While the distribution of RNA polymerase II (PolII) in a variety of complex genomes is correlated with gene expression, the presence of PolII at a gene does not necessarily indicate active expression. Various patterns of PolII binding have been described genome wide; however, whether or not PolII binds at transcriptionally inactive sites remains uncertain. The two X chromosomes in female cells in mammals present an opportunity to examine each of the two alleles of a given locus in both active and inactive states, depending on which X chromosome is silenced by X chromosome inactivation. Here, we investigated PolII occupancy and expression of the associated genes across the active (Xa) and inactive (Xi) X chromosomes in human female cells to elucidate the relationship of gene expression and PolII binding. We find that, while PolII in the pseudoautosomal region occupies both chromosomes at similar levels, it is significantly biased toward the Xa throughout the rest of the chromosome. The general paucity of PolII on the Xi notwithstanding, detectable (albeit significantly reduced) binding can be observed, especially on the evolutionarily younger short arm of the X. PolII levels at genes that escape inactivation correlate with the levels of their expression; however, additional PolII sites can be found at apparently silenced regions, suggesting the possibility of a subset of genes on the Xi that are poised for expression. Consistent with this hypothesis, we show that a high proportion of genes associated with PolII-accessible sites, while silenced in GM12878, are expressed in other female cell lines.

Background

Combinations of histone variants and modifications, conceptually representing a histone code, have been proposed to play a significant role in gene regulation and developmental processes in complex organisms. While various mechanisms have been implicated in establishing and maintaining epigenetic patterns at specific locations in the genome, they are generally believed to be independent of primary DNA sequence on a more global scale.

Results

To address this systematically in the case of the human genome, we have analyzed primary DNA sequences underlying patterns of 19 different methylated histones in human primary T-cells and patterns of three methylated histones across additional human cell lines. We report strong sequence biases associated with most of these histone marks genome-wide in each cell type. Furthermore, the sequence characteristics for such association are distinct for different groups of histone marks.

Conclusions

These findings provide evidence of an influence of genomic sequence on patterns of histone modification associated with gene expression and chromatin programming, and they suggest that the mechanisms responsible for global histone modifications may interpret genomic sequence in various ways.

Background

Centromeres are sites of chromosomal spindle attachment during mitosis and meiosis. While the sequence basis for centromere identity remains a subject of considerable debate, one approach is to examine the genomic organization at these active sites that are correlated with epigenetic marks of centromere function.

Results

We have developed an approach to characterize both satellite and non-satellite centromeric sequences that are missing from current assemblies in complex genomes, using the dog genome as an example. Combining this genomic reference with an epigenetic dataset corresponding to sequences associated with the histone H3 variant centromere protein A (CENP-A), we identify active satellite sequence domains that appear to be both functionally and spatially distinct within the overall definition of satellite families.

Conclusions

These findings establish a genomic and epigenetic foundation for exploring the functional role of centromeric sequences in the previously sequenced dog genome and provide a model for similar studies within the context of less-characterized genomes.

Many essential aspects of genome function, including gene expression and chromosome segregation, are mediated throughout development and differentiation by changes in the chromatin state. Along with genomic signals encoded in the DNA, epigenetic processes regulate heritable gene expression patterns. Genomic signals such as enhancers, silencers, and repetitive DNA, while required for the establishment of alternative chromatin states, have an unclear role in epigenetic processes that underlie the persistence of chromatin states throughout development. Here, we demonstrate in fission yeast that the maintenance and inheritance of ectopic heterochromatin domains are independent of the genomic sequences necessary for their de novo establishment. We find that both structural heterochromatin and gene silencing can be stably maintained over an ∼10-kb domain for up to hundreds of cell divisions in the absence of genomic sequences required for heterochromatin establishment, demonstrating the long-term persistence and stability of this chromatin state. The de novo heterochromatin, despite the absence of nucleation sequences, is also stably inherited through meiosis. Together, these studies provide evidence for chromatin-dependent, epigenetic control of gene silencing that is heritable, stable, and self-sustaining, even in the absence of the originating genomic signals.

