Jorja G. Henikoff

Identification of functional elements and regulatory circuits by Drosophila modENCODE

From Genome to Regulatory Networks For biologists, having a genome in hand is only the beginning—much more investigation is still needed to characterize how the genome is used to help to produce a functional organism (see the Perspective by Blaxter). In this vein, Gerstein et al. (p. 1775) summarize for the Caenorhabditis elegans genome, and The modENCODE Consortium (p. 1787) summarize for the Drosophila melanogaster genome, full transcriptome analyses over developmental stages, genome-wide identification of transcription factor binding sites, and high-resolution maps of chromatin organization. Both studies identified regions of the nematode and fly genomes that show highly occupied targets (or HOT) regions where DNA was bound by more than 15 of the transcription factors analyzed and the expression of related genes were characterized. Overall, the studies provide insights into the organization, structure, and function of the two genomes and provide basic information needed to guide and correlate both focused and genome-wide studies. The Drosophila modENCODE project demonstrates the functional regulatory network of flies. To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

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.

Position-based sequence weights☆

Sequence weighting methods have been used to reduce redundancy and emphasize diversity in multiple sequence alignment and searching applications. Each of these methods is based on a notion of distance between a sequence and an ancestral or generalized sequence. We describe a different approach, which bases weights on the diversity observed at each position in the alignment, rather than on a sequence distance measure. These position-based weights make minimal assumptions, are simple to compute, and perform well in comprehensive evaluations.

Genome-Wide Kinetics of Nucleosome Turnover Determined by Metabolic Labeling of Histones

Everything Changes Nucleosomes package DNA, and their assembly and disassembly regulate access to the genome. The ability to follow these changes is limited to steady-state methods in higher eukaryotes, although direct kinetic analyses are available in yeast. To address this deficiency, Deal et al. (p. 1161) developed a general method for following the genome-wide dynamics of nucleosome assembly and disassembly. High levels of nucleosome turnover were observed across gene bodies and at the sites of epigenetic regulatory elements in fruit fly tissue culture cells. Nucleosomes were replaced multiple times during each 20-hour cell cycle, suggesting that histone modifications themselves are unlikely to transmit epigenetic information. Furthermore, analysis of replication origins indicates that they are determined by chromatin dynamics and not by sequence features. Differences between active and silent chromatin states are critical for maintaining the epigenome. Nucleosome disruption and replacement are crucial activities that maintain epigenomes, but these highly dynamic processes have been difficult to study. Here, we describe a direct method for measuring nucleosome turnover dynamics genome-wide. We found that nucleosome turnover is most rapid over active gene bodies, epigenetic regulatory elements, and replication origins in Drosophila cells. Nucleosomes turn over faster at sites for trithorax-group than polycomb-group protein binding, suggesting that nucleosome turnover differences underlie their opposing activities and challenging models for epigenetic inheritance that rely on stability of histone marks. Our results establish a general strategy for studying nucleosome dynamics and uncover nucleosome turnover differences across the genome that are likely to have functional importance for epigenome maintenance, gene regulation, and control of DNA replication.

Increased coverage of protein families with the Blocks Database servers

The Blocks Database WWW (http://blocks.fhcrc.org ) and Email (blocks@blocks.fhcrc.org ) servers provide tools to search DNA and protein queries against the Blocks+ Database of multiple alignments, which represent conserved protein regions. Blocks+ nearly doubles the number of protein families included in the database by adding families from the Pfam-A, ProDom and Domo databases to those from PROSITE and PRINTS. Other new features include improved Block Searcher statistics, searching with NCBIs IMPALA program and 3D display of blocks on PDB structures.

Blocks+: a non-redundant database of protein alignment blocks derived from multiple compilations.

MOTIVATION As databanks grow, sequence classification and prediction of function by searching protein family databases becomes increasingly valuable. The original Blocks Database, which contains ungapped multiple alignments for families documented in Prosite, can be searched to classify new sequences. However, Prosite is incomplete, and families from other databases are now available to expand coverage of the Blocks Database. RESULTS To take advantage of protein family information present in several existing compilations, we have used five databases to construct Blocks+, a unified database that is built on the PROTOMAT/BLOSUM scoring model and that can be searched using a single algorithm for consistent sequence classification. The LAMA blocks-versus-blocks searching program identifies overlapping protein families, making possible a non-redundant hierarchical compilation. Blocks+ consists of all blocks derived from PROSITE, blocks from Prints not present in PROSITE, blocks from Pfam-A not present in PROSITE or Prints, and so on for ProDom and Domo, for a total of 1995 protein families represented by 8909 blocks, doubling the coverage of the original Blocks Database. A challenge for any procedure aimed at non-redundancy is to retain related but distinct families while discarding those that are duplicates. We illustrate how using multiple compilations can minimize this potential problem by examining the SNF2 family of ATPases, which is detectably similar to distinct families of helicases and ATPases. AVAILABILITY http://blocks.fhcrc.org/

Histone Replacement Marks the Boundaries of cis-Regulatory Domains

Cellular memory is maintained at homeotic genes by cis-regulatory elements whose mechanism of action is unknown. We have examined chromatin at Drosophila homeotic gene clusters by measuring, at high resolution, levels of histone replacement and nucleosome occupancy. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely correspond to nuclease-hypersensitive sites, binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. Our results suggest the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements.

A Comprehensive Map of Insulator Elements for the Drosophila Genome

Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP–chip the genome-wide binding sites of 6 insulator-associated proteins—dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF—to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all classically defined insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw), respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters; divide differentially expressed, alternative, and divergent promoters; act as chromatin boundaries; are associated with chromosomal breakpoints among species; and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units and a framework for understanding insulator function during the development and evolution of Drosophila.

Epigenome characterization at single base-pair resolution

We have combined standard micrococcal nuclease (MNase) digestion of nuclei with a modified protocol for constructing paired-end DNA sequencing libraries to map both nucleosomes and subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including the eviction of nucleosomes. To characterize DNA-binding features of transcription factors, we plotted the length of each fragment versus its position in the genome, which defined the minimal protected region of each factor. This process led to the precise mapping of protected and exposed regions at and around binding sites, and also determination of the degree to which they are flanked by phased nucleosomes and subnucleosome-sized particles. Our protocol and mapping method provide a general strategy for epigenome characterization, including nucleosome phasing and dynamics, ATP-dependent nucleosome remodelers, and transcription factors, from a single-sequenced sample.

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.