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Steven Henikoff

Publications and source records attributed to Steven Henikoff.

At least 37 records · Page 2Linked to original sources
PubMed2005

DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes.

Cytosine DNA methylation in vertebrates is widespread, but methylation in plants is found almost exclusively at transposable elements and repetitive DNA. Within regions of methylation, methylcytosines are typically found in CG, CNG, and asymmetric contexts. CG sites are maintained by a plant homolog of mammalian Dnmt1 acting on hemi-methylated DNA after replication. Methylation of CNG and asymmetric sites appears to be maintained at each cell cycle by other mechanisms. We report a new type of DNA methylation in Arabidopsis, dense CG methylation clusters found at scattered sites throughout the genome. These clusters lack non-CG methylation and are preferentially found in genes, although they are relatively deficient toward the 5' end. CG methylation clusters are present in lines derived from different accessions and in mutants that eliminate de novo methylation, indicating that CG methylation clusters are stably maintained at specific sites. Because 5-methylcytosine is mutagenic, the appearance of CG methylation clusters over evolutionary time predicts a genome-wide deficiency of CG dinucleotides and an excess of C(A/T)G trinucleotides within transcribed regions. This is exactly what we find, implying that CG methylation clusters have contributed profoundly to plant gene evolution. We suggest that CG methylation clusters silence cryptic promoters that arise sporadically within transcription units.

Arabidopsis↗
PubMed2005

Centromeric chromatin: what makes it unique?

Centromeres represent the final frontier of eukaryotic genomes. Although they are defining features of chromosomes--the points at which spindle microtubules attach--the fundamental features that distinguish them from other parts of the chromosome remain mysterious. The function of centromeres is conserved throughout eukaryotic biology, but their DNA sequences are not. Rather, accumulating evidence favors chromatin-based centromeric identification. To understand how centromeric identity is maintained, researchers have studied DNA-protein interactions at native centromeres and ectopic "neocentromeres". Other studies have taken a comparative approach focusing on centromere-specific proteins, of which mammalian CENP-A and CENP-C are the prototypes. Elucidating the assembly and structure of chromatin at centromeres remain key challenges.

Animals↗
PubMed2005

Epigenetic inheritance in Arabidopsis: selective silence.

Eukaryotic organisms have the remarkable ability to inherit states of gene activity without altering the underlying DNA sequence. This epigenetic inheritance can persist over thousands of years, providing an alternative to genetic mutations as a substrate for natural selection. Epigenetic inheritance might be propagated by differences in DNA methylation, post-translational histone modifications, and deposition of histone variants. Mounting evidence also indicates that small interfering RNA (siRNA)-mediated mechanisms play central roles in setting up and maintaining states of gene activity. Much of the epigenetic machinery of many organisms, including Arabidopsis, appears to be directed at silencing viruses and transposable elements, with epigenetic regulation of endogenous genes being mostly derived from such processes.

Arabidopsis↗
PubMed2005

Assembly of variant histones into chromatin.

Chromatin can be differentiated by the deposition of variant histones at centromeres, active genes, and silent loci. Variant histones are assembled into nucleosomes in a replication-independent manner, in contrast to assembly of bulk chromatin that is coupled to replication. Recent in vitro studies have provided the first glimpses of protein machines dedicated to building and replacing alternative nucleosomes. They deposit variant H2A and H3 histones and are targeted to particular functional sites in the genome. Differences between variant and canonical histones can have profound consequences, either for delivery of the histones to sites of assembly or for their function after incorporation into chromatin. Recent studies have also revealed connections between assembly of variant nucleosomes, chromatin remodeling, and histone post-translational modification. Taken together, these findings indicate that chromosome architecture can be highly dynamic at the most fundamental level, with epigenetic consequences.

Animals↗
PubMed2005

Positive selection of Iris, a retroviral envelope-derived host gene in Drosophila melanogaster.

Eukaryotic genomes can usurp enzymatic functions encoded by mobile elements for their own use. A particularly interesting kind of acquisition involves the domestication of retroviral envelope genes, which confer infectious membrane-fusion ability to retroviruses. So far, these examples have been limited to vertebrate genomes, including primates where the domesticated envelope is under purifying selection to assist placental function. Here, we show that in Drosophila genomes, a previously unannotated gene (CG4715, renamed Iris) was domesticated from a novel, active Kanga lineage of insect retroviruses at least 25 million years ago, and has since been maintained as a host gene that is expressed in all adult tissues. Iris and the envelope genes from Kanga retroviruses are homologous to those found in insect baculoviruses and gypsy and roo insect retroviruses. Two separate envelope domestications from the Kanga and roo retroviruses have taken place, in fruit fly and mosquito genomes, respectively. Whereas retroviral envelopes are proteolytically cleaved into the ligand-interaction and membrane-fusion domains, Iris appears to lack this cleavage site. In the takahashii/suzukii species groups of Drosophila, we find that Iris has tandemly duplicated to give rise to two genes (Iris-A and Iris-B). Iris-B has significantly diverged from the Iris-A lineage, primarily because of the "invention" of an intron de novo in what was previously exonic sequence. Unlike domesticated retroviral envelope genes in mammals, we find that Iris has been subject to strong positive selection between Drosophila species. The rapid, adaptive evolution of Iris is sufficient to unambiguously distinguish the phylogenies of three closely related sibling species of Drosophila (D. simulans, D. sechellia, and D. mauritiana), a discriminative power previously described only for a putative "speciation gene." Iris represents the first instance of a retroviral envelope-derived host gene outside vertebrates. It is also the first example of a retroviral envelope gene that has been found to be subject to positive selection following its domestication. The unusual selective pressures acting on Iris suggest that it is an active participant in an ongoing genetic conflict. We propose a model in which Iris has "switched sides," having been recruited by host genomes to combat baculoviruses and retroviruses, which employ homologous envelope genes to mediate infection.

