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Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or clustered regularly interspaced short palindromic repeats (CRISPR)-based tagging, thus prohibiting the use of such assays in cell types that are difficult to transfect or transduce. We recently developed an 'off-the-shelf' proximity biotinylation method that makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait-specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. In principle, any scientist can perform this protocol within 3 days, although generating the proteomics data requires access to a high-end liquid chromatography-mass spectrometry setup. Data analysis and data visualization are relatively straightforward and can be performed using any type of software that converts raw mass spectrometry spectra files into identified and quantified proteins. The protocol has been optimized for nuclear targets but may also be adapted to other subcellular regions of interest.

Many enhancers play a crucial role in regulating gene expression by assembling regulatory factor (RF) clusters, also referred to as condensates. This process is essential for facilitating enhancer communication and establishing cellular identity. However, how DNA sequence and transcription factor (TF) binding instruct the formation of such high RF environments is still poorly understood. To address this, we developed a novel approach leveraging enhancer-centric chromatin accessibility quantitative trait loci (caQTLs) to nominate RF clusters genome-wide. By analyzing TF binding signatures within the context of caQTLs, we discovered a new class of TFs that specifically contributes to establishing cooperative environments. These “context-only” TFs bind promiscuously with cell type-specific pioneers, recruit coactivators, and, like super enhancers, render downstream gene expression sensitive to condensate-disrupting molecules. We further demonstrate that joint context-only and pioneer TF binding explains enhancer compatibility and provides a mechanistic rationale for how a loose TF syntax can still confer regulatory specificity.

Adipose tissue plasticity is orchestrated by molecularly and functionally diverse cells within the stromal vascular fraction (SVF). While several mouse and human adipose SVF cellular subpopulations have now been identified, we still lack an understanding of the cellular and functional variability of adipose stem and progenitor cell (ASPC) populations across human fat depots. To address this, we performed single-cell and bulk RNA-seq analyses of >30 Lin–SVF samples across four human adipose depots, revealing two ubiquitous hASPC subpopulations with distinct proliferative and adipogenic properties but also depot- and BMI-dependent proportions. Furthermore, we identified an omental-specific, highIGFBP2-expressing stromal population that transitions between mesothelial and mesenchymal cell states and inhibits hASPC adipogenesis through IGFBP2 secretion. Our analyses highlight the molecular and cellular uniqueness of different adipose niches while our discovery of an anti-adipogenic IGFBP2+ omental-specific population provides a new rationale for the biomedically relevant, limited adipogenic capacity of omental hASPCs.

Akkermansia muciniphila is considered a key constituent of a healthy gut microbiota. In inflammatory bowel disease (IBD), A. muciniphila has a reduced abundance while other, putative pathogenic, mucus colonizers bloom. We hypothesized that interbacterial competition may contribute to this observation. By screening the supernatants of a panel of enteric bacteria, we discovered that a previously uncharacterized Allobaculum species potently inhibits the growth of A. muciniphila. Mass spectrometry analysis identified a secreted Allobaculum sialidase as inhibitor of A. muciniphila growth. The sialidase targets sialic acids on casein O-glycans, thereby altering the accessibility of nutrients critical for A. muciniphila. The altered glycometabolic niche results in distorted A. muciniphila cell division and efficiently arrests its growth. The identification of a novel mechanism of A. muciniphila growth inhibition by a competing bacterial pathobiont may provide a rationale for interventions aimed at restoring and maintaining a healthy microbiota symbiosis in patients with intestinal disease.

