Fast fluorescence microscopy for imaging the dynamics of embryonic development

Live imaging has gained a pivotal role in developmental biology since it increasingly allows real-time observation of cell behavior in intact organisms. Microscopes that can capture the dynamics of ever-faster biological events, fluorescent markers optimal for in vivo imaging, and, finally, adapted reconstruction and analysis programs to complete data flow all contribute to this success. Focusing on temporal resolution, we discuss how fast imaging can be achieved with minimal prejudice to spatial resolution, photon count, or to reliably and automatically analyze images. In particular, we show how integrated approaches to imaging that combine bright fluorescent probes, fast microscopes, and custom post-processing techniques can address the kinetics of biological systems at multiple scales. Finally, we discuss remaining challenges and opportunities for further advances in this field

NFATc1 supports imiquimod-induced skin inflammation by suppressing IL-10 synthesis in B cells

Epicutaneous application of Aldara cream containing the TLR7 agonist imiquimod (IMQ) to mice induces skin inflammation that exhibits many aspects of psoriasis, an inflammatory human skin disease. Here we show that mice depleted of B cells or bearing interleukin (IL)-10-deficient B cells show a fulminant inflammation upon IMQ exposure, whereas ablation of NFATc1 in B cells results in a suppression of Aldara-induced inflammation. In vitro, IMQ induces the proliferation and IL-10 expression by B cells that is blocked by BCR signals inducing NFATc1. By binding to HDAC1, a transcriptional repressor, and to an intronic site of the Il10 gene, NFATc1 suppresses IL-10 expression that dampens the production of tumour necrosis factor-α and IL-17 by T cells. These data indicate a close link between NFATc1 and IL-10 expression in B cells and suggest NFATc1 and, in particular, its inducible short isoform, NFATc1/αA, as a potential target to treat human psoriasis

The genesis of cerebellar interneurons and the prevention of neural DNA damage require XRCC1

Defective responses to DNA single strand breaks underlie various neurodegenerative diseases. However, the exact role of this repair pathway during the development and maintenance of the nervous system is unclear. Using murine neural-specific inactivation of Xrcc1, a factor that is critical for the repair of DNA single strand breaks, we found a profound neuropathology that is characterized by the loss of cerebellar interneurons. This cell loss was linked to p53-dependent cell cycle arrest and occurred as interneuron progenitors commenced differentiation. Loss of Xrcc1 also led to the persistence of DNA strand breaks throughout the nervous system and abnormal hippocampal function. Collectively, these data detail the in vivo link between DNA single strand break repair and neurogenesis and highlight the diverse consequences of specific types of genotoxic stress in the nervous system

Heavy Ion Carcinogenesis and Human Space Exploration

Prior to the human exploration of Mars or long duration stays on the Earth s moon, the risk of cancer and other diseases from space radiation must be accurately estimated and mitigated. Space radiation, comprised of energetic protons and heavy nuclei, has been show to produce distinct biological damage compared to radiation on Earth, leading to large uncertainties in the projection of cancer and other health risks, while obscuring evaluation of the effectiveness of possible countermeasures. Here, we describe how research in cancer radiobiology can support human missions to Mars and other planets

Identification of a Small TAF Complex and Its Role in the Assembly of TAF-Containing Complexes

TFIID plays a role in nucleating RNA polymerase II preinitiation complex assembly on protein-coding genes. TFIID is a multisubunit complex comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). Another class of multiprotein transcriptional regulatory complexes having histone acetyl transferase (HAT) activity, and containing TAFs, includes TFTC, STAGA and the PCAF/GCN5 complex. Looking for as yet undiscovered subunits by a proteomic approach, we had identified TAF8 and SPT7L in human TFTC preparations. Subsequently, however, we demonstrated that TAF8 was not a stable component of TFTC, but that it is present in a small TAF complex (SMAT), containing TAF8, TAF10 and SPT7L, that co-purified with TFTC. Thus, TAF8 is a subunit of both TFIID and SMAT. The latter has to be involved in a pathway of complex formation distinct from the other known TAF complexes, since these three histone fold (HF)-containing proteins (TAF8, TAF10 and SPT7L) can never be found together either in TFIID or in STAGA/TFTC HAT complexes. Here we show that TAF8 is absolutely necessary for the integration of TAF10 in a higher order TFIID core complex containing seven TAFs. TAF8 forms a heterodimer with TAF10 through its HF and proline rich domains, and also interacts with SPT7L through its C-terminal region, and the three proteins form a complex in vitro and in vivo. Thus, the TAF8-TAF10 and TAF10-SPT7L HF pairs, and also the SMAT complex, seem to be important regulators of the composition of different TFIID and/or STAGA/TFTC complexes in the nucleus and consequently may play a role in gene regulation

Close 3D proximity of evolutionary breakpoints argues for the notion of spatial synteny

<p>Abstract</p> <p>Background</p> <p>Folding and intermingling of chromosomes has the potential of bringing close to each other loci that are very distant genomically or even on different chromosomes. On the other hand, genomic rearrangements also play a major role in the reorganisation of loci proximities. Whether the same loci are involved in both mechanisms has been studied in the case of somatic rearrangements, but never from an evolutionary standpoint.</p> <p>Results</p> <p>In this paper, we analysed the correlation between two datasets: (i) whole-genome chromatin contact data obtained in human cells using the Hi-C protocol; and (ii) a set of breakpoint regions resulting from evolutionary rearrangements which occurred since the split of the human and mouse lineages. Surprisingly, we found that two loci distant in the human genome but adjacent in the mouse genome are significantly more often observed in close proximity in the human nucleus than expected. Importantly, we show that this result holds for loci located on the same chromosome regardless of the genomic distance separating them, and the signal is stronger in gene-rich and open-chromatin regions.</p> <p>Conclusions</p> <p>These findings strongly suggest that part of the 3D organisation of chromosomes may be conserved across very large evolutionary distances. To characterise this phenomenon, we propose to use the notion of spatial synteny which generalises the notion of genomic synteny to the 3D case.</p

Signatures of Selection in Fusion Transcripts Resulting From Chromosomal Translocations in Human Cancer

BACKGROUND: The recurrence and non-random distribution of translocation breakpoints in human tumors are usually attributed to local sequence features present in the vicinity of the breakpoints. However, it has also been suggested that functional constraints might contribute to delimit the position of translocation breakpoints within the genes involved, but a quantitative analysis of such contribution has been lacking. METHODOLOGY: We have analyzed two well-known signatures of functional selection, such as reading-frame compatibility and non-random combinations of protein domains, on an extensive dataset of fusion proteins resulting from chromosomal translocations in cancer. CONCLUSIONS: Our data provide strong experimental support for the concept that the position of translocation breakpoints in the genome of cancer cells is determined, to a large extent, by the need to combine certain protein domains and to keep an intact reading frame in fusion transcripts. Additionally, the information that we have assembled affords a global view of the oncogenic mechanisms and domain architectures that are used by fusion proteins. This can be used to assess the functional impact of novel chromosomal translocations and to predict the position of breakpoints in the genes involved

Cell Cycle-Dependent Induction of Homologous Recombination by a Tightly Regulated I-SceI Fusion Protein

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase

ATM Modulates the Loading of Recombination Proteins onto a Chromosomal Translocation Breakpoint Hotspot

Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots