De novo induction of a DNA-histone H3K9 methylation loop on synthetic human repetitive DNA in cultured tobacco cells.

Genetic modifications in plants are crucial tools for fundamental and applied research. Transgene expression usually varies among independent lines or their progeny and is associated with the chromatin structure of the insertion site. Strategies based on understanding how to manipulate the epigenetic state of the inserted gene cassette would help to ensure transgene expression. Here, we report a strategy for chromatin manipulation by the artificial tethering of epigenetic effectors to a synthetic human centromeric repetitive DNA (alphoid DNA) platform in plant Bright-Yellow-2 (BY-2) culture cells. By tethering DNA-methyltransferase (Nicotiana tabacum DRM1), we effectively induced DNA methylation and histone methylation (H3K9me2) on the alphoid DNA platform. Tethering of the Arabidopsis SUVH9, which has been reported to lack histone methyltransferase activity, also induced a similar epigenetic state on the alphoid DNA in BY-2 cells, presumably by activating the RNA-dependent DNA methylation (RdDM) pathway. Our results emphasize that the interplay between DNA and histone methylation mechanisms is intrinsic to plant cells. We also found that once epigenetic modification states were induced by the tethering of either DRM1 or SUVH9, the modification was maintained even when the direct tethering of the effector was inhibited. Our system enables the analysis of more diverse epigenetic effectors and will help to elucidate the chromatin assembly mechanisms of plant cells.

Combination of CENP-B Box Positive and Negative Synthetic Alpha Satellite Repeats Improves De Novo Human Artificial Chromosome Formation.

Human artificial chromosomes (HACs) can be formed de novo by introducing large (>30 kb) centromeric sequences consisting of highly repeated 171-bp alpha satellite (alphoid) DNA into HT1080 cells. However, only a subset of transformed cells successfully establishes HACs. CENP-A chromatin and heterochromatin assemble on the HACs and play crucial roles in chromosome segregation. The CENP-B protein, which binds a 17-bp motif (CENP-B box) in the alphoid DNA, functions in the formation of alternative CENP-A chromatin or heterochromatin states. A balance in the coordinated assembly of these chromatin states on the introduced alphoid DNA is important for HAC formation. To obtain information about the relationship between chromatin architecture and de novo HAC formation efficiency, we tested combinations of two 60-kb synthetic alphoid sequences containing either tetO or lacO plus a functional or mutated CENP-B box combined with a multiple fusion protein tethering system. The combination of mutated and wild-type CENP-B box alphoid repeats significantly enhanced HAC formation. Both CENP-A and HP1α were enriched in the wild-type alphoid DNA, whereas H3K27me3 was enriched on the mutant alphoid array. The presence or absence of CENP-B binding resulted in differences in the assembly of CENP-A chromatin on alphoid arrays and the formation of H3K9me3 or H3K27me3 heterochromatin.

CENP-B creates alternative epigenetic chromatin states permissive for CENP-A or heterochromatin assembly.

CENP-B binds to CENP-B boxes on centromeric satellite DNAs (known as alphoid DNA in humans). CENP-B maintains kinetochore function through interactions with CENP-A nucleosomes and CENP-C. CENP-B binding to transfected alphoid DNA can induce de novo CENP-A assembly, functional centromere and kinetochore formation, and subsequent human artificial chromosome (HAC) formation. Furthermore, CENP-B also facilitates H3K9 (histone H3 lysine 9) trimethylation on alphoid DNA, mediated by Suv39h1, at ectopic alphoid DNA integration sites. Excessive heterochromatin invasion into centromere chromatin suppresses CENP-A assembly. It is unclear how CENP-B controls such different chromatin states. Here, we show that the CENP-B acidic domain recruits histone chaperones and many chromatin modifiers, including the H3K36 methylase ASH1L, as well as the heterochromatin components Suv39h1 and HP1 (HP1α, ß and γ, also known as CBX5, CBX1 and CBX3, respectively). ASH1L facilitates the formation of open chromatin competent for CENP-A assembly on alphoid DNA. These results indicate that CENP-B is a nexus for histone modifiers that alternatively promote or suppress CENP-A assembly by mutually exclusive mechanisms. Besides the DNA-binding domain, the CENP-B acidic domain also facilitates CENP-A assembly de novo on transfected alphoid DNA. CENP-B therefore balances CENP-A assembly and heterochromatin formation on satellite DNA.

