The intranuclear position of many genes has been correlated with their activity state, suggesting that migration to functional subcompartments may influence gene expression. Indeed, nascent RNA production and RNA polymerase II seem to be localized into discrete foci or 'transcription factories'. Current estimates from cultured cells indicate that multiple genes could occupy the same factory, although this has not yet been observed. Here we show that, during transcription in vivo, distal genes colocalize to the same transcription factory at high frequencies. Active genes are dynamically organized into shared nuclear subcompartments, and movement into or out of these factories results in activation or abatement of transcription. Thus, rather than recruiting and assembling transcription complexes, active genes migrate to preassembled transcription sites.The activity state of a gene can often be correlated with its intranuclear localization to specific subregions or its position relative to constitutive heterochromatin [1][2][3][4][5][6] ; however, it is unclear whether activation or repression of an individual gene is a cause or consequence of intranuclear position. Transcriptionally active genes have been proposed to associate with transcription factories, discrete nuclear sites of nascent RNA production and concentrated transcriptional components, such as RNA polymerase [7][8][9][10][11][12] . Calculations using the number of RNA polymerase II (RNAP II) transcription factories in HeLa cells (several thousand per nucleus) and estimates of the number of transcription units suggest that several active genes could occupy the same factory 10

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.

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.

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their longrange interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.

Transcription in mammalian nuclei is highly compartmentalized in RNA polymerase II-enriched nuclear foci known as transcription factories. Genes in cis and trans can share the same factory, suggesting that genes migrate to preassembled transcription sites. We used fluorescent in situ hybridization to investigate the dynamics of gene association with transcription factories during immediate early (IE) gene induction in mouse B lymphocytes. Here, we show that induction involves rapid gene relocation to transcription factories. Importantly, we find that the Myc proto-oncogene on Chromosome 15 is preferentially recruited to the same transcription factory as the highly transcribed Igh gene located on Chromosome 12. Myc and Igh are the most frequent translocation partners in plasmacytoma and Burkitt lymphoma. Our results show that transcriptional activation of IE genes involves rapid relocation to preassembled transcription factories. Furthermore, the data imply a direct link between the nonrandom interchromosomal organization of transcribed genes at transcription factories and the incidence of specific chromosomal translocations.

The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers-SRR18, SRR107, and SRR111-that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/ Cas9 system and F1 ES cells (Mus musculus 129 3 Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.

Nascent transcription occurs at nuclear foci of concentrated, hyperphosphorylated RNA polymerase II (RNAPII). We investigate RNAPII localization, distal gene coassociation, and Hbb locus conformation during inhibition of transcription. Our results show distal active genes remain associated with RNAPII foci and each other in the absence of elongation. When initiation is inhibited, active genes dissociate from RNAPII foci and each other, suggesting initiation is necessary to tether distal active genes to shared foci. In the absence of transcription RNAPII foci remain, indicating they are not simple accumulations of RNAPII on transcribed genes but exist as independent nuclear subcompartments. Transcription is a fundamental, life-essential process, yet we lack a clear picture of how and where it occurs in vivo. Textbooks tell us that RNA polymerase II (RNAPII) is recruited to the promoters of genes when they become active, suggesting that transcription sites form de novo on individual genes. However, nascent transcription in the nucleus occurs within a limited number of discrete foci containing high concentrations of active RNAPII, termed transcription factories (Jackson et al. 1993;Wansink et al. 1993). Previously, we showed that the Hbb genes are often transcribed in the same factory as other erythroid expressed genes, separated by tens of megabases in cis (Eraf and Uros) or in trans (Hba) (Osborne et al. 2004). This has been confirmed by recent 4C (chromosome conformation capture-on-chip) studies showing associations between Hbb and multiple active genes (Simonis et al. 2006). Furthermore, several studies have shown that individual genes often loop out of their chromosomal territory upon activation, suggesting repositioning commonly occurs with transcriptional activation (Volpi et al. 2000;Chambeyron and Bickmore 2004;Wiblin et al. 2005). Indeed, we showed recently that immediate early gene activation occurs via rapid gene repositioning to preassembled transcription factories (Osborne et al. 2007). Taken together, these data suggest that genes migrate to pre-existing specialized sites in the nucleus for transcription.However, whether transcription factories are simply aggregates of RNAPII on individual active genes or independent structures is still unclear. In this study we assessed RNAPII localization during short-term transcription inhibition to investigate these possibilities. DRB (5,6-Dichlorobenzimidazole 1-␤-D-ribofuranoside) specifically inhibits RNAPII elongation by inhibiting kinases that phosphorylate Ser2 of the C-terminal domain (CTD) of the large subunit of RNAPII (Fraser et al. 1978;Marshall and Price 1995;Marshall et al. 1996;Gribnau et al. 1998). Heat shock at 45°C has been shown to globally inhibit transcription, disrupting both initiation and elongation (Hieda et al. 2005). In mammalian cells, heatshock-induced inhibition of RNAPII occurs specifically through production of B2 RNAs (transcribed from short interspersed elements [SINEs] by RNA polymerase III) that bind to RNAPII preinitiation com...

Transcriptional enhancers are critical for maintaining cell-type-specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes.