Howard Y. Chang

Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis

Large intervening non-coding RNAs (lincRNAs) are pervasively transcribed in the genome yet their potential involvement in human disease is not well understood. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodelling activities. Here we show that lincRNAs in the HOX loci become systematically dysregulated during breast cancer progression. The lincRNA termed HOTAIR is increased in expression in primary breast tumours and metastases, and HOTAIR expression level in primary tumours is a powerful predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb repressive complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. These findings indicate that lincRNAs have active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.

Functional Demarcation of Active and Silent Chromatin Domains in Human HOX Loci by Noncoding RNAs

Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.

Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes

A Lot of HOTAIR The roles of several classes of small (<50 nucleotides) noncoding RNAs are beginning to be defined in molecular detail, whereas the function of most of the long (∼200+ nucleotides), intergenic noncoding (linc)RNAs found in most eukaryotic genomes remains something of a mystery. The HOTAIR lincRNA, which is transcribed from the mouse HOXC locus, binds to the Polycomb Repressive Complex 2 (PRC2) and recruits it to HOXD and other genes, where its histone methylase activity acts to repress gene transcription. Tsai et al. (p. 689, published online 8 July) now show that HOTAIR also binds to a histone demethylase enzyme, LSD1, part of the CoREST/REST repressor complex. LSD1 acts to remove transcription-activating histone marks, reinforcing the repressive activity of the PRC2 complex. HOTAIR thus functions as a platform for the coordinated binding of PRC2 and LSD1-containing complexes to genes, as revealed in a genome-wide analysis of PRC1/CoREST/REST co-regulated genes. The long noncoding RNA HOTAIR binds two distinct protein complexes that modify chromatin and repress transcription. Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here, we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5′ domain of HOTAIR binds polycomb repressive complex 2 (PRC2), whereas a 3′ domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1 and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation. Our results suggest that lincRNAs may serve as scaffolds by providing binding surfaces to assemble select histone modification enzymes, thereby specifying the pattern of histone modifications on target genes.

Genome regulation by long noncoding RNAs.

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. The discovery of extensive transcription of large RNA transcripts that do not code for proteins, termed long noncoding RNAs (lncRNAs), provides an important new perspective on the centrality of RNA in gene regulation. Here, we discuss genome-scale strategies to discover and characterize lncRNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators and then target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, lncRNAs can function as modular scaffolds to specify higher-order organization in RNP complexes and in chromatin states. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs for proper gene control across all kingdoms of life.

Molecular Mechanisms of Long Noncoding RNAs

Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. Here we discuss the emerging archetypes of molecular functions that lncRNAs execute-as signals, decoys, guides, and scaffolds. For each archetype, examples from several disparate biological contexts illustrate the commonality of the molecular mechanisms, and these mechanistic views provide useful explanations and predictions of biological outcomes. These archetypes of lncRNA function may be a useful framework to consider how lncRNAs acquire properties as biological signal transducers and hint at their possible origins in evolution. As new lncRNAs are being discovered at a rapid pace, the molecular mechanisms of lncRNAs are likely to be enriched and diversified.

Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position

We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500–50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4+ T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individuals epigenome on a timescale compatible with clinical decision-making.

A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression

The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3′ to 5′of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5′ tip of the HOXA locus that coordinates the activation of several 5′ HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.

Daxx, a Novel Fas-Binding Protein That Activates JNK and Apoptosis

The Fas cell surface receptor induces apoptosis upon receptor oligomerization. We have identified a novel signaling protein, termed Daxx, that binds specifically to the Fas death domain. Overexpression of Daxx enhances Fas-mediated apoptosis and activates the Jun N-terminal kinase (JNK) pathway. A C-terminal portion of Daxx interacts with the Fas death domain, while a different region activates both JNK and apoptosis. The Fas-binding domain of Daxx is a dominant-negative inhibitor of both Fas-induced apoptosis and JNK activation, while the FADD death domain partially inhibits death but not JNK activation. The Daxx apoptotic pathway is sensitive to both Bcl-2 and dominant-negative JNK pathway components and acts cooperatively with the FADD pathway. Thus, Daxx and FADD define two distinct apoptotic pathways downstream of Fas.

Long noncoding RNAs and human disease

A new class of transcripts, long noncoding RNAs (lncRNAs), has been recently found to be pervasively transcribed in the genome. Multiple lines of evidence increasingly link mutations and dysregulations of lncRNAs to diverse human diseases. Alterations in the primary structure, secondary structure, and expression levels of lncRNAs as well as their cognate RNA-binding proteins underlie diseases ranging from neurodegeneration to cancer. Recent progress suggests that the involvement of lncRNAs in human diseases could be far more prevalent than previously appreciated. We review the evidence linking lncRNAs to diverse human diseases and highlight fundamental concepts in lncRNA biology that still need to be clarified to provide a robust framework for lncRNA genetics.

Long Noncoding RNAs: Cellular Address Codes in Development and Disease

In biology as in real estate, location is a cardinal organizational principle that dictates the accessibility and flow of informational traffic. An essential question in nuclear organization is the nature of the address code--how objects are placed and later searched for and retrieved. Long noncoding RNAs (lncRNAs) have emerged as key components of the address code, allowing protein complexes, genes, and chromosomes to be trafficked to appropriate locations and subject to proper activation and deactivation. lncRNA-based mechanisms control cell fates during development, and their dysregulation underlies some human disorders caused by chromosomal deletions and translocations.