Long-Read DNA Sequencing: Recent Advances and Remaining Challenges.

DNA sequencing has revolutionized medicine over recent decades. However, analysis of large structural variation and repetitive DNA, a hallmark of human genomes, has been limited by short-read technology, with read lengths of 100-300 bp. Long-read sequencing (LRS) permits routine sequencing of human DNA fragments tens to hundreds of kilobase pairs in size, using both real-time sequencing by synthesis and nanopore-based direct electronic sequencing. LRS permits analysis of large structural variation and haplotypic phasing in human genomes and has enabled the discovery and characterization of rare pathogenic structural variants and repeat expansions. It has also recently enabled the assembly of a complete, gapless human genome that includes previously intractable regions, such as highly repetitive centromeres and homologous acrocentric short arms. With the addition of protocols for targeted enrichment, direct epigenetic DNA modification detection, and long-range chromatin profiling, LRS promises to launch a new era of understanding of genetic diversity and pathogenic mutations in human populations.

Noncoding variants alter GATA2 expression in rhombomere 4 motor neurons and cause dominant hereditary congenital facial paresis.

Hereditary congenital facial paresis type 1 (HCFP1) is an autosomal dominant disorder of absent or limited facial movement that maps to chromosome 3q21-q22 and is hypothesized to result from facial branchial motor neuron (FBMN) maldevelopment. In the present study, we report that HCFP1 results from heterozygous duplications within a neuron-specific GATA2 regulatory region that includes two enhancers and one silencer, and from noncoding single-nucleotide variants (SNVs) within the silencer. Some SNVs impair binding of NR2F1 to the silencer in vitro and in vivo and attenuate in vivo enhancer reporter expression in FBMNs. Gata2 and its effector Gata3 are essential for inner-ear efferent neuron (IEE) but not FBMN development. A humanized HCFP1 mouse model extends Gata2 expression, favors the formation of IEEs over FBMNs and is rescued by conditional loss of Gata3. These findings highlight the importance of temporal gene regulation in development and of noncoding variation in rare mendelian disease.

Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres.

Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha satellite DNA and permit analysis of centromeric DNA and chromatin organization. In this study, we present molecular cytogenetic and CENP-A chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique DNA and CENP-A chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated DNA. This is the first example of a neocentromere forming on repetitive DNA, as all other mapped neocentromeres have formed on single copy DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem DNA in centromere structure. The second neocentromere was found on a neocentric ring chromosome that contained the 8q21 tandemly repeated DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the ring chromosome. This neocentromere suggests that chromosomal rearrangement and DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.

Large inverted repeats within Xp11.2 are present at the breakpoints of isodicentric X chromosomes in Turner syndrome.

Turner syndrome (TS) results from whole or partial monosomy X and is mediated by haploinsufficiency of genes that normally escape X-inactivation. Although a 45,X karyotype is observed in half of all TS cases, the most frequent variant TS karyotype includes the isodicentric X chromosome alone [46,X,idic(X)(p11)] or as a mosaic [46,X,idic(X)(p11)/45,X]. Given the mechanism of idic(X)(p11) rearrangement is poorly understood and breakpoint sequence information is unknown, this study sought to investigate the molecular mechanism of idic(X)(p11) formation by determining their precise breakpoint intervals. Karyotype analysis and fluorescence in situ hybridization mapping of eight idic(X)(p11) cell lines and three unbalanced Xp11.2 translocation lines identified the majority of breakpoints within a 5 Mb region, from approximately 53 to 58 Mb, in Xp11.1-p11.22, clustering into four regions. To further refine the breakpoints, a high-resolution oligonucleotide microarray (average of approximately 350 bp) was designed and array-based comparative genomic hybridization (aCGH) was performed on all 11 idic(X)(p11) and Xp11.2 translocation lines. aCGH analyses identified all breakpoint regions, including an idic(X)(p11) line with two potential breakpoints, one breakpoint shared between two idic(X)(p11) lines and two Xp translocations that shared breakpoints with idic(X)(p11) lines. Four of the breakpoint regions included large inverted repeats composed of repetitive gene clusters and segmental duplications, which corresponded to regions of copy-number variation. These data indicate that the rearrangement sites on Xp11.2 that lead to isodicentric chromosome formation and translocations are probably not random and suggest that the complex repetitive architecture of this region predisposes it to rearrangements, some of which are recurrent.

