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

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.

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Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽

10.1242/dev.125.22.4349 ◽

1998 ◽

Vol 125 (22) ◽

pp. 4349-4358 ◽

Cited By ~ 4

Author(s):

J. Charite ◽

W. de Graaff ◽

D. Consten ◽

M.J. Reijnen ◽

J. Korving ◽

...

Keyword(s):

Binding Sites ◽

Hox Genes ◽

Regulatory Element ◽

Ectopic Expression ◽

Positional Information ◽

Regulatory Elements ◽

Gene Products ◽

Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

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Patterning the ascidian nervous system: structure, expression and transgenic analysis of the CiHox3 gene

Development ◽

10.1242/dev.126.21.4737 ◽

1999 ◽

Vol 126 (21) ◽

pp. 4737-4748 ◽

Cited By ~ 1

Author(s):

A. Locascio ◽

F. Aniello ◽

A. Amoroso ◽

M. Manzanares ◽

R. Krumlauf ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Hox Genes ◽

Regional Identity ◽

Regulatory Elements ◽

System Structure ◽

Anteroposterior Patterning ◽

Hox Gene ◽

The Central Nervous System ◽

Group 3

Hox genes play a fundamental role in the establishment of chordate body plan, especially in the anteroposterior patterning of the nervous system. Particularly interesting are the anterior groups of Hox genes (Hox1-Hox4) since their expression is coupled to the control of regional identity in the anterior regions of the nervous system, where the highest structural diversity is observed. Ascidians, among chordates, are considered a good model to investigate evolution of Hox gene, organisation, regulation and function. We report here the cloning and the expression pattern of CiHox3, a Ciona intestinalis anterior Hox gene homologous to the paralogy group 3 genes. In situ hybridization at the larva stage revealed that CiHox3 expression was restricted to the visceral ganglion of the central nervous system. The presence of a sharp posterior boundary and the absence of transcript in mesodermal tissues are distinctive features of CiHox3 expression when compared to the paralogy group 3 in other chordates. We have investigated the regulatory elements underlying CiHox3 neural-specific expression and, using transgenic analysis, we were able to isolate an 80 bp enhancer responsible of CiHox3 activation in the central nervous system (CNS). A comparative study between mouse and Ciona Hox3 promoters demonstrated that divergent mechanisms are involved in the regulation of these genes in vertebrates and ascidians.

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Effects of the maternal factor Zelda on zygotic enhancer activity in the Drosophila embryo

10.1101/385070 ◽

2018 ◽

Author(s):

Xiao-Yong Li ◽

Michael B. Eisen

Keyword(s):

Binding Sites ◽

Expression Patterns ◽

Drosophila Embryo ◽

Temporal Expression ◽

Reporter Construct ◽

Limited Effect ◽

Enhancer Activity ◽

Maternal Factor ◽

Anterior Posterior

AbstractThe maternal factor Zelda is broadly bound to zygotic enhancers during early fly embryogenesis, and has been shown to be important for the expression of a large number of genes. However, its function remains poorly understood. Here, we carried out detailed analysis of the functional role of Zelda on the activities of a group of enhancers that drive patterned gene expression along the anterior -posterior axis. We found that among these enhancers, only one lost its activity entirely when all its Zelda bind sites were mutated. For all others, mutations of all of their Zelda binding sites only had limited effect, which varied temporally and spatially. These results suggest that Zld may exert a quantitative effect on a broad range of enhancers, which presumably is critical to generate highly diverse spatial and temporal expression patterns for different genes in the developmental gene network in fly embryo. Lastly, we found that the observed effect of Zelda site mutations was much stronger when a mutant enhancer was tested using a BAC based reporter construct than a simple reporter construct, suggesting that the effect of Zld is dependent on chromatin environment.

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Human library of cardiac promoters and enhancers

10.1101/2020.06.14.150904 ◽

2020 ◽

Author(s):

Ruslan M. Deviatiiarov ◽

Anna Gams ◽

Roman Syunyaev ◽

Tatiana V. Tatarinova ◽

Oleg Gusev ◽

...

Keyword(s):

Heart Diseases ◽

Association Studies ◽

Expression Patterns ◽

Critical Role ◽

Regulatory Elements ◽

Specific Cell ◽

Genome Wide Association Studies ◽

Specific Expression ◽

Enhancer Activity ◽

Human Donor

AbstractGenome regulatory elements play a critical role during cardiac development and maintenance of normal physiological homeostasis, and genome-wide association studies identified a large number of SNPs associated with cardiovascular diseases localized in intergenic zones. We used cap analysis of gene expression (CAGE) to identify transcription start sites (TSS) with one nucleotide resolution that effectively maps genome regulatory elements in a representative collection of human heart tissues. Here we present a comprehensive and fully annotated CAGE atlas of human promoters and enhancers from four chambers of the non-diseased human donor hearts, including both atria and ventricles. We have identified 10,528 novel regulatory elements, where 2,750 are classified as TSS and 4,258 novel enhancers, which were validated with ChIP-seq libraries and motif enrichment analysis. We found that heart-region specific expression patterns are primarily based on the alternative promoter and specific enhancer activity. Our study significantly increased evidence of the association of regulatory elements-located variants with heart morphology and pathologies. The precise location of cardiac disease-related SNPs within the regulatory regions and their correlation with a specific cell type offers a new understanding of genetic heart diseases.