The methylation of cytosines in CpG dinucleotides is essential for cellular differentiation and the progression of many cancers, and it plays an important role in gametic imprinting. To assess variation and inheritance of genome-wide patterns of DNA methylation simultaneously in humans, we applied reduced representation bisulfite sequencing (RRBS) to somatic DNA from six members of a three-generation family. We observed that 8.1% of heterozygous SNPs are associated with differential methylation in cis, which provides a robust signature for Mendelian transmission and relatedness. The vast majority of differential methylation between homologous chromosomes (>92%) occurs on a particular haplotype as opposed to being associated with the gender of the parent of origin, indicating that genotype affects DNA methylation of far more loci than does gametic imprinting. We found that 75% of genotype-dependent differential methylation events in the family are also seen in unrelated individuals and that overall genotype can explain 80% of the variation in DNA methylation. These events are under-represented in CpG islands, enriched in intergenic regions, and located in regions of low evolutionary conservation. Even though they are generally not in functionally constrained regions, 22% (twice as many as expected by chance) of genes harboring genotype-dependent DNA methylation exhibited allele-specific gene expression as measured by RNA-seq of a lymphoblastoid cell line, indicating that some of these events are associated with gene expression differences. Overall, our results demonstrate that the influence of genotype on patterns of DNA methylation is widespread in the genome and greatly exceeds the influence of imprinting on genome-wide methylation patterns.

Author Summary

DNA methylation is a dynamic epigenetic mark that is essential for mammalian organismal development. DNA methylation levels can be influenced by environment, a chromosome's parental origin, and genome sequence. In this study, we evaluated the impact that DNA sequence has on DNA methylation by analyzing methylation levels in a three-generation family as well as unrelated individuals. By following DNA methylation patterns through the family along with nearby SNPs, we found that allelic differences between chromosomes play a much larger role in determining DNA methylation than the parental origin of the chromosome, indicating that DNA sequence has a larger impact on DNA methylation than gametic imprinting. We also found that allelic differences in DNA methylation found in the family can also be observed in unrelated individuals. In fact, the majority of variation in DNA methylation can be explained by genotype. Our results emphasize the importance of genome sequence in setting patterns of DNA methylation and indicate that genotype will need to be taken into account when assessing DNA methylation in the context of disease.

Centromeric regions in many complex eukaryotic species contain highly repetitive satellite DNAs. Despite the diversity of centromeric DNA sequences among species, the functional centromeres in all species studied to date are marked by CENP-A, a centromere-specific histone H3 variant. Although it is well established that families of multimeric higher-order alpha satellite are conserved at the centromeres of human and great ape chromosomes and that diverged monomeric alpha satellite is found in old and new world monkey genomes, little is known about the organization, function, and evolution of centromeric sequences in more distant primates, including lemurs. Aye-Aye (Daubentonia madagascariensis) is a basal primate and is located at a key position in the evolutionary tree to study centromeric satellite transitions in primate genomes. Using the approach of chromatin immunoprecipitation with antibodies directed to CENP-A, we have identified two satellite families, Daubentonia madagascariensis Aye-Aye 1 (DMA1) and Daubentonia madagascariensis Aye-Aye 2 (DMA2), related to each other but unrelated in sequence to alpha satellite or any other previously described primate or mammalian satellite DNA families. Here, we describe the initial genomic and phylogenetic organization of DMA1 and DMA2 and present evidence of higher-order repeats in Aye-Aye centromeric domains, providing an opportunity to study the emergence of chromosome-specific modes of satellite DNA evolution in primate genomes.

The extent to which variation in chromatin structure and transcription factor binding may influence gene expression, and thus underlie or contribute to variation in phenotype, is unknown. To address this question, we cataloged both individual-to-individual variation and differences between homologous chromosomes within the same individual (allele-specific variation) in chromatin structure and transcription factor binding in lymphoblastoid cells derived from individuals of geographically diverse ancestry. Ten percent of active chromatin sites were individual-specific; a similar proportion were allele-specific. Both individual-specific and allele-specific sites were commonly transmitted from parent to child, which suggests that they are heritable features of the human genome. Our study shows that heritable chromatin status and transcription factor binding differ as a result of genetic variation and may underlie phenotypic variation in humans.

