scholarly journals Comprehensive, high-resolution binding energy landscapes reveal context dependencies of transcription factor binding

2018 ◽  
Vol 115 (16) ◽  
pp. E3702-E3711 ◽  
Author(s):  
Daniel D. Le ◽  
Tyler C. Shimko ◽  
Arjun K. Aditham ◽  
Allison M. Keys ◽  
Scott A. Longwell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Energy ◽  
Dna Sequences ◽  
Large Fraction ◽  
Binding Energies ◽  
Transcriptional Networks ◽  
Binding Behavior ◽  
Biophysical Models ◽  
Quantitative Measurements

Transcription factors (TFs) are primary regulators of gene expression in cells, where they bind specific genomic target sites to control transcription. Quantitative measurements of TF–DNA binding energies can improve the accuracy of predictions of TF occupancy and downstream gene expression in vivo and shed light on how transcriptional networks are rewired throughout evolution. Here, we present a sequencing-based TF binding assay and analysis pipeline (BET-seq, for Binding Energy Topography by sequencing) capable of providing quantitative estimates of binding energies for more than one million DNA sequences in parallel at high energetic resolution. Using this platform, we measured the binding energies associated with all possible combinations of 10 nucleotides flanking the known consensus DNA target interacting with two model yeast TFs, Pho4 and Cbf1. A large fraction of these flanking mutations change overall binding energies by an amount equal to or greater than consensus site mutations, suggesting that current definitions of TF binding sites may be too restrictive. By systematically comparing estimates of binding energies output by deep neural networks (NNs) and biophysical models trained on these data, we establish that dinucleotide (DN) specificities are sufficient to explain essentially all variance in observed binding behavior, with Cbf1 binding exhibiting significantly more nonadditivity than Pho4. NN-derived binding energies agree with orthogonal biochemical measurements and reveal that dynamically occupied sites in vivo are both energetically and mutationally distant from the highest affinity sites.

scholarly journals Comprehensive, high-resolution binding energy landscapes reveal context dependencies of transcription factor binding

2017 ◽  
Author(s):  
Daniel D. Le ◽  
Tyler C. Shimko ◽  
Arjun K. Aditham ◽  
Allison M. Keys ◽  
Yaron Orenstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Large Fraction ◽  
Binding Energies ◽  
Transcriptional Networks ◽  
Binding Behavior ◽  
Biophysical Models ◽  
Quantitative Measurements ◽  
Biochemical Measurements

Transcription factors (TFs) are primary regulators of gene expression in cells, where they bind specific genomic target sites to control transcription. Quantitative measurements of TF-DNA binding energies can improve the accuracy of predictions of TF occupancy and downstream gene expression in vivo and further shed light on how transcriptional networks are rewired throughout evolution. Here, we present a novel sequencing-based TF binding assay and analysis pipeline capable of providing quantitative estimates of binding energies for more than one million DNA sequences in parallel at high energetic resolution. Using this platform, we measured the binding energies associated with all possible combinations of 10 nucleotides flanking the known consensus DNA target for two model yeast TFs, Pho4 and Cbf1. A large fraction of these flanking mutations change overall binding energies by an amount equal to or greater than consensus site mutations, suggesting that current definitions of TF binding sites may be too restrictive. By systematically comparing estimates of binding energies output by deep neural networks (NN) and biophysical models trained on these data, we establish that dinucleotide specificities are sufficient to explain essentially all variance in observed binding behavior, with Cbf1 binding exhibiting significantly more epistasis than Pho4. NN-derived binding energies agree with orthogonal biochemical measurements and reveal that dynamically occupied sites in vivo are both energetically and mutationally distant from the highest-affinity sites.

scholarly journals Location of sequences in polyomavirus DNA that are required for early gene expression in vivo and in vitro.

1984 ◽  
Vol 4 (12) ◽  
pp. 2594-2609 ◽  
Author(s):  
C R Mueller ◽  
A M Mes-Masson ◽  
M Bouvier ◽  
J A Hassell
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Dna Sequences ◽  
Viral Genome ◽  
Simian Virus 40 ◽  
Early Gene ◽  
Wild Type ◽  
Transcription In Vitro

