Mitotic chromosomes fold as compact arrays of chromatin loops. To identify the pathway of mitotic chromosome formation, we combined imaging and Hi-C analysis of synchronous DT40 cell cultures with ...polymer simulations. Here we show that in prophase, the interphase organization is rapidly lost in a condensin-dependent manner, and arrays of consecutive 60-kilobase (kb) loops are formed. During prometaphase, ~80-kb inner loops are nested within ~400-kb outer loops. The loop array acquires a helical arrangement with consecutive loops emanating from a central "spiral staircase" condensin scaffold. The size of helical turns progressively increases to ~12 megabases during prometaphase. Acute depletion of condensin I or II shows that nested loops form by differential action of the two condensins, whereas condensin II is required for helical winding.
The mechanism by which chromatids and chromosomes are segregated during mitosis and meiosis is a major puzzle of biology and biophysics. Using polymer simulations of chromosome dynamics, we show that ...a single mechanism of loop extrusion by condensins can robustly compact, segregate and disentangle chromosomes, arriving at individualized chromatids with morphology observed in vivo. Our model resolves the paradox of topological simplification concomitant with chromosome 'condensation', and explains how enzymes a few nanometers in size are able to control chromosome geometry and topology at micron length scales. We suggest that loop extrusion is a universal mechanism of genome folding that mediates functional interactions during interphase and compacts chromosomes during mitosis.
Topologically associating domains (TADs) are fundamental structural and functional building blocks of human interphase chromosomes, yet the mechanisms of TAD formation remain unclear. Here, we ...propose that loop extrusion underlies TAD formation. In this process, cis-acting loop-extruding factors, likely cohesins, form progressively larger loops but stall at TAD boundaries due to interactions with boundary proteins, including CTCF. Using polymer simulations, we show that this model produces TADs and finer-scale features of Hi-C data. Each TAD emerges from multiple loops dynamically formed through extrusion, contrary to typical illustrations of single static loops. Loop extrusion both explains diverse experimental observations—including the preferential orientation of CTCF motifs, enrichments of architectural proteins at TAD boundaries, and boundary deletion experiments—and makes specific predictions for the depletion of CTCF versus cohesin. Finally, loop extrusion has potentially far-ranging consequences for processes such as enhancer-promoter interactions, orientation-specific chromosomal looping, and compaction of mitotic chromosomes.
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•TADs can be formed by loop extrusion limited by boundary elements•Polymer simulations and genomic analyses were jointly used to test this proposal•Proposed roles of cohesin and CTCF reconcile diverse experimental observations
Topologically associating domains (TADs) are fundamental building blocks of human interphase chromosomes. Fudenberg et al. propose that TADs emerge as a consequence of loop extrusion limited by boundary elements. The authors use polymer simulations and genomic analyses to identify molecular roles for the architectural proteins cohesin and CTCF.
During cell division, chromosomes are compacted in length by more than a 100-fold. A wide range of experiments demonstrated that in their compacted state, mammalian chromosomes form arrays of closely ...stacked consecutive ∼100 kb loops. The mechanism underlying the active process of chromosome compaction into a stack of loops is unknown. Here we test the hypothesis that chromosomes are compacted by enzymatic machines that actively extrude chromatin loops. When such loop-extruding factors (LEF) bind to chromosomes, they progressively bridge sites that are further away along the chromosome, thus extruding a loop. We demonstrate that collective action of LEFs leads to formation of a dynamic array of consecutive loops. Simulations and an analytically solved model identify two distinct steady states: a sparse state, where loops are highly dynamic but provide little compaction; and a dense state, where there are more stable loops and dramatic chromosome compaction. We find that human chromosomes operate at the border of the dense steady state. Our analysis also shows how the macroscopic characteristics of the loop array are determined by the microscopic properties of LEFs and their abundance. When the number of LEFs are used that match experimentally based estimates, the model can quantitatively reproduce the average loop length, the degree of compaction, and the general loop-array morphology of compact human chromosomes. Our study demonstrates that efficient chromosome compaction can be achieved solely by an active loop-extrusion process.
The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the ...auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, our data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization.
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•Acute depletion of endogenous CTCF in ESCs with an auxin-inducible degron•CTCF instructs chromatin loops and TAD insulation genome wide•Disrupting TAD insulation does not impact higher-order genomic compartmentalization•CTCF depletion impacts transcriptional activity but not spread of H3K27me3 domains
Dissecting out the roles of CTCF on genome looping, organization into TADs, and overall chromatin compartmentalization reveals fine layers of regulation.
Extracting biologically meaningful information from chromosomal interactions obtained with genome-wide chromosome conformation capture (3C) analyses requires the elimination of systematic biases. We ...present a computational pipeline that integrates a strategy to map sequencing reads with a data-driven method for iterative correction of biases, yielding genome-wide maps of relative contact probabilities. We validate this ICE (iterative correction and eigenvector decomposition) technique on published data obtained by the high-throughput 3C method Hi-C, and we demonstrate that eigenvector decomposition of the obtained maps provides insights into local chromatin states, global patterns of chromosomal interactions, and the conserved organization of human and mouse chromosomes.
We present Micro-C XL, an improved method for analysis of chromosome folding at mononucleosome resolution. Using long crosslinkers and isolation of insoluble chromatin, Micro-C XL increases ...signal-to-noise ratio. Micro-C XL maps of budding and fission yeast genomes capture both short-range chromosome fiber features such as chromosomally interacting domains and higher order features such as centromere clustering. Micro-C XL provides a single assay to interrogate chromosome folding at length scales from the nucleosome to the full genome.
Pyteomics is a cross-platform, open-source Python library providing a rich set of tools for MS-based proteomics. It provides modules for reading LC-MS/MS data, search engine output, protein sequence ...databases, theoretical prediction of retention times, electrochemical properties of polypeptides, mass and
m/z
calculations, and sequence parsing. Pyteomics is available under Apache license; release versions are available at the Python Package Index
http://pypi.python.org/pyteomics
, the source code repository at
http://hg.theorchromo.ru/pyteomics
, documentation at
http://packages.python.org/pyteomics
. Pyteomics.biolccc documentation is available at
http://packages.python.org/pyteomics.biolccc/
. Questions on installation and usage can be addressed to pyteomics mailing list: pyteomics@googlegroups.com
Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In ...particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses.
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•A subset of self-renewing HSCs in adult bone marrow was genetically marked•HSCs provide a major sustained contribution to endogenous hematopoiesis•HSCs give rise to all immune cell types except B-1a cells and tissue macrophages•Multilineage contribution of HSCs is accelerated by interferon response
The role of hematopoietic stem cells (HSCs) in steady-state hematopoiesis remains controversial. Here, Sawai et al. use lineage tracing to reveal a major contribution of HSCs to all blood cell lineages, including myeloid cells and lymphocytes, throughout adult life.
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