scholarly journals Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome.

2021 ◽  
Author(s):  
Miguel Martinez-Ara ◽  
Federico Comoglio ◽  
Joris van Arensbergen ◽  
Bas van Steensel
Keyword(s):  
Mammalian Cells ◽  
Mouse Genome ◽  
Regulatory Elements ◽  
Genomic Context ◽  
Systematic Analysis ◽  
Chromatin Folding ◽  
Reporter Assays ◽  
Mouse Cells ◽  
Genomic Regions ◽  
Single Promoter

Gene expression is in part controlled by cis-regulatory elements (CREs) such as enhancers and repressive elements. Anecdotal evidence has indicated that a CRE and a promoter need to be biochemically compatible for promoter regulation to occur, but this compatibility has remained poorly characterised in mammalian cells. We used high-throughput combinatorial reporter assays to test thousands of CRE - promoter pairs from three Mb-sized genomic regions in mouse cells. This revealed that CREs vary substantially in their promoter compatibility, ranging from striking specificity for a single promoter to quantitative differences in activation across a broad set of promoters. More than half of the tested CREs exhibit significant promoter selectivity. Housekeeping promoters tend to have similar CRE preferences, but other promoters exhibit a wide diversity of compatibilities. Higher-order TF motif combinations may account for compatibility. CRE-promoter selectivity does not correlate with looping interactions in the native genomic context, suggesting that chromatin folding and compatibility are two orthogonal mechanisms that confer specificity to gene regulation.

scholarly journals Dissection of acute stimulus-inducible nucleosome remodeling in mammalian cells

2019 ◽  
Author(s):  
Federico Comoglio ◽  
Marta Simonatto ◽  
Sara Polletti ◽  
Xin Liu ◽  
Stephen T. Smale ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Elements ◽  
Micrococcal Nuclease ◽  
Nucleosome Remodeling ◽  
Nucleosome Dynamics ◽  
Nucleosome Organization ◽  
Regulatory Information ◽  
Different Types ◽  
Genomic Regions ◽  
The Impact

ABSTRACTAccessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). Whereas stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis-regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq datasets and used it to analyze nucleosome dynamics at stimulus-regulated cis-regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing however different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.

scholarly journals Sensitivity of cohesin–chromatin association to high-salt treatment corroborates non-topological mode of loop extrusion

2021 ◽  
Author(s):  
Arkadiy K. Golov ◽  
Anastasia V. Golova ◽  
Alexey A. Gavrilov ◽  
Sergey V. Razin
Keyword(s):  
Mammalian Cells ◽  
Dna Interactions ◽  
Salt Treatment ◽  
Basic Principles ◽  
Parsimonious Explanation ◽  
Chromatid Cohesion ◽  
Chromatin Folding ◽  
The Stability ◽  
Experimental Findings ◽  
Genomic Regions

Abstract Cohesin is a key organizer of chromatin folding in eukaryotic cells. The two main activities of this ring-shaped protein complex are the maintenance of sister chromatid cohesion and the establishment of long-range DNA–DNA interactions through the process of loop extrusion. Although the basic principles of both cohesion and loop extrusion have been described, we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here, we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed the stability of cohesin-dependent loops and cohesin association with chromatin in high ionic strength conditions in G1-synchronised HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions that correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. Because topologically engaged cohesin is considered to be salt resistant, our data corroborate a non-topological model of loop extrusion. We also propose a model of cohesin activity throughout the interphase, which essentially equates the termination of non-topological loop extrusion with topological loading of cohesin. This theoretical framework enables a parsimonious explanation of various seemingly contradictory experimental findings.

scholarly journals Main constraints for RNAi induced by expressed long dsRNA in mouse cells

2019 ◽  
Vol 2 (1) ◽  
pp. e201800289 ◽  
Author(s):  
Tomas Demeter ◽  
Michaela Vaskovicova ◽  
Radek Malik ◽  
Filip Horvat ◽  
Josef Pasulka ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Interferon Response ◽  
Protein Kinase R ◽  
Systematic Analysis ◽  
Positive Effects ◽  
Rnai Efficiency ◽  
Mouse Cells ◽  
Dicer Cleavage ◽  
Microrna Pathway ◽  
Microrna Function

RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.

scholarly journals Sensitivity of cohesin–chromatin association to high-salt treatment corroborates non-topological mode of loop extrusion