Duplication of the ZIC2 gene is not associated with holoprosencephaly.

Cytogenetic testing using genomic microarrays presents a clinical challenge when data regarding the phenotypic consequences of the genomic alteration are not available. We describe a chromosome 13q32.3 duplication discovered by microarray testing in a fetus with a prenatally detected apparently balanced de novo translocation 46,XY,t(2;13)(q37;q32). Microarray analysis on the fetal DNA showed duplications of 384 and 564 kb at the breakpoint regions on chromosomes 2q37.3 and 13q32.3, respectively. There were no disease-associated genes in the duplicated region on chromosome 2q37. The duplicated region on chromosome 13q contains the ZIC2 gene. Haploinsufficiency of ZIC2 is known to cause holoprosencephaly and other brain malformations. Studies in the mouse models have suggested that over expression of ZIC2 may also lead to brain malformations. Fetal MRI of the brain was normal and the family elected to continue the pregnancy. An apparently normal baby was born at term. At 3 months of age a physical exam showed no abnormalities and no developmental delay. This report shows that duplication of ZIC2 is not necessarily associated with brain malformations. We also describe the phenotype from four additional patients with duplications of the region of chromosome 13 containing ZIC2 and three previously described patients with supernumerary marker chromosomes derived from distal chromosome 13. None of the eight patients had holoprosencephaly or brain malformations, indicating that duplication of ZIC2 is not associated with brain anomalies. This information will be useful for counseling in other occurrences of this duplication identified by microarray.

Analysis of transposon interruptions suggests selection for L1 elements on the X chromosome.

It has been hypothesised that the massive accumulation of L1 transposable elements on the X chromosome is due to their function in X inactivation, and that the accumulation of Alu elements near genes is adaptive. We tested the possible selective advantage of these two transposable element (TE) families with a novel method, interruption analysis. In mammalian genomes, a large number of TEs interrupt other TEs due to the high overall abundance and age of repeats, and these interruptions can be used to test whether TEs are selectively neutral. Interruptions of TEs, which are beneficial for the host, are expected to be deleterious and underrepresented compared with neutral ones. We found that L1 elements in the regions of the X chromosome that contain the majority of the inactivated genes are significantly less frequently interrupted than on the autosomes, while L1s near genes that escape inactivation are interrupted with higher frequency, supporting the hypothesis that L1s on the X chromosome play a role in its inactivation. In addition, we show that TEs are less frequently interrupted in introns than in intergenic regions, probably due to selection against the expansion of introns, but the insertion pattern of Alus is comparable to other repeats.

Biased distributions and decay of long interspersed nuclear elements in the chicken genome.

The genomes of birds are much smaller than mammalian genomes, and transposable elements (TEs) make up only 10% of the chicken genome, compared with the 45% of the human genome. To study the mechanisms that constrain the copy numbers of TEs, and as a consequence the genome size of birds, we analyzed the distributions of LINEs (CR1's) and SINEs (MIRs) on the chicken autosomes and Z chromosome. We show that (1) CR1 repeats are longest on the Z chromosome and their length is negatively correlated with the local GC content; (2) the decay of CR1 elements is highly biased, and the 5'-ends of the insertions are lost much faster than their 3'-ends; (3) the GC distribution of CR1 repeats shows a bimodal pattern with repeats enriched in both AT-rich and GC-rich regions of the genome, but the CR1 families show large differences in their GC distribution; and (4) the few MIRs in the chicken are most abundant in regions with intermediate GC content. Our results indicate that the primary mechanism that removes repeats from the chicken genome is ectopic exchange and that the low abundance of repeats in avian genomes is likely to be the consequence of their high recombination rates.

Analysis of the largest tandemly repeated DNA families in the human genome.

