Peter Fraser

The Air Noncoding RNA Epigenetically Silences Transcription by Targeting G9a to Chromatin

A number of large noncoding RNAs (ncRNAs) epigenetically silence genes through unknown mechanisms. The Air ncRNA is imprinted—monoallelically expressed from the paternal allele. Air is required for allele-specific silencing of the cis-linked Slc22a3, Slc22a2, and Igf2r genes in mouse placenta. We show that Air interacts with the Slc22a3 promoter chromatin and the H3K9 histone methyltransferase G9a in placenta. Air accumulates at the Slc22a3 promoter in correlation with localized H3K9 methylation and transcriptional repression. Genetic ablation of G9a results in nonimprinted, biallelic transcription of Slc22a3. Truncated Air fails to accumulate at the Slc22a3 promoter, which results in reduced G9a recruitment and biallelic transcription. Our results suggest that Air, and potentially other large ncRNAs, target repressive histone-modifying activities through molecular interaction with specific chromatin domains to epigenetically silence transcription.

Nuclear organization of the genome and the potential for gene regulation

Much work has been published on the cis-regulatory elements that affect gene function locally, as well as on the biochemistry of the transcription factors and chromatin- and histone-modifying complexes that influence gene expression. However, surprisingly little information is available about how these components are organized within the three-dimensional space of the nucleus. Technological advances are now helping to identify the spatial relationships and interactions of genes and regulatory elements in the nucleus and are revealing an unexpectedly extensive network of communication within and between chromosomes. A crucial unresolved issue is the extent to which this organization affects gene function, rather than just reflecting it.

Single-cell Hi-C reveals cell-to-cell variability in chromosome structure

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns.

Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells

The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, implying that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hot spots optimized for efficient and coordinated transcriptional control.

No-Nonsense Functions for Long Noncoding RNAs

The mysterious secrets of long noncoding RNAs, often referred to as the Dark Matter of the genome, are gradually coming to light. Several recent papers dig deep to reveal surprisingly complex and diverse functions of these enigmatic molecules.

Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus

Cohesin-mediated sister chromatid cohesion is essential for chromosome segregation and post-replicative DNA repair. In addition, evidence from model organisms and from human genetics suggests that cohesin is involved in the control of gene expression. This non-canonical role has recently been rationalized by the findings that mammalian cohesin complexes are recruited to a subset of DNase I hypersensitive sites and to conserved noncoding sequences by the DNA-binding protein CTCF. CTCF functions at insulators (which control interactions between enhancers and promoters) and at boundary elements (which demarcate regions of distinct chromatin structure), and cohesin contributes to its enhancer-blocking activity. The underlying mechanisms remain unknown, and the full spectrum of cohesin functions remains to be determined. Here we show that cohesin forms the topological and mechanistic basis for cell-type-specific long-range chromosomal interactions in cis at the developmentally regulated cytokine locus IFNG. Hence, the ability of cohesin to constrain chromosome topology is used not only for the purpose of sister chromatid cohesion, but also to dynamically define the spatial conformation of specific loci. This new aspect of cohesin function is probably important for normal development and disease.

Intergenic Transcription and Developmental Remodeling of Chromatin Subdomains in the Human β-globin Locus

Gene activation requires chromatin remodeling complexes, which hyperacetylate histones and enable factor access; however, the targeting mechanisms leading to the establishment and maintenance of large, hyperacetylated DNase-sensitive chromatin domains are unknown. Recent work has shown that histone acetyltransferases are associated with RNA-pol II complexes, suggesting that transcription of chromatin plays a role in chromatin modification. Here we show the human beta-globin locus is divided into three differentially activated chromatin subdomains. Large transcripts precisely delineate the active domains at key cell cycle points associated with chromatin transitions and remodeling. We identify an element that initiates these transcripts, located in a region required for chromatin activation. The results suggest that intergenic transcription is required for chromatin remodeling of chromosomal domains.

Heterochromatin Effects on the Frequency and Duration of LCR-Mediated Gene Transcription

Locus control regions (LCRs) are responsible for initiating and maintaining a stable tissue-specific open chromatin structure of a locus. In transgenic mice, LCRs confer high level expression on linked genes independent of position in the mouse genome. Here we show that an incomplete LCR loses this property when integrated into heterochromatic regions. Two disruption mechanisms were observed. One is classical position-effect variegation, resulting in continuous transcription in a clonal subpopulation of cells. The other is a novel mechanism resulting in intermittent gene transcription in all cells. We conclude that only a complete LCR fully overcomes heterochromatin silencing and that it controls the level of transcription by ensuring activity in all cells at all times rather than directly controlling the rate of transcription.

Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C

Transcriptional control in large genomes often requires looping interactions between distal DNA elements, such as enhancers and target promoters. Current chromosome conformation capture techniques do not offer sufficiently high resolution to interrogate these regulatory interactions on a genomic scale. Here we use Capture Hi-C (CHi-C), an adapted genome conformation assay, to examine the long-range interactions of almost 22,000 promoters in 2 human blood cell types. We identify over 1.6 million shared and cell type–restricted interactions spanning hundreds of kilobases between promoters and distal loci. Transcriptionally active genes contact enhancer-like elements, whereas transcriptionally inactive genes interact with previously uncharacterized elements marked by repressive features that may act as long-range silencers. Finally, we show that interacting loci are enriched for disease-associated SNPs, suggesting how distal mutations may disrupt the regulation of relevant genes. This study provides new insights and accessible tools to dissect the regulatory interactions that underlie normal and aberrant gene regulation.

Detailed analysis of the site 3 region of the human beta-globin dominant control region.

Four DNase I hypersensitive sites characterize the human beta‐globin Dominant Control Region (DCR) providing position independent, high levels of erythroid specific expression to linked homologous and heterologous genes when introduced into cultured cells or in transgenic mice. We have delineated the hypersensitive site located 10.5 kbp upstream of the epsilon‐globin gene by short range DNase I sensitivity mapping to a 600 bp region. Using transgenic mice and MEL cells the functional part of this region was further mapped to a 300 bp central core, which provides position independent, high level expression. It contains a number of ubiquitous and erythroid specific protein binding sites, including the previously described factors NF‐E1 (GF1) and NF‐E2. The latter binds to a dimer of the consensus binding sequence for jun/fos. The presence of this sequence is required for the function of the element, but single or multimerized copies of this site failed to give position independent, high levels of expression in transgenic mice or MEL cells. We therefore conclude that a combination of factor binding sites is necessary to allow site 3 to function as a strong transcriptional activator, resulting in position independent expression of the beta‐globin gene.