scholarly journals Characterization of sequence determinants of enhancer function using natural genetic variation

2021 ◽  
Author(s):  
Marty G. Yang ◽  
Emi Ling ◽  
Christopher J. Cowley ◽  
Michael E. Greenberg ◽  
Thomas Vierbuchen
Keyword(s):  
Binding Sites ◽  
Sequence Variation ◽  
Regulatory Elements ◽  
Human Populations ◽  
Sequence Variant ◽  
Sequence Variants ◽  
Enhancer Activity ◽  
F1 Hybrid ◽  
Enhancer Function ◽  
The Impact

Sequence variation in enhancers, a class of cis-regulatory elements that control cell type-specific gene transcription, contributes significantly to phenotypic variation within human populations. Enhancers are short DNA sequences (~200 bp) composed of multiple binding sites (4-10 bp) for transcription factors (TFs). The transcriptional regulatory activity of an enhancer is encoded by the type, number, and distribution of TF binding sites that it contains. However, the sequence determinants of TF binding to enhancers and the relationship between TF binding and enhancer activity are complex, and thus it remains difficult to predict the effect of any given sequence variant on enhancer function. Here, we generate allele-specific maps of TF binding and enhancer activity in fibroblasts from a panel of F1 hybrid mice that have a high frequency of sequence variants. We identified thousands of enhancers that exhibit differences in TF binding and/or activity between alleles and use these data to define features of sequence variants that are most likely to impact enhancer function. Our data demonstrate a critical role for AP-1 TFs at many fibroblast enhancers, reveal a hierarchical relationship between AP-1 and TEAD TF binding at enhancers, and delineate the nature of sequence variants that contribute to AP-1 TF binding. These data represent one of the most comprehensive assessments to date of the impact of sequence variation on enhancer function in chromatin, with implications for identifying functional cis-regulatory variation in human populations.

scholarly journals Massively parallel discovery of human-specific substitutions that alter enhancer activity

2020 ◽  
Vol 118 (2) ◽  
pp. e2007049118
Author(s):  
Severin Uebbing ◽  
Jake Gockley ◽  
Steven K. Reilly ◽  
Acadia A. Kocher ◽  
Evan Geller ◽  
...  
Keyword(s):  
Human Evolution ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Enhancer Activity ◽  
Genetic Changes ◽  
Gene Regulatory Elements ◽  
Enhancer Function ◽  
Human Accelerated Regions ◽  
The Impact ◽  
Human Specific

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of human traits such as the expansion of the cerebral cortex. However, identifying the particular changes that modified regulatory activity during human evolution remain challenging. Here we used massively parallel enhancer assays in neural stem cells to quantify the functional impact of >32,000 human-specific substitutions in >4,300 human accelerated regions (HARs) and human gain enhancers (HGEs), which include enhancers with novel activities in humans. We found that >30% of active HARs and HGEs exhibited differential activity between human and chimpanzee. We isolated the effects of human-specific substitutions from background genetic variation to identify the effects of genetic changes most relevant to human evolution. We found that substitutions interacted in both additive and nonadditive ways to modify enhancer function. Substitutions within HARs, which are highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions is buffered in enhancers with constrained ancestral functions. Our findings yield insight into how human-specific genetic changes altered enhancer function and provide a rich set of candidates for studies of regulatory evolution in humans.

scholarly journals Massively parallel discovery of human-specific substitutions that alter neurodevelopmental enhancer activity

2019 ◽  
Author(s):  
Severin Uebbing ◽  
Jake Gockley ◽  
Steven K. Reilly ◽  
Acadia A. Kocher ◽  
Evan Geller ◽  
...  
Keyword(s):  
Binding Sites ◽  
Experimental Studies ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Enhancer Activity ◽  
Genetic Changes ◽  
Human Neural Stem Cells ◽  
Human Accelerated Regions ◽  
The Impact ◽  
Human Specific

