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Biological discovery has been driven by advances in throughput and resolution of analysis technologies. They have also created an indelible bias for snapshot-based knowledge. Even though recent methods such as multi-omics single-cell assays have empowered immunological investigations, they still provide snapshots of cellular behaviors and thus, have inherent limitations in reconstructing unsynchronized dynamic events across individual cells. Here, we present a rationale for how NF-κB may convey specificity of contextual information through subtle quantitative features of its signaling dynamics. The next frontier of predictive understanding should involve functional characterization of NF-κB signaling dynamics and their immunological implications. This may help solve the apparent paradox that a ubiquitously activated transcription factor can shape accurate responses to different immune challenges.

The rapid transcriptional response to the transcription factor, glucocorticoid receptor (GR), including gene activation or repression, is mediated by the spatial association of genes with multiple GR binding sites (GBSs) over large genomic distances. However, only a minority of the GBSs have independent GR-mediated activating capacity, and GBSs with independent repressive activity were rarely reported. To understand the positive and negative effects of GR we mapped the regulatory environment of its gene targets. We show that the chromatin interaction networks of GR-activated and repressed genes are spatially separated and vary in the features and configuration of their GBS and other non-GBS regulatory elements. The convergence of the KLF4 pathway in GR-activated domains and the STAT6 pathway in GR-repressed domains, impose opposite transcriptional effects to GR, independent of hormone application. Moreover, the ROR and Rev-erb transcription factors serve as positive and negative regulators, respectively, of GR-mediated gene activation. We found that the spatial crosstalk between GBSs and non-GBSs provides a physical platform for sequestering the Ep300 co-activator from non-GR regulatory loci in both GR-activated and -repressed gene compartments. While this allows rapid gene repression, Ep300 recruitment to GBSs is productive specifically in the activated compartments, thus providing the basis for gene induction.

Cross-talk between estrogen receptors (ER) and glucocorticoid receptors (GR) has been shown to contribute to the development and progression of breast cancer. Importantly, the ER and GR status in breast cancer cells is a significant factor in determining the outcome of the disease. However, mechanistic details defining the cellular interactions between ER and GR are poorly understood. We investigated genome-wide binding profiles for ER and GR upon coactivation and characterized the status of the chromatin landscape. We describe a novel mechanism dictating the molecular interplay between ER and GR. Upon induction, GR modulates access of ER to specific sites in the genome by reorganization of the chromatin configuration for these elements. Binding to these newly accessible sites occurs either by direct recognition of ER response elements or indirectly through interactions with other factors. The unveiling of this mechanism is important for understanding cellular interactions between ER and GR and may represent a general mechanism for cross-talk between nuclear receptors in human disease. Cancer Res; 73(16); 5130–9. ©2013 AACR.

Supplementary Figure 1. ER binds at pre-programmed sites in MCF7 cells and genomic regions for ER and GR ChIPs. Supplementary Figure 2. ER and GR binding modules. Supplementary Figure 3. Changes in gene expression upon treatment of cells with Dex and E2. Supplementary Figure 4. Changes in chromatin accessibility at the different binding modules. Supplementary Figure 5. Motif composition at induced ER and GR binding events. Supplementary Figure 6. Analysis of the ER DNA binding mutant’s ability to bind to specific sites.

In vitro studies suggest that mapping the spatiotemporal complexity of nuclear factor κB (NF-κB) signaling is essential to understanding its function. The lack of tools to directly monitor NF-κB proteins in vivo has hindered such efforts. Here, we introduce reporter mice with the endogenous RelA (p65) or c-Rel labeled with distinct fluorescent proteins and a double knockin with both subunits labeled. Overcoming hurdles in simultaneous live-cell imaging of RelA and c-Rel, we show that quantitative features of signaling reflect the identity of activating ligands, differ between primary and immortalized cells, and shift toward c-Rel in microglia from aged brains. RelA:c-Rel heterodimer is unexpectedly depleted in the nuclei of stimulated cells. Trajectories of subunit co-expression in immune lineages reveal a reduction at key cell maturation stages. These results demonstrate the power of these reporters in gaining deeper insights into NF-κB biology, with the spectral complementarity of the labeled NF-κB proteins enabling diverse applications.

