Dynamics of the 4D genome during in vivo lineage specification and differentiation

Mammalian gene expression patterns are controlled by regulatory elements, which interact within topologically associating domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. Here, we present Tiled-C, a low-input chromosome conformation capture (3C) technique. We use this approach to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. Integrated analysis of chromatin accessibility and single-cell expression data shows that regulatory elements gradually become accessible within pre-existing TADs during early differentiation. This is followed by structural re-organization within the TAD and formation of specific contacts between enhancers and promoters. Our high-resolution data show that these enhancer-promoter interactions are not established prior to gene expression, but formed gradually during differentiation, concomitant with progressive upregulation of gene activity. Together, these results provide new insight into the close, interdependent relationship between chromatin architecture and gene regulation during development

Expansion of the MANAGE Database with Forest and Drainage Studies

The “Measured Annual Nutrient loads from AGricultural Environments” (MANAGE) database was published in 2006 to expand an early 1980s compilation of nutrient export (load) data from cultivated and pasture/range land at the field or farm scale. Then in 2008, MANAGE was updated with 15 additional studies, and nitrogen (N) and phosphorus (P) concentrations in runoff were added. Since then, MANAGE has undergone significant expansion adding N and P water quality along with relevant management and site characteristic data from: (1) 30 runoff studies from forested land uses, (2) 91 drainage water quality studies from drained land, and (3) 12 additional runoff studies from cultivated and pasture/range land uses. In this expansion, an application timing category was added to the existing fertilizer data categories (rate, placement, formulation) to facilitate analysis of 4R Nutrient Stewardship, which emphasizes right fertilizer source, rate, time, and place. In addition, crop yield and N and P uptake data were added, although this information was only available for 21 and 7% of studies, respectively. Inclusion of these additional data from cultivated, pasture/range, and forest land uses as well as artificially drained agricultural land should facilitate expanded spatial analyses and improved understanding of regional differences, management practice effectiveness, and impacts of land use conversions and management techniques

Chromosome looping at the human alpha-globin locus is mediated via the major upstream regulatory element (HS-40)

Previous studies in the mouse have shown that high levels of alpha-globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human alpha cluster and their interactions with the alpha-globin promoter. All of these elements interact with the alpha-globin gene in an erythroid-specific manner. These results were confirmed in a mouse model of human alpha globin expression in which the human cluster replaces the mouse cluster in situ (humanized mouse). We have also shown that expression and all of the long-range interactions depend largely on just one of these elements; removal of the previously characterized major regulatory element (called HS -40) results in loss of all the interactions and alpha-globin expression. Reinsertion of this element at an ectopic location restores both expression and the intralocus interactions. In contrast to other more complex systems involving multiple upstream elements and promoters, analysis of the human alpha-globin cluster during erythropoiesis provides a simple and tractable model to understand the mechanisms underlying long-range gene regulation

Loss of Atrx Affects Trophoblast Development and the Pattern of X-Inactivation in Extraembryonic Tissues

ATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease associated with defects in such proteins. To better understand the role of ATRX in development and the associated abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked) syndrome, we conditionally inactivated the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues. Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the murine trophoblast and present an example of escape from imprinted X chromosome inactivation

Spitzer IRAC observations of newly-discovered planetary nebulae from the Macquarie-AAO-Strasbourg H-alpha Planetary Nebula Project

We compare H-alpha, radio continuum, and Spitzer Space Telescope (SST) images of 58 planetary nebulae (PNe) recently discovered by the Macquarie-AAO-Strasbo- urg H-alpha PN Project (MASH) of the SuperCOSMOS H-alpha Survey. Using InfraRed Array Camera (IRAC) data we define the IR colors of PNe and demonstrate good isolation between these colors and those of many other types of astronomical object. The only substantive contamination of PNe in the color-color plane we illustrate is due to YSOs. However, this ambiguity is readily resolved by the unique optical characteristics of PNe and their environs. We also examine the relationships between optical and MIR morphologies from 3.6 to 8.0um and explore the ratio of mid-infrared (MIR) to radio nebular fluxes, which is a valuable discriminant between thermal and nonthermal emission. MASH emphasizes late evolutionary stages of PNe compared with previous catalogs, enabling study of the changes in MIR and radio flux that attend the aging process. Spatially integrated MIR energy distributions were constructed for all MASH PNe observed by the GLIMPSE Legacy Project, using the H-alpha morphologies to establish the dimensions for the calculations of the Midcourse Space Experiment (MSX), IRAC, and radio continuum (from the Molonglo Observatory Synthesis Telescope and the Very Large Array) flux densities. The ratio of IRAC 8.0-um to MSX 8.3-um flux densities provides a measure of the absolute diffuse calibration of IRAC at 8.0 um. We independently confirm the aperture correction factor to be applied to IRAC at 8.0um to align it with the diffuse calibration of MSX. The result agrees with the recommendations of the Spitzer Science Center and with results from a parallel study of HII regions. These PNe probe the diffuse calibration of IRAC on a spatial scale of 9-77 arcsec.Comment: 48 pages, LaTeX (aastex), incl. 18 PostScript (eps) figures and 3 tables. Accepted by Astrophysical Journa

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

How transcriptional and epigenetic programmes are played out on an individual mammalian gene cluster during lineage commitment and differentiation

In the post-genomic era, a great deal of work has focused on understanding how DNA sequence is used to programme complex nuclear, cellular and tissue functions throughout differentiation and development. There are many approaches to these issues, but we have concentrated on understanding how a single mammalian gene cluster is activated or silenced as stem cells undergo lineage commitment, differentiation and maturation. In particular we have analysed the alpha globin cluster, which is expressed in a cell-type- and developmental stage-specific manner in the haemopoietic system. Our studies include analysis of the transcriptional programme that accompanies globin gene activation, focusing on the expression of relevant transcription factors and cofactors. Binding of these factors to the chromosomal domain containing the alpha globin cluster has been characterized by ChIP (chromatin immunoprecipitation). In addition, we have monitored the epigenetic modifications (e.g. nuclear position, timing of replication, chromatin modification, DNA methylation) that occur as the genes are activated (in erythroid cells) or silenced (e.g. in granulocytes) as haemopoiesis proceeds. Together, these observations provide a uniquely well-characterized model illustrating the mechanisms that regulate and memorize patterns of mammalian gene expression as stem cells undergo lineage specification, differentiation and terminal maturation

Manipulating the Mouse Genome to Engineer Precise Functional Syntenic Replacements with Human Sequence

SummaryWe have devised a strategy (called recombinase-mediated genomic replacement, RMGR) to allow the replacement of large segments (>100 kb) of the mouse genome with the equivalent human syntenic region. The technique involves modifying a mouse ES cell chromosome and a human BAC by inserting heterotypic lox sites to flank the proposed exchange interval and then using Cre recombinase to achieve segmental exchange. We have demonstrated the feasibility of this approach by replacing the mouse α globin regulatory domain with the human syntenic region and generating homozygous mice that produce only human α globin chains. Furthermore, modified ES cells can be used iteratively for functional studies, and here, as an example, we have used RMGR to produce an accurate mouse model of human α thalassemia. RMGR has general applicability and will overcome limitations inherent in current transgenic technology when studying the expression of human genes and modeling human genetic diseases

Generation of bivalent chromatin domains during cell fate decisions

<p>Abstract</p> <p>Background</p> <p>In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined.</p> <p>Results</p> <p>Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3.</p> <p>Conclusions</p> <p>While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different.</p