AbstractGenetic changes that altered the function of gene regulatory elements have been implicated in the evolution of the human brain. However, identifying the particular changes that modified regulatory activity during neurodevelopment remains challenging. Here we used massively parallel enhancer assays in human neural stem cells to measure the impact of 32,776 human-specific substitutions on enhancer activity in 1,363 Human Accelerated Regions (HARs) and 3,027 Human Gain Enhancers (HGEs), which include enhancers with novel activities in humans. We found that 31.9% of active HARs and 36.4% of active HGEs exhibited differential activity between human and chimpanzee. This enabled us to isolate the effects of 401 human-specific substitutions from other types of genetic variation in HARs and HGEs. Substitutions acted in both an additive and non-additive manner to alter enhancer activity. Human-specific substitutions altered predicted binding sites for a specific set of human transcription factors (TFs) that were a subset of TF binding sites associated with enhancer activity in our assay. Substitutions within HARs, which are overall highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions may be buffered in enhancers with constrained ancestral functions. Our findings yield insight into the mechanisms by which human-specific genetic changes impact enhancer function and provide a rich set of candidates for experimental studies of regulatory evolution in humans.

scholarly journals Tissue context determines the penetrance of regulatory DNA variation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jessica M. Halow ◽  
Rachel Byron ◽  
Megan S. Hogan ◽  
Raquel Ordoñez ◽  
Mark Groudine ◽  
...  
Keyword(s):  
Sequence Variation ◽  
Regulatory Elements ◽  
Quantitative Prediction ◽  
Common Disease ◽  
Human Populations ◽  
Cell Type ◽  
Genomic Context ◽  
Cell Type Specificity ◽  
F1 Hybrid ◽  
Dna Accessibility

AbstractFunctional assessment of disease-associated sequence variation at non-coding regulatory elements is complicated by their high degree of context sensitivity to both the local chromatin and nuclear environments. Allelic profiling of DNA accessibility across individuals has shown that only a select minority of sequence variation affects transcription factor (TF) occupancy, yet low sequence diversity in human populations means that no experimental assessment is available for the majority of disease-associated variants. Here we describe high-resolution in vivo maps of allelic DNA accessibility in liver, kidney, lung and B cells from 5 increasingly diverged strains of F1 hybrid mice. The high density of heterozygous sites in these hybrids enables precise quantification of effect size and cell-type specificity for hundreds of thousands of variants throughout the mouse genome. We show that chromatin-altering variants delineate characteristic sensitivity profiles for hundreds of TF motifs. We develop a compendium of TF-specific sensitivity profiles accounting for genomic context effects. Finally, we link maps of allelic accessibility to allelic transcript levels in the same samples. This work provides a foundation for quantitative prediction of cell-type specific effects of non-coding variation on TF activity, which will facilitate both fine-mapping and systems-level analyses of common disease-associated variation in human genomes.

scholarly journals Identification of cellular context sensitive regulatory variation in mouse genomes

2020 ◽  
Author(s):  
Matthew T. Maurano ◽  
Jessica M. Halow ◽  
Rachel Byron ◽  
Mark Groudine ◽  
M. A. Bender ◽  
...  
Keyword(s):  
Sequence Variation ◽  
Regulatory Elements ◽  
Quantitative Prediction ◽  
Common Disease ◽  
Human Populations ◽  
Cell Type ◽  
Genomic Context ◽  
Functional Variation ◽  
F1 Hybrid ◽  
Dna Accessibility

SUMMARYAssessment of the functional consequences of disease-associated sequence variation at non-coding regulatory elements is complicated by their high degree of context sensitivity to both the local chromatin and nuclear environments. Allelic profiling of DNA accessibility across individuals has shown that only a select minority of sequence variation affects transcription factor (TF) occupancy, yet the low sequence diversity in human populations means that no experimental assessment is available for the majority of disease-associated variants. Here we describe high-resolution in vivo maps of allelic DNA accessibility in liver, kidney, lung and B cells from 5 increasingly diverged strains of F1 hybrid mice. The high density of heterozygous sites in these hybrids enables precise quantification of the effect size and cell-type specificity of hundreds of thousands of variants throughout the mouse genome. We show that functional variation delineates characteristic sensitivity profiles for hundreds of TF motifs, representing nearly all important TF families. We develop a compendium of TF-specific sensitivity profiles accounting for genomic context effects. Finally, we link these maps of allelic accessibility to allelic transcript levels in the same samples. This work provides a foundation for quantitative prediction of cell-type specific effects of non-coding variation on TF activity, which will dramatically facilitate both fine-mapping and systems-level analyses of common disease-associated variation in human genomes.