Characterizing how genomic sequence interacts with trans-acting regulatory factors to implement a program of gene expression in eukaryotic organisms is critical to understanding genome function. One means by which patterns of gene expression are achieved is through the differential packaging of DNA into distinct types of chromatin. While chromatin state exerts a major influence on gene expression, the extent to which cis-acting DNA sequences contribute to the specification of chromatin state remains incompletely understood. To address this, we have used a fission yeast sequence element (L5), known to be sufficient to nucleate heterochromatin, to establish de novo heterochromatin domains in the Schizosaccharomyces pombe genome. The resulting heterochromatin domains were queried for the presence of H3K9 di-methylation and Swi6p, both hallmarks of heterochromatin, and for levels of gene expression. We describe a major effect of genomic sequences in determining the size and extent of such de novo heterochromatin domains. Heterochromatin spreading is antagonized by the presence of genes, in a manner that can occur independent of strength of transcription. Increasing the dosage of Swi6p results in increased heterochromatin proximal to the L5 element, but does not result in an expansion of the heterochromatin domain, suggesting that in this context genomic effects are dominant over trans effects. Finally, we show that the ratio of Swi6p to H3K9 di-methylation is sequence-dependent and correlates with the extent of gene repression. Taken together, these data demonstrate that the sequence content of a genomic region plays a significant role in shaping its response to encroaching heterochromatin and suggest a role of DNA sequence in specifying chromatin state.

Author Summary

Epigenetic packaging of DNA sequence into chromatin is a major force in shaping the function of complex genomes. Different types of chromatin have distinct effects on gene expression, and thus chromatin state imparts distinct features on the associated genomic DNA. Our study focuses on the transition between two opposing chromatin states: euchromatin, which generally correlates with gene expression, and heterochromatin, which is typically refractive to gene expression. While heterochromatin is capable of spreading into euchromatic domains, the parameters that influence such spreading are unknown. We established heterochromatin at ectopic sites in the genome and evaluated whether specific DNA sequences affected the extent of heterochromatin spreading and the transition between heterochromatin and euchromatin. We found that the nature of the genomic DNA neighboring the heterochromatic sequence dramatically affected the extent of heterochromatin spreading. In particular, the presence of genes antagonized the spread of heterochromatin, whereas neutral sequence elements were incorporated into the domain. This study demonstrates that genome sequence and chromatin identity are inextricably linked; features of both interact to determine the structural and functional fate of underlying DNA sequences.

Background

In recent years, the completion of the Human Genome Project and other rapid advances in genomics have led to increasing anticipation of an era of genomic and personalized medicine, in which an individual's health is optimized through the use of all available patient data, including data on the individual's genome and its downstream products. Genomic and personalized medicine could transform healthcare systems and catalyze significant reductions in morbidity, mortality, and overall healthcare costs.

Discussion

Critical to the achievement of more efficient and effective healthcare enabled by genomics is the establishment of a robust, nationwide clinical decision support infrastructure that assists clinicians in their use of genomic assays to guide disease prevention, diagnosis, and therapy. Requisite components of this infrastructure include the standardized representation of genomic and non-genomic patient data across health information systems; centrally managed repositories of computer-processable medical knowledge; and standardized approaches for applying these knowledge resources against patient data to generate and deliver patient-specific care recommendations. Here, we provide recommendations for establishing a national decision support infrastructure for genomic and personalized medicine that fulfills these needs, leverages existing resources, and is aligned with the Roadmap for National Action on Clinical Decision Support commissioned by the U.S. Office of the National Coordinator for Health Information Technology. Critical to the establishment of this infrastructure will be strong leadership and substantial funding from the federal government.

Summary

A national clinical decision support infrastructure will be required for reaping the full benefits of genomic and personalized medicine. Essential components of this infrastructure include standards for data representation; centrally managed knowledge repositories; and standardized approaches for leveraging these knowledge repositories to generate patient-specific care recommendations at the point of care.

