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Steven Henikoff

Publications and source records attributed to Steven Henikoff.

At least 19 recordsLinked to original sources
PubMed2025

CUT&TIME captures the history of open chromatin in developing neurons.

Chromatin structure plays a central role in defining cell identity by regulating gene expression. During development, shifts in chromatin structure facilitate changes in gene expression needed to specify distinct cell types. To understand how changes in chromatin structure influence the developmental trajectory of neural progenitor cells, we developed CUT&TIME, a technique that uses a hyperactive 6-methyl adenosine (6mA) methyltransferase pulsed in living cells to map historical chromatin accessibility genome-wide in single cells. We show that CUT&TIME produces a record of the chromatin landscape during neurogenesis in the developing retina, specifically as neural progenitors produce the major projection neuron type, retinal ganglion cells (RGCs). We further show that this method is compatible with single cell profiling technologies, which allows us to visualize and capture the diversity of chromatin states that produce RGCs. Additionally, we identify changes in promoter accessibility associated with the transition from progenitor to RGC. Together, these data demonstrate that CUT&TIME captures a historical record of chromatin structure, which can be used to identify early changes in accessibility associated with cell-fate commitment.

Journal Article↗
PubMed2006

Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription.

Cytosine methylation, a common form of DNA modification that antagonizes transcription, is found at transposons and repeats in vertebrates, plants and fungi. Here we have mapped DNA methylation in the entire Arabidopsis thaliana genome at high resolution. DNA methylation covers transposons and is present within a large fraction of A. thaliana genes. Methylation within genes is conspicuously biased away from gene ends, suggesting a dependence on RNA polymerase transit. Genic methylation is strongly influenced by transcription: moderately transcribed genes are most likely to be methylated, whereas genes at either extreme are least likely. In turn, transcription is influenced by methylation: short methylated genes are poorly expressed, and loss of methylation in the body of a gene leads to enhanced transcription. Our results indicate that genic transcription and DNA methylation are closely interwoven processes.

Arabidopsis↗
PubMed2006

Centromeres put epigenetics in the driver's seat.

A defining feature of chromosomes is the centromere, the site for spindle attachment at mitosis and meiosis. Intriguingly, centromeres of plants and animals are maintained by both sequence-specific and sequence-independent (epigenetic) processes. Epigenetic inheritance might enable kinetochores (the structures that attach centromeres to spindles) to maintain an optimal size. However, centromeres are susceptible to the evolution of "selfish" DNA repeats that bind to kinetochore proteins. We argue that such sequence-specific interactions are evolutionarily unstable because they enable repeat arrays to influence kinetochore size. Changes in kinetochore size could affect the interaction of kinetochores with the spindle and, in principle, skew Mendelian segregation. We propose that key kinetochore proteins have adapted to disrupt such sequence-specific interactions and restore epigenetic inheritance.

Animals↗
PubMed2006

High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling.

Human individuals differ from one another at only approximately 0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease.

Algorithms↗
PubMed2006

Biotin-tag affinity purification of a centromeric nucleosome assembly complex.

Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, we used controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a single chaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. Here, we highlight the benefits of our improved biotin-mediated purification method, and address the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres.

Animals↗
PubMed2006

Chaperone-mediated assembly of centromeric chromatin in vitro.

Every eukaryotic chromosome requires a centromere for attachment to spindle microtubules for chromosome segregation. Although centromeric DNA sequences vary greatly among species, centromeres are universally marked by the presence of a centromeric histone variant, centromeric histone 3 (CenH3), which replaces canonical histone H3 in centromeric nucleosomes. Conventional chromatin is maintained in part by histone chaperone complexes, which deposit the S phase-limited (H3) and constitutive (H3.3) forms of histone 3. However, the mechanism that deposits CenH3 specifically at centromeres and faithfully maintains its chromosome location through mitosis and meiosis is unknown. To address this problem, we have biochemically purified a soluble assembly complex that targets tagged CenH3 to centromeres in Drosophila cells. Two different affinity procedures led to purification of the same complex, which consists of CenH3, histone H4, and a single protein chaperone, RbAp48, a highly abundant component of various chromatin assembly, remodeling, and modification complexes. The corresponding CenH3 assembly complex reconstituted in vitro is sufficient for chromatin assembly activity, without requiring additional components. The simple CenH3 assembly complex is in contrast to the multisubunit complexes previously described for H3 and H3.3, suggesting that centromeres are maintained by a passive mechanism that involves exclusion of the complexes that deposit canonical H3s during replication and transcription.

Animals↗
PubMed2006

The epigenetic progenitor origin of human cancer.

