Peter Fraser - Head of Laboratory
Dynamic re-arrangement of chromatin in transcription regulation
Dynamic changes in chromatin and nuclear structure can regulate patterns of cellular gene expression during differentiation and development. Our research looks at various levels of chromatin and nuclear structure, from individual nucleosome modifications to the dynamic 3D structure of chromosomes and their relationships in the nucleus.
Intergenic transcription
Our work suggests that large regions of chromatin may be opened or maintained in the open state, in part, by the RNA polymerase II complex in a process known as intergenic transcription. The RNA polymerase complex has been shown to contain a number of factors capable of modifying the structure of the chromatin fiber by adding chemical side groups to nucleosomes and other chromatin proteins. These modifications are thought to increase the accessibility of the genes within these regions or domains resulting in augmented binding of transcription factors to the gene and may also serve as a memory of the active state. Recent studies show that a huge proportion of the genome is transcribed; much more than can be accounted for by gene transcription alone.
Long-range chromatin interactions
Many genes require additional regulatory regions of DNA known as enhancers that are often located considerable distances from the gene along the chromatin fiber. We have shown, using RNA TRAP (tagging and recovery of associated proteins), that distant enhancers actually physically contact their target genes in the nucleus by looping out the intervening DNA.
Such long-range interactions between enhancers and genes are powerful switches that turn on transcription of individual genes resulting in high levels of expression. 
Our work suggests that these regulatory interactions between enhancers and genes can only occur if the chromatin containing them is first remodelled to the open state by intergenic transcription.
Preferential associations between co-regulated genes at transcription factories
While the transcriptional machinery has been studied in great detail, much less is known about the organization of transcription in the three-dimensional space of the nucleus. Transcription in higher eukaryotes takes place in dedicated nuclear sub-compartments known as transcription factories.
Transcription factories are sites highly enriched in the hyperphosphorylated form of RNA polymerase II, the enzyme that transcribes genes into messenger RNA. We have shown that genes rapidly migrate to pre-assembled transcription factories upon activation.
The number of transcription factories per cell is severely limited compared to the number of expressed genes, compelling multiple genes to share the same factory. To this end, genes undergo dynamic long-range associations with other genomic loci located on the same chromosome (intra-chromosomal associations or associations in cis), or on other chromosomes (inter-chromosomal associations or associations in trans) at transcription factories. However, it was unknown how widespread chromosomal associations are, or what the underlying molecular mechanism is.
To address these questions, we have developed e4C, a novel assay to map chromosomal associations on a genome-wide basis. Using the mouse Hba and Hbb globin genes in erythroid cells as a model system, we found that chromosomal associations, in cis and in trans, are widespread. The globin genes undergo associations with hundreds of other active genes, located on all chromosomes, at transcription factories.
Remarkably, these associations are non-random, meaning that both globin genes have preferred interaction partners. Among the globin interacting genes, we observed that genes regulated by the transcription factor Klf1 (Kruppel like factor 1) were overrepresented. Furthermore, in cells lacking Klf1, interaction frequencies between the globin genes and specific interacting loci were dramatically reduced.
Thus, our results show that the transcription factor Klf1is not only required for the efficient transcription of target genes, but also for their three-dimensional clustering in nuclear space. The interactions between Klf1-regulated genes take place at a subset of transcription factories, which appear to be specialized for the optimal expression of Klf1-regulated genes. Finally, our results indicate that the organization of the transcribed genome is inherently plastic and flexible, resulting in multiple different genome conformations.
In summary, our data uncover a transcriptional “interactome” of the mouse globin genes in erythroid cells. We predict that the three-dimensional organization of active genes in the nucleus will have a major impact on tissue-specific gene expression programs.
Non-coding RNA and epigenetic silencing of gene expression
Recent progress in high-throughput transcriptome analyses in mammals has revealed an unexpectedly large number of long non-coding RNAs (ncRNA) that do not encode proteins. Most of these ncRNAs remain enigmatic, however some have attracted attention because they appear to be involved in epigenetic regulation of gene expression. The mechanisms by which large non-coding RNAs silence gene expression are unknown.
The Air ncRNA, appears to be required for placental silencing of three cis-linked genes Slc22a3, Slc22a2 and Igf2r, located within 400 kb from the Air transcription unit. To investigate the possibility that Air is targeted to specific genomic regions, we used RNA TRAP (see above) and found marked accumulation of Air at the promoter region of the distal Slc22a3 gene. Taking advantage of the developmental control of Air-dependent Slc22a3 repression, we showed that Air’s association with the Slc22a3 promoter correlated with Slc22a3 repression and repressive H3K9 histone methylation at the promoter.