Heterochromatin formation involves the nucleation and spreading of structural and epigenetic features along the chromatin fiber. Chromatin barriers and associated proteins counteract the spreading of heterochromatin, thereby restricting it to specific regions of the genome. We have performed gene expression studies and chromatin immunoprecipitation on strains in which native centromere sequences have been mutated to study the mechanism by which a tRNAAlanine gene barrier (cen1 tDNAAla) blocks the spread of pericentromeric heterochromatin at the centromere of chromosome 1 (cen1) in the fission yeast, Schizosaccharomyces pombe. Within the centromere, barrier activity is a general property of tDNAs and, unlike previously characterized barriers, requires the association of both transcription factor IIIC and RNA Polymerase III. Although the cen1 tDNAAla gene is actively transcribed, barrier activity is independent of transcriptional orientation. These findings provide experimental evidence for the involvement of a fully assembled RNA polymerase III transcription complex in defining independent structural and functional domains at a eukaryotic centromere.

A significant number of human X-linked genes escape X chromosome inactivation and are thus expressed from both the active and inactive X chromosomes. The basis for escape from inactivation and the potential role of the X chromosome primary DNA sequence in determining a gene's X inactivation status is unclear. Using a combination of the X chromosome sequence and a comprehensive X inactivation profile of more than 600 genes, two independent yet complementary approaches were used to systematically investigate the relationship between X inactivation and DNA sequence features. First, statistical analyses revealed that a number of repeat features, including long interspersed nuclear element (LINE) and mammalian-wide interspersed repeat repetitive elements, are significantly enriched in regions surrounding transcription start sites of genes that are subject to inactivation, while Alu repetitive elements and short motifs containing ACG/CGT are significantly enriched in those that escape inactivation. Second, linear support vector machine classifiers constructed using primary DNA sequence features were used to correctly predict the X inactivation status for >80% of all X-linked genes. We further identified a small set of features that are important for accurate classification, among which LINE-1 and LINE-2 content show the greatest individual discriminatory power. Finally, as few as 12 features can be used for accurate support vector machine classification. Taken together, these results suggest that features of the underlying primary DNA sequence of the human X chromosome may influence the spreading and/or maintenance of X inactivation.

Synopsis

Female mammals have two X chromosomes while males have one X and one Y chromosome. To equalize dosage of X chromosome genes in males and females, one X in female cells is inactivated, repressing the expression of most genes on the chromosome. Despite the chromosome-wide nature of X inactivation, at least 10%–15% of genes “escape” this inactivation in human females and are still expressed on the inactivated X. Whether a gene escapes or is subject to inactivation is thought to be determined epigenetically, and it is unknown to what extent, if at all, the underlying genomic DNA sequence of the chromosome plays a role. In this work, the authors show that the DNA sequence surrounding genes that escape inactivation is significantly different from the sequence surrounding genes that are subject to inactivation. In fact, a small number of DNA sequence features can be used to predict with high accuracy whether a gene will escape or be subject to this silencing. This establishes strong evidence that epigenetic regulation is, at least in part, dependent on genomic sequence and organization and provides a list of candidate sequence features whose role(s) in X inactivation can now be explored.

Advances in genome technology and other fruits of the Human Genome Project are playing a growing role in the delivery of health care. With the development of new technologies and opportunities for large-scale analysis of the genome, transcriptome, proteome and metabolome, the genome sciences are poised to have a profound impact on clinical medicine. Cancer prognostics will be among the first major test cases for a genomic medicine paradigm, given that all cancer is caused by genomic instability, and microarrays allow assessment of patients' entire expressed genomes. Analysis of breast cancer patients' expression patterns can already be highly correlated with recurrence risks. By integrating clinical data with gene expression profiles, imaging, metabolomic profiles and proteomic data, the prospect for developing truly individualized care becomes ever more real. Notwithstanding these promises, daunting challenges remain for genomic medicine. Success will require planning robust prospective trials, analysing health care economic and outcome data, assuaging insurance and privacy concerns, developing health delivery models that are commercially viable and scaling up to meet the needs of the whole population.

Background

Human Artificial Chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of de novo HAC formation. Indeed, to date, only a small number of large (>100 kb) genomic loci have been reported to be successfully packaged into de novo HACs.

