The Experts below are selected from a list of 228 Experts worldwide ranked by ideXlab platform

Welcome Bender - One of the best experts on this subject based on the ideXlab platform.

  • H3K27 modifications define segmental regulatory domains in the Drosophila Bithorax Complex
    eLife, 2014
    Co-Authors: Sarah K. Bowman, Aimee M Deaton, Heber G. Domingues, Peggy I. Wang, Ruslan I. Sadreyev, Robert E. Kingston, Welcome Bender
    Abstract:

    The Bithorax Complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a 'stairstep' pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity.

  • Noncoding RNAs of the UltraBithorax domain of the Drosophila Bithorax Complex.
    Genetics, 2013
    Co-Authors: Benjamin Pease, Ana C. Borges, Welcome Bender
    Abstract:

    RNA transcripts without obvious coding potential are widespread in many creatures, including the fruit fly, Drosophila melanogaster. Several noncoding RNAs have been identified within the Drosophila Bithorax Complex. These first appear in blastoderm stage embryos, and their expression patterns indicate that they are transcribed only from active domains of the Bithorax Complex. It has been suggested that these noncoding RNAs have a role in establishing active domains, perhaps by setting the state of Polycomb Response Elements A comprehensive survey across the proximal half of the Bithorax Complex has now revealed nine distinct noncoding RNA transcripts, including four within the UltraBithorax transcription unit. At the blastoderm stage, the noncoding transcripts collectively span ∼75% of the 135 kb surveyed. Recombination-mediated cassette exchange was used to invert the promoter of one of the noncoding RNAs, a 23-kb transcript from the bxd domain of the Bithorax Complex. The resulting animals fail to make the normal bxd noncoding RNA and show no transcription across the bxd Polycomb Response Element in early embryos. The mutant flies look normal; the regulation of the bxd domain appears unaffected. Thus, the bxd noncoding RNA has no apparent function.

  • The border between the ultraBithorax and abdominal-A regulatory domains in the Drosophila Bithorax Complex.
    Genetics, 2013
    Co-Authors: Welcome Bender, Maura Lucas
    Abstract:

    The Bithorax Complex in Drosophila melanogaster includes three homeobox-containing genes—UltraBithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)—which are required for the proper differentiation of the posterior 10 segments of the body. Each of these genes has multiple distinct regulatory regions; there is one for each segmental unit of the body plan where the genes are expressed. One additional protein- coding gene in the Bithorax Complex, Glut3, a sugar-transporter homolog, can be deleted without phenotype. We focus here on the upstream regulatory region for Ubx, the Bithoraxoid (bxd) domain, and its border with the adjacent infraabdominal-2 (iab-2) domain, which controls abdA. These two domains can be defined by the phenotypes of rearrangement breakpoints, and by the expression patterns of enhancer traps. In D. virilis, the homeotic cluster is split between Ubx and abd-A, and so the border can also be located by a sequence comparison between species. When the border region is deleted in melanogaster, the flies show a dominant phenotype called Front-ultraabdominal (Fub); the first abdominal segment is transformed into a copy of the second abdominal segment. Thus, the border blocks the spread of activation from the bxd domain into the iab-2 domain.

  • The structure of Polycomb-repressed chromatin in the Bithorax Complex
    Epigenetics & Chromatin, 2013
    Co-Authors: Sarah K. Bowman, Welcome Bender, Aimee M Deaton, Heber G. Domingues, Robert E. Kingston
    Abstract:

    The 300 kb of the Drosophila Bithorax Complex (BX-C) is the original model for studying Hox gene repression by Polycomb group proteins. Decades of genetic experiments have led to the hypothesis that Polycomb organizes BX-C chromatin differently in each of the abdominal parasegments, and the structure of the chromatin is critical for maintaining cell identity. Studying the molecular organization of chromatin in individual parasegments has been technically difficult. We solved this problem by developing a sorted nuclei ChIP-seq pipeline. In this system, transgenic embryos produce tagged nuclei in single parasegments. Using FACS, we sorted these tagged nuclei and performed small-scale ChIP-seq. Initial results show that a histone mark catalyzed by Polycomb group proteins, trimethylated histone H3 at lysine 27 (H3K27me3), covers less of the BX-C as we move from PS5 to PS7. The boundaries of the H3K27 methylation correlate precisely with previously identified CTCF binding sites, a protein known to act as a chromatin barrier. Correspondingly, a mark that correlates with gene activation, H3K4me3, appears over the transcription start sites of BX-C genes that are not associated with Polycomb histone methyltransferase activity. Going forward, we will assay the localization of Polycomb group proteins and many other chromatin regulatory proteins to gain a complete picture of the chromatin environment that regulates Hox gene expression in the developing embryo. Still, the current results offer a compelling first look at BX-C chromatin in vivo, and provide molecular support to a long-standing genetic hypothesis.

