Mechanism of EHMT2-mediated genomic imprinting associated with Prader-Willi syndrome.

Prader-Willi Syndrome (PWS) is caused by loss of expression of paternally expressed genes in the human 15q11.2-q13 imprinting domain. A set of imprinted genes that are active on the paternal but silenced on the maternal chromosome are intricately regulated by a bipartite imprinting center (PWS-IC) located in the PWS imprinting domain. In past work, we discovered that euchromatic histone lysine N-methyltransferase-2 (EHMT2/G9a) inhibitors were capable of un-silencing PWS-associated genes by restoring their expression from the maternal chromosome. Here, in mice lacking the Ehmt2 gene, we document unsilencing of the imprinted Snrpn/Snhg14 gene on the maternal chromosome in the late embryonic and postnatal brain. Using PWS and Angelman syndrome patient derived cells with either paternal or maternal deletion of 15q11-q13, we have found that chromatin of maternal PWS-IC is closed and has compact 3D folding confirmation. We further show that a new and distinct noncoding RNA preferentially transcribed from upstream of the PWS-IC interacts with EHMT2 and forms a heterochromatin complex to silence gene expression of SNRPN in CIS on maternal chromosome. Taken together, these findings demonstrate that allele-specific recruitment of EHMT2 is required to maintain the maternal imprints. Our findings provide novel mechanistic insights and support a new model for imprinting maintenance of the PWS imprinted domain.

Longitudinal single-cell transcriptional dynamics throughout neurodegeneration in SCA1.

Neurodegeneration is a protracted process involving progressive changes in myriad cell types that ultimately results in the death of vulnerable neuronal populations. To dissect how individual cell types within a heterogeneous tissue contribute to the pathogenesis and progression of a neurodegenerative disorder, we performed longitudinal single-nucleus RNA sequencing of mouse and human spinocerebellar ataxia type 1 (SCA1) cerebellar tissue, establishing continuous dynamic trajectories of each cell population. Importantly, we defined the precise transcriptional changes that precede loss of Purkinje cells and, for the first time, identified robust early transcriptional dysregulation in unipolar brush cells and oligodendroglia. Finally, we applied a deep learning method to predict disease state accurately and identified specific features that enable accurate distinction of wild-type and SCA1 cells. Together, this work reveals new roles for diverse cerebellar cell types in SCA1 and provides a generalizable analysis framework for studying neurodegeneration.

Perturb-tracing enables high-content screening of multiscale 3D genome regulators.

Three-dimensional (3D) genome organization becomes altered during development, aging, and disease1-23, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multiscale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screen, a new cellular barcode readout method (BARC-FISH), and chromatin tracing. We performed a loss-of-function 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 discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop-extrusion and A-B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. We found that the ATP-dependent helicase CHD7, the loss of which causes the congenital neural crest syndrome CHARGE24 and a chromatin remodeler previously shown to promote local chromatin openness25-27, counter-intuitively compacts chromatin over long range in different genomic contexts and cell backgrounds including neural crest cells, and globally represses gene expression. The DNA compaction effect of CHD7 is independent of its chromatin remodeling activity and does not require other protein partners. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome functions, such as global gene and nuclear regulation, in health and disease.

New mechanism of chromatin compartmentalization by BRD2.

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.

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations.

BACKGROUND: Topologically 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. RESULTS: Here 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. CONCLUSIONS: In sum, this study suggests that single-cell domains are genome architecture building blocks independent of the tested major epigenetic components.

Chromatin tracing and multiplexed imaging of nucleome architectures (MINA) and RNAs in single mammalian cells and tissue.

The genome is hierarchically organized into several 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 have been 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 have 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 d for experimental work and 2-3 d for data analysis. We expect these developments to be broadly applicable and to affect 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.

ProbeDealer is a convenient tool for designing probes for highly multiplexed fluorescence in situ hybridization.

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.

Dynamic acetylation of the kinetochore-associated protein HEC1 ensures accurate microtubule-kinetochore attachment.

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.

Solution for the nonuniformity correction of infrared focal plane arrays.

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.