Human artificial chromosome: Chromatin assembly mechanisms and CENP-B.

The centromere is a specialized chromosomal locus required for accurate chromosome segregation. Heterochromatin also assembles around centromere chromatin and forms a base that supports sister chromatid cohesion until anaphase begins. Both centromere chromatin and heterochromatin assemble on a centromeric DNA sequence, a highly repetitive sequence called alphoid DNA (α-satellite DNA) in humans. Alphoid DNA can form a de novo centromere and subsequent human artificial chromosome (HAC) when introduced into the human culture cells HT1080. HAC is maintained stably as a single chromosome independent of other human chromosomes. For de novo centromere assembly and HAC formation, the centromere protein CENP-B and its binding sites, CENP-B boxes, are required in the repeating units of alphoid DNA. CENP-B has multiple roles in de novo centromere chromatin assembly and stabilization and in heterochromatin formation upon alphoid DNA introduction into the cells. Here we review recent progress in human artificial chromosome construction and centromere/heterochromatin assembly and maintenance, focusing on the involvement of human centromere DNA and CENP-B protein.

Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies.

Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in human cells. This HAC was termed the alphoidtetO-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoidtetO-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoidtetO-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci. Importantly, the phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. AlphoidtetO-HAC-based technology has also been used to develop new drug screening and assessment strategies to manipulate the CIN phenotype in cancer cells. In summary, the alphoidtetO-HAC is proving to be a versatile tool for studying human chromosome transactions and structure as well as for genome and cancer studies.

KAT7/HBO1/MYST2 Regulates CENP-A Chromatin Assembly by Antagonizing Suv39h1-Mediated Centromere Inactivation.

Centromere chromatin containing histone H3 variant CENP-A is required for accurate chromosome segregation as a foundation for kinetochore assembly. Human centromere chromatin assembles on a part of the long α-satellite (alphoid) DNA array, where it is flanked by pericentric heterochromatin. Heterochromatin spreads into adjacent chromatin and represses gene expression, and it can antagonize centromere function or CENP-A assembly. Here, we demonstrate an interaction between CENP-A assembly factor M18BP1 and acetyltransferase KAT7/HBO1/MYST2. Knocking out KAT7 in HeLa cells reduced centromeric CENP-A assembly. Mitotic chromosome misalignment and micronuclei formation increased in the knockout cells and were enhanced when the histone H3-K9 trimethylase Suv39h1 was overproduced. Tethering KAT7 to an ectopic alphoid DNA integration site removed heterochromatic H3K9me3 modification and was sufficient to stimulate new CENP-A or histone H3.3 assembly. Thus, KAT7-containing acetyltransferases associating with the Mis18 complex provides competence for histone turnover/exchange activity on alphoid DNA and prevents Suv39h1-mediated heterochromatin invasion into centromeres.

CENP-C and CENP-I are key connecting factors for kinetochore and CENP-A assembly.

Although it is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity, the pathways leading to the formation and maintenance of centromere chromatin remain unclear. We previously generated human artificial chromosomes (HACs) whose centromeres contain a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator (alphoid(tetO)). We also obtained cell lines bearing the alphoid(tetO) array at ectopic integration sites on chromosomal arms. Here, we have examined the regulation of CENP-A assembly at centromeres as well as de novo assembly on the ectopic arrays by tethering tetracycline repressor (tetR) fusions of substantial centromeric factors and chromatin modifiers. This analysis revealed four classes of factors that influence CENP-A assembly. Interestingly, many kinetochore structural components induced de novo CENP-A assembly at the ectopic site. We showed that these components work by recruiting CENP-C and subsequently recruiting M18BP1. Furthermore, we found that CENP-I can also recruit M18BP1 and, as a consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C. Thus, we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly.