  • MicroRNAs in the Drosophila Bithorax Complex
    Genes & development, 2008
    Co-Authors: Welcome Bender
    Abstract:

    The iab-4 noncoding RNA from the Drosophila Bithorax Complex is the substrate for a microRNA (miRNA). Gene conversion was used to delete the hairpin precursor of this miRNA; flies homozygous for this deletion are sterile. Surprisingly, this mutation complements with rearrangement breakpoint mutations that disrupt the iab-4 RNA but fails to complement with breaks mapping in the iab-5 through iab-7 regulatory regions. These breaks disrupt the iab-8 RNA, transcribed from the opposite strand. This iab-8 RNA also encodes a miRNA, detected on Northern blots, derived from the hairpin complementary to the iab-4 precursor hairpin. UltraBithorax is a target of both miRNAs, although its repression is subtle in both cases.

Robert A. Drewell - One of the best experts on this subject based on the ideXlab platform.

  • Transcription factor binding site redundancy in embryonic enhancers of the Drosophila Bithorax Complex.
    G3 (Bethesda Md.), 2011
    Co-Authors: Robert A. Drewell
    Abstract:

    The molecular control of gene expression in development is mediated through the activity of embryonic enhancer cis-regulatory modules. This activity is determined by the combination of repressor and activator transcription factors that bind at specific DNA sequences in the enhancer. A proposed mechanism to ensure a high fidelity of transcriptional output is functional redundancy between closely spaced binding sites within an enhancer. Here I show that at the Bithorax Complex in Drosophila there is selective redundancy for both repressor and activator factor binding sites in vivo. The absence of compensatory binding sites is responsible for two rare gain-of-function mutations in the Complex.

  • Non-genic transcription at the Drosophila Bithorax Complex functional activity of the dark matter of the genome.
    The International journal of developmental biology, 2009
    Co-Authors: Benjamin J. Schiller, Sara E. Goetz, Robert A. Drewell
    Abstract:

    Drosophila melanogaster is a powerful model system for the study of gene regulation due to its short generation time, high fertility and the availability of various genetic tools to manipulate the genome. Investigation into the regulation of homeotic genes and their role in embryonic patterning during development was pioneered in Drosophila. Recently, the molecular mechanisms responsible for regulating gene expression in the Bithorax Complex have been the focus of active study. Many of these studies have pointed to the importance of cis-regulatory modules, genetic sequences that direct the temporal and spatial patterns of gene expression over large genomic distances. Additional components of the regulatory code have emerged beyond the primary DNA sequence. In particular, non-genic transcription is an important mechanism for controlling gene expression either through direct transcriptional mechanisms that mediate dynamic epigenetic control of the chromatin environment or through functional activity of the RNA products.

  • A novel promoter-tethering element regulates enhancer-driven gene expression at the Bithorax Complex in the Drosophila embryo
    Development (Cambridge England), 2007
    Co-Authors: Omar S. Akbari, Esther Bae, Holly Johnsen, Alfred Villaluz, Debbie Wong, Robert A. Drewell
    Abstract:

    A key question in our understanding of the cis-regulation of gene expression during embryonic development has been the molecular mechanism that directs enhancers to specific promoters within a gene Complex. Promoter competition and insulators are thought to play a role in regulating these interactions. In the Bithorax Complex of Drosophila , the IAB5 enhancer is located 55 kb 3′ of the Abdominal-B ( Abd-B ) promoter and 48 kb 5′ of the abdominal-A ( abd-A ) promoter. Although roughly equidistant from the two promoters, IAB5 specifically interacts only with the Abdominal-B promoter, even though the enhancer and promoter are separated by at least two insulators. Here we demonstrate that a 255 bp element, located 40 bp 5′ of the Abd-B transcriptional start site, has a novel cis-regulatory activity as it is able to tether IAB5 to the Abd-B promoter in transgenic embryos. The tethering element is sufficient to direct IAB5 to an ectopic promoter in competition assays. Deletion of the promoter-tethering element results in the redirection of enhancer-driven gene expression on transgenes. Taken together, these results provide evidence that specific long-range enhancer-promoter interactions in the Bithorax Complex are regulated by a tethering element 5′ of the Abd-B promoter. We discuss a bioinformatic analysis of the tethering element across different Drosophila species and a possible molecular mechanism by which this element functions. We also examine existing evidence that this novel class of cis-regulatory elements might regulate enhancer-promoter specificity at other gene Complexes.