Conserved and distinct roles of kreisler in regulation of the paralogous Hoxa3 and Hoxb3 genes

Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 759-769 ◽  
Author(s):  
M. Manzanares ◽  
S. Cordes ◽  
L. Ariza-McNaughton ◽  
V. Sadl ◽  
K. Maruthainar ◽  
...  
Keyword(s):  
Binding Sites ◽  
Hox Genes ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Enhancer Activity ◽  
Anteroposterior Patterning ◽  
Endogenous Gene ◽  
Transgenic Embryos ◽  
The Individual ◽  
Segmental Expression

During anteroposterior patterning of the developing hindbrain, the anterior expression of 3′ Hox genes maps to distinct rhombomeric boundaries and, in many cases, is upregulated in specific segments. Paralogous genes frequently have similar anterior boundaries of expression but it is not known if these are controlled by common mechanisms. The expression of the paralogous Hoxa3 and Hoxb3 genes extends from the posterior spinal cord up to the rhombomere (r) 4/5 boundary and both genes are upregulated specifically in r5. However, in this study, we have found that Hoxa3 expression is also upregulated in r6, showing that there are differences in segmental expression between paralogues. We have used transgenic analysis to investigate the mechanisms underlying the pattern of segmental expression of Hoxa3. We found that the intergenic region between Hoxa3 and Hoxa4 contains several enhancers, which summed together mediate a pattern of expression closely resembling that of the endogenous Hoxa3 gene. One enhancer specifically directs expression in r5 and r6, in a manner that reflects the upregulation of the endogenous gene in these segments. Deletion analysis localized this activity to a 600 bp fragment that was found to contain a single high-affinity binding site for the Maf bZIP protein Krml1, encoded by the kreisler gene. This site is necessary for enhancer activity and when multimerized it is sufficient to direct a kreisler-like pattern in transgenic embryos. Furthermore the r5/r6 enhancer activity is dependent upon endogenous kreisler and is activated by ectopic kreisler expression. This demonstrates that Hoxa3, along with its paralog Hoxb3, is a direct target of kreisler in the mouse hindbrain. Comparisons between the Krml1-binding sites in the Hoxa3 and Hoxb3 enhancers reveal that there are differences in both the number of binding sites and way that kreisler activity is integrated and restricted by these two control regions. Analysis of the individual sites revealed that they have different requirements for mediating r5/r6 and dorsal roof plate expression. Therefore, the restriction of Hoxb3 to r5 and Hoxa3 to r5 and r6, together with expression patterns of Hoxb3 in other vertebrate species suggests that these regulatory elements have a common origin but have later diverged during vertebrate evolution.

scholarly journals Regulation of the human T-cell receptor alpha gene enhancer: multiple ubiquitous and T-cell-specific nuclear proteins interact with four hypomethylated enhancer elements.

1990 ◽  
Vol 10 (9) ◽  
pp. 4720-4727 ◽  
Author(s):  
I C Ho ◽  
J M Leiden
Keyword(s):  
T Cell ◽  
Protein Binding ◽  
Binding Sites ◽  
Nuclear Protein ◽  
Cell Receptor ◽  
Enhancer Activity ◽  
Protein Binding Sites ◽  
Human T Cell ◽  
Alpha Gene ◽  
Enhancer Function

Transcription of human T-cell receptor (TCR) alpha genes is regulated by a T-cell-specific transcriptional enhancer that is located 4.5 kilobases 3' of the C alpha gene segment. Previous studies have demonstrated that this enhancer contains at least five nuclear protein-binding sites called T alpha 1 to T alpha 5. In the studies described in this report, we have determined the molecular requirements for human TCR alpha enhancer function. In vitro mutagenesis and deletion analyses demonstrated that full enhancer activity is retained in a 116-base-pair fragment containing the T alpha 1 and T alpha 2 nuclear protein-binding sites and that both of these sites are required for full enhancer function. Functional enhancer activity requires that the T alpha 1 and T alpha 2 binding sites be separated by more than 15 and fewer than 85 base pairs. However, the sequence of this spacer region and the relative phase of the two binding sites on the DNA helix do not affect enhancer function. Deletion and mutation analyses demonstrated that the T alpha 3 and T alpha 4 nuclear protein-binding sites are not necessary or sufficient for TCR alpha enhancer activity. However, a fragment containing these two sites was able to compensate for T alpha 1 and T alpha 2 mutations that otherwise abolished enhancer activity. Electrophoretic mobility shift analyses of the TCR alpha enhancer binding proteins revealed that the T alpha 1, T alpha 3, and T alpha 4 binding proteins are expressed in a variety of T-cell and non-T-cell tumor cell lines. In contrast, one of the two T alpha 2 binding activities was detected only in T-cell nuclear extracts. The activity of the TCR alpha enhancer does not appear to be regulated solely at the level of DNA methylation on that the enhancer sequences were found to be identically hypomethylated in B and T cells as compared with fibroblasts. Taken together, these results suggest that TCR alpha enhancer activity is regulated by the interaction of multiple T-cell-specific and ubiquitous nuclear proteins with partially redundant cis-acting enhancer elements that are hypomethylated in cells of the lymphoid lineage.