Results

We have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. We report here the creation of a novel, bimolecular system based on bacterial artificial chromosomes (BACs) for the construction of HACs incorporating any defined genomic region. We have utilized this vector system to rapidly design, construct and validate multiple de novo HACs containing large (100–200 kb) genomic loci including therapeutically significant genes for human growth hormone (HGH), polycystic kidney disease (PKD1) and ß-globin. We report significant differences in the ability of different genomic loci to support de novo HAC formation, suggesting possible effects of cis-acting genomic elements. Finally, as a proof of principle, we have observed sustained ß-globin gene expression from HACs incorporating the entire 200 kb ß-globin genomic locus for over 90 days in the absence of selection.

Conclusion

Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus. We have evaluated the impact of different genomic loci on the frequency of HAC formation and identified segments of genomic DNA that appear to facilitate de novo HAC formation. These genomic loci may be useful for identifying discrete functional elements that may be incorporated into future generations of HAC vectors.

Efficient construction of BAC-based human artificial chromosomes (HACs) requires optimization of each key functional unit as well as development of techniques for the rapid and reliable manipulation of high-molecular weight BAC vectors. Here, we have created synthetic chromosome 17-derived alpha-satellite arrays, based on the 16-monomer repeat length typical of natural D17Z1 arrays, in which the consensus CENP-B box elements are either completely absent (0/16 monomers) or increased in density (16/16 monomers) compared to D17Z1 alpha-satellite (5/16 monomers). Using these vectors, we show that the presence of CENP-B box elements is a requirement for efficient de novo centromere formation and that increasing the density of CENP-B box elements may enhance the efficiency of de novo centromere formation. Furthermore, we have developed a novel, high-throughput methodology that permits the rapid conversion of any genomic BAC target into a HAC vector by transposon-mediated modification with synthetic alpha-satellite arrays and other key functional units. Taken together, these approaches offer the potential to significantly advance the utility of BAC-based HACs for functional annotation of the genome and for applications in gene transfer.

An assay of the formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha satellite DNA revealed that only a small amount of heterochromatin may be required for centromere function and that replication late in S phase is not a requirement for centromere function.

Background

Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.

Results

Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.

Conclusions

Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.

One of several features acquired by chromatin of the inactive X chromosome (Xi) is enrichment for the core histone H2A variant macroH2A within a distinct nuclear structure referred to as a macrochromatin body (MCB). In addition to localizing to the MCB, macroH2A accumulates at a perinuclear structure centered at the centrosome. To better understand the association of macroH2A1 with the centrosome and the formation of an MCB, we investigated the distribution of macroH2A1 throughout the somatic cell cycle. Unlike Xi-specific RNA, which associates with the Xi throughout interphase, the appearance of an MCB is predominantly a feature of S phase. Although the MCB dissipates during late S phase and G2 before reforming in late G1, macroH2A1 remains associated during mitosis with specific regions of the Xi, including at the X inactivation center. This association yields a distinct macroH2A banding pattern that overlaps with the site of histone H3 lysine-4 methylation centered at the DXZ4 locus in Xq24. The centrosomal pool of macroH2A1 accumulates in the presence of an inhibitor of the 20S proteasome. Therefore, targeting of macroH2A1 to the centrosome is likely part of a degradation pathway, a mechanism common to a variety of other chromatin proteins.

Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope–tagged or green fluorescent protein–tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease–digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

Chromatin on the inactive X chromosome (Xi) of female mammals is enriched for the histone variant macroH2A that can be detected at interphase as a distinct nuclear structure referred to as a macro chromatin body (MCB). Green fluorescent protein-tagged and Myc epitope-tagged macroH2A readily form an MCB in the nuclei of transfected female, but not male, cells. Using targeted disruptions, we have identified two macrochromatin domains within macroH2A that are independently capable of MCB formation and association with the Xi. Complete removal of the non-histone C-terminal tail does not reduce the efficiency of association of the variant histone domain of macroH2A with the Xi, indicating that the histone portion alone can target the Xi. The non-histone domain by itself is incapable of MCB formation. However, when directed to the nucleosome by fusion to core histone H2A or H2B, the non-histone tail forms an MCB that appears identical to that of the endogenous protein. Mutagenesis of the non-histone portion of macroH2A localized the region required for MCB formation and targeting to the Xi to an ∼190 amino acid region.