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Arkadiy K. Golov ◽  
Anastasia V. Golova ◽  
Alexey A. Gavrilov ◽  
Sergey V. Razin
Keyword(s):  
Mammalian Cells ◽  
Dna Interactions ◽  
Salt Treatment ◽  
Basic Principles ◽  
Parsimonious Explanation ◽  
Chromatid Cohesion ◽  
Chromatin Folding ◽  
The Stability ◽  
Experimental Findings ◽  
Genomic Regions

AbstractCohesin is a key organizer of chromatin folding in eukaryotic cells. The two main activities of this ring-shaped protein complex are the maintenance of sister chromatid cohesion and the establishment of long-range DNA–DNA interactions through the process of loop extrusion. Although the basic principles of both cohesion and loop extrusion have been described, we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here, we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed the stability of cohesin-dependent loops and cohesin association with chromatin in high-ionic-strength conditions in G1-synchronized HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions that correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. Because topologically engaged cohesin is considered to be salt resistant, our data corroborate a non-topological model of loop extrusion. We also propose a model of cohesin activity throughout the interphase, which essentially equates the termination of non-topological loop extrusion with topological loading of cohesin. This theoretical framework enables a parsimonious explanation of various seemingly contradictory experimental findings.

scholarly journals CONTEXT-INDEPENDENT FUNCTION OF A CHROMATIN BOUNDARY IN VIVO

2021 ◽  
Author(s):  
Andréa Willemin ◽  
Lucille Lopez-Delisle ◽  
Christopher Chase Bolt ◽  
Marie-Laure Gadolini ◽  
Denis Duboule ◽  
...  
Keyword(s):  
Boundary Element ◽  
Gene Cluster ◽  
Target Genes ◽  
Regulatory Elements ◽  
Nuclear Space ◽  
Genomic Context ◽  
Mouse Development ◽  
Architectural Proteins ◽  
Genomic Regions ◽  
The Impact

ABSTRACTMammalian genomes are partitioned into sub-megabase to megabase-sized units of preferential interactions called topologically associating domains or TADs, which are likely important for the proper implementation of gene regulatory processes. These domains provide structural scaffolds for distant cis regulatory elements to interact with their target genes within the three-dimensional nuclear space and architectural proteins such as CTCF as well as the cohesin complex participate in the formation of the boundaries between them. However, the importance of the genomic context in providing a given DNA sequence the capacity to act as a boundary element remains to be fully investigated. To address this question, we randomly relocated a topological boundary functionally associated with the mouse HoxD gene cluster and show that it can indeed act similarly outside its initial genomic context. In particular, the relocated DNA segment recruited the required architectural proteins and induced a significant depletion of contacts between genomic regions located across the integration site. The host chromatin landscape was re-organized, with the splitting of the TAD wherein the boundary had integrated. These results provide evidence that topological boundaries can function independently of their site of origin, under physiological conditions during mouse development.AUTHOR SUMMARYDuring development, enhancer sequences tightly regulate the spatio-temporal expression of target genes often located hundreds of kilobases away. This complex process is made possible by the folding of chromatin into domains, which are separated from one another by specific genomic regions referred to as boundaries. In order to understand whether such boundary sequences require their particular genomic contexts to achieve their isolating effect, we analyzed the impact of introducing one such boundary, taken from the HoxD gene cluster, into a distinct topological domain. We show that this ectopic boundary splits the host domain into two sub-domains and affects the expression levels of a neighboring gene. We conclude that this sequence can work independently from its genomic context and thus carries all the information necessary to act as a boundary element.

scholarly journals Exploring functionally annotated transcriptional consensus regulatory elements with CONREL

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Davide Dalfovo ◽  
Samuel Valentini ◽  
Alessandro Romanel
Keyword(s):  
Cell Lines ◽  
Web Application ◽  
Regulatory Networks ◽  
Regulatory Elements ◽  
Levels Of Abstraction ◽  
Binding Motifs ◽  
Extensive Collection ◽  
Transcription Factor Binding Motifs ◽  
Gene Regulatory ◽  
Genomic Regions

Abstract Understanding the interaction between human genome regulatory elements and transcription factors is fundamental to elucidate the structure of gene regulatory networks. Here we present CONREL, a web application that allows for the exploration of functionally annotated transcriptional ‘consensus’ regulatory elements at different levels of abstraction. CONREL provides an extensive collection of consensus promoters, enhancers and active enhancers for 198 cell-lines across 38 tissue types, which are also combined to provide global consensuses. In addition, 1000 Genomes Project genotype data and the ‘total binding affinity’ of thousands of transcription factor binding motifs at genomic regulatory elements is fully combined and exploited to characterize and annotate functional properties of our collection. Comparison with other available resources highlights the strengths and advantages of CONREL. CONREL can be used to explore genomic loci, specific genes or genomic regions of interest across different cell lines and tissue types. The resource is freely available at https://bcglab.cibio.unitn.it/conrel.