These data reveal a specific interaction between the large ncRNA and promoter chromatin, rather than a uniform ‘coating’ mechanism throughout the entire gene cluster. Next we showed by RNA-IP that Air interacts with the histone methyltransferase G9a, which has been implicated in H3K9 methylation. We showed that Air was required to target G9a to the Slc22a3 promoter to epigenetically silence gene expression. In addition, a critical role for G9a in silencing Slc22a3 was confirmed in G9a knockout mice.
Taken together, this work provides answers for two important questions: where and how large ncRNAs work to achieve epigenetic regulation on chromatin. Our results suggest that Air recruits G9a to the Slc22a3 promoter to epigenetically silence gene expression. However it is also possible that Air creates a repressive nuclear compartment that recruits G9a and the Slc22a3 promoter, to modify its chromatin structure and restrict its access to transcription factories (as has been suggested for Xist). This work reveals a potentially vast, previously unappreciated, regulatory network of large ncRNAs that play important roles in regulating gene expression.
Recent, selected publications
Schoenfelder S, Sexton T, Chakalova L, Cope NF, Horton A, Andrews S, Kurukuti S, Mitchell JA, Umlauf D, Dimitrova DS, Eskiw CH, Luo Y, Wei C-L, Ruan Y, Bieker JJ, Fraser P (2010) Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells.
Nature Genetics 42 53-61
http://dx.doi.org/10.1038/ng.496
Hadjur S, Williams LM, Ryan NK, Cobb BS, Sexton T, Fraser P, Fisher AG, Merkenschlager M (2009) Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus.
Nature 460 410-413
http://dx.doi.org/10.1038/nature08079
Nagano T, Mitchell JA, Sanz LA, Pauler FM, Ferguson-Smith AC, Feil R, Fraser P (2008) The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin.
Science 322 1717-1720
http://dx.doi.org/10.1126/science.1163802
Jennifer Mitchell and Peter Fraser
Transcription factories are nuclear subcompartments that remain in the absence of transcription
Genes & Development 22: 20 - 25, 2008
http://dx.doi.org/10.1101/gad.454008
Fraser P, Bickmore W (2007) Nuclear organization of the genome and the potential for gene regulation.
Nature 447 413-417
http://dx.doi.org/10.1038/nature05916
Osborne CS, Chakalova L, Mitchell JA, Horton AM, Wood AL, Bolland DJ, Corcoran AE, Fraser P (2007) Myc dynamically and preferentially relocates to a transcription factory occupied by Igh.
PLoS Biology 5 e192, 1763-1772
http://dx.doi.org/10.1371/journal.pbio.0050192
Chakalova L, Debrand E, Mitchell JA, Osborne CS, Fraser P (2005) Replication and transcription: shaping the landscape of the genome.
Nature Reviews Genetics 6 669-677
http://dx.doi.org/10.1038/nrg1673
Bolland DJ, Wood AL, Johnston CM, Bunting SF, Morgan G, Chakalova L, Fraser P, Corcoran AE (2004) Antisense intergenic transcription in V(D)J recombination.
Nature Immunology 5 630-637
http://dx.doi.org/10.1038/ni1068
Osborne CS, Chakalova L, Brown KE, Carter D, Horton A, Debrand E, Goyenechea B, Mitchell JA, Lopes S, Reik W, Fraser P (2004) Active genes dynamically co-localize to shared sites of ongoing transcription.
Nature Genetics 36 1065-1071
http://dx.doi.org/10.1038/ng1423
Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon E, Sorensen R, Forster A, Fraser P, Cohen JI, De Saint Basile G, Alexander I, Wintergerst U, Frebourg T, Aurias A, Stoppa-Lyonnet D, Romana S, Radford-Weiss I, Gross F, Valensi F, Delabesse E, Macintyre E, Sigaux F, Soulier J, Leiva LE, Wissler M, Prinz C, Rabbitts TH, Le Deist F, Fischer A, Cavazzana-Calvo M (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.
Science 302 415-419
http://dx.doi.org/10.1126/science.1088547
Carter D, Chakalova L, Osborne CS, Dai Y-F, Fraser P (2002) Long-range chromatin regulatory interactions in vivo.
Nature Genetics 32 623-626
http://dx.doi.org/10.1038/ng1051
Gribnau J, Diderich K, Pruzina S, Calzolari R, Fraser P (2000) Intergenic transcription and developmental remodeling of chromatin subdomains in the human β-globin locus.
Molecular Cell 5 377-386
http://dx.doi.org/10.1016/S1097-2765(00)80432-3
Group Members
Postdocs
Dr Daniela Dimitrova
Dr Christopher Eskiw
Dr Takashi Nagano
Dr Stefan Schoenfelder
PhD Students
Ieuan Clay
Nathan Cope
Luke Edelman
Harris Lazaris
Catherine Moir
