scholarly journals High affinity enhancer-promoter interactions can bypass CTCF/cohesin-mediated insulation and contribute to phenotypic robustness

2022 ◽  
Author(s):  
Shreeta Chakraborty ◽  
Nina Kopitchinski ◽  
Ariel Eraso ◽  
Parirokh Awasthi ◽  
Raj Chari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Structure ◽  
Transcriptional Control ◽  
Physical Structure ◽  
Allelic Series ◽  
Sox2 Expression ◽  
High Affinity ◽  
Mouse Mutants ◽  
Phenotypic Robustness ◽  
The Impact

Transcriptional control by distal enhancers is an integral feature of gene regulation. To understand how enhancer-promoter interactions arise and assess the impact of disrupting 3D chromatin structure on gene expression, we generated an allelic series of mouse mutants that perturb the physical structure of the Sox2 locus. We show that in the epiblast and in neuronal tissues, CTCF-mediated loops are neither required for the interaction of the Sox2 promoter with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs between Sox2 and its distal enhancers generated ectopic loops with varying degrees of insulation that directly correlated with reduced transcriptional output. Yet, even the mutants exhibiting the strongest insulation, with six CTCF motifs in divergent orientation, could not fully abolish activation by distal enhancers, and failed to disrupt implantation and neurogenesis. In contrast, cells of the anterior foregut were more susceptible to chromatin structure disruption with no detectable SOX2 expression in mutants with the strongest CTCF-mediated boundaries. These animals phenocopied loss of SOX2 in the anterior foregut, failed to separate trachea from esophagus and died perinatally. We propose that baseline transcription levels and enhancer density may influence the tissue-specific ability of distal enhancers to overcome physical barriers and maintain faithful gene expression. Our work suggests that high affinity enhancer-promoter interactions that can overcome chromosomal structural perturbations, play an essential role in maintaining phenotypic robustness.

BCL6 Transcriptionally Reprograms the DLBCL Genome by Forming Distinct Biochemically Active Complexes on Genes Corresponding to Different Pathways.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2646-2646
Author(s):  
Jose M. Polo ◽  
Katerina Chatzi ◽  
Tania Dell’Oso ◽  
Paola Lev ◽  
Ari Melnick
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Chromatin Structure ◽  
Transcriptional Control ◽  
Target Genes ◽  
Biological Effects ◽  
Hematologic Malignancies ◽  
Specific Gene ◽  
Control Mechanisms ◽  
Biochemical Mechanisms

Abstract Aberrant gene expression is a hallmark of cancer, and so it is not surprising that the most common category of oncogenes and tumor suppressors involved in hematologic malignancies are transcription factors. These factors mediate their effects by nucleating biochemically active cofactor complexes to modify the chromatin structure of their respective target genes. BCL6 is a transcriptional repressor and the most commonly involved oncogene in diffuse large B-cell lymphomas. BCL6 represses genes by recruiting several corepressor complexes including SMRT, N-CoR, BCoR; all of which bind to BCL6 through its BTB domain. Each of these complexes has different biochemical functions (e.g. BCoR forms a polycomb complex vs. SMRT which forms an HDAC3 complex). Moreover, our preliminary data suggested that BCL6 uses different sets of corepressors to mediate distinct biological effects, possibly by using different biochemical mechanisms at specific sets of target genes. Therefore, we hypothesized that BCL6 regulates its target genes using different biochemical tools, allowing it to exquisitely fine tune gene expression and provide specific control mechanisms for different biological functions. In order to test this hypothesis we first identified the direct target genes of BCL6 SMRT, N-CoR and BCoR by ChIP-on-chip in DLBCL cells (Ly1 cells) in multiple replicates, and examined whether the overlapping sets of genes corresponded to different gene pathways. We used a 24,000 promoter microarray representing 1.5 KB of sequence for each gene. The results show reproducible binding of BCL6 at 940 promoters, While BCoR bound to 770, SMRT to 545 and N-CoR to 487 promoters respectively. BCL6 and BCoR overlapped at 400 genes, preferentially involved in involve in cell cycle, cell death chromatin structure, ubiquitin dependent process and chemotaxis. BCL6 and SMRT overlapped on 376 genes, involved in immune response, cell motility and also as BCOR cell death, while N-CoR and BCL6 overlapped on 100 genes including transcriptional control and cell death pathways. The overlap between BCoR and SMRT was at 200 genes, BCoR and N-CoR at 60 genes and SMRT and N-CoR at 85 genes. All three overlapped at 50 genes. We also examined whether these corepressors were associated with specific combinations of histone modifications including H3K9 acetylation, H3K9 methylation, H3K4 methylation, H3K27 methylation, H4K16 acetylation and H3K36 acetylation. Taken together, the data indicate that specific subsets of BCL6 target genes are dependent on distinct biochemical mechanisms, suggesting that additional layers of biochemical complexity govern formation of gene repression complexes in DLBCL cells and providing opportunities for highly specific therapeutic targeting of specific gene programs.