Summary Polycomb Repressive Complex 2 (PRC2) plays an essential role in gene repression during development, catalyzing H3 lysine 27 trimethylation (H3K27me3). MTF2 in the PRC2.1 sub-complex, and JARID2 in PRC2.2, are central in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). To investigate how PRC2.1 and PRC2.2 cooperate, we combined Polycomb mutant mESCs with chemical inhibition of binding to H3K27me3. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, which are mutually reinforced. Whereas PRC2.1 recruitment is mediated by MTF2 binding to DNA, JARID2-containing PRC2.2 recruitment is more dependent on PRC1. Both recruitment axes are supported by core subunit EED binding to H3K27me3, but EED inhibition exhibits a more pronounced effect in Jarid2 null cells. Finally, we show that PRC1 and PRC2 enhance reciprocal binding. Together, these data disentangle the interdependent interactions that are important for PRC2 recruitment., Graphical Abstract, Highlights • Systematic analysis of PRC2 genomic binding in mouse ESCs • Mutations and chemical inhibition uncover roles of EED and other interactions • Contributions of MTF2 to PRC2.1 binding and of JARID2 and PRC1 to PRC2.2 binding • PRC1, PRC2.1, and PRC2.2 are all mutually interdependent for binding to chromatin, Veenstra and Marks and colleagues systematically analyze the contributions of Polycomb Repressive Complexes PRC2.1, PRC2.2, and PRC1 in ESCs, using null mutations in combination with chemical inhibition of EED, the core subunit that interacts with H3K27me3. The mutations and the EED inhibition uncover the functional overlap, and the compensatory and highly interdependent binding of PRC2.1, PRC2.2, and PRC1.

Understanding how chromatin is regulated is essential to fully grasp genome biology, and establishing the locus-specific protein composition is a major step toward this goal. Here we explain why the isolation and analysis of a specific chromatin segment are technically challenging, independently of the method. We then describe the published strategies and discuss their advantages and limitations. We conclude by discussing why significant technology developments are required to unambiguously describe the composition of small single loci.

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals. Neagu, van Genderen and Escudero et al. show that simultaneous inhibition of WNT and MEK signalling maintains a naive-primed intermediate pluripotency state in vitro, which displays features of the mouse embryonic rosette.

Cell cycle transitions arise from collective changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs), but conceptual and mechanistic explanations for the abrupt cellular reorganisation that occurs upon mitotic entry are lacking. Specific interactions between distinct CDK-cyclin complexes and sequence motifs encoded in substrates might result in highly ordered phosphorylation1, while bistability in the mitotic CDK1 control network can trigger switch-like phosphorylation2. Yet the dynamics of mitotic phosphorylation has not been demonstrated in vivo, and the roles of most cell cycle-regulated phosphorylations are unclear. Here, we show evidence that switch-like phosphorylation of intrinsically disordered proteins (IDPs) by CDKs contributes to mitotic cellular reorganisation by controlling protein-protein interactions and phase separation. We studied protein phosphorylation in single Xenopus embryos throughout synchronous cell cycles, performed parallel assignment of cell cycle phases using egg extracts, and analysed dynamics of mitotic phosphorylation using quantitative targeted phosphoproteomics. This provided a high-resolution map of dynamic phosphosites from the egg to the 16-cell embryo and showed that mitotic phosphorylation occurs on entire protein complexes involved in diverse subcellular processes and is switch-like in vivo. Most cell cycle-regulated phosphosites occurred in CDK consensus motifs and located to intrinsically disordered regions. We found that substrates of CDKs and other cell cycle kinases are significantly more disordered than phosphoproteins in general, a principle conserved from yeast to humans, while around half are components of membraneless organelles (MLOs), whose assembly is thought to involve phase separation. Analytical modelling predicts modulation of homotypic IDP interactions by CDK-mediated phosphorylation, which was confirmed by biophysical and biochemical analysis of a model IDP, Ki-67. These results highlight the dynamic control of intrinsic disorder as a conserved hallmark of the cell cycle and suggest a mechanism for CDK-mediated mitotic cellular reorganisation.

SummarySwitch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34

Summary Mouse embryonic stem cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1 phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here, we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and that is required for the elongated G1 phase characteristic of ground state ESCs. RNA sequencing and chromatin immunoprecipitation sequencing analyses reveal that P53 directly regulates the expression of the retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveal a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation., Highlights • The P53-P21 pathway is activated upon adaptation of ESCs to their pluripotent ground state. • P53 is required for the elongated G1-phase characteristic to 2i ESCs. • P53 binds the promoter and activates Rb1 expression., Compared with somatic cells, mouse embryonic stem cells (ESCs) grown in serum-rich conditions display a shortened G1 phase and lack activity of the P53-P21 pathway. Stunnenberg and colleagues show that P53 is activated upon adaptation of ESCs to their pluripotent ground state in serum-free conditions. P53 modulates elongation of the G1 phase not via P21, but through transcriptional activation of the RB protein.