Amino Acid Sequence↗
PubMed2004

Silencing of transposons in plant genomes: kick them when they're down.

Recent progress in understanding the silencing of transposable elements in the model plant Arabidopsis has revealed an interplay between DNA methylation, histone methylation and small interfering RNAs. DNA and histone methylation are not always sufficient to maintain silencing, and RNA-based reinforcement can be needed to maintain as well as initiate it.

Animals↗
PubMed2004

Adaptive evolution of centromere proteins in plants and animals.

BACKGROUND: Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif). RESULTS: Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection. CONCLUSIONS: CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi.

Adaptation, Physiological↗
PubMed2004

Discovery of induced point mutations in maize genes by TILLING.

BACKGROUND: Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community. RESULTS: We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious. CONCLUSIONS: Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.

Ethyl Methanesulfonate↗
PubMed2004

Adaptive evolution of the histone fold domain in centromeric histones.

Centromeric DNA, being highly repetitive, has been refractory to molecular analysis. However, centromeric structural proteins are encoded by single-copy genes, and these can be analyzed by using standard phylogenetic tools. The centromere-specific histone, CenH3, replaces histone H3 in centromeric nucleosomes, and is required for the proper distribution of chromosomes during cell division. Whereas histone H3s are nearly identical between species, CenH3s are divergent, with an N-terminal tail that is highly variable in length and sequence. Both the N-terminal tail and histone fold domain (HFD) are subject to adaptive evolution in Drosophila. Similarly, comparisons between Arabidopsis thaliana and Arabidopsis arenosa detected adaptive evolution, but only in the N-terminal tail. We have extended our evolutionary analyses of CenH3s to other members of the Brassicaceae, which allowed the detection of positive selection in both the N-terminal tail and in the HFD. We find that adaptively evolving sites in the HFD can potentially interact with DNA, including sites in the loop 1 region of the HFD that are required for centromeric targeting in Drosophila. Other adaptively evolving sites in the HFD can be localized on the structure of the nucleosome core particle, revealing an extended surface in addition to loop 1 in which conformational changes might alter histone-DNA contacts or water bridges. The identification of adaptively evolving sites provides a structural basis for the interaction between centromeric DNA and the protein that is thought to underlie the evolution of centromeres and the accumulation of pericentric heterochromatin.

Amino Acid Sequence↗
PubMed2004

TILLING. Traditional mutagenesis meets functional genomics.

Most of the genes of an organism are known from sequence, but most of the phenotypes are obscure. Thus, reverse genetics has become an important goal for many biologists. However, reverse-genetic methodologies are not similarly applicable to all organisms. In the general strategy for reverse genetics that we call TILLING (for Targeting Induced Local Lesions in Genomes), traditional chemical mutagenesis is followed by high-throughput screening for point mutations. TILLING promises to be generally applicable. Furthermore, because TILLING does not involve transgenic modifications, it is attractive not only for functional genomics but also for agricultural applications. Here, we present an overview of the status of TILLING methodology, including Ecotilling, which entails detection of natural variation. We describe public TILLING efforts in Arabidopsis and other organisms, including maize (Zea mays) and zebrafish. We conclude that TILLING, a technology developed in plants, is rapidly being adopted in other systems.

Agriculture↗
PubMed2004

Mismatch cleavage by single-strand specific nucleases.

We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S1) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using higher pH, temperature and divalent cation conditions than are routinely used for digestion of single-stranded DNA. Surprisingly, crude plant extracts performed as well as the highly purified preparations for this application. These observations suggest that diverse members of the S1 family of sss nucleases act similarly in cleaving non-specifically at bulges in heteroduplexes, and single-base mismatches are the least accessible because they present the smallest single-stranded region for enzyme binding. We conclude that a variety of sss nucleases and extracts can be effectively used for high-throughput mutation and polymorphism discovery.

Base Pair Mismatch↗
PubMed2004

Visualizing gene expression: an unfolding story.