scholarly journals Spatial Organization of Chromatin: Transcriptional Control of Adaptive Immune Cell Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Jagan M. R. Pongubala ◽  
Cornelis Murre
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Chromatin Structure ◽  
Transcriptional Control ◽  
Spatial Organization ◽  
Structural Integrity ◽  
Immune Cell ◽  
Three Dimensional ◽  
Spatial Proximity ◽  
Gene Promoters

Higher-order spatial organization of the genome into chromatin compartments (permissive and repressive), self-associating domains (TADs), and regulatory loops provides structural integrity and offers diverse gene regulatory controls. In particular, chromatin regulatory loops, which bring enhancer and associated transcription factors in close spatial proximity to target gene promoters, play essential roles in regulating gene expression. The establishment and maintenance of such chromatin loops are predominantly mediated involving CTCF and the cohesin machinery. In recent years, significant progress has been made in revealing how loops are assembled and how they modulate patterns of gene expression. Here we will discuss the mechanistic principles that underpin the establishment of three-dimensional (3D) chromatin structure and how changes in chromatin structure relate to alterations in gene programs that establish immune cell fate.

The -Globin mRNA Stabilization Complex Is Assembled in the Nucleus Supporting CP Integration of Nuclear and Cytoplasmic Controls over -Globin Gene Expression. ααα.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1205-1205
Author(s):  
Xinjun Ji ◽  
Jian Kong ◽  
Russ P. Carstens ◽  
Stephen A. Liebhaber
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Binding Sites ◽  
Globin Gene ◽  
Splice Acceptor Site ◽  
Globin Mrna ◽  
High Affinity ◽  
Intron 1 ◽  
The Impact ◽  
Globin Gene Expression

Abstract Stability of human -globin mRNA in erythroid cells is critically dependent on the function of a 3UTR RNA-protein (RNP) complex ( complex ). This complex is assembled by the high-affinity binding of a polyC-specific RNA binding protein (CP) to a defined C-rich segment in the 3UTR. Recently we observed that certain CP protein isoforms contain nuclear localization signals, co-localize with nuclear speckles, and shuttle between nuclear and cytoplasmic compartments. These data suggested that the -complex may initially assemble on nascent nuclear transcripts and may have nuclear as well as cytoplasmic functions. To test this model, we immuno-enriched CP-RNP complexes from K562 nuclear extracts and probed these complexes for -globin transcripts. We found that these CP-containing nuclear RNP complexes were enriched for intron-containing -globin transcripts as well as mature, spliced -globin mRNAs. The association of mature -globin mRNA in the complex was entirely dependent on the presence of the 3UTR CP binding site. In contrast, the unspliced -globin transcripts remained associated with CP complexes in the absence of the 3 UTR binding site. These data suggested that one or more previously unrecognized CP binding sites are present within and specific to unspliced -globin transcripts. Visual inspection of -globin intronic sequences revealed two prominent C-rich elements. These sequences are in an open secondary structure and conform to established parameters for high-affinity CP binding sites. RNA EMSA confirmed two high-affinity CP binding sites within intron 1; the stronger site was coincident with the pyrimidine-rich tract of the splice-acceptor site. In vitro splicing assays were used to explore nuclear functions of the a complexes. Incubation of full-length -globin transcripts with HeLa cell nuclear extract resulted in the efficient removal of both introns. Elimination of the 3UTR complex slowed removal of intron 1 but accelerated intron 2 excision. The impact of the intron-1 complexes on splicing was also studied. Incubation of an -globin transcript segment extending from exon 1 through intron 1 and into exon 2 resulted in efficient intron excision. Affinity-depletion of CP from the HeLa extract enhanced intron 1 excision from this substrate. This splicing enhancement was reversed by the addition of recombinant CP to the depleted extract. In summary, we report that assembly of the complexes on -globin transcripts takes place in the nucleus, that these RNP complexes assemble at sites within intron 1 as well as at the previously described 3UTR binding site, and that the both sets of a complexes have the potential to alter the efficiency of -globin intron excision. The fully processed -globin mRNA, containing the remaining 3UTR complex, is then exported to the cytoplasm in a pre-packaged form where it is stabilized by the UTR complex and is robustly translated. We propose that CP mediates and integrates nuclear as well as cytoplasmic post-transcriptional controls over human -globin gene expression. α′’α’ αα′αααααααα′ααα′αααααα′ααααααα′αα′α“”′ααα

scholarly journals Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

2016 ◽  
Vol 199 (3) ◽  
Author(s):  
Jiandong Chen ◽  
Susan Gottesman
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Bacterial Species ◽  
High Capacity ◽  
Constitutive Expression ◽  
Receptor Protein ◽  
Control Of Gene Expression ◽  
Content Type ◽  
High Affinity