Polycomb Repressive Complex 2 (PRC2) plays an essential role in development by catalysing trimethylation of histone H3 lysine 27 (H3K27me3), resulting in gene repression. PRC2 consists of two sub-complexes, PRC2.1 and PRC2.2, in which the PRC2 core associates with distinct ancillary subunits such as MTF2 and JARID2, respectively. Both MTF2, present in PRC2.1, and JARID2, present in PRC2.2, play a role in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). However, it remains unclear how these distinct sub-complexes cooperate to establish Polycomb domains. Here, we combine a range of Polycomb mutant mESCs with chemical inhibition of PRC2 catalytic activity, to systematically dissect their relative contributions to PRC2 binding to target loci. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, with mutually reinforced binding. Part of the cross-talk between PRC2.1 and PRC2.2 occurs via their catalytic product H3K27me3, which is bound by the PRC2 core-subunit EED, thereby mediating a positive feedback. Strikingly, removal of either JARID2 or H3K27me3 only has a minor effect on PRC2 recruitment, whereas their combined ablation largely attenuates PRC2 recruitment. This strongly suggests an unexpected redundancy between JARID2 and EED-H3K27me3-mediated recruitment of PRC2. Furthermore, we demonstrate that all core PRC2 recruitment occurs through the combined action of MTF2-mediated recruitment of PRC2.1 to DNA and PRC1-mediated recruitment of JARID2-containing PRC2.2. Both axes of binding are supported by EED-H3K27me3 positive feedback, but to a different degree. Finally, we provide evidence that PRC1 and PRC2 mutually reinforce reciprocal binding. Together, these data disentangle the interdependent and cooperative interactions between Polycomb complexes that are important to establish Polycomb repression at target sites.HighlightsSystematic analysis of Polycomb complex binding to target loci in mESCs using null mutations and chemical inhibition.PRC1, PRC2.1 and PRC2.2 are all mutually dependent for binding to chromatin, mediated in part by H3K27me3.PRC2.1 recruitment is dependent on MTF2PRC2.2 recruitment by JARID2 is dependent on PRC1 and largely redundant with recruitment by H3K27me3

Background Genomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we apply allele-specific RNA-seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic tissues between E3.5 and E7.25 and in pluripotent cell lines to evaluate maintenance of imprinted gene expression. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos and from embryos obtained after nuclear transfer (NT) or parthenogenetic activation (PGA). Results As homozygous genomic regions of PGA-derived cells are not compatible with allele-specific RNA-seq, we developed an RNA-seq-based genotyping strategy allowing identification of informative heterozygous regions. Global analysis shows that proper imprinted gene expression as observed in embryonic tissues is largely lost in the ESC lines included in this study, which mainly consisted of female ESCs. Differentiation of ESC lines to embryoid bodies or NPCs does not restore monoallelic expression of imprinted genes, neither did reprogramming of the serum-cultured ESCs to the pluripotent ground state by the use of 2 kinase inhibitors. Fertilized EpiSC and EpiSC-NT lines largely maintain imprinted gene expression, as did EpiSC-PGA lines that show known paternally expressed genes being silent and known maternally expressed genes consistently showing doubled expression. Notably, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. With respect to female EpiSC, most of the lines displayed completely skewed X inactivation suggesting a (near) clonal origin. Conclusions Altogether, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential. Electronic supplementary material The online version of this article (10.1186/s13072-019-0259-8) contains supplementary material, which is available to authorized users.