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The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila

10.1101/373308 ◽

2018 ◽

Author(s):

Arya Zandvakili ◽

Juli Uhl ◽

Ian Campbell ◽

Yuntao Charlie Song ◽

Brian Gebelein

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Dna Sequences ◽

Binding Sites ◽

Target Genes ◽

Hox Genes ◽

Regulatory Elements ◽

Protease Gene ◽

Precise Integration

AbstractHox genes encode a family of transcription factors that, despite having similar in vitro DNA binding preferences, regulate distinct genetic programs along the metazoan anterior-posterior axis. To better define mechanisms of Hox specificity, we compared and contrasted the ability of abdominal Hox factors to regulate two cis-regulatory elements within the Drosophila embryo. Both the Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox factors form cooperative complexes with the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the distal-less leg selector gene via the DCRE, whereas only Abd-A interacts with Exd and Hth on the RhoA element to activate a rhomboid serine protease gene that stimulates Epidermal Growth Factor secretion. By swapping binding sites between these elements, we found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can also convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE, but only in one orientation. We further show that the orientation and spacing of Hox sites relative to additional transcription factor binding sites within the RhoA and DCRE elements is critical to mediate appropriate cell- and segment-specific output. These results indicate that the interaction between Hox, Exd, and Hth neither determines activation vs repression specificity nor defines Ubx vs Abd-A specificity. Instead the precise integration of Hox sites with additional TF inputs is required for accurate transcriptional output. Taken together, these studies provide new insight into the mechanisms of Hox target and regulatory specificity as well as the constraints placed on regulatory elements to convey appropriate outputs.Author SummaryThe Hox genes encode a family of transcription factors that give cells within each region along the developing body plan a unique identity in animals from worms to mammals. Surprisingly, however, most of the Hox factors bind the same or highly similar DNA sequences. These findings raise a paradox: How can proteins that have highly similar DNA binding properties perform different functions in the animal by regulating different sets of target genes? In this study, we address this question by studying how two Hox factors regulate the expression of target genes that specify leg development and the making of liver-like cells in the developing fly. By comparing and contrasting how Hox target genes are activated and/or repressed, we found that the same Hox binding sites can mediate either activation or repression in a manner that depends upon context. In addition, we found that a Hox binding site that is normally regulated by only one Hox factor, can also be used by more than one Hox factor swapped into another target gene. These findings indicate that the specificity of a Hox factor to regulate target genes does not rely solely upon DNA binding specificity but also requires regulatory specificity.

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Human atlas of cardiac promoters and enhancers reveals important role of regulatory elements in heritable diseases

10.21203/rs.3.rs-37530/v1 ◽

2020 ◽

Author(s):

Ruslan Deviatiiarov ◽

Anna Gams ◽

Roman Syunyaev ◽

Tatiana Tatarinova ◽

Oleg Gusev ◽

...

Keyword(s):

Heart Diseases ◽

Association Studies ◽

Expression Patterns ◽

Critical Role ◽

Regulatory Elements ◽

Specific Cell ◽

Genome Wide Association Studies ◽

Specific Expression ◽

Enhancer Activity ◽

Human Donor

Abstract Genome regulatory elements play a critical role during cardiac development and maintenance of normal physiological homeostasis, and genome-wide association studies identified a large number of SNPs associated with cardiovascular diseases localized in intergenic zones. We used cap analysis of gene expression (CAGE) to identify transcription start sites (TSS) with one nucleotide resolution that effectively maps genome regulatory elements in a representative collection of human heart tissues. Here we present a comprehensive and fully annotated CAGE atlas of human promoters and enhancers from four chambers of the non-diseased human donor hearts, including both atria and ventricles. We have identified 10,528 novel regulatory elements, where 2,750 are classified as TSS and 4,258 novel enhancers, which were validated with ChIP-seq libraries and motif enrichment analysis. We found that heart-region specific expression patterns are primarily based on the alternative promoter and specific enhancer activity. Our study significantly increased evidence of the association of regulatory elements-located variants with heart morphology and pathologies. The precise location of cardiac disease-related SNPs within the regulatory regions and their correlation with a specific cell type offers a new understanding of genetic heart diseases.

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Distal and proximal cis-regulatory elements sense X-chromosomal dosage and developmental state at the Xist locus

10.1101/2021.03.29.437476 ◽

2021 ◽

Author(s):

Rutger A.F. Gjaltema ◽

Till Schwämmle ◽

Pauline Kautz ◽

Michael Robson ◽

Robert Schöpflin ◽

...