  • The abdominal-B promoter tethering element mediates promoter-enhancer specificity at the Drosophila Bithorax Complex.
    Fly, 2007
    Co-Authors: Omar S. Akbari, Ben J. Schiller, Sara E. Goetz, Esther Bae, Robert A. Drewell
    Abstract:

    At the Drosophila Bithorax Complex many distinct classes of cis-regulatory modules work collectively during development to control gene expression. Abdominal-B (Abd-B) is one of three homeotic genes in the BX-C and is expressed in specific presumptive abdominal segments in the embryo. The transcription of Abd-B is tightly controlled by an array of cis-regulatory modules that direct its expression over extended genomic distances. These regulatory modules include promoters, insulators, silencers, enhancers, promoter targeting sequences and the recently identified promoter tethering element (PTE). To activate gene expression at the endogenous Complex, enhancers located >50 kb away must bypass intervening insulators to interact with the Abd-B promoter. The molecular mechanisms that allow enhancers to bypass insulators are not currently well understood. In this short article, we report on a novel mechanism for insulator bypass involving the PTE. In addition, we use bioinformatic analysis across twelve Drosophila genomes to identify putative cis-regulatory sequences that may be capable of facilitating specific promoter-enhancer interactions at the Bithorax Complex and propose a model for their molecular function during development.

  • Characterization of the intergenic RNA profile at abdominal-A and Abdominal-B in the Drosophila Bithorax Complex.
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Esther Bae, E. B. Lewis, Michael S. Levine, Vincent C. Calhoun, Robert A. Drewell
    Abstract:

    The correct spatial expression of two Drosophila Bithorax Complex (BX-C) genes, abdominal-A (abdA) and Abdominal-B (AbdB), is dependent on the 100-kb intergenic infraabdominal (iab) region. The iab region is known to contain a number of different domains (iab2 through iab8) that harbor cis-regulatory elements responsible for directing expression of abdA and AbdB in the second through eighth abdominal segments. Here, we use in situ hybridization to perform high-resolution mapping of the transcriptional activity in the iab control regions. We show that transcription of the control regions themselves is abundant and precedes activation of the abdA and AbdB genes. As with the homeotic genes of the BX-C, the transcription patterns of the RNAs from the iab control regions demonstrate colinearity with the sequence of the iab regions along the chromosome and the domains in the embryo under the control of the specific iab regions. These observations suggest that the intergenic RNAs may play a role in initiating cis regulation at the BX-C early in development.

François Karch - One of the best experts on this subject based on the ideXlab platform.

  • Mutations in the Bithorax Complex used in this study.
    2018
    Co-Authors: Robert K. Maeda, Jessica L. Sitnik, Yohan Frei, Elodie Prince, Dragan Gligorov, Mariana F. Wolfner, François Karch
    Abstract:

    The top line shows a scale map of the region of the Drosophila Bithorax Complex from abd-A to Abd-B with the parasegment specific cis-regulatory domains bracketed above the line. The exonic structure of the abd-A and Abd-B primary transcripts are shown as broken arrows below the line, connected with lines that indicate their splice patterns. Shown as a thinner broken arrow under the Bithorax Complex DNA is the most prominent splice version of the iab-8 ncRNA. The expected msa transcript is shown beneath the iab-8 transcript. Under the primary transcripts, are some of the mutations used in this work. Deletions are indicated by () separating two horizontal lines. The msa inversion is also shown and labeled accordingly. The arrows above the Bithorax map indicate the positions of chromosomal rearrangement breaks used in this work. The chromosomal breaks are label and are color-coded (red for breaks that do not complement Df(P9) in the accessory gland, and blue for breaks that complement the Df(P9) in the accessory gland).