Age-associated low-grade sterile inflammation, commonly referred to as inflammaging, is a recognized hallmark of aging, which contributes to many age-related diseases. While tissue-resident macrophages are innate immune cells that secrete many types of inflammatory cytokines in response to various stimuli, it is not clear whether they have a role in driving inflammaging. Here we characterized the transcriptional changes associated with physiological aging in mouse resident macrophage populations across different tissues and sexes. Although the age-related transcriptomic signatures of resident macrophages were strikingly tissue-specific, the differentially expressed genes were collectively enriched for those with important innate immune functions such as antigen presentation, cytokine production, and cell adhesion. The brain-resident microglia had the most wide-ranging age-related alterations, with compromised expression of tissue-specific genes and relatively exaggerated responses to endotoxin stimulation. Despite the tissue-specific patterns of aging transcriptomes, components of the hedgehog (Hh) signaling pathway were decreased in aged macrophages across multiple tissues. In vivo suppression of Hh signaling in young animals increased the expression of pro-inflammatory cytokines, while in vitro activation of Hh signaling in old macrophages, in turn, suppressed the expression of these inflammatory cytokines. This suggests that hedgehog signaling could be a potential intervention axis for mitigating age-associated inflammation and related diseases. Overall, our data represent a resourceful catalog of tissue-specific and sex-specific transcriptomic changes in resident macrophages of peritoneum, liver, and brain, during physiological aging.

The complex signaling dynamics of transcription factors can encode both qualitative and quantitative information about the extracellular environment, which increases the information transfer capacity and potentially supports accurate cellular decision-making. An important question is how these signaling dynamics patterns are translated into functionally appropriate gene regulation programs. To address this question for transcription factors of the nuclear factor κB (NF-κB) family, we profiled the single-cell dynamics of two major NF-κB subunits, RelA and c-Rel, induced by a panel of pathogen-derived stimuli in immune and nonimmune cellular contexts. Diverse NF-κB-activating ligands produced different patterns of RelA and c-Rel signaling dynamic features, such as variations in duration or time-integrated activity. Analysis of nascent transcripts delineated putative direct targets of NF-κB as compared to genes controlled by other transcriptional and posttranscriptional mechanisms and showed that the transcription of more than half of the induced genes was tightly linked to specific dynamic features of NF-κB signaling. Fibroblast and macrophage cell lines shared a cluster of such "NF-κB dynamics-decoding" genes, as well as cell type-specific decoding genes. Dissecting the subunit specificity of dynamics-decoding genes suggested that target genes were most often linked to both RelA and c-Rel or to RelA alone. Thus, our analysis reveals the cell type-specific interpretation of pathogenic information through the signaling dynamics of NF-κB.

The transcription factor glucocorticoid receptor (GR) is a key mediator of stress response and a broad range of physiological processes. How can GR rapidly activate the expression of some genes while repress others, remains an open question due to the challenge to associate GR binding sites (GBSs) to their distant gene targets. Mapping the full 3D scope of GR-responsive promoters using high-resolution 4C-seq unravelled spatial separation between chromatin interaction networks of GR-activated and repressed genes. Analysing GR binding sites and other regulatory loci in their functional 3D context revealed that GR sequesters the co-activator Ep300 from active non-GBS enhancers in both activated and repressed gene compartments. While this is sufficient for rapid gene repression, gene activation is countered by productive recruitment of Ep300 to GBS. Importantly, in GR-activated compartments Klf4 binding at non-GBS regulatory elements cluster in 3D with GBS and antagonizes GR activation. In addition, we revealed ROR and Rev-erb transcription factors as novel co-regulators for GR-mediated gene expression.

