Promoter-promoter Interactions Research Articles

A Bidirectional Non-Coding RNA Promoter Mediates Long-Range Gene Expression Regulation.

Recent evidence suggests that human gene promoters display gene expression regulatory mechanisms beyond the typical single gene local transcription modulation. In mammalian genomes, genes with an associated bidirectional promoter are abundant; bidirectional promoter architecture serves as a regulatory hub for a gene pair expression. However, it has been suggested that its contribution to transcriptional regulation might exceed local transcription initiation modulation. Despite their abundance, the functional consequences of bidirectional promoter architecture remain largely unexplored. This work studies the long-range gene expression regulatory role of a long non-coding RNA gene promoter using chromosome conformation capture methods. We found that this particular bidirectional promoter contributes to distal gene expression regulation in a target-specific manner by establishing promoter-promoter interactions. In particular, we validated that the promoter-promoter interactions of this regulatory element with the promoter of distal gene BBX contribute to modulating the transcription rate of this gene; removing the bidirectional promoter from its genomic context leads to a rearrangement of BBX promoter-enhancer interactions and to increased gene expression. Moreover, long-range regulatory functionality is not directly dependent on its associated non-coding gene pair expression levels.

Regulatory architecture of housekeeping genes is driven by promoter assemblies

Genes that are key to cell identity are generally regulated by cell-type-specific enhancer elements bound by transcription factors, some of which facilitate looping to distant gene promoters. In contrast, genes that encode housekeeping functions, whose regulation is essential for normal cell metabolism and growth, generally lack interactions with distal enhancers. We find that Ronin (Thap11) assembles multiple promoters of housekeeping and metabolic genes to regulate gene expression. This behavior is analogous to how enhancers are brought together with promoters to regulate cell identity genes. Thus, Ronin-dependent promoter assemblies provide a mechanism to explain why housekeeping genes can forgo distal enhancer elements and why Ronin is important for cellular metabolism and growth control. We propose that clustering ofregulatory elements is a mechanism common to cell identity and housekeeping genes but is accomplished by different factors binding distinct control elements to establish enhancer-promoter or promoter-promoter interactions, respectively.

Enhancer-promoter contact formation requires RNAPII and antagonizes loop extrusion.

Homotypic chromatin interactions and loop extrusion are thought to be the two main drivers of mammalian chromosome folding. Here we tested the role of RNA polymerase II (RNAPII) across different scales of interphase chromatin organization in a cellular system allowing for its rapid, auxin-mediated degradation. We combined Micro-C and computational modeling to characterize subsets of loops differentially gained or lost upon RNAPII depletion. Gained loops, extrusion of which was antagonized by RNAPII, almost invariably formed by engaging new or rewired CTCF anchors. Lost loops selectively affected contacts between enhancers and promoters anchored by RNAPII, explaining the repression of most genes. Surprisingly, promoter-promoter interactions remained essentially unaffected by polymerase depletion, and cohesin occupancy was sustained. Together, our findings reconcile the role of RNAPII in transcription with its direct involvement in setting-up regulatory three-dimensional chromatin contacts genome wide, while also revealing an impact on cohesin loop extrusion.

GAGA-associated factor fosters loop formation in the Drosophila genome

The impact of genome organization on the control of gene expression persists as a major challenge in regulatory biology. Most efforts have focused on the role of CTCF-enriched boundary elements and TADs, which enable long-range DNA-DNA associations via loop extrusion processes. However, there is increasing evidence for long-range chromatin loops between promoters and distal enhancers formed through specific DNA sequences, including tethering elements, which bind the GAGA-associated factor (GAF). Previous studies showed that GAF possesses amyloid properties invitro, bridging separate DNA molecules. In this study, we investigated whether GAF functions as a looping factor in Drosophila development. We employed Micro-C assays to examine the impact of defined GAF mutants on genome topology. These studies suggest that the N-terminal POZ/BTB oligomerization domain is important for long-range associations of distant GAGA-rich tethering elements, particularly those responsible for promoter-promoter interactions that coordinate the activities of distant paralogous genes.

DeepPHiC: predicting promoter-centered chromatin interactions using a novel deep learning approach.