We have measured and characterized methylmalonyl coenzyme A (CoA) mutase activity in extracts of cultured human fibroblasts from 23 patients with inherited deficiency of the mutase apoenzyme and from eight obligate heterozygotes for this defect. The mutant cell lines fall into two categories. Those without detectable residual mutase activity in cell extracts (>0.1% of control), and whose ability to utilize propionate in intact cells is refractory to supplementation of the culture medium with hydroxocobalamin, are designated mut° mutants. Those with detectable residual activity in cell extracts (∼0.5-50% of control), and whose ability to utilize propionate in intact cells in markedly increased by hydroxocobalamin supplementation, are designated mut− mutants. The mutant enzyme in the mut− mutants exhibits a 50- to 5,000-fold elevated Michaelis constant (Km) for adenosylcobalamin in vitro, a normal Km for methylmalonyl CoA, and a strikingly reduced thermal stability at 45°C relative to control. Mutase from one mut− mutant turns over at a rate three to four times that of control enzyme when cells are grown in hydroxocobalamin-supplemented medium.

To detect heterozygous carriers of mutant mut alleles, we compared mutase activity in fibroblast extracts from four controls with that from eight parents of either mut° or mut− mutants. After cell growth in either unsupplemented or hydroxocobalamin-supplemented medium, activity in cell lines from heterozygotes was reduced to 47 or 37% of the mean control activities, respectively. We also examined the effect of adenosylcobalamin concentration on reaction kinetics of mutase from heterozygote cell lines. All four cell lines from parents of mut− mutants exhibited complex enzyme kinetics; ∼80% of mutase activity demonstrated a Km indistinguishable from control, whereas a smaller component of activity exhibited a Km similar to the abnormal Km expressed by the mut− propositus in each family. In two families with a mut° propositus, mutase from three of the four parents exhibited only the normal Km for adenosylcobalamin, whereas mutase from one parent displayed complex kinetics, indicating expression of both a normal allele (mut+) and a mutant allele with an abnormal Km. From these studies, we conclude that mut mutants reflect mutations at the autosomal gene locus for the methylmalonyl CoA mutase apoenzyme; that mut°, mut−, and mut+ alleles at this locus are codominantly expressed; and that some mut mutants may be genetic compounds, inheriting two different mut° or mut− alleles from their parents.

We have studied the intracellular binding of radioactive cobalamin by normal cultured human fibroblasts grown in medium containing [57Co]-cobalamin. We have also assessed the significance of defects in this binding activity exhibited by two classes of human mutants (cbl C and cbl D) each characterized by pleiotropic deficiencies in the accumulation and retention of cobalamin, in the synthesis of cobalamin coenzymes, and accordingly, in the holoenzyme activities of both cobalamin-dependent enzymes, 5-methyltetrahydrofolate:homocysteine methyltransferase and methylmalonyl-CoA mutase. Based on the coincidence of [57Co]cobalamin binding and cobalamin-dependent enzyme activities after Sephadex G-150 chromatography and polyacrylamide gel electrophoresis, we conclude that, as in rat liver, the intracellular binding of labeled cobalamin by normal fibroblasts reflects the attachment of the vitamin to the cobalamin-dependent methyltransferase and mutase. Whereas cbl C cells are completely deficient in the binding of [57Co]cobalamin to either enzyme, fibroblasts which bear the phenotypically similar but genetically distinct cbl D mutation retain some binding activity, and accordingly, have higher holomethyltransferase and holomutase activities than do cbl C cells. The defect in [57Co]-cobalamin binding exhibited by both cbl C and cbl D fibroblasts is almost certainly not a result of mutations which affect the methyltransferase or mutase apoenzymes, since the electrophoretic mobilities and the affinities of these enzymes for their respective cobalamin coenzymes are indistinguishable from those in control cell extracts. These results suggest that both the cbl C and cbl D mutations affect some enzymatic step(s) which converts newly taken up cobalamin to a form capable of being bound by the two cobalamin-dependent enzymes.