To define the DNA sequences required for the expression of the polyomavirus early transcription unit, we cloned part of the viral genome in a plasmid vector, isolated mutants bearing lesions introduced in vitro within DNA sequences upstream of the transcriptional start site, and measured the capacity of these various mutant genomes to transform cells and to function as templates for transcription in vitro by comparison with wild-type DNA. One set of mutants bore 5' unidirectional deletions beginning at position -810 and extending downstream to position +4. Another set of mutants bore 3' undirectional deletions starting at position +4 and progressing upstream to position -311. The last set of mutants bore internal deletions between positions -810 and +4. Analyses of the properties of these mutant DNAs led us to conclude that the region between positions -403 and -311 includes an enhancer of gene expression. Deletion of this area from the viral genome reduced gene expression in vivo to 1 to 2% of wild-type levels, as measured by transformation assays. Moreover, this region increased the frequency of transformation of thymidine kinase-negative Rat-2 cells by the herpes simplex virus thymidine kinase (tk) gene from 5- to 20-fold. This occurred only if the polyomavirus sequences were covalently linked to the tk gene and then occurred independently of their orientation or position relative to the tk gene. A second transcriptional element is located downstream of the enhancer between positions -311 and -213. This element together with the enhancer was sufficient to bring about transformation of Rat-1 cells at nearly wild-type frequencies, and together these elements constitute the minimal sequences required for gene expression in vivo. The sequences making up the second element may be functionally duplicated downstream of position -165 (between positions -165 and -60). This was revealed by the characterization of mutant genomes with deletions between positions -349 and -60. The role of these redundant elements is not known; however, they may be analogous to the 21-base-pair repeats of simian virus 40. Finally, sequences between positions -57 and -1 were required for accurate and efficient transcription in vitro. However, this DNA stretch, which includes the TATA box and major transcriptional start sites, was not absolutely required for gene expression in vivo. We conclude that the polyomavirus promoter comprises multiple functional elements which are distributed across a DNA stretch of about 400 base pairs.

Molecular pathophysiology of glucagon-SV40 T antigen transgenic mice

1994 ◽  
Vol 267 (5) ◽  
pp. E629-E635 ◽  
Author(s):  
D. J. Drucker
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Dna Sequences ◽  
Large Bowel ◽  
Biological Activities ◽  
T Antigen ◽  
Sv40 Large T Antigen ◽  
Molecular Determinants

The gene encoding proglucagon is expressed in the pancreas, intestine, and brain. The molecular determinants of proglucagon gene expression and the biological activities of the proglucagon-derived peptides (PGDPs) have been examined using transgenic mice harboring a glucagon-SV40 large T antigen (GLUTag) transgene. These experiments have delineated DNA sequences important for intestinal-specific proglucagon gene transcription. GLUTag mice develop neuroendocrine tumors of the pancreas and large bowel, leading to elevated plasma levels of the PGDPs and suppression of endogenous proglucagon gene expression. Transplantation of the large bowel tumors subcutaneously into nude mice provides additional evidence for inhibition of endogenous pancreatic proglucagon gene expression by one or more of the tumor-derived PGDPs. The large bowel GLUTag tumors exhibit abnormalities in the posttranslational processing of proglucagon. GLUTag tumors may be passaged in vivo and in vitro and have been used to generate stable cell lines that express the proglucagon gene at high levels. Taken together, these studies highlight the utility of transgenic systems for the physiological analysis of hormone action and the molecular determinants of peptide hormone gene expression.

Gene expression profiles of RAW264.7 macrophages stimulated with preparations of LPS differing in isolation and purity

2011 ◽  
Vol 18 (1) ◽  
pp. 80-88 ◽  
Author(s):  
Holly R Rutledge ◽  
Weiwen Jiang ◽  
Jun Yang ◽  
Laura A Warg ◽  
David A Schwartz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Differentially Expressed ◽  
Transcriptional Networks ◽  
Molecular Pathways ◽  
Raw264.7 Macrophages ◽  
Lps Treatment

Lipopolysaccharide is a major component of the cell wall of Gram-negative bacteria and a potent stimulator of innate immune response via TLR4. Studies on the LPS action both in vivo and in vitro have used different preparations of LPS, including ultra-pure LPS (LIST) and a less pure but less expensive form (Sigma) isolated from Escherichia coli serotype O111:B4. The difference between the effects of these compounds has not been well studied although this information is important in understanding TLR stimulation. In this study, we compared response of RAW264.7 macrophage cells treated LIST or Sigma LPS for 6 h and 24 h. Gene expression data were analyzed to identify specific genes and pathways that are in common and unique to the two LPS preparations. Seven hundred fifty-five genes were differentially expressed at 6 h in response to Sigma LPS and 973 were differentially expressed following LIST LPS treatment, with 503 in common. At 24 h, Sigma LPS induced or repressed 901 genes while 1646 genes were differentially regulated by LIST LPS treatment; 701 genes were shared by two forms of LPS. Although considerably more genes were differentially expressed in response to LIST LPS, similar molecular pathways and transcriptional networks were activated by the two LPS preparations. We also treated bone marrow-derived macrophages (BMMs) from three strains of mice with different concentrations of LIST and Sigma LPS and showed that BMMs produced more IL-6 and TNF-α in response to LIST LPS at low LPS concentrations but, at higher LPS concentrations, more cytokines were produced in response to stimulation by Sigma LPS. Together, these findings suggest that, despite activation of similar molecular pathways by LIST and Sigma LPS preparations, residual protein impurities in the Sigma LPS preparation may nevertheless influence the transcriptional profile attributed to TLR4 stimulation.