International audience; The pluripotent ground state is defined as a basal state free of epigenetic restrictions, which influence lineage specification. While naive embryonic stem cells (ESCs) can be maintained in a hypomethylated state with open chromatin when grown using two small-molecule inhibitors (2i)/leukemia inhibitory factor (LIF), in contrast to serum/LIF-grown ESCs that resemble early post-implantation embryos, broader features of the ground-state pluripotent epigenome are not well understood. We identified epigenetic features of mouse ESCs cultured using 2i/LIF or serum/LIF by proteomic profiling of chromatin-associated complexes and histone modifications. Polycomb-repressive complex 2 (PRC2) and its product H3K27me3 are highly abundant in 2i/LIF ESCs, and H3K27me3 is distributed genome-wide in a CpG-dependent fashion. Consistently, PRC2-deficient ESCs showed increased DNA methylation at sites normally occupied by H3K27me3 and increased H4 acetylation. Inhibiting DNA methylation in PRC2-deficient ESCs did not affect their viability or transcriptome. Our findings suggest a unique H3K27me3 configuration protects naive ESCs from lineage priming, and they reveal widespread epigenetic crosstalk in ground-state pluripotency.

Pluripotency can be captured in vitro in the form of Embryonic Stem Cells (ESCs). These ESCs can be either maintained in the unrestricted "naïve" state of pluripotency, adapted to developmentally more constrained "primed" pluripotency or differentiated towards each of the three germ layers. Epigenetic protein complexes and transcription factors have been shown to specify and instruct transitions from ESCs to distinct cell states. In this study, proteomic profiling of the chromatin landscape by chromatin enrichment for proteomics (ChEP) is used in mouse naive pluripotent ESCs, primed pluripotent Epiblast stem cells (EpiSCs), and cells in early stages of differentiation. A comprehensive overview of epigenetic protein complexes associated with the chromatin is provided and proteins associated with the maintenance and loss of pluripotency are identified. The data reveal major compositional alterations of epigenetic complexes during priming and differentiation of naïve pluripotent ESCs. These results contribute to the understanding of ESC differentiation and provide a framework for future studies of lineage commitment of ESCs.

Regulation of gene expression is essential for the functioning of all eukaryotic organisms. Understanding gene expression regulation requires determining which proteins interact with regulatory elements in chromatin. MS-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation, as it allows studying protein function and protein complex formation in their in vivo chromatin-bound context. Total chromatin isolated from cells can be directly analyzed using MS or further fractionated into transcriptionally active and inactive chromatin prior to MS-based analysis. Newly formed chromatin that is assembled during DNA replication can also be specifically isolated and analyzed. Furthermore, capturing specific chromatin domains facilitates the identification of previously unknown transcription factors interacting with these domains. Finally, in recent years, advances have been made toward identifying proteins that interact with a single genomic locus of interest. In this review, we highlight the power of chromatin proteomics approaches and how these provide complementary alternatives compared with conventional affinity purification methods. Furthermore, we discuss the biochemical challenges that should be addressed to consolidate and expand the role of chromatin proteomics as a key technology in the context of gene expression regulation and epigenetics research in health and disease., Graphical abstract, Highlights • An overview of proteomics methods to study chromatin and gene regulation. • Strength and limitations of the different approaches are highlighted. • An outlook on the outstanding challenges for chromatin proteomics. • Future directions for chromatin proteomics are discussed., In Brief MS-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation. Total chromatin isolated from cells can be directly analyzed using MS, further fractionated into transcriptionally active and inactive chromatin, enriched for specific compartment or regions, and potentially used for single-locus isolation. This review highlights recent advances and discusses current challenges that should be addressed to further advance the field of chromatin proteomics.

Synovial sarcoma tumours contain a characteristic fusion protein, SS18-SSX, which drives disease development. Targeting oncogenic fusion proteins presents an attractive therapeutic opportunity. However, SS18-SSX has proven intractable for therapeutic intervention. Using a domain-focused CRISPR screen we identified the bromodomain of BRD9 as a critical functional dependency in synovial sarcoma. BRD9 is a component of SS18-SSX containing BAF complexes in synovial sarcoma cells; and integration of BRD9 into these complexes is critical for cell growth. Moreover BRD9 and SS18-SSX co-localize extensively on the synovial sarcoma genome. Remarkably, synovial sarcoma cells are highly sensitive to a novel small molecule degrader of BRD9, while other sarcoma subtypes are unaffected. Degradation of BRD9 induces downregulation of oncogenic transcriptional programs and inhibits tumour progression in vivo. We demonstrate that BRD9 supports oncogenic mechanisms underlying the SS18-SSX fusion in synovial sarcoma and highlight targeted degradation of BRD9 as a potential therapeutic opportunity in this disease.