scholarly journals Evolution of mouse circadian enhancers from transposable elements

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Julius Judd ◽  
Hayley Sanderson ◽  
Cédric Feschotte
Keyword(s):  
Gene Expression ◽  
Transposable Elements ◽  
Binding Sites ◽  
Mouse Liver ◽  
Integration Site ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Circadian Gene ◽  
Binding Motifs ◽  
Reporter Assays

Abstract Background Transposable elements are increasingly recognized as a source of cis-regulatory variation. Previous studies have revealed that transposons are often bound by transcription factors and some have been co-opted into functional enhancers regulating host gene expression. However, the process by which transposons mature into complex regulatory elements, like enhancers, remains poorly understood. To investigate this process, we examined the contribution of transposons to the cis-regulatory network controlling circadian gene expression in the mouse liver, a well-characterized network serving an important physiological function. Results ChIP-seq analyses reveal that transposons and other repeats contribute ~ 14% of the binding sites for core circadian regulators (CRs) including BMAL1, CLOCK, PER1/2, and CRY1/2, in the mouse liver. RSINE1, an abundant murine-specific SINE, is the only transposon family enriched for CR binding sites across all datasets. Sequence analyses and reporter assays reveal that the circadian regulatory activity of RSINE1 stems from the presence of imperfect CR binding motifs in the ancestral RSINE1 sequence. These motifs matured into canonical motifs through point mutations after transposition. Furthermore, maturation occurred preferentially within elements inserted in the proximity of ancestral CR binding sites. RSINE1 also acquired motifs that recruit nuclear receptors known to cooperate with CRs to regulate circadian gene expression specifically in the liver. Conclusions Our results suggest that the birth of enhancers from transposons is predicated both by the sequence of the transposon and by the cis-regulatory landscape surrounding their genomic integration site.

scholarly journals Position Effects Are Influenced by the Orientation of a Transgene with Respect to Flanking Chromatin

2001 ◽  
Vol 21 (1) ◽  
pp. 298-309 ◽  
Author(s):  
Yong-Qing Feng ◽  
Matthew C. Lorincz ◽  
Steve Fiering ◽  
John M. Greally ◽  
Eric E. Bouhassira
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Methylation Status ◽  
Regulatory Elements ◽  
Cell Populations ◽  
Methylation Analysis ◽  
Position Effects ◽  
Methylation State ◽  
Cassette Exchange

ABSTRACT We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

scholarly journals Evolutionary Analysis of Transcriptional Regulation Mediated by Cdx2 in Rodents

2021 ◽  
Author(s):  
Weizheng Liang ◽  
Guipeng Li ◽  
Huanhuan Cui ◽  
Yukai Wang ◽  
Wencheng Wei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Phenotypic Diversity ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Evolutionary Analysis ◽  
Protein Sequence Analysis ◽  
Systematic Analysis

AbstractDifferences in gene expression, which can arise from divergence in cis-regulatory elements or alterations in transcription factors binding specificity, are one of the most important causes of phenotypic diversity during evolution. By protein sequence analysis, we observed high sequence conservation in the DNA binding domain (DBD) of the transcription factor Cdx2 across many vertebrates, whereas three amino acid changes were exclusively found in mouse Cdx2 (mCdx2), suggesting potential positive selection in the mouse lineage. Multi-omics analyses were then carried out to investigate the effects of these changes. Surprisingly, there were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Finally, we used rat-mouse allodiploid embryonic stem cells (RMES) to study the cis effects of Cdx2-mediated gene regulation between the two rodents. Interestingly, whereas Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.Author summaryOur study 1) represented a first systematic analysis of species-specific adaptation in DNA binding pattern of transcription factor. Although the mouse-specific amino acid changes did not manifest functional impact in our system, several explanations may account for it (See Discussion part for the detail); 2) represented a first study of cis-regulation between two reproductively isolated species by using a novel allodiploid system; 3) demonstrated a higher conservation of transcriptional output than that of DNA binding, suggesting the evolvability/plasticity of the latter; 4) finally provided a rich data resource for Cdx2 mediated regulation, including gene expression, chromatin accessibility and DNA binding etc.

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