STAT (Signal transducer and activator of transcription) is a potent transcription factor and its aberrant activation by phosphorylation is associated with human cancers [1][2][3][4] . We have shown previously that overactivation of JAK, which phosphorylates STAT 5,6 , disrupts heterochromatin formation globally in Drosophila melanogaster 7 . However, it remains unclear how this effect is mediated and whether STAT is involved. Here, we demonstrate that Drosophila STAT (STAT92E) is involved in controlling heterochromatin protein 1 (HP1) distribution and heterochromatin stability. We found, unexpectedly, that loss of STAT92E, had the same effects as overactivation of JAK in disrupting heterochromatin formation and heterochromatic gene silencing, whereas overexpression of STAT92E had the opposite effects. We have further shown that the unphosphorylated or 'transcriptionally inactive' form of STAT92E is localized on heterochromatin in association with HP1, and is required for stabilizing HP1 localization and histone H3 Lys 9 methylation (H3mK9). However, activation by phosphorylation reduces heterochromatin-associated STAT92E, causing HP1 displacement and heterochromatin destabilization. Thus, reducing levels of unphosphorylated STAT92E, either by loss of STAT92E or increased phosphorylation, causes heterochromatin instability. These results suggest that activation of STAT by phosphorylation controls both access to chromatin and activity of the transcription machinery.To understand the molecular mechanism underlying JAK/STAT-mediated tumour formation, we have previously investigated the role of JAK in a Drosophila leukaemia model, in which a hyperactive mutant form of JAK (Tum-1) causes leukaemia-like overproliferation of blood cells 5,8 . We have demonstrated that oncogenic JAK disrupts heterochromatin formation globally, allowing transcriptional activation of genes that are not necessarily direct targets of STAT 7,9 . The molecular mechanism underlying the effects of Hopscotch (Hop, Drosophila JAK) on heterochromatin remains unclear. It may be mediated by phosphorylation of STAT92E, as in the canonical JAK/STAT pathway 10,11 . Alternatively, Hop may activate cellular targets other than STAT92E 9 .To investigate whether disruption of heterochromatin induced by Hop-activation 7 is mediated by STAT92E, we examined the effects of reducing stat92E + dosage on heterochromatic gene silencing, which can be measured by position-effect variegation (PEV) 12 We next examined the epistatic relationship between HP1 and STAT92E ( Fig. 1d-k). HP1, encoded by Su(var)205, is a constitutive component of heterochromatin and is essential for heterochromatic gene silencing 12,13 . Consistent with the results described above, we found that increasing stat92E + dosage by a chromosomal duplication (referred to as 3 × stat92E + , Fig. 1f) or a stat92E + transgene ( Supplementary Information, Fig. S1) enhanced heterochromatic gene silencing, resulting in complete silencing of the variegated white + gene. This effect was antagonized,...

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A single pulse interferometric coherent anti-Stokes Raman (CARS) spectroscopy method is used to obtain broadband CARS spectra and microscopy images of liquid and polymer samples. The pump, Stokes, and probe pulses are all selected inside a single broadband ultrafast pulse by a phase- and polarization-controlled pulse shaping technique and used to generate two spectral interference CARS signals simultaneously. The normalized difference of these two signals provides an amplified background-free broadband resonant CARS spectrum over the 400-1500 cm(-1) range with 35 cm(-1) spectral resolution. Chemically selective microscopy images of multicomponent polymer and liquid samples are investigated with this new CARS method. Multiplex CARS spectra at 10,000 spatial points are measured within a few minutes, and used to construct chemically selective microscopy images with a spatial resolution of 400 nm. The spectral bandwidth limits, sensitivity, homodyne amplification advantages, spatial resolution, depolarization, chromatic aberration, and chemical imaging aspects of this new technique are discussed in detail.

A novel Fourier transform spectral interferometric (FTSI) multiplex coherent anti-Stokes Raman scattering (CARS) technique is developed to extract the vibrational spectrum equivalent to the spontaneous Raman scattering. The conventional FTSI method is modified to use the internal nonresonant CARS signal as a local oscillator to perform spectral interferometry. Utilizing the causality of the coherent vibration (i.e., there should be no signal before the laser excitation), this new FTSI method recovers the entire complex vibrational spectral parameters. We demonstrate this technique with a previously reported single-pulse multiplex CARS method that uses a single phase-controlled broadband ultrafast laser pulse.

We present a significant sensitivity improvement of interferometric multiplex coherent anti-Stokes Raman scattering (CARS) by optimizing the power, bandwidth and phase of the pump, Stokes, and probe pulses independently. Fourier transform spectral interferometry (FTSI) is used to retrieve the entire complex quantity of the CARS spectrum by utilizing the non-resonant background as a local oscillator. Background-free spontaneous Raman-like vibrational spectra can be measured over the 500-1400 cm -1 range with 20 cm -1 spectral resolution within a tens of microseconds time scale. Chemically selective microscopy of a multicomponent polymer film is performed to demonstrate the feasibility of its microscopy application. A systematic analysis of the signal recovery method and several technical issues are discussed.

Heterochromatin is a form of highly compacted chromatin associated with epigenetic gene silencing and chromosome organization. We have previously shown that unphosphorylated nuclear signal transducer and activator of transcription (STAT) physically interacts with heterochromatin protein 1 (HP1) to promote heterochromatin stability. To understand whether STAT and heterochromatin are important for maintenance of genome stability, we genetically manipulated the levels of unphosphorylated STAT and HP1 [encoded by Su(var)205] in Drosophila and examined the effects on chromosomal morphology and resistance to DNA damage under conditions of genotoxic stress. Here we show that, compared with wild-type controls, Drosophila mutants with reduced levels of unphosphorylated STAT or heterochromatin are more sensitive to radiation-induced cell cycle arrest, have higher levels of spontaneous and radiation-induced DNA damage, and exhibit defects in chromosomal compaction and segregation during mitosis. Conversely, animals with increased levels of heterochromatin exhibit less DNA damage and increased survival rate after irradiation. These results suggest that maintaining genome stability by heterochromatin formation and correct chromosomal packaging is essential for normal cellular functions and for survival of animals under genotoxic stress.

We present a new coherent anti-Stokes Raman scattering (CARS) method that can perform background-free microscopy and microspectroscopy at the vibrational fingerprint region. Chirped broad-band pulses from a single Ti:sapphire laser generate CARS signals over 800-1700 cm -1 with a spectral resolution of 20 cm -1 . Fast modulation of the time delay between the pump and Stokes pulses coupled with lock-in signal detection not only removes the nonresonant background but also produces Raman-like CARS signals. Chemical imaging and microspectroscopy are demonstrated with various samples such as edible oils, lipid membranes, skin tissue, and plant cell walls. Systematic studies of the signal generation mechanism and several fundamental aspects are discussed.

Chromatin insulators are functionally conserved DNA–protein complexes situated throughout the genome that organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the precise mechanisms are not yet understood. Here we identify the exosome, the highly conserved major cellular 3′ to 5′ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. Genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32 and CTCF insulator proteins. Colocalization occurs mainly at promoters but also boundary elements such as Mcp, Fab-8, scs and scs′, which overlaps with a promoter. Surprisingly, exosome associates primarily with promoters but not gene bodies of active genes, arguing against simple cotranscriptional recruitment to RNA substrates. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is required specifically for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel positive relationship between exosome and chromatin insulators throughout the genome.