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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↗
PubMed2003

SIFT: Predicting amino acid changes that affect protein function.

Single nucleotide polymorphism (SNP) studies and random mutagenesis projects identify amino acid substitutions in protein-coding regions. Each substitution has the potential to affect protein function. SIFT (Sorting Intolerant From Tolerant) is a program that predicts whether an amino acid substitution affects protein function so that users can prioritize substitutions for further study. We have shown that SIFT can distinguish between functionally neutral and deleterious amino acid changes in mutagenesis studies and on human polymorphisms. SIFT is available at http://blocks.fhcrc.org/sift/SIFT.html.

Amino Acid Substitution↗
PubMed2003

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design.

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).

Amino Acid Sequence↗
PubMed2003

Maintenance of chromatin states: an open-and-shut case.

The traditional view of chromatin envisions two states: one is 'active' and accessible to nucleases, whereas the other is 'silent' and relatively inaccessible. Recent evidence that combinations of diverse histone tail modifications represent a spectrum of chromatin states challenges this simple view. Here, we examine inter-relationships between chromatin remodeling, histone modification, DNA methylation, RNA interference, and nucleosome assembly activities. We find that the two-state view can accommodate these new findings, and that nucleosome assembly pathways may ultimately maintain euchromatic and heterochromatic states.

Animals↗
PubMed2003

Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of Arabidopsis thaliana centromeres.

The centromeres of Arabidopsis thaliana chromosomes contain megabases of complex DNA consisting of numerous types of repetitive DNA elements. We developed a chromatin immunoprecipitation (ChIP) technique using an antibody against the centromeric H3 histone, HTR12, in Arabidopsis. ChIP assays showed that the 180-bp centromeric satellite repeat was precipitated with the antibody, suggesting that this repeat is the key component of the centromere/kinetochore complex in Arabidopsis.

Arabidopsis↗
PubMed2003

Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis.

Chemical mutagenesis has been the workhorse of traditional genetics, but it has not been possible to determine underlying rates or distributions of mutations from phenotypic screens. However, reverse-genetic screens can be used to provide an unbiased ascertainment of mutation statistics. Here we report a comprehensive analysis of approximately 1900 ethyl methanesulfonate (EMS)-induced mutations in 192 Arabidopsis thaliana target genes from a large-scale TILLING reverse-genetic project, about two orders of magnitude larger than previous such efforts. From this large data set, we are able to draw strong inferences about the occurrence and randomness of chemically induced mutations. We provide evidence that we have detected the large majority of mutations in the regions screened and confirm the robustness of the high-throughput TILLING method; therefore, any deviations from randomness can be attributed to selectional or mutational biases. Overall, we detect twice as many heterozygotes as homozygotes, as expected; however, for mutations that are predicted to truncate an encoded protein, we detect a ratio of 3.6:1, indicating selection against homozygous deleterious mutations. As expected for alkylation of guanine by EMS, >99% of mutations are G/C-to-A/T transitions. A nearest-neighbor bias around the mutated base pair suggests that mismatch repair counteracts alkylation damage.

Arabidopsis↗
PubMed2003

Large-scale discovery of induced point mutations with high-throughput TILLING.

TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.

Alkylating Agents↗
PubMed2003

Epigenomic profiling using microarrays.

Genes occupy only a minor fraction of genomes such as ours; however, histone and nonhistone chromosomal proteins and methylated DNA bases are distributed over both genic and nongenic regions. These widespread "epigenomic" features can be mapped and characterized by alternative applications of the same microarray technologies that have been used for conventional transcriptional profiling. Here we describe diverse microarray-based strategies for profiling patterns of DNA methylation, DNA replication, DNA binding, and chromatin-associated proteins and histone modifications. The rapid progress that is being made in developing and applying epigenomic profiling methods and the increasing availability of microarrays mean that epigenomic profiling is likely to become a standard research tool for understanding chromatin structure and gene expression during development.

Animals↗
PubMed2002

Epigenetic consequences of nucleosome dynamics.

Current models for epigenetic gene silencing envision a static relationship between histone modifications and transcription. However, evidence for nucleosome mobility and replacement favors a dynamic model that may explain phenomena ranging from variegation to the neural restriction of Rett syndrome.

Animals↗
PubMed2002

Tech.Sight. Phage display. Affinity selection from biological libraries.