scholarly journals Mapping DNA sequence to transcription factor binding energy in vivo

2018 ◽  
Author(s):  
Stephanie L. Barnes ◽  
Nathan M. Belliveau ◽  
William T. Ireland ◽  
Justin B. Kinney ◽  
Rob Phillips
Keyword(s):  
Transcription Factor ◽  
Binding Energy ◽  
Dna Sequence ◽  
Binding Sites ◽  
Gene Networks ◽  
Transcription Factor Binding ◽  
Binding Energies ◽  
Regulatory Sequences ◽  
Factor Binding

AbstractDespite the central importance of transcriptional regulation in systems biology, it has proven difficult to determine the regulatory mechanisms of individual genes, let alone entire gene networks. It is particularly difficult to analyze a promoter sequence and identify the locations, regulatory roles, and energetic properties of binding sites for transcription factors and RNA polymerase. In this work, we present a strategy for interpreting transcriptional regulatory sequences using in vivo methods (i.e. the massively parallel reporter assay Sort-Seq) to formulate quantitative models that map a transcription factor binding site’s DNA sequence to transcription factor-DNA binding energy. We use these models to predict the binding energies of transcription factor binding sites to within 1 kBT of their measured values. We further explore how such a sequence-energy mapping relates to the mechanisms of trancriptional regulation in various promoter contexts. Specifically, we show that our models can be used to design specific induction responses, analyze the effects of amino acid mutations on DNA sequence preference, and determine how regulatory context affects a transcription factor’s sequence specificity.

scholarly journals Parallel functional testing identifies enhancers active in early postnatal mouse brain

2021 ◽  
Author(s):  
Jason T. Lambert ◽  
Linda Su-Feher ◽  
Karol Cichewicz ◽  
Tracy L. Warren ◽  
Iva Zdilar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Brain ◽  
Dna Sequences ◽  
Regulatory Element ◽  
Linkage Disequilibrium Block ◽  
Regulatory Elements ◽  
Functional Screening ◽  
Functional Testing ◽  
Enhancer Activity

ABSTRACTCis-regulatory elements such as enhancers play critical regulatory roles in modulating developmental transcription programs and driving cell-type specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted the self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identify putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in normal and pathogenic brain development.

scholarly journals In vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

2019 ◽  
Author(s):  
Nathan J. VanDusen ◽  
Julianna Y. Lee ◽  
Weiliang Gu ◽  
Isha Sethi ◽  
Yanjiang Zheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regenerative Medicine ◽  
Forward Genetics ◽  
Transcriptional Networks ◽  
Epigenetic Mark ◽  
High Resource ◽  
Organ Systems ◽  
Forward Genetic Screens ◽  
Somatic Mutagenesis

ABSTRACTBetween birth and adulthood cardiomyocytes (CMs) undergo dramatic changes in size, ultrastructure, metabolism, and gene expression, in a process collectively referred to as CM maturation. The transcriptional network that coordinates CM maturation is poorly understood, creating a bottleneck for cardiac regenerative medicine. Forward genetic screens are a powerful, unbiased method to gain novel insights into transcriptional networks, yet this approach has rarely been used in vivo in mammals because of high resource demands. Here we utilized somatic mutagenesis to perform the first reported in vivo CRISPR genetic screen within a mammalian heart. We discovered and validated several novel transcriptional regulators of CM maturation. Among them were RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicated that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

scholarly journals A family of transcription factors that limit lifespan: ETS factors have conserved roles in longevity

2018 ◽  
Author(s):  
Adam J. Dobson ◽  
Richard Boulton-McDonald ◽  
Lara Houchou ◽  
Ziyu Ren ◽  
Mimoza Hoti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcriptional Networks ◽  
Regulation Of Transcription ◽  
Detailed Knowledge ◽  
Extended Lifespan ◽  
Ets Family ◽  
Ets Factors

