Long-range chromatin contacts reveal a role for the pluripotency and Polycomb networks in genome organization

The spatial organization of the genome is linked to its biological function. However, the relationship between specific gene regulatory networks that govern cell identity and large-scale organization of genomes remains unclear. To investigate the basis of distal chromatin interactions occurring between genomic regions mega-bases away on the same or different chromosomes, we mapped long-range chromatin interactions in embryonic stem cells (ESCs) using 4C-seq, and examined the genomic features of the interacting regions. Confirming previous results, we show that open, accessible versus closed chromatin character is the primary determinant of distal chromatin interaction preferences, where interacting regions exhibit very similar open/closed chromatin character. We extend these results by demonstrating that genomic regions highly enriched for binding by the pluripotency transcription factors Oct4, Sox2, and Nanog preferentially co-localize, as do regions strongly enriched for Polycomb proteins and trimethylation of histone H3 at lysine 27 (H3K27me3), which include the Hox clusters. Consistent with a spatial segregation of these transcriptional networks, we find that Nanog and Polycomb proteins occupy distinct spaces in the nucleus. Importantly, loss of the Polycomb protein Eed and H3K27me3 diminishes the preferential interactions between regions normally highly enriched for Polycomb proteins and H3K27me3 without dramatically changing long-range chromatin interactions related to the open/closed chromatin state. Finally, a comparison of interactomes in ESCs and fibroblasts uncovered an ESC-specific spatial organization to the mouse genome that is gradually re-established upon reprogramming to induced pluripotent stem cells (iPSCs). Together, our data suggest that transcriptional networks that govern ESC identity play a role in determining genome-organization. We propose the existence of a hierarchy in the organization of chromatin contacts wherein, at the largest scale, the open/closed chromatin character defines an interaction space and overall chromosome conformation; on a finer scale, cell type-specific transcriptional networks direct preferential distal interactions, which we speculate are critical for efficient regulation of transcription and establishment of local chromatin environments

Long-range chromatin contacts reveal a role for the pluripotency and Polycomb networks in genome organization

The spatial organization of the genome is linked to its biological function. However, the relationship between specific gene regulatory networks that govern cell identity and large-scale organization of genomes remains unclear. To investigate the basis of distal chromatin interactions occurring between genomic regions mega-bases away on the same or different chromosomes, we mapped long-range chromatin interactions in embryonic stem cells (ESCs) using 4C-seq, and examined the genomic features of the interacting regions. Confirming previous results, we show that open, accessible versus closed chromatin character is the primary determinant of distal chromatin interaction preferences, where interacting regions exhibit very similar open/closed chromatin character. We extend these results by demonstrating that genomic regions highly enriched for binding by the pluripotency transcription factors Oct4, Sox2, and Nanog preferentially co-localize, as do regions strongly enriched for Polycomb proteins and trimethylation of histone H3 at lysine 27 (H3K27me3), which include the Hox clusters. Consistent with a spatial segregation of these transcriptional networks, we find that Nanog and Polycomb proteins occupy distinct spaces in the nucleus. Importantly, loss of the Polycomb protein Eed and H3K27me3 diminishes the preferential interactions between regions normally highly enriched for Polycomb proteins and H3K27me3 without dramatically changing long-range chromatin interactions related to the open/closed chromatin state. Finally, a comparison of interactomes in ESCs and fibroblasts uncovered an ESC-specific spatial organization to the mouse genome that is gradually re-established upon reprogramming to induced pluripotent stem cells (iPSCs). Together, our data suggest that transcriptional networks that govern ESC identity play a role in determining genome-organization. We propose the existence of a hierarchy in the organization of chromatin contacts wherein, at the largest scale, the open/closed chromatin character defines an interaction space and overall chromosome conformation; on a finer scale, cell type-specific transcriptional networks direct preferential distal interactions, which we speculate are critical for efficient regulation of transcription and establishment of local chromatin environments

A mechanistic link between gene regulation and genome architecture in mammalian development

The organization of chromatin within the nucleus and the regulation of transcription are tightly linked. Recently, mechanisms underlying this relationship have been uncovered. By defining the organizational hierarchy of the genome, determining changes in chromatin organization associated with changes in cell identity, and describing chromatin organization within the context of linear genomic features (such as chromatin modifications and transcription factor binding) and architectural proteins (including Cohesin, CTCF, and Mediator), a new paradigm in genome biology was established wherein genomes are organized around gene regulatory factors that govern cell identity. As such, chromatin organization plays a central role in establishing and maintaining cell state during development, with gene regulation and genome organization being mutually dependent effectors of cell identity

Plasma Cell Fate Is Orchestrated by Elaborate Changes in Genome Compartmentalization and Inter-chromosomal Hubs.

