Vladimir Larionov

Inactivation of a Human Kinetochore by Specific Targeting of Chromatin Modifiers

Summary We 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.

Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore.

Kinetochores assemble on distinct ‘centrochromatin’ containing the histone H3 variant CENP‐A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4–K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine‐specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α‐satellite DNA and to no longer efficiently recruit HJURP, the CENP‐A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP‐A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α‐satellite transcription, maintenance of CENP‐A levels and kinetochore stability.

Accelerated evolution of the ASPM gene controlling brain size begins prior to human brain expansion.

Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in ASPM, which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM. Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.

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.

Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae

Here, we describe a protocol for the selective isolation of any genomic fragment or gene of interest up to 250 kb in size from complex genomes as a circular yeast artificial chromosome (YAC). The method is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae between genomic DNA and a linearized TAR cloning vector containing targeting sequences homologous to a region of interest. Recombination between the vector and homologous sequences in the co-transformed mammalian DNA results in the establishment of a YAC that is able to propagate, segregate and be selected for in yeast. Yield of gene-positive clones varies from 1% to 5%. The entire procedure takes 2 weeks to complete once the TAR vector is constructed and genomic DNA is prepared. The TAR cloning method has a broad application in functional and comparative genomics, long-range haplotyping and characterization of chromosomal rearrangements, including copy number variations.

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.

TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution

The structural and functional analysis of mammalian genomes would benefit from the ability to isolate from multiple DNA samples any targeted chromosomal segment that is the size of an average human gene. A cloning technique that is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae satisfies this need. It is a unique tool to selectively recover chromosome segments that are up to 250 kb in length from complex genomes. In addition, TAR cloning can be used to characterize gene function and genome variation, including polymorphic structural rearrangements, mutations and the evolution of gene families, and for long-range haplotyping.

The human telomerase gene: complete genomic sequence and analysis of tandem repeat polymorphisms in intronic regions

In this work, the full-length hTERT gene was isolated and the sequence of the previously unknown region in intron 6 as well as that of upstream and downstream hTERT regions was determined. We have shown that intron 6 includes a variable number of tandem repeats (VNTR) of a 38 bp sequence, (hTERT-VNTR 6-1). Eight alleles of hTERT-VNTR 6-1 were identified among 103 unrelated individuals, ranging from 27 to 47 repeats. hTERT-VNTR 2-2 is another new 61 bp minisatellite repeat found in intron 2 of hTERT. At least four alleles of hTERT-VNTR 2-2 can be distinguished. Previous studies have described polymorphisms for minisatellites hTERT-VNTR 2-1, a 42 bp repeat in intron 2, and hTERT-VNTR 6-2, a 36 bp repeat in intron 6. These, together with another minisatellite found in intron 12, add up to five such structures within the hTERT gene. The segregation of hTERT minisatellites was analysed in families, revealing that the VNTRs are transmitted through meiosis following a Mendelian inheritance. Minisatellites in hTERT were also analysed in matching normal and cancer tissues from patients with tumors; in one patient with a kidney tumor, the two VNTRs in intron 6 had undergone concomitant rearrangements. This observation suggests that chromosomal rearrangements implicating these VNTRs may be associated with the activation of telomerase expression in cancer cells.

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.

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function.

Human kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated ‘centrochromatin’, despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-κB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ~10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ~150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity – kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.