BACKGROUND: Tandemly Repeated DNA represents a large portion of the human genome, and accounts for a significant amount of copy number variation. Here we present a genome wide analysis of the largest tandem repeats found in the human genome sequence. RESULTS: Using Tandem Repeats Finder (TRF), tandem repeat arrays greater than 10 kb in total size were identified, and classified into simple sequence e.g. GAATG, classical satellites e.g. alpha satellite DNA, and locus specific VNTR arrays. Analysis of these large sequenced regions revealed that several "simple sequence" arrays actually showed complex domain and/or higher order repeat organization. Using additional methods, we further identified a total of 96 additional arrays with tandem repeat units greater than 2 kb (the detection limit of TRF), 53 of which contained genes or repeated exons. The overall size of an array of tandem 12 kb repeats which spanned a gap on chromosome 8 was found to be 600 kb to 1.7 Mbp in size, representing one of the largest non-centromeric arrays characterized. Several novel megasatellite tandem DNA families were observed that are characterized by repeating patterns of interspersed transposable elements that have expanded presumably by unequal crossing over. One of these families is found on 11 different chromosomes in >25 arrays, and represents one of the largest most widespread megasatellite DNA families. CONCLUSION: This study represents the most comprehensive genome wide analysis of large tandem repeats in the human genome, and will serve as an important resource towards understanding the organization and copy number variation of these complex DNA families.

Evolutionary history of mammalian transposons determined by genome-wide defragmentation.

The constant bombardment of mammalian genomes by transposable elements (TEs) has resulted in TEs comprising at least 45% of the human genome. Because of their great age and abundance, TEs are important in comparative phylogenomics. However, estimates of TE age were previously based on divergence from derived consensus sequences or phylogenetic analysis, which can be unreliable, especially for older more diverged elements. Therefore, a novel genome-wide analysis of TE organization and fragmentation was performed to estimate TE age independently of sequence composition and divergence or the assumption of a constant molecular clock. Analysis of TEs in the human genome revealed approximately 600,000 examples where TEs have transposed into and fragmented other TEs, covering >40% of all TEs or approximately 542 Mbp of genomic sequence. The relative age of these TEs over evolutionary time is implicit in their organization, because newer TEs have necessarily transposed into older TEs that were already present. A matrix of the number of times that each TE has transposed into every other TE was constructed, and a novel objective function was developed that derived the chronological order and relative ages of human TEs spanning >100 million years. This method has been used to infer the relative ages across all four major TE classes, including the oldest, most diverged elements. Analysis of DNA transposons over the history of the human genome has revealed the early activity of some MER2 transposons, and the relatively recent activity of MER1 transposons during primate lineages. The TEs from six additional mammalian genomes were defragmented and analyzed. Pairwise comparison of the independent chronological orders of TEs in these mammalian genomes revealed species phylogeny, the fact that transposons shared between genomes are older than species-specific transposons, and a subset of TEs that were potentially active during periods of speciation.

DNA modification and functional delivery into human cells using Escherichia coli DH10B.

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a approximately 200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd(-) rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.

CP248, a derivative of exisulind, causes growth inhibition, mitotic arrest, and abnormalities in microtubule polymerization in glioma cells.

Exisulind (sulindac sulfone) and two potent derivatives, CP248 and CP461, have been shown previously to cause growth inhibition and apoptosis in several types of human carcinoma cell lines. These and related compounds have not been previously studied with respect to glioma cell lines. In the present study, we found that these three compounds caused marked growth inhibition in four rat glioma and eight human glioma cell lines, with IC50 values of 150, 1, and 0.075 microm, respectively. When studied at these concentrations exisulind and CP461 had no significant effect on the cell cycle profile of glioma cells, but CP248 caused marked arrest in mitosis. Detailed studies of CP248 in the 9L rat gliosarcoma cell line indicated that treatment with 0.075 microM CP248 caused abnormalities in the spindle apparatus and activation of the spindle assembly check point. In interphase glioma cells, CP248 stabilized microtubules (MTs) at low concentrations (0.075 microM) and depolymerized MTs at higher concentrations (0.2-0.4 microM). In NIH 3T3 fibroblasts, 0.1 microM CP248 caused extensive MT depolymerization. CP248 also caused MT depolymerization when added to assembled MTs in vitro, which indicated that it can directly affect MTs, perhaps because it shares certain structural similarities with Colcemid. In glioma cells, the effects of CP248 on MTs were independent of the previously reported effects of this compound on activation of protein kinase G. Therefore, CP248 is a novel MT-active agent that may be useful in the treatment of glioblastoma, and possibly other types of cancer, because of its dual effects on protein kinase G and MTs.

