caused the loss of CENP-A, CENP-B, CENP-C, and H3K4me2 from the centromere accompanied by an accumulation of histone H3K9me3. Our results reveal that a dynamic balance between centromeric chromatin and heterochromatin is essential for vertebrate kinetochore activity.

Despite the efforts that bioengineers have exerted in designing and constructing biological processes that function according to a predetermined set of rules, their operation remains fundamentally circumstantial. The contextual situation in which molecules and single-celled or multi-cellular organisms find themselves shapes the way they interact, respond to the environment and process external information. Since the birth of the field, synthetic biologists have had to grapple with contextual issues, particularly when the molecular and genetic devices inexplicably fail to function as designed when tested in vivo. In this review, we set out to identify and classify the sources of the unexpected divergences between design and actual function of synthetic systems and analyze possible methodologies aimed at controlling, if not preventing, unwanted contextual issues.

Eukaryotic mRNA precursors (pre-mRNAs) 1 are synthesized and processed in the nucleus prior to their export to the cytoplasm where they serve as templates for protein synthesis. Transcription is coupled spatially and temporally to the capping of the pre-mRNA at the 5Ј-end, splicing, and 3Ј-end formation. The mature 3Ј-ends of most eukaryotic mRNAs are generated by endonucleolytic cleavage of the primary transcript followed by the addition of a poly(A) tail to the upstream cleavage product (for reviews see Refs. 1 and 2). In mammals, these reactions are catalyzed by a large multicomponent complex that is assembled in a cooperative manner on specific cis-acting sequence elements in the pre-mRNA. The cleavage and polyadenylation specificity factor (CPSF) (3) recognizes the highly conserved hexanucleotide AAUAAA, whereas the cleavage stimulation factor (CstF) (4) binds a more degenerate GUor U-rich element downstream of the poly(A) site. It has been suggested that in vivo CPSF and CstF may become associated with each other prior to pre-mRNA binding, recognizing the two elements in a concerted manner (5). In addition, the cleavage reaction requires mammalian cleavage factor I (CF I m ), cleavage factor II m (CF II m ), and poly(A) polymerase (PAP). After the first step of 3Ј-end processing, CPSF remains bound to the upstream cleavage fragment and tethers PAP to the 3Ј-end of the pre-mRNA (6). In the presence of the nuclear poly(A)-binding protein 1 (PABPN1), PAP elongates the poly(A) tail in a processive manner (6). These factors are both necessary and sufficient to reconstitute cleavage and polyadenylation in vitro. However, the other proteins involved in either transcription, such as the C-terminal domain of RNA polymerase II, or capping (nuclear cap-binding complex) and splicing (U2AF65) have been shown to greatly enhance the efficiency of the first step of the reaction (7-9).Three major polypeptides of 25, 59, and 68 kDa and one minor polypeptide of 72 kDa copurify with CF I m activity from HeLa cell nuclear extract (10). Reconstitution of CF I m activity with recombinant proteins suggests that CF I m is a heterodimer consisting of the 25-kDa subunit and one of the larger polypeptides (11). All of the three larger proteins appear to be highly related in their amino acid sequence. Moreover, all of the CF I m subunits are only present in metazoan organisms. The primary sequence of the 25-kDa polypeptide contains a NUDIX motif (12). The amino acid composition of the 68-kDa protein has a domain organization that is reminiscent of spliceosomal SR proteins. Members of the SR family of splicing factors contain one or more N-terminal RNA recognition motifs (RRMs) that function in sequence-specific RNA binding and a C-terminal domain rich in alternating arginine and serine residues, referred to as RS domain, which is required for proteinprotein interactions with other RS domains (13). In the 68-kDa protein, the RRM and the RS-like domain are separated by a

We previously used a human artificial chromosome (HAC) with a synthetic kinetochore that could be targeted with chromatin modifiers fused to tetracycline repressor to show that targeting of the transcriptional repressor tTS within kinetochore chromatin disrupts kinetochore structure and function. Here we show that the transcriptional corepressor KAP1, a downstream effector of the tTS, can also inactivate the kinetochore. The disruption of kinetochore structure by KAP1 subdomains does not simply result from loss of centromeric CENP-A nucleosomes. Instead it reflects a hierarchical disruption of the outer kinetochore, with CENP-C levels falling before CENP-A levels and, in certain instances, CENP-H being lost more readily than CENP-C. These results suggest that this novel approach to kinetochore dissection may reveal new patterns of protein interactions within the kinetochore. INTRODUCTIONThe centromere/kinetochore is one of the most complex cellular substructures, with more than 80 protein components described to date (reviewed in Carroll and Straight, 2006;Cheeseman and Desai, 2008;Fukagawa, 2008;Vagnarelli et al., 2008). These components perform the complex job of attaching chromosomes to the mitotic spindle; ensuring that those attachments are correct; signaling to delay mitotic progression if they are not, and regulating the movements of the chromosomes toward the spindle poles in anaphase.The kinetochore is assembled at a unique locus on each natural chromosome. However, for organisms with regional centromeres (Pluta et al., 1995), this reflects only a preference¤, and not an absolute requirement for particular DNA sequences. Kinetochores can form on a wide range of singlecopy and repeated DNA sequences, leading to the conclusion that the ultimate determinants of kinetochore assembly are epigenetic. The long-term purpose of our studies is to determine the epigenetic "landscape" that promotes kinetochore assembly and its maintenance during cell divisions.Experiments including yeast genetics, RNA interference (RNAi) studies in mammalian cells, and gene knockout analysis in mouse and chicken DT40 cells have revealed that kinetochores assemble on a foundation of specialized chromatin containing the kinetochore-specific histone H3 variant CENP-A (Earnshaw and Rothfield, 1985). CENP-A is upstream of almost all other known components in the kinetochore assembly pathway. However, that pathway is multiplex, as recent studies in chicken and Drosophila show that inner kinetochore proteins CENP-H and -C are required for normal CENP-A loading or retention (Okada et al., 2006;Goshima et al., 2007;Erhardt et al., 2008).Our work was inspired by an approach first developed a number of years ago in which cloned fragments of human centromeric DNA were used to form human artificial chromosomes (HACs) in HT1080 fibrosarcoma cells (Harrington et al., 1997;Ikeno et al., 1998). Originally, HAC formation was only achieved with regular repeated arrays of ␣-satellite DNA containing CENP-B boxes Ohzeki et al., 2002;Okada et al., 2007)....