Coregulated human globin genes are frequently in spatial proximity when active

The organization of genes within the nucleus may influence transcription. We have analyzed the nuclear positioning of the coordinately regulated α- and β-globin genes and show that the gene-dense chromatin surrounding the human α-globin genes is frequently decondensed, independent of transcription. Against this background, we show the frequent juxtaposition of active α- and β-globin genes and of homologous α-globin loci that occurs at nuclear speckles and correlates with transcription. However, we did not see increased colocalization of signals, which would be expected with direct physical interaction. The same degree of proximity does not occur between human β-globin genes or between murine globin genes, which are more constrained to their chromosome territories. Our findings suggest that the distribution of globin genes within erythroblast nuclei is the result of a self-organizing process, involving transcriptional status, diffusional ability of chromatin, and physical interactions with nuclear proteins, rather than a directed form of higher-order control

Association between active genes occurs at nuclear speckles and is modulated by chromatin environment

Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the mouse α-globin gene cluster in situ with its human counterpart, we demonstrate a direct effect of the regional chromatin environment on the frequency of association, whereas nascent transcription from the human α-globin gene appears unaffected. We see no evidence that cotranscribed erythroid genes associate at shared transcription foci, but we do see stochastic clustering of active genes around common nuclear SC35-enriched speckles (hence the apparent nonrandom association between genes). Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles

Global gene expression analysis of human erythroid progenitors

This article is available open access through the publisher’s website. Copyright @ 2011 American Society of Hematology. This article has an erratum: http://bloodjournal.hematologylibrary.org/content/118/26/6993.3.Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html.National Health Service Blood and Transplant, National Institute for Health Research Biomedical Research Center Program, and National Institute for Health Research

SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith–Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267–1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides

De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder

BACKGROUND: Autism spectrum disorders (ASDs) are common and have a strong genetic basis, yet the cause of ∼70-80% ASDs remains unknown. By clinical cytogenetic testing, we identified a family in which two brothers had ASD, mild intellectual disability and a chromosome 22 pericentric inversion, not detected in either parent, indicating de novo mutation with parental germinal mosaicism. We hypothesised that the rearrangement was causative of their ASD and localised the chromosome 22 breakpoints. METHODS: The rearrangement was characterised using fluorescence in situ hybridisation, Southern blotting, inverse PCR and dideoxy-sequencing. Open reading frames and intron/exon boundaries of the two physically disrupted genes identified, TCF20 and TNRC6B, were sequenced in 342 families (260 multiplex and 82 simplex) ascertained by the International Molecular Genetic Study of Autism Consortium (IMGSAC). RESULTS: IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families. Through exome sequencing in another project, we independently identified a de novo frameshifting mutation of TCF20 in a woman with ASD and moderate intellectual disability. We did not identify a significant association of TNRC6B mutations with ASD. CONCLUSIONS: TCF20 encodes a transcriptional coregulator (also termed SPBP) that is structurally and functionally related to RAI1, the critical dosage-sensitive protein implicated in the behavioural phenotypes of the Smith-Magenis and Potocki-Lupski 17p11.2 deletion/duplication syndromes, in which ASD is frequently diagnosed. This study provides the first evidence that mutations in TCF20 are also associated with ASD

DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

A biophysical model for transcription factories

Summary Transcription factories are nuclear domains where gene transcription takes place although the molecular basis for their formation and maintenance are unknown. In this study, we explored how the properties of chromatin as a polymer may contribute to the structure of transcription factories. We found that transcriptional active chromatin contains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Single fibre analysis showed that this modification spans the entire body of the gene. Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active and inactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in the chromatin fibre. The result of this change in flexibility is that chromatin could behave like a multi-block copolymer with repetitions of stiff-flexible (active-inactive chromatin) components. Copolymers with such structure self-organize through spontaneous phase separation into microdomains. Consistent with such model H4K16ac chromatin form foci that associates with nascent transcripts. We propose that transcription factories are the result of the spontaneous concentration of H4K16ac chromatin that are in proximity, mainly in cis.To: Ministerio de Economia y Competitividad, (Spain) (Grant number: BFU2009-10792) and The Medical Research Council (UK), for supporting this work. We also thank Fundação Ciência e Tecnologia, Portugal for funding RPN.Peer Reviewe

Expanded GAA repeats impair FXN gene expression and reposition the FXN locus to the nuclear lamina in single cells

© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] expanded DNA repeats are associated with several neurodegenerative diseases. In Friedreich's ataxia (FRDA), expanded GAA repeats in intron 1 of the frataxin gene (FXN) reduce FXN mRNA levels in averaged cell samples through a poorly understood mechanism. By visualizing FXN expression and nuclear localization in single cells, we show that GAA-expanded repeats decrease the number of FXN mRNA molecules, slow transcription, and increase FXN localization at the nuclear lamina (NL). Restoring histone acetylation reverses NL positioning. Expanded GAA-FXN loci in FRDA patient cells show increased NL localization with increased silencing of alleles and reduced transcription from alleles positioned peripherally. We also demonstrate inefficiencies in transcription initiation and elongation from the expanded GAA-FXN locus at single-cell resolution. We suggest that repressive epigenetic modifications at the expanded GAA-FXN locus may lead to NL relocation, where further repression may occur.This work was supported by Ataxia UK (fellowship no. 7125 to M.M.L.), the Friedreich's Ataxia Research Alliance (FARA) (to M.M.L. and R.W.-M.), BabelFAmily (to M.M.L.), Associazione Italiana per la lotta alle Sindromi Atassiche (AISA) (to M.M.L.), the European Union 7th Framework Program EFACTS (grant agreement no. 242193) (to R.W.-M.). M.M.L. is an Ataxia UK research fellow co-funded by FARA and AISA. A.M.S. was supported by Fundação para a Ciência e a Tecnologia PhD studentship (SFRH/BD/61048/2009). Advanced microscopy at Micron Oxford was supported by a Wellcome Trust Strategic Award (091911).info:eu-repo/semantics/publishedVersio