Unique case of mosaicism involving two morphologically similar marker chromosomes of different centric origin in a patient with developmental delay.

A five-year-old Caucasian male presented with developmental delay, minor dysmorphic features, and hyperactivity. Cytogenetic analysis showed the presence of a marker chromosome in the majority of cells analyzed. Fluorescence in situ hybridization (FISH) analyses using several alpha satellite probes, including D13Z1/D21Z1, did not reveal any signal on the marker chromosome. Subsequent multicolor FISH (M-FISH) indicated the marker to be derived from chromosome 13, and FISH with a chromosome 13 paint confirmed this finding. The absence of D13Z1/D21Z1 signal on the marker suggested that it was analphoid in nature. Comparative genomic hybridization (CGH) was utilized to further characterize the region of chromosome 13 from which the marker originated, and unexpectedly revealed a gain of chromosomal material at both the centromeric regions of chromosomes 3 and 13. In view of the CGH results, extensive FISH studies with D3Z1 and D13Z1/D21Z1 were performed and revealed the presence of four cell lines comprising one normal cell line (50.5%), a cell line with a chromosome 3 derived marker (19%), a cell line containing a marker derived from chromosome 13 (20%), and a cell line with both markers (10.5%). As the two markers appeared morphologically similar by GTG banding, all 47,XY metaphases in the initial analysis were thought to comprise only a single marker. This is the first report, to our knowledge, of the presence of a chromosome 3 and a chromosome 13 marker in mosaic condition in a congenital disorder. In light of our experience, we urge caution in interpreting karyotypes with marker chromosomes. Our case illustrates the limitations of fluorescent DNA probes and sampling errors.

Identification of a neocentromere in a rearranged Y chromosome with no detectable DYZ3 centromeric sequence.

An 18-year-old woman was evaluated because of primary amenorrhea and hypogonadism. Chromosome analysis from peripheral blood lymphocytes revealed a nonmosaic 46,X,+mar constitution. The marker was shown to be a rearranged Y chromosome consisting of an inverted duplication of the long arm: rea(Y)(qter-q11::q11-qter). Deletion mapping analysis with Y-specific STS showed that the marker lacked Yp and Y-centromeric (DYZ3) sequences, but it was positive for Yq sequences tested. Fluorescence in situ hybridization analysis with Y and X chromosome centromeric and pancentromeric probes showed no hybridization signals. The marker chromosome is present in 100% of the cells; therefore, it is mitotically stable despite the absence of DYZ3 centromeric sequence. Hybridization with CENP-A and CENP-C specific antibodies localized a neocentromere close to the breakpoint.

Chromosome 13q neocentromeres: molecular cytogenetic characterization of three additional cases and clinical spectrum.

We report three new cases of chromosome 13 derived marker chromosomes, found in unrelated patients with dysmorphisms and/or developmental delay. Molecular cytogenetic analysis was performed using fluorescence in situ hybridization (FISH) with chromosome-specific painting probes, alpha satellite probes, and physically mapped probes from chromosome 13q, as well as comparative genomic hybridization (CGH). This analysis demonstrated that these markers consisted of inversion duplications of distal portions of chromosome 13q that have separated from the endogenous chromosome 13 centromere and contain no detectable alpha satellite DNA. The presence of a functional neocentromere on these marker chromosomes was confirmed by immunofluorescence with antibodies to centromere protein-C (CENP-C). The cytogenetic location of a neocentromere in band 13q32 was confirmed by simultaneous FISH with physically mapped YACs from 13q32 and immunofluorescence with anti-CENP-C. The addition of these three new cases brings the total number of described inv dup 13q neocentic chromosomes to 11, representing 21% (11/52) of the current overall total of 52 described cases of human neocentric chromosomes. This higher than expected frequency suggests that chromosome 13q may have an increased propensity for neocentromere formation. The clinical spectrum of all 11 cases is presented, representing a unique collection of polysomy for different portions of chromosome 13q without aneuploidies for additional chromosomal regions. The complexity and variability of the phenotypes seen in these patients does not support a simple reductionist view of phenotype/genotype correlation with polysomy for certain chromosomal regions.