Phage display is a simple yet powerful technology that is used to rapidly characterize protein-protein interactions from amongst billions of candidates. This widely practiced technique is used to map antibody epitopes, create vaccines and to engineer peptides, antibodies and other proteins as both diagnostic tools and as human therapeutics. We overview the history of phage display and several recent applications.

Animals↗
PubMed2002

Histone H3 variants specify modes of chromatin assembly.

Histone variants have been known for 30 years, but their functions and the mechanism of their deposition are still largely unknown. Drosophila has three versions of histone H3. H3 packages the bulk genome, H3.3 marks active chromatin and may be essential for gene regulation, and Cid is the characteristic structural component of centromeric chromatin. We have characterized the properties of these histones by using a Drosophila cell-line system that allows precise analysis of both DNA replication and histone deposition. The deposition of H3 is restricted to replicating DNA. In striking contrast, H3.3 and Cid deposit throughout the cell cycle. Deposition of H3.3 occurs without any corresponding DNA replication. To confirm that the deposition of Cid is also replication-independent (RI), we examined centromere replication in cultured cells and neuroblasts. We found that centromeres replicate out of phase with heterochromatin and display replication patterns that may limit H3 deposition. This confirms that both variants undergo RI deposition, but at different locations in the nucleus. How variant histones accomplish RI deposition is unknown, and raises basic questions about the stability of nucleosomes, the machinery that accomplishes nucleosome assembly, and the functional organization of the nucleus. The different in vivo properties of H3, H3.3, and Cid set the stage for identifying the mechanisms by which they are differentially targeted. Here we suggest that local effects of "open" chromatin and broader effects of nuclear organization help to guide the two different H3 variants to their target sites.

Centromere↗
PubMed2002

Recurrent evolution of DNA-binding motifs in the Drosophila centromeric histone.

All eukaryotes contain centromere-specific histone H3 variants (CenH3s), which replace H3 in centromeric chromatin. We have previously documented the adaptive evolution of the Drosophila CenH3 (Cid) in comparisons of Drosophila melanogaster and Drosophila simulans, a divergence of approximately 2.5 million years. We have proposed that rapidly changing centromeric DNA may be driving CenH3's altered DNA-binding specificity. Here, we compare Cid sequences from a phylogenetically broader group of Drosophila species to suggest that Cid has been evolving adaptively for at least 25 million years. Our analysis also reveals conserved blocks not only in the histone-fold domain but also in the N-terminal tail. In several lineages, the N-terminal tail of Cid is characterized by subgroup-specific oligopeptide expansions. These expansions resemble minor groove DNA binding motifs found in various histone tails. Remarkably, similar oligopeptides are also found in N-terminal tails of human and mouse CenH3 (Cenp-A). The recurrent evolution of these motifs in CenH3 suggests a packaging function for the N-terminal tail, which results in a unique chromatin organization at the primary constriction, the cytological marker of centromeres.

Amino Acid Sequence↗
PubMed2002

Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3.

DNA methylation has been implicated in a variety of epigenetic processes, and abnormal methylation patterns have been seen in tumors. Analysis of methylation patterns has traditionally been conducted either by using Southern analysis after cleavage with methyl-sensitive restriction endonucleases or by bisulfite sequencing. However, neither method is practical for analyzing more than a few genes. Here, we describe a simple technique for genome-wide mapping of DNA methylation patterns. Fragmentation by a methyl-sensitive restriction endonuclease is followed by size fractionation and hybridization to microarrays. We demonstrate the utility of this method by characterizing methylation patterns in Arabidopsis methylation mutants. This analysis reveals that CHROMOMETHYLASE3 (CMT3), which was previously shown to maintain CpXpG methylation, preferentially methylates transposons, even when they are present as single copies within the genome. Methylation profiling has potential applications in disease research and diagnostic screening.

Arabidopsis↗
PubMed2002

Near the edge of a chromosome's "black hole".

The inability of current technology to determine contiguous sequence for highly repetitive regions means that centromeres fall within multi-megabase gaps, analogous to black holes from which no information escapes. However, high-resolution mapping of a human X-centromere border reveals a remarkable structure, including a gradient of alpha-satellite divergence up to the edge of the youngest and most homogeneous repeats.

Centromere↗