Stable complex formation of CENP-B with the CENP-A nucleosome.

CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.

Genetic and epigenetic regulation of centromeres: a look at HAC formation.

The centromere is a specialized chromosomal locus required for accurate chromosome segregation. A specific histone H3 variant, CENP-A, assembles at centromeres. CENP-A is required for kinetochore protein assembly and is an epigenetic marker for the maintenance of a functional centromere. Human CENP-A chromatin normally assembles on α-satellite DNA (alphoid DNA), a centromeric repetitive sequence. Using alphoid DNA arrays, human artificial chromosomes (HACs) have been constructed in human HT1080 cells and used to dissect the requirements for CENP-A assembly on DNA sequence. However, centromere formation is not a simple genetic event. In other commonly used human cell lines, such as HeLa and U2OS cells, no functional de novo centromere formation occurs efficiently with the same centromeric alphoid DNA sequences. Recent studies using protein tethering combined with the HAC system and/or genetic manipulation have revealed that epigenetic chromatin regulation mechanisms are also involved in the CENP-A chromatin assembly pathway and subsequent centromere/kinetochore formation. We summarize the DNA sequence requirements for CENP-A assembly and discuss the epigenetic regulation of human centromeres.

Nap1 regulates proper CENP-B binding to nucleosomes.

CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1.

Breaking the HAC Barrier: histone H3K9 acetyl/methyl balance regulates CENP-A assembly.

The kinetochore is responsible for accurate chromosome segregation. However, the mechanism by which kinetochores assemble and are maintained remains unclear. Here we report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENP-A assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar results are obtained following tethering of CENP-A deposition factors hMis18α or HJURP. HAT tethering bypasses the need for hMis18α, but HJURP is still required for de novo kinetochore assembly. In contrast, H3K9 methylation following tethering of H3K9 tri-methylase (Suv39h1) to the array prevents de novo CENP-A assembly and kinetochore formation. CENP-A arrays assembled de novo by this mechanism can form human artificial chromosomes (HACs) that are propagated indefinitely in human cells.

Human gamma-satellite DNA maintains open chromatin structure and protects a transgene from epigenetic silencing.

The role of repetitive DNA sequences in pericentromeric regions with respect to kinetochore/heterochromatin structure and function is poorly understood. Here, we use a mouse erythroleukemia cell (MEL) system for studying how repetitive DNA assumes or is assembled into different chromatin structures. We show that human gamma-satellite DNA arrays allow a transcriptionally permissive chromatin conformation in an adjacent transgene and efficiently protect it from epigenetic silencing. These arrays contain CTCF and Ikaros binding sites. In MEL cells, this gamma-satellite DNA activity depends on binding of Ikaros proteins involved in differentiation along the hematopoietic pathway. Given our discovery of gamma-satellite DNA in pericentromeric regions of most human chromosomes and a dynamic chromatin state of gamma-satellite arrays in their natural location, we suggest that gamma-satellite DNA represents a unique region of the functional centromere with a possible role in preventing heterochromatin spreading beyond the pericentromeric region.

CENP-B controls centromere formation depending on the chromatin context.

The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.

A minimal CENP-A core is required for nucleation and maintenance of a functional human centromere.

Chromatin clusters containing CENP-A, a histone H3 variant, are found in centromeres of multicellular eukaryotes. This study examines the ability of alpha-satellite (alphoid) DNA arrays in different lengths to nucleate CENP-A chromatin and form functional kinetochores de novo. Kinetochore assembly was followed by measuring human artificial chromosome formation in cultured human cells and by chromatin immunoprecipitation analysis. The results showed that both the length of alphoid DNA arrays and the density of CENP-B boxes had a strong impact on nucleation, spreading and/or maintenance of CENP-A chromatin, and formation of functional kinetochores. These effects are attributed to a change in the dynamic balance between assembly of chromatin containing trimethyl histone H3-K9 and chromatin containing CENP-A/C. The data presented here suggest that a functional minimum core stably maintained on 30-70 kb alphoid DNA arrays represents an epigenetic memory of centromeric chromatin.