Keyword(s):

Expression Patterns ◽

Embryonic Stem ◽

Developmental State ◽

Regulatory Elements ◽

Developmental Time ◽

Proximal Region ◽

Spatial Proximity ◽

Enhancer Activity ◽

Regulatory Regions ◽

Spatio Temporal

AbstractDevelopmental genes such as Xist, the master regulator of X-chromosome inactivation (XCI), are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatio-temporal expression patterns. Xist integrates information on X-chromosomal dosage and developmental stage to trigger XCI at the primed pluripotent state in females only. Through a pooled CRISPR interference screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist during the onset of random XCI. By quantifying how enhancer activity is modulated by X-dosage and differentiation, we find that X-dosage controls the promoter-proximal region in a binary switch-like manner. By contrast, differentiation cues activate a series of distal elements and bring them into closer spatial proximity of the Xist promoter. The strongest distal element is part of an enhancer cluster ∼200 kb upstream of the Xist gene which is associated with a previously unannotated Xist-enhancing regulatory transcript, we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. Our study is the first step to disentangle how multiple, functionally distinct regulatory regions interact to generate complex expression patterns in mammals.

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Molecular competition can shape enhancer activity in the Drosophila embryo

10.1101/2021.05.07.443186 ◽

2021 ◽

Author(s):

Rachel Waymack ◽

Mario Gad ◽

Zeba Wunderlich

Keyword(s):

Gene Expression ◽

Binding Sites ◽

Inhibitory Effect ◽

Drosophila Embryo ◽

Enhancer Activity ◽

Endogenous Gene ◽

Endogenous Genes ◽

Regulatory Dna ◽

The Impact

Transgenic reporters allow the measurement of regulatory DNA activity in vivo and consequently have long been useful tools in the study of enhancers. Despite the utility of transgenic reporters, few studies have investigated the potential effects these reporters have on the expression of other transgenic reporters or endogenous genes. A full understanding of the impacts transgenic reporters have on expression is required for accurate interpretation of transgenic reporter data and characterization of gene regulatory mechanisms. Here, we investigate the impact transgenic reporters have on the expression of other transgenic reporters and endogenous genes. By measuring the expression of Kruppel (Kr) enhancer reporters in live Drosophila embryos that contain either one or two copies of identical reporters, we find reporters have an inhibitory effect on one another's expression. Further, expression of a nearby endogenous gene is decreased in the presence of a Kr enhancer reporter. Through the use of competitor binding site arrays, we present evidence that reporters, and potentially endogenous genes, are competing for transcription factors (TFs). Increasing the number of competitor Bcd binding sites decreases the peak levels and spatial extent of Bcd-regulated enhancer reporters' expression. To understand how small numbers of added TF binding sites could impact gene expression to the extent we observe, we develop a simple thermodynamic model of our system. Our model predicts competition of the measured magnitude specifically if TF binding is restricted to distinct nuclear subregions, underlining the importance of the non-homogenous nature of the nucleus in regulating gene expression.

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Characterization of sequence determinants of enhancer function using natural genetic variation

10.1101/2021.12.17.473050 ◽

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.

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Control of early cardiac-specific transcription of Nkx2-5 by a GATA-dependent enhancer

Development ◽

10.1242/dev.126.1.75 ◽

1999 ◽

Vol 126 (1) ◽

pp. 75-84 ◽

Cited By ~ 12

Author(s):

C.L. Lien ◽

C. Wu ◽

B. Mercer ◽

R. Webb ◽

J.A. Richardson ◽

...

Keyword(s):

Homeobox Gene ◽

Regulatory Elements ◽

Upstream Region ◽

Transcriptional Networks ◽

Heart Tube ◽

Enhancer Activity ◽

Endogenous Gene ◽

Developing Heart ◽

High Affinity Binding Site ◽

Cardiac Lineage

The homeobox gene Nkx2-5 is the earliest known marker of the cardiac lineage in vertebrate embryos. Nkx2-5 expression is first detected in mesodermal cells specified to form heart at embryonic day 7.5 in the mouse and expression is maintained throughout the developing and adult heart. In addition to the heart, Nkx2-5 is transiently expressed in the developing pharynx, thyroid and stomach. To investigate the mechanisms that initiate cardiac transcription during embryogenesis, we analyzed the Nkx2-5 upstream region for regulatory elements sufficient to direct expression of a lacZ transgene in the developing heart of transgenic mice. We describe a cardiac enhancer, located about 9 kilobases upstream of the Nkx2-5 gene, that fully recapitulates the expression pattern of the endogenous gene in cardiogenic precursor cells from the onset of cardiac lineage specification and throughout the linear and looping heart tube. Thereafter, as the atrial and ventricular chambers become demarcated, enhancer activity becomes restricted to the developing right ventricle. Transcription of Nkx2-5 in pharynx, thyroid and stomach is controlled by regulatory elements separable from the cardiac enhancer. This distal cardiac enhancer contains a high-affinity binding site for the cardiac-restricted zinc finger transcription factor GATA4 that is essential for transcriptional activity. These results reveal a novel GATA-dependent mechanism for activation of Nkx2-5 transcription in the developing heart and indicate that regulation of Nkx2-5 is controlled in a modular manner, with multiple regulatory regions responding to distinct transcriptional networks in different compartments of the developing heart.

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