ABSTRACTIncreasing average population age, and the accompanying burden of ill health, is one of the public health crises of our time. Understanding the basic biology of the ageing process may help ameliorate the pathologies that characterise old age. Ageing can be modulated, often through changes in gene expression where regulation of transcription plays a pivotal role. Activities of Forkhead transcription factors (TFs) are known to extend lifespan, but detailed knowledge of the broader transcriptional networks that promote longevity is lacking. This study focuses on the E twenty-six (ETS) family of TFs. This family of TFs is large, conserved across metazoa, and known to play roles in development and cancer, but the role of its members in ageing has not been studied extensively. InDrosophila, an ETS transcriptional repressor,Aop, and an ETS transcriptional activator,Pnt, are known to genetically interact withFoxoand activatingAopis sufficient to extend lifespan. Here, it is shown thatAopandFoxoeffect a related gene-expression programme. Additionally,Aopcan modulateFoxo’s transcriptional output to moderate or synergise withFoxoactivity depending on promoter context, bothin vitroandin vivo.In vivogenome-wide mRNA expression analysis in response toAop,PntorFoxoindicated, and further experiments confirmed, that combinatorial activities of the three TFs dictate metabolic status, and that direct reduction ofPntactivity is sufficient to promote longevity. The role of ETS factors in longevity was not limited toPntandAop. Knockdown ofEts21corEip74EFin distinct cell types also extended lifespan, revealing that lifespan is limited by transcription from the ETS binding site in multiple cellular contexts. Reducing the activity of theC. elegansETS TFLin-1also extended lifespan, a finding that corroborates established evidence of roles of this TF family in ageing. Altogether, these results reveal the ETS family of TFs as pervasive and evolutionarily conserved brokers of longevity.

scholarly journals MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome

2015 ◽  
Vol 112 (17) ◽  
pp. 5509-5514 ◽  
Author(s):  
Lin Chen ◽  
Kaifu Chen ◽  
Laura A. Lavery ◽  
Steven Andrew Baker ◽  
Chad A. Shaw ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Rett Syndrome ◽  
Dna Sequences ◽  
Neurodevelopmental Disorder ◽  
Adult Brain ◽  
Gene Encoding ◽  
Dna Elements ◽  
Timing Of Onset

Epigenetic mechanisms, such as DNA methylation, regulate transcriptional programs to afford the genome flexibility in responding to developmental and environmental cues in health and disease. A prime example involving epigenetic dysfunction is the postnatal neurodevelopmental disorder Rett syndrome (RTT), which is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2). Despite decades of research, it remains unclear how MeCP2 regulates transcription or why RTT features appear 6–18 months after birth. Here we report integrated analyses of genomic binding of MeCP2, gene-expression data, and patterns of DNA methylation. In addition to the expected high-affinity binding to methylated cytosine in the CG context (mCG), we find a distinct epigenetic pattern of substantial MeCP2 binding to methylated cytosine in the non-CG context (mCH, where H = A, C, or T) in the adult brain. Unexpectedly, we discovered that genes that acquire elevated mCH after birth become preferentially misregulated in mouse models of MeCP2 disorders, suggesting that MeCP2 binding at mCH loci is key for regulating neuronal gene expression in vivo. This pattern is unique to the maturing and adult nervous system, as it requires the increase in mCH after birth to guide differential MeCP2 binding among mCG, mCH, and nonmethylated DNA elements. Notably, MeCP2 binds mCH with higher affinity than nonmethylated identical DNA sequences to influence the level of Bdnf, a gene implicated in the pathophysiology of RTT. This study thus provides insight into the molecular mechanism governing MeCP2 targeting and sheds light on the delayed onset of RTT symptoms.

Binding of a factor to an enhancer element responsible for the tissue-specific expression of the chicken alpha A-crystallin gene

Development ◽  
1991 ◽  
Vol 113 (2) ◽  
pp. 539-550 ◽  
Author(s):  
I. Matsuo ◽  
M. Kitamura ◽  
K. Okazaki ◽  
K. Yasuda
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Nuclear Protein ◽  
Fusion Gene ◽  
Regulatory Region ◽  
Binding Activity ◽  
Flanking Sequence ◽  
Specific Expression ◽  
Enhancer Element

We have characterized a regulatory region of the chicken alpha A-crystallin gene using transfection assays, which revealed that a 84 base pair element (−162 to −79) in the 5′ flanking sequence is necessary and sufficient for lens-specific expression. A multimer of this element functions as lens-specific enhancer and synergistically activates transcription from chicken alpha A-crystallin or beta-actin basal promoters fused to the CAT gene. In vivo competition experiments demonstrated that DNA sequences containing the 84 bp element reduced alpha A-crystallin-CAT fusion gene expression. A nuclear factor present exclusively in lens cells binds to the 84 bp element in the region between positions −165 and −140. Southwestern blot analysis showed that 61,000 Mr (61 × 10(3) Mr) lens nuclear protein exhibited DNA-binding activity specific to the 84 bp element. Our data suggested that the 61 × 10(3) Mr nuclear protein, and the 84 bp element that it interacts with, may be involved in regulating the alpha A-crystallin gene expression in vivo.

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