Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

SummaryCell proliferation changes concomitantly with fate transitions during reprogramming, differentiation, regeneration, and oncogenesis. Methods to resolve cell cycle length heterogeneity in real-time are currently lacking. Here, we describe a genetically encoded fluorescent reporter that captures live cell cycle speed using a single measurement. This reporter is based on the color-changing Fluorescent Timer (FT) protein, which emits blue fluorescence when newly synthesized before maturing into a red fluorescent protein. We generated a mouse strain expressing an H2B-FT fusion reporter from a universally active locus, and demonstrate that faster-cycling cells can be distinguished from slower-cycling ones based on the intracellular fluorescence ratio between the FT’s blue and red states. Using this reporter, we reveal the native cell cycle speed distributions of fresh hematopoietic cells, and demonstrate its utility in analyzing cell proliferation in solid tissues. This system is broadly applicable for dissecting functional heterogeneity associated with cell cycle dynamics in complex tissues.

The isotopic evolution of the Kohistan Ladakh arc from subduction initiation to continent arc collision

Magmatic arcs associated with subduction zones are the dominant active locus of continental crust formation, and evolve in space and time towards magmatic compositions comparable to that of continental crust. Accordingly, the secular evolution of magmatic arcs is crucial to the understanding of crust formation processes. In this paper we present the first comprehensive U–Pb, Hf, Nd and Sr isotopic dataset documenting c. 120 myr of magmatic evolution in the Kohistan-Ladakh paleo-island arc. We found a long-term magmatic evolution that is controlled by the overall geodynamic of the Neo-Tethys realm. Apart from the post-collisionnal melts, the intra-oceanic history of the arc shows two main episodes (150–80 Ma and 80–50 Ma) of distinct geochemical signatures involving the slab and the sub-arc mantle components that are intimately linked to the slab dynamics.

The Country, the City, and Visions of Modernity in 1930s China

The 1930s saw new research and extended debates over the nature of the countryside in China and its socio-economic and historical role. Those on different sides of these debates, many drawing on historical materialism, tended to recreate modernist assumptions. These included assumptions that history was unilinear, and that what was Western was also modern, universal, urban, and objective, while what was not Western was traditional, local, rural, and subjective. The modern nation state, made up of modern citizens, was the outcome of, and the active subject in, a progressive history.Yet other approaches, focusing on the role of the countryside in China's development, challenged not only aspects of modernity but the assumptions in which the debates were couched. The philosopher and social activist Liang Shuming (1893–1988) focused on the countryside as the creative field in China. He envisioned a new form of community that, instead of the state, would be the active locus for change. Liang's new, village communities would draw on but not reproduce cultural traditions across a ruptured history and form the basis of a non-capitalist industrialisation. Rather than appropriate the dominant image of modernity, Liang revisited its basic assumptions. In so doing, he generated fresh visions, opening up a different set of possibilities and recasting the relation of the country to the city, the nation and the future.

A 220-nucleotide deletion of the intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy chain locus activation

A tissue-specific transcriptional enhancer, Eμ, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Eμ results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Eμ-independent and Eμ-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression.

Distinct Functions of Dispersed GATA Factor Complexes at an Endogenous Gene Locus

ABSTRACT The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a “GATA switch” abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at +9.5 kb. Despite indistinguishable GATA-1 occupancy at −77 kb and +9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the −77 kb region. The −77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1- and GATA-2-expressing cells. By contrast, the +9.5 kb region exhibited considerably stronger enhancer activity in GATA-2- than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1- or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the −77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.

What the genome sequence is revealing about trypanosome antigenic variation

African trypanosomes evade humoral immunity through antigenic variation, whereby they switch expression of the gene encoding their VSG (variant surface glycoprotein) coat. Switching proceeds by duplication of silent VSG genes into a transcriptionally active locus. The genome project has revealed that most of the silent archive consists of hundreds of subtelomeric VSG tandem arrays, and that most of these are not functional genes. Precedent suggests that they can contribute combinatorially to the formation of expressed, functional genes through segmental gene conversion. These findings from the genome project have major implications for evolution of the VSG archive and for transmission of the parasite in the field.

A Human Globin Enhancer Causes both Discrete and Widespread Alterations in Chromatin Structure

ABSTRACT Gene activation requires alteration of chromatin structure to facilitate active transcription complex formation at a gene promoter. Nucleosome remodeling complexes and histone modifying complexes each play unique and interdependent roles in bringing about these changes. The role of distant enhancers in these structural alterations is not well understood. We studied nucleosome remodeling and covalent histone modification mediated by the β-globin locus control region HS2 enhancer at nucleosome-level resolution throughout a 5.5-kb globin gene model locus in vivo in K562 cells. We compared the transcriptionally active locus to one in which HS2 was inactivated by mutations in the core NF-E2 sites. In contrast to inactive templates, nucleosomes were mobilized in discrete areas of the active locus, including the HS2 core and the proximal promoter. Large differences in restriction enzyme accessibility between the active and inactive templates were limited to the regions of nucleosome mobilization, which subsumed the DNase I hypersensitive sites. In contrast to this discrete pattern, histone H3 and H4 acetylation and H3 K4 methylation were elevated across the entire active locus, accompanied by depletion of linker histone H1. The coding region of the gene differed from the regulatory regions, demonstrating both nucleosome mobilization and histone hyperacetylation, but lacked differences in restriction enzyme accessibility between transcriptionally active and inactive genes. Thus, although the histone modification pattern we observe is consistent with the spreading of histone modifying activity from the distant enhancer, the pattern of nucleosome mobilization is more compatible with direct contact between an enhancer and promoter.