The ground state of pluripotency is defined as a minimal unrestricted epigenetic state as present in the Inner Cell Mass. Mouse embryonic stem cells (ESCs) grown in a defined serum-free medium with two kinase inhibitors (“2i ESCs”) have been postulated to reflect ground-state pluripotency, whereas ESCs grown in the presence of serum (“serum ESCs”) share more similarities with post-implantation epiblast cells. Pluripotency results from an intricate interplay between cytoplasmic, nuclear and chromatin-associated proteins. Here, we perform quantitative subcellular proteomics to gain insight in the molecular mechanisms sustaining the pluripotent states reflected by 2i and serum ESCs. We describe a full SILAC workflow and quality controls for proteomic comparison of 2i and serum ESCs, allowing subcellular proteomics of the cytoplasm, nucleoplasm and chromatin. The obtained quantitative information revealed increased levels of naïve pluripotency factors on the chromatin of 2i ESCs. Surprisingly, the cytoplasmic proteome suggests that 2i and serum ESCs utilize distinct metabolic programs, which include upregulation of free radical buffering by the glutathione pathway in 2i ESCs. Through induction of intracellular radicals, we show that the altered metabolic environment renders 2i ESCs less sensitive to oxidative stress. Altogether, this work provides novel insights into the proteomic landscape underlying ground state pluripotency. Keywords: Embryonic stem cells, Metabolism, Naïve pluripotency, SILAC

Polycomb-mediated repression of gene expression is essential for development, with a pivotal role played by trimethylation of histone H3 lysine 27 (H3K27me3), which is deposited by Polycomb Repressive Complex 2 (PRC2). The mechanism by which PRC2 is recruited to target genes has remained largely elusive, particularly in vertebrates. Here we demonstrate that MTF2, one of the three vertebrate homologs of Drosophila melanogaster Polycomblike, is a DNA-binding, methylation-sensitive PRC2 recruiter in mouse embryonic stem cells. MTF2 directly binds to DNA and is essential for recruitment of PRC2 both in vitro and in vivo. Genome-wide recruitment of the PRC2 catalytic subunit EZH2 is abrogated in Mtf2 knockout cells, resulting in greatly reduced H3K27me3 deposition. MTF2 selectively binds regions with a high density of unmethylated CpGs in a context of reduced helix twist, which distinguishes target from non-target CpG islands. These results demonstrate instructive recruitment of PRC2 to genomic targets by MTF2.

After the publication of this work [1], we noticed there was an error in Fig. 5 where −1,0 and 1 are incorrectly displayed in the y-axis in panel b. Please see the corrected Fig. 5 below. We apologize for this error. Fig. 5 llele-specific RNA-seq on three NPC lines identifies three distal regions of genes that escape XCI. a Ratio of Xi/Xa (y-axis; for each of the three NPC lines sorted from highest to lowest) for genes showing a log2 ratio of at least −5. We set ...

Background During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. Results Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. Conclusions The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0698-x) contains supplementary material, which is available to authorized users.

Regulation of gene expression is a complex but highly guided process. While genomic technologies and computational approaches have allowed high-throughput mapping of cis-regulatory elements (CREs) and their interactions in 3D, their precise role in regulating gene expression remains obscure. Recent complementary observations revealed that interactions between CREs frequently result in the formation of small-scale functional modules within topologically associating domains. Such chromatin modules likely emerge from a complex interplay between regulatory machineries assembled at CREs, including site-specific binding of transcription factors. Here, we review the methods that allow identifying chromatin modules, summarize possible mechanisms that steer CRE interactions within these modules, and discuss outstanding challenges to uncover how chromatin modules fit in our current understanding of the functional 3D genome.