Aim: Long noncoding RNAs (lncRNAs) have been reported to influence multiple gene regulatory processes. Technological advances in RNA-seq platforms allow detection of low-abundance RNA species such as lncRNAs. This study examined the relationship between expression of lncRNAs and their putative partner mRNAs. Methods: We analyzed total RNA-seq data from mouse macrophages under various inflammatory and intervention conditions. Results: The macrophage expression of lncRNAs is strongly regulated by an inflammatory stimulus. Moreover, the expression of a majority of lncRNAs was correlated or anti-correlated with the partner mRNA(s), across the different treatment conditions. This relationship was maintained even in cells from a distinct genotype. Conclusion: These results suggest a previously unappreciated tight coupling of lncRNA and mRNA expression during macrophage responses to various microenvironmental perturbations.

Additional file 2. Supplementary figures.

The Perspective by Sung, Baek and Hager discusses consensus, remaining issues and hurdles for genomic footprinting. High-throughput sequencing technologies have allowed many gene locus–level molecular biology assays to become genome-wide profiling methods. DNA-cleaving enzymes such as DNase I have been used to probe accessible chromatin. The accessible regions contain functional regulatory sites, including promoters, insulators and enhancers. Deep sequencing of DNase-seq libraries and computational analysis of the cut profiles have been used to infer protein occupancy in the genome at the nucleotide level, a method introduced as 'digital genomic footprinting'. The approach has been proposed as an attractive alternative to the analysis of transcription factors (TFs) by chromatin immunoprecipitation followed by sequencing (ChIP-seq), and in theory it should overcome antibody issues, poor resolution and batch effects. Recent reports point to limitations of the DNase-based genomic footprinting approach and call into question the scope of detectable protein occupancy, especially for TFs with short-lived chromatin binding. The genomics community is grappling with issues concerning the utility of genomic footprinting and is reassessing the proposed approaches in terms of robust deliverables. Here we summarize the consensus as well as different views emerging from recent reports, and we describe the remaining issues and hurdles for genomic footprinting.

Aging is associated with changes in gene expression. Age-related changes in chromatin structure may contribute to alterations in gene expression and to aging phenotypes in human. Macrophages play essential roles in tissue homeostasis and repair. Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor (TF) families. We previously showed that chromatin accessibility of macrophages is massively increased after LPS treatment. Similar programs may also contribute to the chronically elevated inflammatory state in aging. Therefore, it is important to understand how macrophages are epigenetically and functionally altered during aging. To address the age-associated differences in the transcriptome and chromatin landscape of macrophages, we have performed RNA-seq and ATAC-seq of tissue-resident macrophages in the presence or absence of LPS. Our results revealed that age effect in macrophages seems cell type-specific with the strongest effect in microglia. The majority of age-dependent TF footprints in macrophages showed reduced TF occupancy, but with either reduced or increased chromatin accessibility. We are investigating the functional relevance of the candidate TFs identified in our analysis. Our data highlight the tissue-specificity of aging features that occur in resident macrophages in vivo.

Technological advances are continually improving our ability to obtain more accurate views about the inner workings of biological systems. One such rapidly evolving area is single cell biology, and in particular gene expression and its regulation by transcription factors in response to intrinsic and extrinsic factors. Regarding the study of transcription factors, we discuss some of the promises and pitfalls associated with investigating how individual cells regulate gene expression through modulation of transcription factor activities. Specifically, we discuss four leading experimental approaches, the data that can be obtained from each, and important considerations that investigators should be aware of when drawing conclusions from such data.

Transition from resting to cell cycle in response to antigenic stimulation is an essential step for naive CD8+ T cells to differentiate to effector and memory cells. Leaving the resting state requires dramatic changes of chromatin status in the key cell cycle inhibitors but the details of these concerted events are not fully elucidated. Here, we showed that Ezh2, an enzymatic component of polycomb repressive complex 2 (PRC2) catalyzing the trimethylation of lysine 27 on histone 3 (H3K27me3), regulates activation induced naive CD8+ T cells proliferation and apoptosis. Upon deletion of Ezh2 during thymocyte development (Ezh2fl/flCD4Cre+ mice), naive CD8+ T cells displayed impaired proliferation and increased apoptosis in response to antigen stimulation. However, naive CD8+ T cells only had impaired proliferation but no increase in apoptosis when Ezh2 was deleted after activation (Ezh2fl/flGzmBCre+ mice), suggesting cell cycle and apoptosis are temporally separable events controlled by Ezh2. We then showed that deletion of Ezh2 resulted in the increase in expression of cyclin-dependent kinase inhibitors Cdkn2a (p16 and Arf) and Cdkn1c (p57) in activated naive CD8+ T cells as the consequence of reduced levels of H3K27me3 at these two gene loci. Finally, with real time imaging, we observed prolonged cell division times of naive CD8+ T cells in the absence of Ezh2 post in vitro stimulation. Together, these findings reveal that repression of Cdkn1c and Cdkn2a by Ezh2 plays an essential role in execution of activation-induced CD8+ T cell proliferation.

Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1−/− macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1+/− mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.

Three-dimensional (3-D) genome organization in the nuclear space affects various genomic functions. Circular chromosome conformation capture (4C-seq) is a powerful technique that allows researchers to measure long-range chromosomal interactions with a locus of interest across the entire genome. This method relies on enzymatic cleavage of cross-linked chromatin and consecutive ligation to create ligation junctions between physically adjacent loci, followed by PCR amplification of locus-specific associating loci. The enzymes used must meet 4C standards because variations in their efficiency and performance may affect the quality of the obtained data. Here we systematically compare the efficiency and reliability of different T4 DNA ligases and PCR DNA polymerases, assessing the most critical and technically challenging steps in 4C. The results of this analysis enable the use of cost-effective enzymes with superior specificity and efficiency for 4C and save time in screening for appropriate primers. This information provides users with flexibility in their experimental design and guidelines for adapting and testing any enzyme of choice for obtaining standardized results.

Although physiological steroid levels are often pulsatile (ultradian), the genomic effects of this pulsatility are poorly understood. By utilizing glucocorticoid receptor (GR) signaling as a model system, we uncovered striking spatiotemporal relationships between receptor loading, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We found that hormone-induced DHSs were enriched within ±50 kb of GR-responsive genes and displayed a broad spectrum of lifetimes upon hormone withdrawal. These lifetimes dictate the strength of the DHS interactions with gene targets and contribute to gene regulation from a distance. Our results demonstrate that pulsatile and constant hormone stimulations induce unique, treatment-specific patterns of gene and regulatory element activation. These modes of activation have implications for corticosteroid function in vivo and for steroid therapies in various clinical settings.

The cytokines interleukin 1β and 6 (IL-1β, IL-6) mediate the acute phase response (APR). In liver, they regulate the secretion of acute phase proteins. Using RNA-seq in primary hepatocytes, we show that these cytokines regulate transcription in a bifurcated manner, leading to both synergistic and antagonistic gene expression. By mapping changes in enhancer landscape and transcription factor occupancy (using ChIP-seq), we show that synergistic gene induction is achieved by assisted loading of STAT3 on chromatin by NF-κB. With IL-6 treatment alone, STAT3 does not efficiently bind 20% of its coordinated binding sites. In the presence of IL-1β, NF-κB is activated, binds a subset of enhancers and primes their activity, as evidenced by increasing H3K27ac. This facilitates STAT3 binding and synergistic gene expression. Our findings reveal an enhancer-specific crosstalk whereby NF-κB enables STAT3 binding at some enhancers while perturbing it at others. This model reconciles seemingly contradictory reports of NF-κB-STAT3 crosstalk., The cytokines IL-1β and IL-6 mediate the systemic acute phase response (APR). Here, the authors provide evidence that these cytokines lead to both synergistic and antagonistic gene expression during APR; synergistic induction occurs by assisted loading of STAT3 on chromatin by NF-κB.