The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein.

The most common cause of primary autosomal recessive microcephaly (MCPH) appears to be mutations in the ASPM gene which is involved in the regulation of neurogenesis. The predicted gene product contains two putative N-terminal calponin-homology (CH) domains and a block of putative calmodulin-binding IQ domains common in actin binding cytoskeletal and signaling proteins. Previous studies in mouse suggest that ASPM is preferentially expressed in the developing brain. Our analyses reveal that ASPM is widely expressed in fetal and adult tissues and upregulated in malignant cells. Several alternatively spliced variants encoding putative ASPM isoforms with different numbers of IQ motifs were identified. The major ASPM transcript contains 81 IQ domains, most of which are organized into a higher order repeat (HOR) structure. Another prominent spliced form contains an in-frame deletion of exon 18 and encodes 14 IQ domains not organized into a HOR. This variant is conserved in mouse. Other spliced variants lacking both CH domains and a part of the IQ motifs were also detected, suggesting the existence of isoforms with potentially different functions. To elucidate the biochemical function of human ASPM, we developed peptide specific antibodies to the N- and C-termini of ASPM. In a western analysis of proteins from cultured human and mouse cells, the antibodies detected bands with mobilities corresponding to the predicted ASPM isoforms. Immunostaining of cultured human cells with antibodies revealed that ASPM is localized in the spindle poles during mitosis. This finding suggests that MCPH is the consequence of an impairment in mitotic spindle regulation in cortical progenitors due to mutations in ASPM.

The role of CENP-B and alpha-satellite DNA: de novo assembly and epigenetic maintenance of human centromeres.

The centromere is an essential functional domain responsible for the correct inheritance of eukaryotic chromosomes during cell division. Eukaryotic centromeres include the highly conserved centromere-specific histone H3 variant, CENP-A, which has provided a powerful tool for investigating the recruitment of centromere components. However, the trigger that targets CENP-A to a specific genomic locus during centromere assembly remains unknown. Although, on rare occasions, CENP-A chromatin may assemble at non-centromeric DNA, all normal human centromeres are assembled and maintained on alpha-satellite (alphoid) DNA. The importance of alphoid DNA and CENP-B binding sites (CENP-B boxes), typical of normal human centromere DNA configurations, has been demonstrated through their requirement in de novo centromere assembly and Human Artificial Chromosome (HAC) assays. Mechanisms to link the centromere tightly to specific genomic sequences exist in humans and the two yeast species.

Epigenetic assembly of centromeric chromatin at ectopic alpha-satellite sites on human chromosomes.

To investigate the mechanism of chromatin assembly at human centromeres, we isolated cultured human cell lines in which a transfected alpha-satellite (alphoid) YAC was integrated ectopically into the terminal region of host chromosome 16, where it was stably maintained. Centromere activity of the alphoid YAC was suppressed at ectopic locations on the host chromosome, as indicated by the absent or reduced assembly of CENP-A and -C. However, long-term culture in selective medium, or short-term treatment with the histone deacetylase inhibitor Trichostatin A (TSA), promoted the re-assembly of CENPA, -B and -C at the YAC site and the release of minichromosomes containing the YAC integration site. Chromatin immunoprecipitation analyses of the re-formed minichromosome and the alphoid YAC-based stable human artificial chromosome both indicated that CENP-A and CENP-B assembled only on the inserted alphoid array but not on the YAC arms. On the YAC arms at the alphoid YAC integration sites, TSA treatment increased both the acetylation level of histone H3 and the transcriptional level of a marker gene. An increase in the level of transcription was also observed after long-term culture in selective medium. These activities, which are associated with changes in chromatin structure, might reverse the suppressed chromatin state of the YAC at ectopic loci, and thus might be involved in the epigenetic change of silent centromeres on ectopic alphoid loci.

CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA.

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.