Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Summary Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which impact cell differentiation, gene regulation and other key cellular processes. We present a genome-wide chromatin landscape for Drosophila melanogaster based on 18 histone modifications, summarized by 9 prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNaseI hypersensitivity, GRO-seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements, and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions, and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

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.

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Summary The Drosophila MSL complex associates with active genes specifically on the male X chromosome to acetylate histone H4 at lysine 16, and increase expression approximately two-fold. To date, no DNA sequence has been discovered to explain the specificity of MSL binding. We hypothesized that sequence-specific targeting occurs at “chromatin entry sites”, but the majority of sites are sequence-independent. Here we characterize 150 potential entry sites by ChIP-chip and ChIP-seq and discover a GA-rich MSL recognition element (MRE). The motif is only slightly enriched on the X chromosome (~2 fold), but this is doubled when considering its preferential location within or 3’ to active genes (>4 fold enrichment). When inserted on an autosome, a newly identified site can direct local MSL spreading to flanking active genes. These results provide strong evidence for both sequence-dependent and -independent steps in MSL targeting of dosage compensation to the male X chromosome.

The multisubunit MSL dosage compensation complex binds to hundreds of sites along the Drosophila single male X chromosome, mediating its hypertranscription. The male X chromosome is also coated with noncoding roX RNAs. When either msl3, mle, or mof is mutant, a partial MSL complex is bound at only approximately 35 unusual sites distributed along the X. We show that two of these sites are the roX1 and roX2 genes and postulate that one of their functions is to provide entry sites for the MSL complex to recognize the X chromosome. The roX1 gene provides a nucleation site for extensive spreading of the MSL complex into flanking chromatin even when moved to an autosome. The spreading can occur in cis or in trans between paired homologs. We present a model for how the dosage compensation complex recognizes X chromatin.

Long noncoding RNAs (lncRNAs) have important regulatory roles and can function at the level of chromatin. To determine where lncRNAs bind to chromatin, we developed capture hybridization analysis of RNA targets (CHART), a hybridization-based technique that specifically enriches endogenous RNAs along with their targets from reversibly cross-linked chromatin extracts. CHART was used to enrich the DNA and protein targets of endogenous lncRNAs from flies and humans. This analysis was extended to genomewide mapping of roX2, a well-studied ncRNA involved in dosage compensation in Drosophila. CHART revealed that roX2 binds at specific genomic sites that coincide with the binding sites of proteins from the male-specific lethal complex that affects dosage compensation. These results reveal the genomic targets of roX2 and demonstrate how CHART can be used to study RNAs in a manner analogous to chromatin immunoprecipitation for proteins.chromatin-associated RNAs | chromatin-modifying complexes | RNase H mapping G enerating cellular diversity from genetic information requires the regulatory interplay between cis-acting elements encoded at specific loci in chromatin and trans-acting factors that bind them (1). Although the importance of trans-acting proteins (e.g., transcription factors) has long been appreciated, there is growing interest in the role of long noncoding RNAs (lncRNAs) (2) as factors that can regulate specific chromatin loci. This interest is enhanced by the recent discovery that the majority of eukaryotic genomes are transcribed (3) and that many of the resulting transcripts are developmentally regulated (4) but do not encode proteins. Although the functional scope of these RNAs remains unknown (5-7), several lncRNAs play important regulatory roles at the level of chromatin (8). Determining where these ncRNAs bind on the genome is central to determining their function.Examples of lncRNAs that influence chromatin include the roX ncRNAs in flies and Xist in mammals, both having wellestablished roles in dosage compensation (8, 9); Kcnq1ot1 and Air ncRNAs, which are expressed from genomically imprinted loci and affect chromatin silencing (10-13); Evf2, HSR1, and other ncRNAs that positively regulate transcription (14-16); lncRNAs that target the dihydrofolate reductase promoter and the rDNA promoters through triplex formation (17, 18); and the human HOTAIR and HOTTIP lncRNAs, which regulate polycomb-repressed and trithorax-activated chromatin, respectively (19,20). Dysregulation of several of these lncRNAs has been associated with disease (21, 22). Our understanding of the biochemical roles of these RNAs comes largely from their interactions with specific proteins-insights gained from classical biochemical techniques developed for studying translation and RNA-processing complexes and also more recent technological advances using RNA immunoprecipitation (23) and cross-linking and immunoprecipitation (24)(25)(26). These experiments suggest that several lncRNAs specifically interact with chromatin-m odifying machine...

We report testing of the specificity and utility of over 200 antibodies raised against 57 different histone modifications, in Drosophila melanogaster, Caenorhabditis elegans and human cells. While most antibodies performed well, over 25% failed specificity tests by dot blot or western blot. Among specific antibodies, over 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use and provide a website for posting new test results.