Search

BackgroundTopologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs has been shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing lead to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains, which are highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies. Recent computational modeling studies suggest that epigenetic interactions may underlie the formation of the single-cell domains.ResultsHere we use chromatin tracing to visualize in female human cells the fine-scale chromatin folding of inactive and active X chromosomes, which are known to have distinct global epigenetic landscapes and distinct population-averaged TAD profiles, with inactive X chromosomes largely devoid of TADs and cohesin. We show that both inactive and active X chromosomes possess highly variant single-cell domains across the same genomic region despite the fact that only active X chromosomes show clear TAD structures at the population level. These X chromosome single-cell domains exist in distinct cell lines. Perturbations of major epigenetic components and transcription mostly do not affect the frequency or strength of the single-cell domains. Increased chromatin compaction of inactive X chromosomes occurs at a length scale above that of the single-cell domains.ConclusionsIn sum, this study suggests that single-cell domains are genome architecture building blocks independent of the tested major epigenetic components.

The molecular mechanism underlying 3D genome compartmentalization remains a mystery. Xie et al. found that the bromodomain and extraterminal (BET) family scaffold protein BRD2 promotes the compartmentalization of the accessible chromatin domains after cohesin loss. An antagonistic interplay between loop extrusion and compartmentalization modulates chromatin folding.

Three-dimensional (3D) genome organization becomes altered during development, aging, and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of long-range chromatin organization. Here, we developed an image-based high-content CRISPR screening platform that combines a new FISH-based barcode readout method (BARC-FISH) with chromatin tracing. We performed a pooled loss-of-function genetic screen in human cells and visualized alterations to their genome organization from 13,000 imaging target-perturbation combinations, alongside perturbation-paired barcode readout in the same single cells. Using 1.4 million 3D positions along chromosome traces, we identified 26 regulators of chromatin architectures at different length scales. We found that the ATP-dependent helicase CHD7, the loss of which causes the congenital syndrome CHARGE, cooperates with CTCF to promote large-scale chromatin compaction. Altogether, our method enables scalable, high-throughput identification of chromatin topology regulators that will stimulate new insights into the 3D genome.One-Sentence SummaryIn situCRISPR screen, cellular barcoding, and chromatin tracing identify regulators of higher-order chromatin organization.

Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore–microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore–microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80–microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1–80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80–microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore–microtubule attachment in the maintenance of genomic stability during mitosis.

Fluorescence in situ hybridization (FISH) is a powerful method to visualize the spatial positions of specific genomic loci and RNA species. Recent technological advances have leveraged FISH to visualize these features in a highly multiplexed manner. Notable examples include chromatin tracing, RNA multiplexed error-robust FISH (MERFISH), multiplexed imaging of nucleome architectures (MINA), and sequential single-molecule RNA FISH. However, one obstacle to the broad adoption of these methods is the complexity of the multiplexed FISH probe design. In this paper, we introduce an easy-to-use, versatile, and all-in-one application called ProbeDealer to design probes for a variety of multiplexed FISH techniques and their combinations. ProbeDealer offers a one-stop shop for multiplexed FISH design needs of the research community.

The genome is hierarchically organized into multiple three-dimensional (3D) architectures including chromatin loops, domains, compartments, and regions associated with nuclear lamina and nucleoli. Changes in these architectures have been associated with normal development, aging, and a wide range of diseases. Despite its critical importance, understanding how the genome is spatially organized in single cells, how organization varies in different cell types in mammalian tissue, and how organization affects gene expression remains a major challenge. Previous approaches are limited by a lack of capacity to directly trace chromatin folding in 3D, and to simultaneously measure genomic organization in relation to other nuclear components and gene expression in the same, single cells. We developed an image-based 3D genomics technique termed chromatin tracing, which enables direct 3D tracing of chromatin folding along individual chromosomes in single cells. More recently, we also developed Multiplexed Imaging of Nucleome Architectures (MINA), which enables simultaneous measurements of multiscale chromatin folding, associations of genomic regions with nuclear lamina and nucleoli, and copy numbers of numerous RNA species in the same, single cells in mammalian tissue. Here we provide detailed protocols for chromatin tracing in cell lines and MINA in mammalian tissue, which take 3–4 days for experimental work and 2–3 days for data analysis. We expect these developments to be broadly applicable and to impact many lines of research on 3D genomics by depicting multiscale genomic architectures associated with gene expression, in different types of cells and tissue undergoing different biological processes.

