CTCF-binding locations represent regulatory sequences that are highly constrained over the course of evolution. To gain insight into how these DNA elements are conserved and spread through the ...genome, we defined the full spectrum of CTCF-binding sites, including a 33/34-mer motif, and identified over five thousand highly conserved, robust, and tissue-independent CTCF-binding locations by comparing ChIP-seq data from six mammals. Our data indicate that activation of retroelements has produced species-specific expansions of CTCF binding in rodents, dogs, and opossum, which often functionally serve as chromatin and transcriptional insulators. We discovered fossilized repeat elements flanking deeply conserved CTCF-binding regions, indicating that similar retrotransposon expansions occurred hundreds of millions of years ago. Repeat-driven dispersal of CTCF binding is a fundamental, ancient, and still highly active mechanism of genome evolution in mammalian lineages.
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► CTCF-binding locations are highly conserved across mammals ► New locations for CTCF binding are carried by SINE repeats in many mammals ► Ancient and newly born CTCF-binding events similarly demarcate chromatin barriers ► Retroelements can reposition organizing elements throughout the genome
Evolutionary analysis of six divergent mammals uncovers both highly conserved and species-specific binding locations of the chromatin organizer, CTCF. This study reveals that activation of retroelements drives genome evolution by repositioning genome-organizing elements.
Adipocyte development and differentiation have an important role in the aetiology of obesity and its co-morbidities
. Although multiple studies have investigated the adipogenic stem and precursor ...cells that give rise to mature adipocytes
, our understanding of their in vivo origin and properties is incomplete
. This is partially due to the highly heterogeneous and unstructured nature of adipose tissue depots
, which has proven difficult to molecularly dissect using classical approaches such as fluorescence-activated cell sorting and Cre-lox lines based on candidate marker genes
. Here, using the resolving power of single-cell transcriptomics
in a mouse model, we reveal distinct subpopulations of adipose stem and precursor cells in the stromal vascular fraction of subcutaneous adipose tissue. We identify one of these subpopulations as CD142
adipogenesis-regulatory cells, which can suppress adipocyte formation in vivo and in vitro in a paracrine manner. We show that adipogenesis-regulatory cells are refractory to adipogenesis and that they are functionally conserved in humans. Our findings point to a potentially critical role for adipogenesis-regulatory cells in modulating adipose tissue plasticity, which is linked to metabolic control, differential insulin sensitivity and type 2 diabetes.
Transcription factors (TFs) direct gene expression by binding to DNA regulatory regions. To explore the evolution of gene regulation, we used chromatin immunoprecipitation with high-throughput ...sequencing (ChIP-seq) to determine experimentally the genome-wide occupancy of two TFs, CCAAT/enhancer-binding protein alpha and hepatocyte nuclear factor 4 alpha, in the livers of five vertebrates. Although each TF displays highly conserved DNA binding preferences, most binding is species-specific, and aligned binding events present in all five species are rare. Regions near genes with expression levels that are dependent on a TF are often bound by the TF in multiple species yet show no enhanced DNA sequence constraint. Binding divergence between species can be largely explained by sequence changes to the bound motifs. Among the binding events lost in one lineage, only half are recovered by another binding event within 10 kilobases. Our results reveal large interspecies differences in transcriptional regulation and provide insight into regulatory evolution.
The cohesin protein complex holds sister chromatids in dividing cells together and is essential for chromosome segregation. Recently, cohesin has been implicated in mediating transcriptional ...insulation, via its interactions with CTCF. Here, we show in different cell types that cohesin functionally behaves as a tissue-specific transcriptional regulator, independent of CTCF binding. By performing matched genome-wide binding assays (ChIP-seq) in human breast cancer cells (MCF-7), we discovered thousands of genomic sites that share cohesin and estrogen receptor alpha (ER) yet lack CTCF binding. By use of human hepatocellular carcinoma cells (HepG2), we found that liver-specific transcription factors colocalize with cohesin independently of CTCF at liver-specific targets that are distinct from those found in breast cancer cells. Furthermore, estrogen-regulated genes are preferentially bound by both ER and cohesin, and functionally, the silencing of cohesin caused aberrant re-entry of breast cancer cells into cell cycle after hormone treatment. We combined chromosomal interaction data in MCF-7 cells with our cohesin binding data to show that cohesin is highly enriched at ER-bound regions that capture inter-chromosomal loop anchors. Together, our data show that cohesin cobinds across the genome with transcription factors independently of CTCF, plays a functional role in estrogen-regulated transcription, and may help to mediate tissue-specific transcriptional responses via long-range chromosomal interactions.
Single-cell RNA-sequencing (scRNA-seq) allows whole transcriptome profiling of thousands of individual cells, enabling the molecular exploration of tissues at the cellular level. Such analytical ...capacity is of great interest to many research groups in the world, yet these groups often lack the expertise to handle complex scRNA-seq datasets.
