Christopher H. Eskiw

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.

PML bodies: a meeting place for genomic loci?

Promyelocytic leukemia (PML) bodies have been implicated in a variety of cellular processes, such as cell-cycle regulation, apoptosis, proteolysis, tumor suppression, DNA repair and transcription. Despite this, the function of PML bodies is still unknown. Direct and indirect evidence supports the hypothesis that PML bodies interact with specific genes or genomic loci. This includes the finding that the stability of PML bodies is affected by cell stress and changes in chromatin structure. PML bodies also facilitate the transcription and replication of double-stranded DNA viral genomes. Moreover, PML bodies associate with specific regions of high transcriptional activity in the cellular genome. We propose that PML bodies functionally interact with chromatin and are important for the regulation of gene expression.

Chromatin Contributes to Structural Integrity of Promyelocytic Leukemia Bodies through a SUMO-1-independent Mechanism

Promyelocytic leukemia (PML) protein is implicated in transcriptional regulation, apoptosis, DNA repair, and tumor suppression. It is not known, however, whether PML and other components of PML bodies function within the vicinity of the bodies or elsewhere in the nucleoplasm. In this study, we demonstrate that chromatin organization around PML bodies influences their morphology, dynamics, and structural integrity by a SUMO-1-independent mechanism. Following transcriptional inhibition and during early apoptosis, chromatin retracts from the periphery of PML bodies, coinciding with the formation of new PML-containing structures through fission of supramolecular PML-containing microbodies. Both fission and fusion of microbodies with parental PML bodies indicate a loss of structural integrity of the bodies, dependent on the state of the surrounding chromatin. This is supported by the observation that treatment of live cells with DNase I could reproduce the structural instability of PML bodies. In addition, PML bodies, which are normally surrounded by chromatin and are positionally stable, become more dynamic following these treatments, presumably due to the loss of chromatin contacts. Overexpression of SUMO-1, a modification required for PML body formation, did not prevent PML body fission, indicating that chromatin-based integrity of PML body structure occurs through a SUMO-1-independent mechanism.

RNA polymerase II activity is located on the surface of protein-rich transcription factories.

We used electron spectroscopic imaging to map nucleoplasmic transcription sites in human cells at unprecedented resolution. HeLa cells were permeabilised, nascent transcripts were extended in BrUTP by ∼40 nucleotides and the resulting BrRNA immunolabelled with gold particles before structures were viewed. Nascent RNA is almost invariably associated with polymorphic and nitrogen-rich (but phosphorus-poor) structures with a diameter of ∼87 nm and mass of 10 MDa (calculated by reference to nucleosomes with known numbers of phosphorus and nitrogen atoms). Structures with similar atomic signatures and diameters were observed using correlative microscopy and in unpermeabilised cells. Our results are consistent with RNA synthesis occurring on the surface of these huge protein-rich transcription factories.

Mitotic accumulations of PML protein contribute to the re-establishment of PML nuclear bodies in G1

Although the mechanism of chromosomal segregation is well known, it is unclear how other nuclear compartments such as promyelocytic leukemia (PML) nuclear bodies partition during mitosis and re-form in G1. We demonstrate that PML nuclear bodies partition via mitotic accumulations of PML protein (MAPPs), which are distinct from PML nuclear bodies in their dynamics, biochemistry and structure. During mitosis PML nuclear bodies lose biochemical components such as SUMO-1 and Sp100. We demonstrate that MAPPs are also devoid of Daxx and these biochemical changes occur prior to chromatin condensation and coincide with the loss of nuclear membrane integrity. MAPPs are highly mobile, yet do not readily exchange PML protein as demonstrated by fluorescence recovery after photo-bleaching (FRAP). A subset of MAPPs remains associated with mitotic chromosomes, providing a possible nucleation site for PML nuclear body formation in G1. As the nuclear envelope reforms in late anaphase, these nascent PML nuclear bodies accumulate components sequentially, for example Sp100 and SUMO-1 before Daxx. After cytokinesis, MAPPs remain in the cytoplasm long after the reincorporation of splicing components and their disappearance coincides with new PML nuclear body formation even in the absence of new protein synthesis. The PML protein within MAPPs is not degraded during mitosis but is recycled to contribute to the formation of new PML nuclear bodies in daughter nuclei. The recycling of PML protein from one cell cycle to the next via mitotic accumulations may represent a common mechanism for the partitioning of other nuclear bodies during mitosis.

Farnesyltransferase inhibitor treatment restores chromosome territory positions and active chromosome dynamics in Hutchinson-Gilford progeria syndrome cells

BackgroundHutchinson-Gilford progeria syndrome (HGPS) is a premature ageing syndrome that affects children leading to premature death, usually from heart infarction or strokes, making this syndrome similar to normative ageing. HGPS is commonly caused by a mutation in the A-type lamin gene, LMNA (G608G). This leads to the expression of an aberrant truncated lamin A protein, progerin. Progerin cannot be processed as wild-type pre-lamin A and remains farnesylated, leading to its aberrant behavior during interphase and mitosis. Farnesyltransferase inhibitors prevent the accumulation of farnesylated progerin, producing a less toxic protein.ResultsWe have found that in proliferating fibroblasts derived from HGPS patients the nuclear location of interphase chromosomes differs from control proliferating cells and mimics that of control quiescent fibroblasts, with smaller chromosomes toward the nuclear interior and larger chromosomes toward the nuclear periphery. For this study we have treated HGPS fibroblasts with farnesyltransferase inhibitors and analyzed the nuclear location of individual chromosome territories. We have found that after exposure to farnesyltransferase inhibitors mis-localized chromosome territories were restored to a nuclear position akin to chromosomes in proliferating control cells. Furthermore, not only has this treatment afforded chromosomes to be repositioned but has also restored the machinery that controls their rapid movement upon serum removal. This machinery contains nuclear myosin 1β, whose distribution is also restored after farnesyltransferase inhibitor treatment of HGPS cells.ConclusionsThis study not only progresses the understanding of genome behavior in HGPS cells but demonstrates that interphase chromosome movement requires processed lamin A.

The yin and yang of chromatin spatial organization

Spatial organization of the genome is non-random. Preferential chromatin interactions, both in cis and in trans and between transcriptionally active and silent regions, influence organization.

Transcription Factories and Nuclear Organization of the Genome

The dynamic compartmental organization of the transcriptional machinery in mammalian nuclei places particular constraints on the spatial organization of the genome. The clustering of active RNA polymerase I transcription units from several chromosomes at nucleoli is probably the best-characterized and universally accepted example. RNA polymerase II localization in mammalian nuclei occurs in distinct concentrated foci that are several-fold fewer in number compared to the number of active genes and transcription units. Individual transcribed genes cluster at these shared transcription factories in a nonrandom manner, preferentially associating with heterologous, coregulated genes. We suggest that the three-dimensional (3D) conformation and relative arrangement of chromosomes in the nucleus has a major role in delivering tissue-specific gene-expression programs.

Transcriptional regulation is affected by subnuclear targeting of reporter plasmids to PML nuclear bodies.

ABSTRACT Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.

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.