The three dimensional folding of mammalian genomes is cell type specific and difficult to alter suggesting that it is an important component of gene regulation. However, given the multitude of chromatin-associating factors, the mechanisms driving the colocalization of active chromosomal domains and the role of this organization in regulating the transcription program in adipocytes are not clear. Analysis of genome-wide chromosomal associations revealed cell type-specific spatial clustering of adipogenic genes in 3T3-L1 cells. Time course analysis demonstrated that the adipogenic 'hub', sampled by PPARγ and Lpin1, undergoes orchestrated reorganization during adipogenesis. Coupling the dynamics of genome architecture with multiple chromatin datasets indicated that among all the transcription factors (TFs) tested, RXR is central to genome reorganization at the beginning of adipogenesis. Interestingly, at the end of differentiation, the adipogenic hub was shifted to an H3K27me3-repressive environment in conjunction with attenuation of gene transcription. We propose a stage-specific hierarchy for the activity of TFs contributing to the establishment of an adipogenic genome architecture that brings together the adipogenic genetic program. In addition, the repositioning of this network in a H3K27me3-rich environment at the end of differentiation may contribute to the stabilization of gene transcription levels and reduce the developmental plasticity of these specialized cells.DATABASE: All sequence data reported in this paper have been deposited at GEO (http://www.ncbi.nlm.nih.gov/geo/) (GSE92475).

Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements.

SUMMARY Genomic footprinting has emerged as an unbiased discovery method for transcription factor (TF) occupancy at cognate DNA in vivo. A basic premise of footprinting is that sequence-specific TF-DNA interactions are associated with localized resistance to nucleases, leaving observable signatures of cleavage within accessible chromatin. This phenomenon is interpreted to imply protection of the critical nucleotides by the stably bound protein factor. However, this model conflicts with previous reports of many TFs exchanging with specific binding sites in living cells on a timescale of seconds. We show that TFs with short DNA residence times have no footprints at bound motif elements. Moreover, the nuclease cleavage profile within a footprint originates from the DNA sequence in the factor-binding site, rather than from the protein occupying specific nucleotides. These findings suggest a revised understanding of TF footprinting and reveal limitations in comprehensive reconstruction of the TF regulatory network using this approach.

Background As the cost of high-throughput sequencing technologies decreases, genome-wide chromatin accessibility profiling methods such as the assay of transposase-accessible chromatin using sequencing (ATAC-seq) are employed widely, with data accumulating at an unprecedented rate. However, accurate inference of protein occupancy requires higher-resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints. Results Here we introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. Mild cross-linking improved the detection of DNase-based footprints of dynamic TFs but interfered with ATAC-based footprinting of the same TFs. Conclusions XL-DNase-seq may help extract novel gene regulatory circuits involving previously undetectable TFs. The DNase-seq and ATAC-seq data generated in our systematic comparison of various cross-linking conditions also represent an unprecedented-scale resource derived from activated mouse macrophage-like cells which share many features of inflammatory macrophages. Electronic supplementary material The online version of this article (10.1186/s13072-019-0277-6) contains supplementary material, which is available to authorized users.

ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4, and Snf2h. The localization patterns of all three proteins significantly overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome-wide, and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization, and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes.

Nuclear bodies contribute to non-random organization of the human genome and nuclear function. Using a major prototypical nuclear body, the Cajal body, as an example, we suggest that these structures assemble at specific gene loci located across the genome as a result of high transcriptional activity. Subsequently, target genes are physically clustered in close proximity in Cajal body-containing cells. However, Cajal bodies are observed in only a limited number of human cell types, including neuronal and cancer cells. Ultimately, Cajal body depletion perturbs splicing kinetics by reducing target small nuclear RNA (snRNA) transcription and limiting the levels of spliceosomal snRNPs, including their modification and turnover following each round of RNA splicing. As such, Cajal bodies are capable of shaping the chromatin interaction landscape and the transcriptome by influencing spliceosome kinetics. Future studies should concentrate on characterizing the direct influence of Cajal bodies upon snRNA gene transcriptional dynamics. Also see the video abstract here.