ABSTRACT The l-arabinose-inducible araBAD promoter (PBAD) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where PBAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA regulates PBAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of PBAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system and has implications for improving its usage for tunable gene expression.

Nutrient-regulated gene expression in eukaryotes

2006 ◽  
Vol 73 ◽  
pp. 85-96 ◽  
Author(s):  
Richard J. Reece ◽  
Laila Beynon ◽  
Stacey Holden ◽  
Amanda D. Hughes ◽  
Karine Rébora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Direct Interaction ◽  
Signalling Pathways ◽  
A Cell ◽  
One Step ◽  
Higher Eukaryotes ◽  
Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

ASSESSMENT OF HYDROLOGICAL CHANGES IN THE ŠUŠVĖ RIVER

2015 ◽  
Author(s):  
Gražina ŽIBIENĖ ◽  
Alvydas ŽIBAS ◽  
Goda BLAŽAITYTĖ
Keyword(s):  
Hydrological Regime ◽  
Maximum Flow ◽  
Environmental Flow ◽  
Physical Structure ◽  
River Channels ◽  
Hydrologic Alteration ◽  
Water Conditions ◽  
Flow Components ◽  
The Impact ◽  
Large Floods

The construction of dams in rivers negatively affects ecosystems because dams violate the continuity of rivers, transform the biological and physical structure of the river channels, and the most importantly – alter the hydrological regime. The impact on the hydrology of the river can occur through reducing or increasing flows, altering seasonality of flows, changing the frequency, duration and timing of flow events, etc. In order to determine the extent of the mentioned changes, The Indicators of Hydrologic Alteration (IHA) software was used in this paper. The results showed that after the construction of Angiriai dam, such changes occurred in IHA Parameters group as: the water conditions of April month decreased by 31 %; 1-day, 3-days, 7-days and 30-days maximum flow decreased; the date of minimum flow occurred 21 days later; duration of high and low pulses and the frequency of low pulses decreased, but the frequency of high pulses increased, etc. The analysis of the Environmental Flow Components showed, that the essential differences were recorded in groups of the small and large floods, when, after the establishment of the Šušvė Reservoir, the large floods no longer took place and the probability of frequency of the small floods didn’t exceed 1 time per year.

Post-Transcriptional Regulation of Cardiac Sarcomere Protein Titin Through 3’ Untranslated Regions

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Structural Features ◽  
Neonatal Rat ◽  
Regulatory Control ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Regulatory Effects

Titin is the largest known protein in the human body, and forms the backbone of all striated muscle sarcomeres. The elastic nature of titin is an important component of muscle compliance and functionality. A significant amount of energy is expended to synthesize titin, thus we postulate that titin gene expression is under strict regulatory control in order to conserve cellular resources. In general, gene expression is mediated in part by post-transcriptional control elements located within the 5’ and 3’ untranslated regions (UTRs) of mature mRNA. The 3’UTR in particular contains structural features that affect binding capacity to other RNA components, such as MicroRNA, which control mRNA localization, translation, and degradation. The degree and significance of the regulatory effects mediated by two determined variants of titin’s 3’ UTR were evaluated in Neonatal Rat Ventricular Myocyte and Human Embryonic Kidney cell lines. Recombinant plasmids to transfect these cells lines were engineered by insertion of the variant titin 3’UTR 431- and 1047-base pairs sequences into luciferase reporter vectors. Expression due to an unaltered reporter vector served as the control. Quantitative changes in luciferase activity due to the recombinants proportionally represented the effect titin’s respective 3’UTR conferred on downstream post-transcriptional expression relative to the control. The effect due to titin’s shorter 3’UTR sequence was inconclusive; however, results illustrated that titin’s longer 3’UTR sequence caused a 35 percent decrease in protein expression. Secondary structural analysis of the two sequences revealed differential folding patterns that affect the stability and degree of MicroRNA-binding within titin’s variant 3’UTR sequences.

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