We developed a fully integrated, web-based platform aimed at the complete analysis of scRNA-seq data post genome alignment: from the parsing, filtering and normalization of the input count data files, to the visual representation of the data, identification of cell clusters, differentially expressed genes (including cluster-specific marker genes), and functional gene set enrichment. This Automated Single-cell Analysis Pipeline (ASAP) combines a wide range of commonly used algorithms with sophisticated visualization tools. Compared with existing scRNA-seq analysis platforms, researchers (including those lacking computational expertise) are able to interact with the data in a straightforward fashion and in real time. Furthermore, given the overlap between scRNA-seq and bulk RNA-seq analysis workflows, ASAP should conceptually be broadly applicable to any RNA-seq dataset. As a validation, we demonstrate how we can use ASAP to simply reproduce the results from a single-cell study of 91 mouse cells involving five distinct cell types.
The tool is freely available at asap.epfl.ch and R/Python scripts are available at github.com/DeplanckeLab/ASAP.
bart.deplancke@epfl.ch.
Supplementary data are available at Bioinformatics online.
Expansion and differentiation of antigen-experienced PD-1
TCF-1
stem-like CD8
T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade
. Hashimoto et al. have ...shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8
T cells similar to those generated in an acute infection
. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor β- and γ-chain (IL-2Rβγ)-biased agonists are currently being developed
. Here we show that IL-2Rβγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rβγ on the same cell recovers the ability to differentiate stem-like CD8
T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.
The cohesin protein complex contributes to transcriptional regulation in a CTCF-independent manner by colocalizing with master regulators at tissue-specific loci. The regulation of transcription ...involves the concerted action of multiple transcription factors (TFs) and cohesin's role in this context of combinatorial TF binding remains unexplored. To investigate cohesin-non-CTCF (CNC) binding events in vivo we mapped cohesin and CTCF, as well as a collection of tissue-specific and ubiquitous transcriptional regulators using ChIP-seq in primary mouse liver. We observe a positive correlation between the number of distinct TFs bound and the presence of CNC sites. In contrast to regions of the genome where cohesin and CTCF colocalize, CNC sites coincide with the binding of master regulators and enhancer-markers and are significantly associated with liver-specific expressed genes. We also show that cohesin presence partially explains the commonly observed discrepancy between TF motif score and ChIP signal. Evidence from these statistical analyses in wild-type cells, and comparisons to maps of TF binding in Rad21-cohesin haploinsufficient mouse liver, suggests that cohesin helps to stabilize large protein-DNA complexes. Finally, we observe that the presence of mirrored CTCF binding events at promoters and their nearby cohesin-bound enhancers is associated with elevated expression levels.
Adipose tissue in the mammary gland undergoes dramatic remodeling during reproduction. Adipocytes are replaced by mammary alveolar structures during pregnancy and lactation, then reappear upon ...weaning. The fate of the original adipocytes during lactation and the developmental origin of the re-appearing adipocyte post involution are unclear. Here, we reveal that adipocytes in the mammary gland de-differentiate into Pdgfrα+ preadipocyte- and fibroblast-like cells during pregnancy and remain de-differentiated during lactation. Upon weaning, de-differentiated fibroblasts proliferate and re-differentiate into adipocytes. This cycle occurs over multiple pregnancies. These observations reveal the potential of terminally differentiated adipocytes to undergo repeated cycles of de-differentiation and re-differentiation in a physiological setting.
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•Adipocytes de-differentiate in the mammary gland during lactation•These de-differentiated adipocytes assume a preadipocyte phenotype•Adipocyte-derived preadipocytes re-differentiate during involution•This de- and re-differentiation occurs repeatedly with multiple pregnancies
Adipocytes in the mammary gland disappear during lactation. Wang et al. show that these mammary adipocytes fully de-differentiate into preadipocytes during lactation and readily re-differentiate during involution. The same adipocytes are therefore “recycled” over multiple rounds of pregnancies. De-differentiation constitutes a new possible fate for terminally differentiated adipocytes.
At least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse ...strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it.
► A mouse carrying human chromosome 21 fails to repress primate-specific repeats ► The lack of repression was revealed by H3K4me3 and transcription factor binding ► Activation corresponded to a decrease in CpG methylation ► Primate-specific repeats activated in human testes were activated in the Tc1 mouse
Recruitment of brite/beige cells, known as browning of white adipose tissue (WAT), is an efficient way to turn an energy-storing organ into an energy-dissipating one and may therefore be of ...therapeutic value in combating obesity. However, a comprehensive understanding of the regulatory mechanisms mediating WAT browning is still lacking. Here, we exploit the large natural variation in WAT browning propensity between inbred mouse strains to gain an inclusive view of the core regulatory network coordinating this cellular process. Combining comparative transcriptomics, perturbation-based validations, and gene network analyses, we present a comprehensive gene regulatory network of inguinal WAT browning, revealing up to four distinct regulatory modules with key roles for uncovered transcriptional factors, while also providing deep insights into the genetic architecture of brite adipogenesis. The presented findings therefore greatly increase our understanding of the molecular drivers mediating the intriguing cellular heterogeneity and plasticity of adipose tissue.
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•The browning capacity of white adipose tissue is under complex genetic control•Genetic architecture of brite adipogenesis is built from several genetic models•Dozens of Ucp1 regulating factors are validated•A core browning regulatory network with four distinct regulatory modules is revealed
Browning of white fat, which turns an energy-storing organ into an energy-dissipating one, holds promise for the development of novel therapeutics against obesity. By exploiting intrinsic mouse strain variation and applying a systems-genetics approach, Li et al. reveal a comprehensive gene regulatory network that controls this browning process.
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