scholarly journals Direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

2019 ◽  
Vol 116 (20) ◽  
pp. 9893-9902 ◽  
Author(s):  
Christopher M. Uyehara ◽  
Daniel J. McKay
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Nuclear Receptor ◽  
Binding Sites ◽  
Transcriptional Responses ◽  
Enhancer Activity ◽  
Developmental Transitions ◽  
Experimental Systems ◽  
The Impact

The ecdysone pathway was among the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. Here, we use the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone response genes. However, the majority of its binding sites reside at genes with wing-specific functions. We also find that EcR binding is temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.

scholarly journals Systematic dissection of genomic features determining transcription factor binding and enhancer function

2017 ◽  
Vol 114 (7) ◽  
pp. E1291-E1300 ◽  
Author(s):  
Sharon R. Grossman ◽  
Xiaolan Zhang ◽  
Li Wang ◽  
Jesse Engreitz ◽  
Alexandre Melnikov ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Binding Assays ◽  
Genomic Locus ◽  
Enhancer Activity ◽  
Genomic Features ◽  
Motif Site ◽  
Reporter Assays ◽  
Enhancer Function

Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.

Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY

Development ◽  
2002 ◽  
Vol 129 (16) ◽  
pp. 3887-3899 ◽  
Author(s):  
Jonathan Gilthorpe ◽  
Marie Vandromme ◽  
Tim Brend ◽  
Alejandro Gutman ◽  
Dennis Summerbell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Specific Interaction ◽  
Transcriptional Regulators ◽  
Heterologous Promoter ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Hox Gene ◽  
Two Factors ◽  
Enhancer Function

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. As these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site.

scholarly journals Type B Leukemogenic Virus Has a T-Cell-Specific Enhancer That Binds AML-1

2001 ◽  
Vol 75 (5) ◽  
pp. 2174-2184 ◽  
Author(s):  
Jennifer A. Mertz ◽  
Farah Mustafa ◽  
Shari Meyers ◽  
Jaquelin P. Dudley
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Protein Complexes ◽  
Regulatory Elements ◽  
Single Mutation ◽  
Type B ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Enhancer Function ◽  
T Cell Lines

ABSTRACT Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell tumors in mice. TBLV is highly related to mouse mammary tumor virus (MMTV) except that TBLV long terminal repeats (LTRs) have a deletion of negative regulatory elements and a triplication of sequences flanking the deletion. To determine if the LTR triplication represents a viral enhancer element, we inserted the triplication upstream and downstream in either orientation relative to the thymidine kinase promoter linked to the luciferase gene. These experiments showed that upregulation of reporter gene activity by the TBLV triplication was relatively orientation independent, consistent with the activity of eukaryotic enhancer elements. TBLV enhancer activity was observed in T-cell lines but not in fibroblasts, B cells, or mammary cells, suggesting that enhancer function is cell type dependent. To analyze the transcription factor binding sites that are important for TBLV enhancer function, we prepared substitution mutations in a reconstituted C3H MMTV LTR that recapitulates the deletion observed in the TBLV LTR. Transient transfections showed that a single mutation (556M) decreased TBLV enhancer activity at least 20-fold in two different T-cell lines. This mutation greatly diminished AML-1 (recently renamed RUNX1) binding in gel shift assays with a mutant oligonucleotide, whereas AML-1 binding to a wild-type TBLV oligomer was specific, as judged by competition and supershift experiments. The 556 mutation also reduced TBLV enhancer binding of two other protein complexes, called NF-A and NF-B, that did not appear to be related to c-Myb or Ets. AML-1overexpression in a mammary cell line enhanced expression from the TBLV LTR approximately 30-fold. These data suggest that binding of AML-1 to the TBLV enhancer, likely in combination with other factors, is necessary for optimal enhancer function.

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