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

Myc dynamically and preferentially relocates to a transcription factory occupied by Igh.

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

An intergenic non-coding RNA promoter required for histone modifications in the human ß-globin chromatin domain

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human ß-globin genes. Mutational analyses in ß-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult [delta]- and ß-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring [epsilon]- and [gamma]-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of ß-like globin gene transcription during development

Intergenic Transcription, Cell-Cycle and the Developmentally Regulated Epigenetic Profile of the Human Beta-Globin Locus

Several lines of evidence have established strong links between transcriptional activity and specific post-translation modifications of histones. Here we show using RNA FISH that in erythroid cells, intergenic transcription in the human β-globin locus occurs over a region of greater than 250 kb including several genes in the nearby olfactory receptor gene cluster. This entire region is transcribed during S phase of the cell cycle. However, within this region there are ∼20 kb sub-domains of high intergenic transcription that occurs outside of S phase. These sub-domains are developmentally regulated and enriched with high levels of active modifications primarily to histone H3. The sub-domains correspond to the β-globin locus control region, which is active at all developmental stages in erythroid cells, and the region flanking the developmentally regulated, active globin genes. These results correlate high levels of non-S phase intergenic transcription with domain-wide active histone modifications to histone H3

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 γ(A) and γ(G) 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 γ(A) methylation during development. DNA methylation causes a 20-fold repression of γ(A) 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

Efficient Detection of Mediterranean β-Thalassemia Mutations by Multiplex Single-Nucleotide Primer Extension

<div><p>β-Thalassemias and abnormal hemoglobin variants are among the most common hereditary abnormalities in humans. Molecular characterization of the causative genetic variants is an essential part of the diagnostic process. In geographic areas with high hemoglobinopathy prevalence, such as the Mediterranean region, a limited number of genetic variants are responsible for the majority of hemoglobinopathy cases. Developing reliable, rapid and cost-effective mutation-specific molecular diagnostic assays targeting particular populations greatly facilitates routine hemoglobinopathy investigations. We developed a one-tube single-nucleotide primer extension assay for the detection of eight common Mediterranean β-thalassemia mutations: Codon 5 (-CT); CCT(Pro)->C–, Codon 6 (-A); GAG(Glu)->G-G, Codon 8 (-AA); AAG(Lys)->–G, IVS-I-1 (G->A), IVS-I-6 (T->C), IVS-I-110 (G->A), Codon 39 (C->T), and IVS-II-745 (C->G), as well as the hemoglobin S variant beta 6(A3) Glu>Val. We validated the new assay using previously genotyped samples obtaining 100% agreement between independent genotyping methods. Our approach, applicable in a range of Mediterranean countries, offers a combination of high accuracy and rapidity exploiting standard techniques and widely available equipment. It can be further adapted to particular populations by including/excluding assayed mutations. We facilitate future modifications by providing detailed information on assay design.</p></div

Developing the single-nucleotide primer extension assay.

<p>(A) Principle of the single-nucleotide primer extension method illustrated through analysis of a sample carrying a point mutation of interest. Four template DNA strands from the maternal (M) and paternal (P) chromosomes are shown (variable nucleotide lettered). The template is interrogated by two extension primers (thick arrows) giving rise to normal and mutant extension products and peaks. ‘+’ and ‘−’ indicate strand specificity of the primers; the fluorescently labeled nucleotides incorporated into extension products are bold and colored as they appear on the electropherogram. N+, normal peak generated from ‘+’ primer; M+, mutant peak generated from ‘+’ primer; N−, normal peak generated from ‘−’ primer; M−, mutant peak generated from ‘−’ primer. (B) Normal DNA electropherogram profile obtained with the optimized primer set: primer extension product peaks are labeled with the corresponding primer names as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048167#pone-0048167-t002" target="_blank">Table 2</a>.</p

Primer extension set.

<p>Primer extension set.</p

Reference chromosomes used for validation of the single-nucleotide primer extension assay.

<p>Reference chromosomes used for validation of the single-nucleotide primer extension assay.</p