Cancer is widely perceived as a heterogeneous group of disorders with markedly different biological properties, which are caused by a series of clonally selected genetic changes in key tumour-suppressor genes and oncogenes. However, recent data suggest that cancer has a fundamentally common basis that is grounded in a polyclonal epigenetic disruption of stem/progenitor cells, mediated by 'tumour-progenitor genes'. Furthermore, tumour cell heterogeneity is due in part to epigenetic variation in progenitor cells, and epigenetic plasticity together with genetic lesions drives tumour progression. This crucial early role for epigenetic alterations in cancer is in addition to epigenetic alterations that can substitute for genetic variation later in tumour progression. Therefore, non-neoplastic but epigenetically disrupted stem/progenitor cells might be a crucial target for cancer risk assessment and chemoprevention.

Animals↗
PubMed2006

Spreading of silent chromatin: inaction at a distance.

One of the oldest unsolved problems in genetics is the observation that gene silencing can 'spread' along a chromosome. Although spreading has been widely perceived as a process of long-range assembly of heterochromatin proteins, such 'oozing' might not apply in most cases. Rather, long-range silencing seems to be a dynamic process, involving local diffusion of histone-modifying enzymes from source binding sites to low-affinity sites nearby. Discontinuous silencing might reflect looping interactions, whereas the spreading of continuous silencing might be driven by the processive movement of RNA or DNA polymerases. We review the evidence for the spreading of silencing in many contexts and organisms and conclude that multiple mechanisms have evolved that silence genes at a distance.

Animals↗
PubMed2006

TILLING: practical single-nucleotide mutation discovery.

In the post-genomic sequencing era, an expanding portfolio of genomic technologies has been applied to the study of gene function. Reverse genetics approaches that provide targeted inactivation of genes identified by sequence analysis include TILLING (for Targeting Local Lesions IN Genomes). TILLING searches the genomes of mutagenized organisms for mutations in a chosen gene, typically single base-pair substitutions. This review covers practical aspects of the technology, ranging from building the mutagenized population to mutation discovery, and discusses possible improvements to current protocols and the impact of new genomic methods for mutation discovery in relation to the future of the TILLING approach.

Mutation↗
PubMed2006

Predicting the effects of amino acid substitutions on protein function.

Nonsynonymous single nucleotide polymorphisms (nsSNPs) are coding variants that introduce amino acid changes in their corresponding proteins. Because nsSNPs can affect protein function, they are believed to have the largest impact on human health compared with SNPs in other regions of the genome. Therefore, it is important to distinguish those nsSNPs that affect protein function from those that are functionally neutral. Here we provide an overview of amino acid substitution (AAS) prediction methods, which use sequence and/or structure to predict the effect of an AAS on protein function. Most methods predict approximately 25-30% of human nsSNPs to negatively affect protein function, and such nsSNPs tend to be rare in the population. We discuss the utility of AAS prediction methods for Mendelian and complex diseases as well as their broader applications for understanding protein function.

Amino Acid Sequence↗
PubMed2006

Histone H3.3 variant dynamics in the germline of Caenorhabditis elegans.

Germline chromatin undergoes dramatic remodeling events involving histone variants during the life cycle of an organism. A universal histone variant, H3.3, is incorporated at sites of active transcription throughout the cell cycle. The presence of H3.3 in chromatin indicates histone turnover, which is the energy-dependent removal of preexisting histones and replacement with new histones. H3.3 is also incorporated during decondensation of the Drosophila sperm pronucleus, indicating a direct role in chromatin remodeling upon fertilization. Here we present a system to monitor histone turnover and chromatin remodeling during Caenorhabditis elegans development by following the developmental dynamics of H3.3. We generated worm strains expressing green fluorescent protein- or yellow fluorescent protein-fused histone H3.3 proteins, HIS-71 and HIS-72. We found that H3.3 is retained in mature sperm chromatin, raising the possibility that it transmits epigenetic information via the male germline. Upon fertilization, maternal H3.3 enters both male and female pronuclei and is incorporated into paternal chromatin, apparently before the onset of embryonic transcription, suggesting that H3.3 can be incorporated independent of transcription. In early embryos, H3.3 becomes specifically depleted from primordial germ cells. Strikingly, the X chromosome becomes deficient in H3.3 during gametogenesis, indicating a low level of histone turnover. These results raise the possibility that the asymmetry in histone turnover between the X chromosome and autosomes is established during gametogenesis. H3.3 patterns are similar to patterns of H3K4 methylation in the primordial germ cells and on the X chromosome during gametogenesis, suggesting that histone turnover and modification are coupled processes. Our demonstration of dynamic H3.3 incorporation in nondividing cells provides a mechanistic basis for chromatin changes during germ cell development.

Animals↗
PubMed2006

High-throughput TILLING for Arabidopsis.

Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

Arabidopsis↗
PubMed2005

Transcription and histone modifications in the recombination-free region spanning a rice centromere.