The transition from the follicular B to the plasma cell stage is associated with large-scale changes in cell morphology. Here, we examine whether plasma cell development is also associated with changes in nuclear architecture. We find that the onset of plasma cell development is concomitant with a decline in remote genomic interactions; a gain in euchromatic character at loci encoding for factors that specify plasma cell fate, including Prdm1 and Atf4; and establishment of de novo inter-chromosomal hubs. We find that, in developing plasma cells and concurrent with transcriptional silencing, the Ebf1 locus repositions from an euchromatic to peri-centromeric heterochromatic environment. Finally, we find that inter-chromosomal hubs are enriched for the deposition of either H3K27Ac or H3K27me3. These data indicate that plasma cell fate is orchestrated by elaborate changes in genome topology and that epigenetic marks, linked with super-enhancers or transcriptionally repressed regions, are enriched at inter-chromosomal hubs

Long-Range Chromatin Contacts in Embryonic Stem Cells Reveal a Role for Pluripotency Factors and Polycomb Proteins in Genome Organization

The relationship between 3D organization of the genome and gene-regulatory networks is poorly understood. Here, we examined long-range chromatin interactions genome-wide in mouse embryonic stem cells (ESCs), iPSCs, and fibroblasts and uncovered a pluripotency-specific genome organization that is gradually reestablished during reprogramming. Our data confirm that long-range chromatin interactions are primarily associated with the spatial segregation of open and closed chromatin, defining overall chromosome conformation. Additionally, we identified two further levels of genome organization in ESCs characterized by colocalization of regions with high pluripotency factor occupancy and strong enrichment for Polycomb proteins/H3K27me3, respectively. Underlining the independence of these networks and their functional relevance for genome organization, loss of the Polycomb protein Eed diminishes interactions between Polycomb-regulated regions without altering overarching chromosome conformation. Together, our data highlight a pluripotency-specific genome organization in which pluripotency factors such as Nanog and H3K27me3 occupy distinct nuclear spaces and reveal a role for cell-type-specific gene-regulatory networks in genome organization

Immunogenicity of Heterologous Recombinant Adenovirus Prime-Boost Vaccine Regimens Is Enhanced by Circumventing Vector Cross-Reactivity

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations has led to the development of recombinant adenovirus (rAd) vectors derived from rare Ad serotypes as vaccine candidates for human immunodeficiency virus type 1 and other pathogens. Vaccine vectors have been constructed from Ad subgroup B, including rAd11 and rAd35, as well as from Ad subgroup D, including rAd49. However, the optimal combination of vectors for heterologous rAd prime-boost vaccine regimens and the extent of cross-reactive vector-specific neutralizing antibodies (NAbs) remain poorly defined. We have shown previously that the closely related vectors rAd11 and rAd35 elicited low levels of cross-reactive NAbs. Here we show that these cross-reactive NAbs correlated with substantial sequence homology in the hexon hypervariable regions (HVRs) and suppressed the immunogenicity of heterologous rAd prime-boost regimens. In contrast, vectors with lower hexon HVR homology, such as rAd35 and rAd49, did not elicit detectable cross-reactive vector-specific NAbs. Consistent with these findings, rAd35-rAd49 vaccine regimens proved more immunogenic than both rAd35-rAd5 and rAd35-rAd11 regimens in mice with anti-Ad5 immunity. These data suggest that optimal heterologous rAd prime-boost regimens should include two vectors that are both rare in human populations to circumvent preexisting antivector immunity as well as sufficiently immunologically distinct to avoid cross-reactive antivector immunity

A mechanistic link between gene regulation and genome architecture in mammalian development

The organization of chromatin within the nucleus and the regulation of transcription are tightly linked. Recently, mechanisms underlying this relationship have been uncovered. By defining the organizational hierarchy of the genome, determining changes in chromatin organization associated with changes in cell identity, and describing chromatin organization within the context of linear genomic features (such as chromatin modifications and transcription factor binding) and architectural proteins (including Cohesin, CTCF, and Mediator), a new paradigm in genome biology was established wherein genomes are organized around gene regulatory factors that govern cell identity. As such, chromatin organization plays a central role in establishing and maintaining cell state during development, with gene regulation and genome organization being mutually dependent effectors of cell identity

Magnitude and Phenotype of Cellular Immune Responses Elicited by Recombinant Adenovirus Vectors and Heterologous Prime-Boost Regimens in Rhesus Monkeys▿

Recombinant adenovirus serotype 5 (rAd5) vaccine vectors for human immunodeficiency virus type 1 (HIV-1) and other pathogens have been shown to elicit antigen-specific cellular immune responses. Rare serotype rAd vectors have also been constructed to circumvent preexisting anti-Ad5 immunity and to facilitate the development of novel heterologous rAd prime-boost regimens. Here we show that rAd5, rAd26, and rAd48 vectors elicit qualitatively distinct phenotypes of cellular immune responses in rhesus monkeys and can be combined as potent heterologous prime-boost vaccine regimens. While rAd5-Gag induced primarily gamma interferon-positive (IFN-γ+) and IFN-γ+/tumor necrosis factor alpha+ (TNF-α+) T-lymphocyte responses, rAd26-Gag and rAd48-Gag induced higher proportions of interleukin-2+ (IL-2+) and polyfunctional IFN-γ+/TNF-α+/IL-2+ T-lymphocyte responses. Priming with the rare serotype rAd vectors proved remarkably effective for subsequent boosting with rAd5 vectors. These data demonstrate that the rare serotype rAd vectors elicited T-lymphocyte responses that were phenotypically distinct from those elicited by rAd5 vectors and suggest the functional relevance of polyfunctional CD8+ and CD4+ T-lymphocyte responses. Moreover, qualitative differences in cellular immune responses may prove critical in determining the overall potency of heterologous rAd prime-boost regimens