The octamer is the major form of CENP-A nucleosomes at human centromeres.

The centromere is the chromosomal locus that ensures fidelity in genome transmission at cell division. Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location independently of DNA sequence. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes. Here we show that the predominant form of the CENP-A particle at human centromeres is an octameric nucleosome. CENP-A nucleosomes are very highly phased on α-satellite 171-base-pair monomers at normal centromeres and also display strong positioning at neocentromeres. At either type of functional centromere, CENP-A nucleosomes exhibit similar DNA-wrapping behavior, as do octameric CENP-A nucleosomes reconstituted with recombinant components, having looser DNA termini than those on conventional nucleosomes containing canonical histone H3. Thus, the fundamental unit of the chromatin that epigenetically specifies centromere location in mammals is an octameric nucleosome with loose termini.

Neocentric X-chromosome in a girl with Turner-like syndrome.

BACKGROUND: Neocentromeres are rare human chromosomal aberrations in which a new centromere has formed in a previously non-centromeric location. We report the finding of a structurally abnormal X chromosome with a neocentromere in a 15-year-old girl with clinical features suggestive of Turner syndrome, including short stature and primary amenorrhea. RESULT: G-banded chromosome analysis revealed a mosaic female karyotype involving two abnormal cell lines. One cell line (84% of analyzed metaphases) had a structurally abnormal X chromosome (duplication of the long arm and deletion of the short arm) and a normal X chromosome. The other cell line (16% of cells) exhibited monosomy X. C-banding studies were negative for the abnormal X chromosome. FISH analysis revealed lack of hybridization of the abnormal X chromosome with both the X centromere-specific probe and the "all human centromeres" probe, a pattern consistent with lack of the X chromosome endogenous centromere. A FISH study using an XIST gene probe revealed the presence of two XIST genes, one on each long arm of the iso(Xq), required for inactivation of the abnormal X chromosome. R-banding also demonstrated inactivation of the abnormal X chromosome. An assay for centromeric protein C (CENP-C) was positive on both the normal and the abnormal X chromosomes. The position of CENP-C in the abnormal X chromosome defined a neocentromere, which explains its mitotic stability. The karyotype is thus designated as 46,X,neo(X)(qter- > q12::q12- > q21.2- > neo- > q21.2- > qter)[42]/45,X[8], which is consistent with stigmata of Turner syndrome. The mother of this patient has a normal karyotype; however, the father was not available for study. CONCLUSION: To our knowledge, this is the first case of mosaic Turner syndrome involving an analphoid iso(Xq) chromosome with a proven neocentromere among 90 previously described cases with a proven neocentromere.

A paucity of heterochromatin at functional human neocentromeres.

BACKGROUND: Centromeres are responsible for the proper segregation of replicated chromatids during cell division. Neocentromeres are fully functional ectopic human centromeres that form on low-copy DNA sequences and permit analysis of centromere structure in relation to the underlying DNA sequence. Such structural analysis is not possible at endogenous centromeres because of the large amounts of repetitive alpha satellite DNA present. RESULTS: High-resolution chromatin immunoprecipitation (ChIP) on CHIP (microarray) analysis of three independent neocentromeres from chromosome 13q revealed that each neocentromere contained approximately 100 kb of centromere protein (CENP)-A in a two-domain organization. Additional CENP-A domains were observed in the vicinity of neocentromeres, coinciding with CpG islands at the 5' end of genes. Analysis of histone H3 dimethylated at lysine 4 (H3K4me2) revealed small domains at each neocentromere. However, these domains of H3K4me2 were also found in the equivalent non-neocentric chromosomes. A surprisingly minimal (approximately 15 kb) heterochromatin domain was observed at one of the neocentromeres, which formed in an unusual transposon-free region distal to the CENP-A domains. Another neocentromere showed a distinct absence of nearby significant domains of heterochromatin. A subtle defect in centromere cohesion detected at these neocentromeres may be due to the paucity of heterochromatin domains. CONCLUSIONS: This high-resolution mapping suggests that H3K4me2 does not seem sufficiently abundant to play a structural role at neocentromeres, as proposed for endogenous centromeres. Large domains of heterochromatin also do not appear necessary for centromere function. Thus, this study provides important insight into the structural requirements of human centromere function.

