Transcription factor binding site orientation and order are major drivers of gene regulatory activity

Georgakopoulos-Soares, Ilias; Deng, Chengyu; Agarwal, Vikram; Chan, Charles; Jin, Zhao; Inoue, Fumitaka; Ahituv, Nadav

doi:10.1038/s41467-023-37960-5

Cited by 24 publications

(12 citation statements)

References 46 publications

(55 reference statements)

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“…3f ), indicating that the combination of up- and downstream GEMs allows a more precise expression fine-tuning. Interestingly, inversions of the building blocks generally resulted in minor changes in fluorescence, with the inverted orientation sometimes leading to enhanced expression, confirming that the orientation of promoter elements can influence gene expression 79 . Surprisingly, the sequencing data also revealed that expression levels were not only influenced by the 3’ GEM-block that was closest to the ORF.…”

Section: Resultsmentioning

confidence: 72%

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast

Cautereels,

Smets,

Bircham

et al. 2024

Nat Commun

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Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.

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Section: Resultsmentioning

confidence: 72%

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast

Cautereels,

Smets,

Bircham

et al. 2024

Nat Commun

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show abstract

“…58,59 These studies focus on the binding affinity of TF and verify in vitro by evaluating the relative concentration of repressor dimers 56 or reporter gene expression. 58 Recently, massively parallel reporter assays (MPRAs) has been utilized to study the function of HCTs, [60][61][62] however, this approach may not recapitulate the truth at the native locus. We determined the cooperative binding of ZNF410 to the CHD4 enhancer regions through both reporter assays and in vivo mutant motif knock-in clones that only replace 2 nucleotides within the ZNF410 motif to disrupt its binding.…”

Section: Discussionmentioning

confidence: 99%

Mechanistic dissection of theCHD4enhancers reveals cooperative functions among the homotypic ZNF410 clustered motifs

Xu,

Ren,

Peng

et al. 2024

Preprint

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SUMMARYTranscription factors often regulate numerous target genes. However, ZNF410 controls only a single gene,CHD4,in human erythroid cells by its highly restricted chromatin occupancy to theCHD4locus via two clusters of ZNF410 binding motifs. Here, we uncover that ZNF410 controls chromatin accessibility and activity of the twoCHD4enhancer regions. Combining CRISPR/Cas9 genomic deletion with CRISPRi approaches, we demonstrate that both enhancer regions additively contribute toCHD4gene expression. Mutations of the di-adenine nucleotides within the ZNF410 binding motif fully disrupt ZNF410-DNA interaction. Luciferase assays, ChIP-seq, and ATAC-seq studies reveal that the homotypic clustered motifs within theCHD4enhancers are recognized by ZNF410 in a collaborative fashion. Especially, the three ZNF410 motifs located in the 3’ end of the distal enhancer act as “switch motifs” to control the chromatin accessibility and activity of the whole distal enhancer region. Together, our findings expose a complex functional hierarchy of motifs, where clustered motifs bound by the same transcription factor cooperate to fine-tune the expression of target gene.HighlightsZNF410 maintains the accessibility of theCHD4enhancer regionsThe two enhancer elements additively modulateCHD4gene expressionThe dinucleotide “AA” is the key bases within ZNF410 binding motifZNF410 binds to homotypic clustered motifs in a cooperative manner

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“…Many phenotypic differences among individuals may be elicited by alterations in gene expression and the underlying transcriptional regulation, and the expression of genes can be regulated by TF binding to TFBSs [35,36]. The SNP located in the TFBS may affect the binding of TF, resulting in differences in gene expression among individuals with different genotypes [37,38].…”

Section: Discussionmentioning

confidence: 99%

Genetic Association of APOA5 and AKT3 Genes with Milk Production Traits in Chinese Holstein Cows

Guo,

Du,

Han

et al. 2024

Agriculture

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Genome selection (GS) technology is an important means to improve the genetic improvement of dairy cows, and the mining and application of functional genes and loci for important traits is one of the important bases for accelerating genetic improvement. Our previous study found that the apolipoprotein A5 (APOA5) and AKT serine/threonine kinase 3 (AKT3) genes were differentially expressed in the liver tissue of Chinese Holstein cows at different lactation stages and influenced milk component synthesis and metabolism, so we considered these two genes as the candidates affecting milk production traits. In this study, we found in total six single nucleotide polymorphisms (SNPs), three in APOA5 and three in AKT3. Subsequent association analysis showed that the six SNPs were significantly associated with milk yield, fat yield, protein yield, or fat percentage (p ≤ 0.05). Three SNPs in APOA5 formed a haplotype block, which was found to be significantly associated with milk yield, fat yield, and protein yield (p ≤ 0.05). In addition, four SNPs were proposed to be functional mutations affecting the milk production phenotype, of which three, 15:g.27446527C>T and 15:g.27447741A>G in APOA5 and 16:g.33367767T>C in AKT3, might change the transcription factor binding sites (TFBSs), and one is a missense mutation, 15:g.27445825T>C in APOA5, which could alter the secondary structure and stability of mRNA and protein. In summary, we demonstrated the genetic effects of APOA5 and AKT3 on milk production traits, and the valuable SNPs could be used as available genetic markers for dairy cattle’s GS.

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Transcription factor binding site orientation and order are major drivers of gene regulatory activity

Cited by 24 publications

References 46 publications

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast

Mechanistic dissection of theCHD4enhancers reveals cooperative functions among the homotypic ZNF410 clustered motifs

Genetic Association of APOA5 and AKT3 Genes with Milk Production Traits in Chinese Holstein Cows

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