Sheila Q. Xie

Polycomb Associates Genome-wide with a Specific RNA Polymerase II Variant, and Regulates Metabolic Genes in ESCs

Summary Polycomb repressor complexes (PRCs) are important chromatin modifiers fundamentally implicated in pluripotency and cancer. Polycomb silencing in embryonic stem cells (ESCs) can be accompanied by active chromatin and primed RNA polymerase II (RNAPII), but the relationship between PRCs and RNAPII remains unclear genome-wide. We mapped PRC repression markers and four RNAPII states in ESCs using ChIP-seq, and found that PRC targets exhibit a range of RNAPII variants. First, developmental PRC targets are bound by unproductive RNAPII (S5p+S7p−S2p−) genome-wide. Sequential ChIP, Ring1B depletion, and genome-wide correlations show that PRCs and RNAPII-S5p physically bind to the same chromatin and functionally synergize. Second, we identify a cohort of genes marked by PRC and elongating RNAPII (S5p+S7p+S2p+); they produce mRNA and protein, and their expression increases upon PRC1 knockdown. We show that this group of PRC targets switches between active and PRC-repressed states within the ESC population, and that many have roles in metabolism.

Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation

Mammalian chromosomes fold into arrays of megabase‐sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher‐order organization remains elusive. Here, we investigate TAD higher‐order interactions with Hi‐C through neuronal differentiation and show that they form a hierarchy of domains‐within‐domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree‐like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency.

Complex multi-enhancer contacts captured by genome architecture mapping

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

Poised Transcription Factories Prime Silent uPA Gene Prior to Activation

The association of poised genes with transcription factories may contribute to rapid transcriptional activation in response to stimuli and to silencing when genes are located at the interior of their chromosome territories.

Proteomic Analysis of Mitotic RNA Polymerase II Reveals Novel Interactors and Association With Proteins Dysfunctional in Disease

RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology.

Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells

Gene expression states influence the 3D conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features that define promoter activation states or binding sites for the transcription factor CTCF. Single-cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fiber fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions.

Applying an Adaptive Watershed to the Tissue Cell Quantification During T-Cell Migration and Embryonic Development

Cell and particle quantification is one of the frequently used techniques in biology and clinical study. Variations of cell/particle population and/or protein expression level can provide information on many biological processes. In this chapter, we propose an image-based automatic quantification approach that can be applied to images from both fluorescence and electron microscopy. The algorithm uses local maxima to identify labelling targets and uses watershed segmentation to define their boundaries. The method is able to provide information on size, intensity centroids and average intensity within the labelling partitions. Further developed from this method, we demonstrated its applications in four different research projects, including recruitment enumeration of circulating T cell in non-lymphoid tissues, cell clustering in the early development of the chick embryo, gold particle localization and clustering in electron microscopy, and registration/co-localization of transcription factors in neural tube development of early chick embryo. The advantages and limitations of the method are also discussed.

CryoFISH: Fluorescence In Situ Hybridization on Ultrathin Cryosections

The visualization of cellular structures and components has become an invaluable tool in biological and medical sciences. Imaging subcellular compartments and single molecules within a cell has prompted the development of a wide range of sample preparation techniques as well as various microscope devices to obtain images with increased spatial resolution. Here, we present cryoFISH, a method for fluorescence in situ hybridization (FISH) on thin ( approximately 150 nm thick) cryosections from sucrose-embedded fixed cells or tissues. CryoFISH can be used in combination with immunodetection (IF) of other cellular components. The main advantages of cryoFISH and cryoIF over whole-cell labeling methods are increased spatial resolution with confocal microscopy, greater sensitivity of detection due to increased probe accessibility, and better image contrast. CryoFISH and cryoIF methods typically used on samples fixed in conditions that preserve ultrastructure, are compatible with the labeling of cells in their tissue context and are ideal for correlative studies that compare fluorescence with electron microscopy.