Eukaryotic transcription is a dynamic process in which RNA polymerases read DNA templates that are tightly wrapped around histones. In this issue of Cell, Janicki et al. (2004) provide a glimpse of the events that follow transcriptional induction, using an integrated cytological system observed in real time. They find that local decondensation of the template is accompanied by profound changes in chromatin composition.

Animals↗
PubMed2004

Maize centromeres: organization and functional adaptation in the genetic background of oat.

Centromeric DNA sequences in multicellular eukaryotes are often highly repetitive and are not unique to a specific centromere or to centromeres at all. Thus, it is a major challenge to study the fine structure of individual plant centromeres. We used a DNA fiber-fluorescence in situ hybridization approach to study individual maize (Zea mays) centromeres using oat (Avena sativa)-maize chromosome addition lines. The maize centromere-specific satellite repeat CentC in the addition lines allowed us to delineate the size and organization of centromeric DNA of individual maize chromosomes. We demonstrate that the cores of maize centromeres contain mainly CentC arrays and clusters of a centromere-specific retrotransposon, CRM. CentC and CRM sequences are highly intermingled. The amount of CentC/CRM sequence varies from approximately 300 to >2800 kb among different centromeres. The association of CentC and CRM with centromeric histone H3 (CENH3) was visualized by a sequential detection procedure on stretched centromeres. The analysis revealed that CENH3 is always associated with CentC and CRM but that not all CentC or CRM sequences are associated with CENH3. We further demonstrate that in the chromosomal addition lines in which two CenH3 genes were present, one from oat and one from maize, the oat CENH3 was consistently incorporated by the maize centromeres.

Adaptation, Physiological↗
PubMed2004

Histone H3.3 is enriched in covalent modifications associated with active chromatin.

Chromatin states can be distinguished by differential covalent modifications of histones or by utilization of histone variants. Chromatin associated with transcriptionally active loci becomes enriched for histones with particular lysine modifications and accumulates the H3.3 histone variant, the substrate for replication-independent nucleosome assembly. However, studies of modifications at particular loci have not distinguished between histone variants, so the relationship among modifications, histone variants, and nucleosome assembly pathways is unclear. To address this uncertainty, we have quantified the relative abundance of H3 and H3.3 and their lysine modifications. Using a Drosophila cell line system in which H3.3 has been shown to specifically package active loci, we found that H3.3 accounts for approximately 25% of total histone 3 in bulk chromatin, enough to package essentially all actively transcribed genes. MS and antibody characterization of separated histone 3 fractions revealed that H3.3 is relatively enriched in modifications associated with transcriptional activity and deficient in dimethyl lysine-9, which is abundant in heterochromatin. To explain enrichment on alternative variants, we propose that histone modifications are tied to the alternative nucleosome assembly pathways that use primarily H3 at replication forks and H3.3 at actively transcribed genes in a replication-independent manner.

Animals↗
PubMed2004

Sequencing of a rice centromere uncovers active genes.

Centromeres are the last frontiers of complex eukaryotic genomes, consisting of highly repetitive sequences that resist mapping, cloning and sequencing. The centromere of rice Chromosome 8 (Cen8) has an unusually low abundance of highly repetitive satellite DNA, which allowed us to determine its sequence. A region of approximately 750 kb in Cen8 binds rice CENH3, the centromere-specific H3 histone. CENH3 binding is contained within a larger region that has abundant dimethylation of histone H3 at Lys9 (H3-Lys9), consistent with Cen8 being embedded in heterochromatin. Fourteen predicted and at least four active genes are interspersed in Cen8, along with CENH3 binding sites. The retrotransposons located in and outside of the CENH3 binding domain have similar ages and structural dynamics. These results suggest that Cen8 may represent an intermediate stage in the evolution of centromeres from genic regions, as in human neocentromeres, to fully mature centromeres that accumulate megabases of homogeneous satellite arrays.

Centromere↗
PubMed2004

Efficient discovery of DNA polymorphisms in natural populations by Ecotilling.

We have adapted the mutation detection technology used in Targeting Induced Local Lesions in Genomes (TILLING) to the discovery of polymorphisms in natural populations. The genomic DNA of a queried individual is mixed with a reference DNA and used to amplify a target 1-kbp region of DNA with asymmetrically labeled fluorescent primers. After heating and annealing, heteroduplexes are nicked at mismatched sites by the endonuclease CEL I and cut strands are visualized using Li-cor gel analyzers. Putative polymorphisms detected in one fluorescence channel can be verified by appearance of the opposite cut strand in the other channel. We demonstrated the efficiency of this technology, called Ecotilling, by the discovery in 150+ individuals of 55 haplotypes in five genes, ranging from sequences differing by a single nucleotide polymorphism to those representing complex haplotypes. The discovered polymorphisms were confirmed by sequencing and included base-pair changes, small insertions and deletions, and variation in microsatellite repeat number. Ecotilling allows the rapid detection of variation in many individuals and is cost effective because only one individual for each haplotype needs to be sequenced. The technology is applicable to any organism including those that are heterozygous and polyploid.

DNA, Plant↗