Despite the widespread use of glucocorticoids (GCs), their anti-inflammatory effects are not understood mechanistically. Numerous investigations have examined the effects of glucocorticoid receptor (GR) activation prior to inflammatory challenges. However, clinical situations are emulated by a GC intervention initiated in the midst of rampant inflammatory responses. To characterize the effects of a late GC treatment, we profiled macrophage transcriptional and chromatinscapes with Dexamethasone (Dex) treatment before or after stimulation by lipopolysaccharide (LPS). The late activation of GR had a similar gene-expression profile as from GR pre-activation, while ameliorating the disruption of metabolic genes. Chromatin occupancy of GR was not predictive of Dex-regulated gene expression, contradicting the "trans-repression by tethering" model. Rather, GR activation resulted in genome-wide blockade of NF-κB interaction with chromatin and directly induced inhibitors of NF-κB and AP-1. Our investigation using GC treatments with clinically relevant timing highlights mechanisms underlying GR actions for modulating the "inflamed epigenome."

High-throughput sequencing technologies have made it possible for biologists to generate genome-wide profiles of chromatin features at the nucleotide resolution. Enzymes such as nucleases or transposes have been instrumental as a chromatin-probing agent due to their ability to target accessible chromatin for cleavage or insertion. On the scale of a few hundred base pairs, preferential action of the nuclear enzymes on accessible chromatin allows mapping of cell state-specific accessibility in vivo. Such accessible regions contain functionally important regulatory sites, including promoters and enhancers, which undergo active remodeling for cells adapting in a dynamic environment. DNase-seq and the more recent ATAC-seq are two assays that are gaining popularity. Deep sequencing of DNA libraries from these assays, termed genomic footprinting, has been proposed to enable the comprehensive construction of protein occupancy profiles over the genome at the nucleotide level. Recent studies have discovered limitations of genomic footprinting which reduce the scope of detectable proteins. In addition, the identification of putative factors that bind to the observed footprints remains challenging. Despite these caveats, the methodology still presents significant advantages over alternative techniques such as ChIP-seq or FAIRE-seq. Here we describe computational approaches and tools for analysis of chromatin accessibility and genomic footprinting. Proper experimental design and assay-specific data analysis ensure the detection sensitivity and maximize retrievable information. The enzyme-based chromatin profiling approaches represent a powerful and evolving methodology which facilitates our understanding of how the genome is regulated.

Higher-order genome organization shows tissue-specific patterns. However, functional relevance and the mechanisms shaping the genome architecture are poorly understood. Here we report a profound shift from promiscuous to highly selective genome organization that accompanies the effector lineage choice of differentiating T cells. As multipotent naive cells receive antigenic signals and commit to a T helper (Th) pathway, the genome-wide contacts of a lineage-specific cytokine locus are preferentially enriched for functionally relevant genes. Despite the establishment of divergent interactomes and global reprogramming of transcription in Th1 versus Th2, the overall expression status of the contact genes is surprisingly similar between the two lineages. Importantly, during differentiation, the genomic contacts are retained and strengthened precisely at DNA binding sites of the specific lineage-determining STAT transcription factor. In cells from the specific STAT knock-out mouse, the signature cytokine locus is unable to shed the promiscuous contacts established in the naive T cells, indicating the importance of genomic STAT binding. Altogether, the global aggregation of STAT binding loci from genic and nongenic regions highlights a new role for differentiation-promoting transcription factors in direct specification of higher-order nuclear architecture through interacting with regulatory regions. Such subnuclear environments have significant implications for efficient functioning of the mature effector lymphocytes.

The kinesin motor protein KIF4 performs essential functions in mitosis. Like other mitotic kinesins, loss of KIF4 causes spindle defects, aneuploidy, genomic instability and ultimately tumor formation. However, KIF4 is unique among molecular motors in that it resides in the cell nucleus throughout interphase, suggesting a non-mitotic function as well. Here we identify a novel cellular function for a molecular motor protein by demonstrating that KIF4 acts as a modulator of large-scale chromatin architecture during interphase. KIF4 binds globally to chromatin and its absence leads to chromatin decondensation and loss of heterochromatin domains. KIF4-dependent chromatin decondensation has functional consequences by causing replication defects and global mis-regulation of gene expression programs. KIF4 exerts its function in chromatin architecture via regulation of ADP-ribosylation of core and linker histones and by physical interaction and recruitment of chromatin assembly proteins during S-phase. These observations document a novel function for a molecular motor protein in establishment and maintenance of higher order chromatin structure.