In order to make the system design meet the requirements of practical ghost imaging, the impact of mechanical vibration on the ghost imaging is analyzed. In ghost imaging system, the light field modulated by a digital micromirror device (DMD) is used to illuminate the target and the transmission or scattering light is detected by a single pixel detector. The target is reconstructed by combining the results of the detector and the intensity distribution of light field, so the modulation matrices of light field play a vital role in ghost imaging. By considering the form of imaging system to vibration and taking the modulation transfer function as an evaluation function, this paper quantitatively analyzes the impacts of various forms of mechanical vibration on the intensity distribution of light field. Combining engineering practice, several solutions are proposed to reduce the impact of vibration on the imaging quality. The results of simulation and experiment indicated that the analysis is correct and usable.

Gradient ghost imaging (GGI) is a new imaging method that can directly obtain the edge information of an unknown object from the correlation of light intensity fluctuations. However, this technique faces the problems of poor directionality and continuity. In this paper, we propose a multidirectional gradient ghost imaging scheme (MGGI), where Kirsch operator with 8-directional masks is first used to generate structured illuminations, and then a fast algorithm based on the connection of the 8 masks is applied in the sampling process. Compared with GGI based on Sobel operator, both simulation and experimental results demonstrate that the proposed method has the advantages of imaging quality and edge continuity even in a noisy environment. Furthermore, the MGGI does not increase the imaging time and thus promotes the practical development of ghost imaging.

In order to improve the visualization of the output data of gaseous pollutants monitoring system and study pseudo-color image generation technology, this research combines the column concentration data of polluted gas with spatial position parameter to design a grey-color conversion method based on visual pseudo-color coding, generates the pseudo-color images of column concentration of the distribution of polluted gas and evaluates the pseudo-color coding scheme designed in HSI color space. The evaluation results show that the designed coding scheme can effectively conduct pseudo-color display for the concentration section of polluted gas.

Gradient ghost imaging (GGI) is a new imaging method that can directly extract the edge of a target from the correlation of light intensity fluctuations. However, there are problems of poor image quality and practicability in GGI. The corner point is an important target feature and has important applications in the fields of image processing and machine vision. In this paper, we propose a corner detection scheme that combines a corner detection algorithm based on curvature scale space with a GGI system, which can directly extract corner points from the reconstruction results of ghost imaging. A simulation and experimental results show that our method can acquire precise and robust corner information of an unknown target in undersampled cases, which promotes the practical development of ghost imaging.

Ghost imaging is an indirect system that allows the imaging of an object without directly seeing the object. The speckle pattern that contains the information about light and objects has increasingly become a popular topic in pseudothermal light ghost imaging. However, existing research still has encountered problems of poor imaging quality and slow sampling speeds. We propose a ghost imaging method based on N-order speckle patterns to recover the object (NSGI). The N-order speckle patterns combine N independent laser speckles individually produced by passing an expanded and collimated He–Ne laser through a digital micromirror device (DMD). The sampling frequency can be improved by controlling the trigger signals of different DMDs. The results of the simulation and experiment have verified that our method can increase sampling speed and reconstruction accuracy. In addition, NSGI can be applied to more applications by designing multiple independent speckles with different properties.

In this paper, we address the problem of tracking a randomly moving target in the similar background. Traditional color particle filter algorithm cannot actively judge the interference created by similar background, and cause that particle filter estimate by mistakes. So we propose and implement the tracking by using structural similarity as a standard for judging tracking states. We establish unified color scale for background to extract color histogram so that we can real-time track moving target. Further we calculate the average structural similarity between the target and background, which is weighed by particles weight, to establish tracking model. The performance of the considered algorithm which is illustrated through computer simulations shows the improved color particle filter that is more effective when both the moving target and the background are very similar.

Based on the S-curve model of the detector response of infrared focal plan arrays (IRFPAs), an improved two-point correction algorithm is presented. The algorithm first transforms the nonlinear image data into linear data and then uses the normal two-point algorithm to correct the linear data. The algorithm can effectively overcome the influence of nonlinearity of the detector's response, and it enlarges the correction precision and the dynamic range of the response. A real-time imaging-signal-processing system for IRFPAs that is based on a digital signal processor and field-programmable gate arrays is also presented. The nonuniformity correction capability of the presented solution is validated by experimental imaging procedures of a 128 x 128 pixel IRFPA camera prototype.