Centromeres are sites of spindle attachment for chromosome segregation. During meiosis, recombination is absent at centromeres and surrounding regions. To understand the molecular basis for recombination suppression, we have comprehensively annotated the 3.5-Mb region that spans a fully sequenced rice centromere. Although transcriptional analysis showed that the 750-kb CENH3-containing core is relatively deficient in genes, the recombination-free region differs little in gene density from flanking regions that recombine. Likewise, the density of transposable elements is similar between the recombination-free region and flanking regions. We also measured levels of histone H4 acetylation and histone H3 methylation at 176 genes within the 3.5-Mb span. Active genes showed enrichment of H4 acetylation and H3K4 dimethylation as expected, including genes within the core. Our inability to detect sequence or histone modification features that distinguish recombination-free regions from flanking regions that recombine suggest that recombination suppression is an epigenetic feature of centromeres maintained by the assembly of CENH3-containing nucleosomes within the core. CENH3-containing centrochromatin does not appear to be distinguished by a unique combination of H3 and H4 modifications. Rather, the varied distribution of histone modifications might reflect the composition and abundance of sequence elements that inhabit centromeric DNA.

Centromere↗
PubMed2005

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis.

BACKGROUND: DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known. RESULTS: We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants. CONCLUSION: We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements.

Arabidopsis↗
PubMed2005

Genome-scale profiling of histone H3.3 replacement patterns.

Histones of multicellular organisms are assembled into chromatin primarily during DNA replication. When chromatin assembly occurs at other times, the histone H3.3 variant replaces canonical H3. Here we introduce a new strategy for profiling epigenetic patterns on the basis of H3.3 replacement, using microarrays covering roughly one-third of the Drosophila melanogaster genome at 100-bp resolution. We identified patterns of H3.3 replacement over active genes and transposons. H3.3 replacement occurred prominently at sites of abundant RNA polymerase II and methylated H3 Lys4 throughout the genome and was enhanced on the dosage-compensated male X chromosome. Active genes were depleted of histones at promoters and were enriched in H3.3 from upstream to downstream of transcription units. We propose that deposition and inheritance of actively modified H3.3 in regulatory regions maintains transcriptionally active chromatin.

Animals↗
PubMed2005

Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila.

Heterochromatin comprises a significant component of many eukaryotic genomes. In comparison to euchromatin, heterochromatin is gene poor, transposon rich, and late replicating. It serves many important biological roles, from gene silencing to accurate chromosome segregation, yet little is known about the evolutionary constraints that shape heterochromatin. A complementary approach to the traditional one of directly studying heterochromatic DNA sequence is to study the evolution of proteins that bind and define heterochromatin. One of the best markers for heterochromatin is the heterochromatin protein 1 (HP1), which is an essential, nonhistone chromosomal protein. Here we investigate the molecular evolution of five HP1 paralogs present in Drosophila melanogaster. Three of these paralogs have ubiquitous expression patterns in adult Drosophila tissues, whereas HP1D/rhino and HP1E are expressed predominantly in ovaries and testes respectively. The HP1 paralogs also have distinct localization preferences in Drosophila cells. Thus, Rhino localizes to the heterochromatic compartment in Drosophila tissue culture cells, but in a pattern distinct from HP1A and lysine-9 dimethylated H3. Using molecular evolution and population genetic analyses, we find that rhino has been subject to positive selection in all three domains of the protein: the N-terminal chromo domain, the C-terminal chromo-shadow domain, and the hinge region that connects these two modules. Maximum likelihood analysis of rhino sequences from 20 species of Drosophila reveals that a small number of residues of the chromo and shadow domains have been subject to repeated positive selection. The rapid and positive selection of rhino is highly unusual for a gene encoding a chromosomal protein and suggests that rhino is involved in a genetic conflict that affects the germline, belying the notion that heterochromatin is simply a passive recipient of "junk DNA" in eukaryotic genomes.

Journal Article↗
PubMed2005

Automated band mapping in electrophoretic gel images using background information.

Some popular methods for polymorphism and mutation discovery involve ascertainment of novel bands by the examination of electrophoretic gel images. Although existing strategies for mapping bands work well for specific applications, such as DNA sequencing, these strategies are not well suited for novel band detection. Here, we describe a general strategy for band mapping that uses background banding patterns to facilitate lane calling and size calibration. We have implemented this strategy in GelBuddy, a user-friendly Java-based program for PC and Macintosh computers, which includes several utilities to assist discovery of mutations and polymorphisms. We demonstrate the use of GelBuddy in applications based on single-base mismatch cleavage of heteroduplexed PCR products. Use of software designed to facilitate novel band detection can significantly shorten the time needed for image analysis and data entry in a high-throughput setting. Furthermore, the interactive strategy implemented in GelBuddy has been successfully applied to DNA fingerprinting applications, such as AFLP. GelBuddy promises to make electrophoretic gel analysis a viable alternative to DNA resequencing for discovery of mutations and polymorphisms.

Algorithms↗