Lyubomira Chakalova

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.

Antisense intergenic transcription in V(D)J recombination.

Antigen receptor genes undergo variable, diversity and joining (V(D)J) recombination, which requires ordered large-scale chromatin remodeling. Here we show that antisense transcription, both genic and intergenic, occurs extensively in the V region of the immunoglobulin heavy chain locus. RNA fluorescence in situ hybridization demonstrates antisense transcription is strictly developmentally regulated and is initiated during the transition from DJH to VDJH recombination and terminates concomitantly with VDJH recombination. Our data show antisense transcription is specific to the V region and suggest transcripts extend across several genes. We propose that antisense transcription remodels the V region to facilitate VH-to-DJH recombination. These findings have wider implications for V(D)J recombination of other antigen receptor loci and developmental regulation of multigene loci.

REPLICATION AND TRANSCRIPTION: SHAPING THE LANDSCAPE OF THE GENOME

As the relationship between nuclear structure and function begins to unfold, a picture is emerging of a dynamic landscape that is centred on the two main processes that execute the regulated use and propagation of the genome. Rather than being subservient enzymatic activities, the replication and transcriptional machineries provide potent forces that organize the genome in three-dimensional nuclear space. Their activities provide opportunities for epigenetic changes that are required for differentiation and development. In addition, they impose physical constraints on the genome that might help to shape its evolution.

Kcnq1ot1/Lit1 Noncoding RNA Mediates Transcriptional Silencing by Targeting to the Perinucleolar Region

ABSTRACT The Kcnq1ot1 antisense noncoding RNA has been implicated in long-range bidirectional silencing, but the underlying mechanisms remain enigmatic. Here we characterize a domain at the 5′ end of the Kcnq1ot1 RNA that carries out transcriptional silencing of linked genes using an episomal vector system. The bidirectional silencing property of Kcnq1ot1 maps to a highly conserved repeat motif within the silencing domain, which directs transcriptional silencing by interaction with chromatin, resulting in histone H3 lysine 9 trimethylation. Intriguingly, the silencing domain is also required to target the episomal vector to the perinucleolar compartment during mid-S phase. Collectively, our data unfold a novel mechanism by which an antisense RNA mediates transcriptional gene silencing of chromosomal domains by targeting them to distinct nuclear compartments known to be rich in heterochromatic machinery.

The pre‐B‐cell receptor induces silencing of VpreB and λ5 transcription

The pre‐B‐cell receptor (pre‐BCR), composed of Ig heavy and surrogate light chain (SLC), signals pre‐BII‐cell proliferative expansion. We have investigated whether the pre‐BCR also signals downregulation of the SLC genes (VpreB and λ5), thereby limiting this expansion. We demonstrate that, as BM cells progress from the pre‐BI to large pre‐BII‐cell stage, there is a shift from bi‐ to mono‐allelic λ5 transcription, while the second allele is silenced in small pre‐BII cells. A VpreB1‐promoter‐driven transgene shows the same pattern, therefore suggesting that VpreB1 is similarly regulated and thereby defines the promoter as a target for transcriptional silencing. Analyses of pre‐BCR‐deficient mice show a temporal delay in λ5 downregulation, thereby demonstrating that the pre‐BCR is essential for monoallelic silencing at the large pre‐BII‐cell stage. Our data also suggest that SLP‐65 is one of the signaling components important for this process. Furthermore, the VpreB1/λ5 alleles undergo dynamic changes with respect to nuclear positioning and heterochromatin association, thereby providing a possible mechanism for their transcriptional silencing.

Fine tuning of globin gene expression by DNA methylation.

Expression patterns in the globin gene cluster are subject to developmental regulation in vivo. While the γA and γG genes are expressed in fetal liver, both are silenced in adult erythrocytes. In order to decipher the role of DNA methylation in this process, we generated a YAC transgenic mouse system that allowed us to control γA methylation during development. DNA methylation causes a 20-fold repression of γA both in non-erythroid and adult erythroid cells. In erythroid cells this modification works as a dominant mechanism to repress γ gene expression, probably through changes in histone acetylation that prevent the binding of erythroid transcription factors to the promoter. These studies demonstrate that DNA methylation serves as an elegant in vivo fine-tuning device for selecting appropriate genes in the globin locus. In addition, our findings provide a mechanism for understanding the high levels of γ-globin transcription seen in patients with Hereditary Persistence of Fetal Hemoglobin, and help explain why 5azaC and butyrate compounds stimulate γ-globin expression in patients with β-hemoglobinopathies.

RNA fluorescence in situ hybridization tagging and recovery of associated proteins to analyze in vivo chromatin interactions.

Publisher Summary This chapter presents a method designed to detect tertiary chromatin interactions between specific DNA sequences in vivo. This method is used to show that the distal β-globin locus control region is intimately associated with an actively transcribed β-globin gene in erythroid cells. The technique, called RNA FISH TRAP (fluorescence in situ hybridization tagging and recovery of associated proteins), utilizes the targeting power of in situ hybridization to tag proteins near a specific, transcriptionally active gene locus. A hapten-labeled, antisense DNA probe is hybridized to the intron sequences of a nascent primary transcript associated with an actively transcribed gene in formaldehyde-fixed cells. A hapten-specific antibody conjugated with horseradish peroxidase (HRP) is then directed to the DNA probe, thereby localizing HRP activity to the specific gene locus. The HRP is then used to catalyze the covalent attachment of a tag (in this case, a biotinylated tyramide) to proteins in the immediate vicinity of the gene. This tag can then be used in affinity purification procedures to recover proteins and chromatin complexes near the site of transcription. This technique helps to bridge the gap between light microscopy and electron microscopy in that it allows the recovery and analysis of sequences that are engaged in functional interactions or specifically juxtaposed to a defined gene locus in vivo. Permutations of this technique will undoubtedly aid in the understanding of higher order chromatin structure and the role of chromatin interactions in various nuclear processes.

Developmental Regulation of the β-Globin Gene Locus

The β-globin genes have become a classical model for studying regulation of gene expression. Wide-ranging studies have revealed multiple levels of epigenetic regulation that coordinately ensure a highly specialised, tissue- and stage-specific gene transcription pattern. Key players include cis-acting elements involved in establishing and maintaining specific chromatin conformations and histone modification patterns, elements engaged in the transcription process through long-range regulatory interactions, trans-acting general and tissue-specific factors. On a larger scale, molecular events occurring at the locus level take place in the context of a highly dynamic nucleus as part of the cellular epigenetic programme.

Nuclear RNA sequencing of the mouse erythroid cell transcriptome

In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq) in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq) of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A)-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

Brushed aside and silenced.

Mammalian genomes are highly organized in the 3D space of cell nuclei, but whether this affects gene function is unclear. Three papers now show that spatial relocation of a gene directly affects expression, and surprisingly, that of its neighbors.