H4K16ac activates the transcription of transposable elements and contributes to their cis-regulatory function.

Mammalian genomes harbor abundant transposable elements (TEs) and their remnants, with numerous epigenetic repression mechanisms enacted to silence TE transcription. However, TEs are upregulated during early development, neuronal lineage, and cancers, although the epigenetic factors contributing to the transcription of TEs have yet to be fully elucidated. Here, we demonstrate that the male-specific lethal (MSL)-complex-mediated histone H4 acetylation at lysine 16 (H4K16ac) is enriched at TEs in human embryonic stem cells (hESCs) and cancer cells.

Satellite repeat transcripts modulate heterochromatin condensates and safeguard chromosome stability in mouse embryonic stem cells.

Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1ɑ. Mouse heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs).

Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal "snapshots" of how proteins are arranged on chromatin within mammalian nuclei.

Enhancer-Promoter Communication: It's Not Just About Contact.

-regulatory elements such as enhancers can be located even a million base pairs away from their cognate promoter and yet modulate gene transcription. Indeed, the 3D organisation of chromatin enables the establishment of long-range enhancer-promoter communication. The observation of long-range enhancer-promoter chromatin loops at active genes originally led to a model in which enhancers and promoters form physical contacts between each other to control transcription.

RNA, Genome Output and Input.

RNA, the transcriptional output of genomes, not only templates protein synthesis or directly engages in catalytic functions, but can feed back to the genome and serve as regulatory input for gene expression. Transcripts affecting the RNA abundance of other genes act by mechanisms similar to and in concert with protein factors that control transcription. Through recruitment or blocking of activating and silencing complexes to specific genomic loci, RNA and protein factors can favor transcription or lower the local gene expression potential.

Citrullination of HP1γ chromodomain affects association with chromatin.

Background: Stem cell differentiation involves major chromatin reorganisation, heterochromatin formation and genomic relocalisation of structural proteins, including heterochromatin protein 1 gamma (HP1γ). As the principal reader of the repressive histone marks H3K9me2/3, HP1 plays a key role in numerous processes including heterochromatin formation and maintenance.

Results: We find that HP1γ is citrullinated in mouse embryonic stem cells (mESCs) and this diminishes when cells differentiate, indicating that it is a dynamically regulated post-translational modification during stem cell differentiation.

FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells.

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.

Calculation of 3D genome structures for comparison of chromosome conformation capture experiments with microscopy: An evaluation of single-cell Hi-C protocols.

Single-cell chromosome conformation capture approaches are revealing the extent of cell-to-cell variability in the organization and packaging of genomes. These single-cell methods, unlike their multi-cell counterparts, allow straightforward computation of realistic chromosome conformations that may be compared and combined with other, independent, techniques to study 3D structure. Here we discuss how single-cell Hi-C and subsequent 3D genome structure determination allows comparison with data from microscopy.

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function.

Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells.

A microfluidic platform for trapping, releasing and super-resolution imaging of single cells.

A multi-layer device, combining hydrodynamic trapping with microfluidic valving techniques, has been developed for on-chip manipulation and imaging of single cells and particles. Such a device contains a flow layer with trapping channels to capture single particles or cells and a control layer with valve channels to selectively control the trap and release processes. Particles and cells have been successfully trapped and released using the proposed device.

Dynamic monitoring of single cell lysis in an impedance-based microfluidic device.

A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis.

Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localisation Microscopy.

Single-molecule localisation microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal 'snapshots' of how proteins are arranged on chromatin within mammalian nuclei.

Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device.

Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents.

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy.

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts.

Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet.

Single-molecule imaging of transcription factor binding to DNA in live mammalian cells.

Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror.

Label-free live-cell imaging of nucleic acids using stimulated Raman scattering microscopy.

Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks.

The polarity protein Par6 induces cell proliferation and is overexpressed in breast cancer.

The polarity protein complex Par6/atypical protein kinase (aPKC)/Cdc42 regulates polarization processes during epithelial morphogenesis, astrocyte migration, and axon specification. We, as well as others, have shown that this complex is also required for disruption of apical-basal polarity during the oncogene ErbB2-induced transformation and transforming growth factor beta-induced epithelial-mesenchymal transition of mammary epithelial cells. Here, we report that expression of Par6 by itself in mammary epithelial cells induces epidermal growth factor-independent cell proliferation and development of hyperplastic three-dimensional acini without affecting apical-basal polarity.