Recombineering linear DNA that replicate stably in E. coli.

The advent of recombineering technology in Escherichia coli has revolutionized the way recombinant DNA molecules are constructed. We present a novel application of recombineering to linearize DNA by capping their ends with individual telomeres derived from bacteriophage N15, which exists as a linear prophage in E. coli. The N15 telomerase occupancy site was recombined into circular DNA and resolved into individual telomeres by the phage N15 protelomerase enzyme. We demonstrate this technique by assembling linear BACs that replicate stably in their host strain E. coli DH10B. Correct linearization of the BACs was confirmed by restriction mapping using pulsed field gel electrophoresis. The linear BAC DNA can be easily purified using standard plasmid isolation methods and resist degradation from RecBCD nuclease in vitro and in vivo owing to the presence of telomeres. Transfection of a linear 100 kb BAC containing the human beta-globin gene cluster into HT1080 cells produced accurately spliced transcripts, demonstrating that the linear DNA will be useful for subsequent functional studies. This novel recombineering technique may be particularly useful for building large linear constructs for assembling artificial chromosomes with telomeres, and may provide a unique means to clone and study large linear viral genomes that contain hairpin ends.

The mouse X chromosome is enriched for multicopy testis genes showing postmeiotic expression.

According to the prevailing view, mammalian X chromosomes are enriched in spermatogenesis genes expressed before meiosis and deficient in spermatogenesis genes expressed after meiosis. The paucity of postmeiotic genes on the X chromosome has been interpreted as a consequence of meiotic sex chromosome inactivation (MSCI)--the complete silencing of genes on the XY bivalent at meiotic prophase. Recent studies have concluded that MSCI-initiated silencing persists beyond meiosis and that most genes on the X chromosome remain repressed in round spermatids. Here, we report that 33 multicopy gene families, representing approximately 273 mouse X-linked genes, are expressed in the testis and that this expression is predominantly in postmeiotic cells. RNA FISH and microarray analysis show that the maintenance of X chromosome postmeiotic repression is incomplete. Furthermore, X-linked multicopy genes exhibit a similar degree of expression as autosomal genes. Thus, not only is the mouse X chromosome enriched for spermatogenesis genes functioning before meiosis, but in addition, approximately 18% of mouse X-linked genes are expressed in postmeiotic cells.

Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres.

BACKGROUND: Mammalian centromere formation is dependent on chromatin that contains centromere protein (CENP)-A, which is the centromere-specific histone H3 variant. Human neocentromeres have acquired CENP-A chromatin epigenetically in ectopic chromosomal locations on low-copy complex DNA. Neocentromeres permit detailed investigation of centromeric chromatin organization that is not possible in the highly repetitive alpha satellite DNA present at endogenous centromeres. RESULTS: We have examined the distribution of CENP-A, as well as two additional centromeric chromatin-associated proteins (CENP-C and CENP-H), across neocentromeric DNA using chromatin immunoprecipitation (ChIP) on CHIP assays on custom genomic microarrays at three different resolutions. Analysis of two neocentromeres using a contiguous bacterial artificial chromosome (BAC) microarray spanning bands 13q31.3 to 13q33.1 shows that both CENP-C and CENP-H co-localize to the CENP-A chromatin domain. Using a higher resolution polymerase chain reaction (PCR)-amplicon microarray spanning the neocentromere, we find that the CENP-A chromatin is discontinuous, consisting of a major domain of about 87.8 kilobases (kb) and a minor domain of about 13.2 kb, separated by an approximately 158 kb region devoid of CENPs. Both CENP-A domains exhibit co-localization of CENP-C and CENP-H, defining a distinct inner kinetochore chromatin structure that is consistent with higher order chromatin looping models at centromeres. The PCR microarray data suggested varying density of CENP-A nucleosomes across the major domain, which was confirmed using a higher resolution oligo-based microarray. CONCLUSION: Centromeric chromatin consists of several CENP-A subdomains with highly discontinuous CENP-A chromatin at both the level of individual nucleosomes and at higher order chromatin levels, raising questions regarding the overall structure of centromeric chromatin.