Promoter-centered chromatin interactions, which include promoter-enhancer (PE) and promoter-promoter (PP) interactions, are important to decipher gene regulation and disease mechanisms. The development of next-generation sequencing technologies such as promoter capture Hi-C (pcHi-C) leads to the discovery of promoter-centered chromatin interactions. However, pcHi-C experiments are expensive and thus may be unavailable for tissues/cell types of interest. In addition, these experiments may be underpowered due to insufficient sequencing depth or various artifacts, which results in a limited finding of interactions. Most existing computational methods for predicting chromatin interactions are based on in situ Hi-C and can detect chromatin interactions across the entire genome. However, they may not be optimal for predicting promoter-centered chromatin interactions. We develop a supervised multi-modal deep learning model, which utilizes a comprehensive set of features such as genomic sequence, epigenetic signal, anchor distance, evolutionary features and DNA structural features to predict tissue/cell type-specific PE and PP interactions. We further extend the deep learning model in a multi-task learning and a transfer learning framework and demonstrate that the proposed approach outperforms state-of-the-art deep learning methods. Moreover, the proposed approach can achieve comparable prediction performance using predefined biologically relevant tissues/cell types compared to using all tissues/cell types in the pretraining especially for predicting PE interactions. The prediction performance can be further improved by using computationally inferred biologically relevant tissues/cell types in the pretraining, which are defined based on the common genes in the proximity of two anchors in the chromatin interactions. https://github.com/lichen-lab/DeepPHiC. Supplementary data are available at Bioinformatics online.

HiCAR is a robust and sensitive method to analyze open-chromatin-associated genome organization

The long-range interactions of cis-regulatory elements (cREs) play a central role in gene regulation. cREs can be characterized as accessible chromatin sequences. However, it remains technically challenging to comprehensively identify their spatial interactions. Here, we report a new method HiCAR (Hi-C on accessible regulatory DNA), which utilizes Tn5 transposase and chromatin proximity ligation, for the analysis of open-chromatin-anchored interactions with low-input cells. By applying HiCAR in human embryonic stem cells and lymphoblastoid cells, we demonstrate that HiCAR identifies high-resolution chromatin contacts with an efficiency comparable with that of in situ Hi-C over all distance ranges. Interestingly, we found that the "poised" gene promoters exhibit silencer-like function to repress the expression of distal genes via promoter-promoter interactions. Lastly, we applied HiCAR to 30,000 primary human muscle stem cells and demonstrated that HiCAR is capable of analyzing chromatin accessibility and looping using low-input primary cells and clinical samples.

Promoter-anchored chromatin interactions predicted from genetic analysis of epigenomic data

Promoter-anchored chromatin interactions (PAIs) play a pivotal role in transcriptional regulation. Current high-throughput technologies for detecting PAIs, such as promoter capture Hi-C, are not scalable to large cohorts. Here, we present an analytical approach that uses summary-level data from cohort-based DNA methylation (DNAm) quantitative trait locus (mQTL) studies to predict PAIs. Using mQTL data from human peripheral blood (n = 1980), we predict 34,797 PAIs which show strong overlap with the chromatin contacts identified by previous experimental assays. The promoter-interacting DNAm sites are enriched in enhancers or near expression QTLs. Genes whose promoters are involved in PAIs are more actively expressed, and gene pairs with promoter-promoter interactions are enriched for co-expression. Integration of the predicted PAIs with GWAS data highlight interactions among 601 DNAm sites associated with 15 complex traits. This study demonstrates the use of mQTL data to predict PAIs and provides insights into the role of PAIs in complex trait variation.

Histone Modifications in Development and Malignancy

Non-Hodgkin lymphoma (NHL) development is driven by the accumulations of multiple genetic, epigenetic, and chromosomal alterations. These lesions can lead to modifications of the chromatin architecture. To identify novel oncogenic interactions driven by modifications of the chromatin 3D organization, we combined high-throughput chromatin conformation capture data (Hi-C) in lymphoma cells with whole genome sequencing (WGS) and epigenetic and transcriptional profiles of primary patient samples and cell lines. Recently, we found that a significant interplay exists between the compartmentalization of the genome into topologically associating domains (TADs) and epigenetic and transcriptional changes mediated by mutated EZH2. Indeed, EZH2 mutations drive aberrant increase of H3K27me3 within specific TADs, resulting in loss of promoter-promoter interactions, synergistic silencing of multiple tumor suppressors and, thus, increased B-cell proliferation and tumor aggressiveness1. Now, we are further investigating how chromosomal amplifications and translocations forms new long-range interactions between enhancers and promoters to support oncogene expression. Importantly, we observed that epigenetic editing of enhancers and pharmacological depletion of H3K27Ac reduces the number of interactions between specific enhancers and promoters and this loss of interactions directly affects gene expression and cell differentiation. These results indicate that is possible to block the oncogenic effect of chromosomal alterations by targeting epigenetic modification in enhancer regions and preventing the formation of oncogenic chromatin interactions. Overall, our results demonstrate that it is important to contextualize the effects of genomic alterations in the 3D organization of the chromatin, to uncover new oncogenic mechanisms and improve the design of new therapeutic approaches. Donaldson-Collier MC, Sungalee S, Zufferey M, et al. EZH2 oncogenic mutations drive epigenetic, transcriptional, and structural changes within chromatin domains. Nat Genet. 2019;51(3):517. doi:10.1038/s41588-018-0338-y Disclosures No relevant conflicts of interest to declare.

DeepTACT: predicting 3D chromatin contacts via bootstrapping deep learning.

Interactions between regulatory elements are of crucial importance for the understanding of transcriptional regulation and the interpretation of disease mechanisms. Hi-C technique has been developed for genome-wide detection of chromatin contacts. However, unless extremely deep sequencing is performed on a very large number of input cells, which is technically limited and expensive, current Hi-C experiments do not have high enough resolution to resolve contacts between regulatory elements. Here, we develop DeepTACT, a bootstrapping deep learning model, to integrate genome sequences and chromatin accessibility data for the prediction of chromatin contacts between regulatory elements. DeepTACT can infer not only promoter–enhancer interactions, but also promoter–promoter interactions. In tests based on promoter capture Hi-C data, DeepTACT shows better performance over existing methods. DeepTACT analysis also identifies a class of hub promoters, which are correlated with transcriptional activation across cell lines, enriched in housekeeping genes, functionally related to fundamental biological processes, and capable of reflecting cell similarity. Finally, the utility of chromatin contacts in the study of human diseases is illustrated by the association of IFNA2 to coronary artery disease via an integrative analysis of GWAS data and interactions predicted by DeepTACT.

Dynamic Reorganization of Extremely Long-Range Promoter-Promoter Interactions between Two States of Pluripotency

Serum-to-2i interconversion of mouse embryonic stem cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum-grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon their further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary, but not sufficient, to establish these interactions, as confirmed by Capture Hi-C on Eed(-/-) serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation.

Highly interactive nature of flower-specific enhancers and promoters, and its potential impact on tissue-specific expression and engineering of multiple genes or agronomic traits.

Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co-existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer-promoter and promoter-promoter interactions in transgenic plants and demonstrated that three of four flower-specific enhancer/promoters were capable of distantly activating a pollen- and stigma-specific Pps promoter (fused to the cytotoxic DT-A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen- and carpel-specific DT-A expression, thus resulting in tissue ablation in an orientation-independent manner; this activation was completely abolished by the insertion of an enhancer-blocking insulator (EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue-specific DT-A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant-derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue-specific engineering of multiple traits using a single-vector stacking approach. Therefore, our work highlights the importance of adopting enhancer-blocking insulators in transformation vectors to minimize promoter-promoter interactions. The practical and fundamental significance of these findings will be discussed.

Promoter cross-talk via a shared enhancer explains paternally biased expression of Nctc1 at the Igf2/H19/Nctc1 imprinted locus

Developmentally regulated transcription often depends on physical interactions between distal enhancers and their cognate promoters. Recent genomic analyses suggest that promoter–promoter interactions might play a similarly critical role in organizing the genome and establishing cell-type-specific gene expression. The Igf2/H19 locus has been a valuable model for clarifying the role of long-range interactions between cis-regulatory elements. Imprinted expression of the linked, reciprocally imprinted genes is explained by parent-of-origin-specific chromosomal loop structures between the paternal Igf2 or maternal H19 promoters and their shared tissue-specific enhancer elements. Here, we further analyze these loop structures for their composition and their impact on expression of the linked long non-coding RNA, Nctc1. We show that Nctc1 is co-regulated with Igf2 and H19 and physically interacts with the shared muscle enhancer. In fact, all three co-regulated genes have the potential to interact not only with the shared enhancer but also with each other via their enhancer interactions. Furthermore, developmental and genetic analyses indicate functional significance for these promoter–promoter interactions. Altogether, we present a novel mechanism to explain developmental specific imprinting of Nctc1 and provide new information about enhancer mechanisms and about the role of chromatin domains in establishing gene expression patterns.

Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.

Promoter-promoter interactions influencing transcription of the yeast mitochondrial gene, Oli 1, coding for ATPase subunit 9. Cis and trans effects.

The gene for ATPase subunit 9 of yeast mitochondria (Oli 1) contains two promoter sequences (Op1 and Op2) separated by 78 nucleotides. Both promoters are transcribed in vivo and in vitro though with different efficiency. The upstream promoter (Op1) is 12-15 times stronger than the downstream promoter (Op2), and this difference in promoter activity is partly attributable to the influence of the +2 nucleotide (Biswas, T. K., and Getz, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 270-274). In addition, the presence of the strong promoter (Op1) in close proximity to the weak promoter (Op2) partially inhibits the expression of the latter (Op2). The relative orientation of the two promoters has no influence on these inhibitory effects. When both promoters are present in the same reaction mixture, the strong promoter always competes effectively with the weak promoter for limited RNA polymerase (trans or competition effect). When the two promoters are present in the same plasmid, there is an inhibitory interaction between them that decreases as the distance between the two promoters increases (cis or position effect). Thus, the difference between the activities of a strong and a weak mitochondrial promoter in tandem is a function of two effects, the trans or competition effect and the cis or position-related effect. A model for promoter-promoter interactions is proposed.