  • Different Evolutionary Strategies To Conserve Chromatin Boundary Function in the Bithorax Complex.
    Genetics, 2017
    Co-Authors: Fabienne Cléard, François Karch, Daniel Wolle, Tsutomu Aoki, Girish Deshpande, Andrew M Taverner, Peter Andolfatto, Paul Schedl
    Abstract:

    Chromatin boundary elements subdivide chromosomes in multicellular organisms into physically independent domains. In addition to this architectural function, these elements also play a critical role in gene regulation. Here we investigated the evolution of a Drosophila Bithorax Complex boundary element called Fab-7 , which is required for the proper parasegment specific expression of the homeotic Abd-B gene. Using a “gene” replacement strategy, we show that Fab-7 boundaries from two closely related species, D . erecta and D. yakuba , and a more distant species, D. pseudoobscura , are able to substitute for the melanogaster boundary. Consistent with this functional conservation, the two known Fab-7 boundary factors, Elba and LBC, have recognition sequences in the boundaries from all species. However, the strategies used for maintaining binding and function in the face of sequence divergence is different. The first is conventional, and depends upon conservation of the 8 bp Elba recognition sequence. The second is unconventional, and takes advantage of the unusually large and flexible sequence recognition properties of the LBC boundary factor, and the deployment of multiple LBC recognition elements in each boundary. In the former case, binding is lost when the recognition sequence is altered. In the latter case, sequence divergence is accompanied by changes in the number, relative affinity, and location of the LBC recognition elements.

  • REVIEW The open for businessmodel of the Bithorax Complex in Drosophila
    2016
    Co-Authors: Robert K. Maeda, François Karch
    Abstract:

    his 1978 landmark paper in which he described the analysis of a series of mutations that affect the identity of the segments that form along the anterior-posterior (AP) axis of the fly (Lewis 1978). The mutations behaved in a non-canonical fashion in complementation tests, forming what Ed Lewis called a Bpseudo-allelic ^ series. Because of this, he never thought that the mutations represented segment-specific genes. As all of these mutations were grouped to a particular area of the Drosophila third chromosome, the locus became known of as the Bithorax Complex (BX-C). One of the key findings of Lewis ’ article was that it revealed for the first time, to a wide scientific audience, that there was a remarkable correlation between the order of the segment-specific muta-tions along the chromosome and the order of the segments they affected along the AP axis. In Ed Lewis ’ eyes, the mu

  • functional requirements for fab 7 boundary activity in the Bithorax Complex
    Molecular and Cellular Biology, 2015
    Co-Authors: Daniel Wolle, Paul Schedl, Fabienne Cléard, Tsutomu Aoki, Girish Deshpande, François Karch
    Abstract:

    Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila Bithorax Complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa Complex, the late boundary Complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

  • Cis-Regulation in the Drosophila Bithorax Complex
    Advances in experimental medicine and biology, 2010
    Co-Authors: Robert K. Maeda, François Karch
    Abstract:

    The discovery of the first homeotic mutation by Calvin Bridges in 19151 profoundly influenced the way we think about developmental processes. Although many mutations modify or deform morphological structures, homeotic mutations cause a spectacular phenotype in which a morphological structure develops like a copy of a structure that is normally found elsewhere on an organism’s body plan. This is best illustrated in Drosophila where homeotic mutations were first discovered. For example, Antennapedia mutants have legs developing on their head instead of antennae. Because a mutation in a single gene creates such complete structures, homeotic genes were proposed to be key “selector genes” regulating the initiation of a developmental program.2 According to this model, once a specific developmental program is initiated (i.e., antenna or leg), it can be executed by downstream “realizator genes” independent of its location along the body axis. Consistent with this idea, homeotic genes have been shown to encode transcription factor proteins that control the activity of the many downstream targets to “realize” a developmental program. Here, we will review the first and perhaps, best characterized homeotic Complex, the Bithorax Complex (BX-C).

Paul Schedl - One of the best experts on this subject based on the ideXlab platform.

  • boundaries mediate long distance interactions between enhancers and promoters in the drosophila Bithorax Complex
    PLOS Genetics, 2018
    Co-Authors: Nikolay Postika, P. G. Georgiev, Paul Schedl, Martin Muller, Mario Metzler, Markus Affolter, O. V. Kyrchanova
    Abstract:

    Drosophila Bithorax Complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously. Paradoxically, seven of the BX-C regulatory domains are separated from their gene target by at least one boundary, and must "jump over" the intervening boundaries. To understand the jumping mechanism, the Mcp boundary was replaced with Fab-7 and Fab-8. Mcp is located between the iab-4 and iab-5 domains, and defines the border between the set of regulatory domains controlling abd-A and Abd-B. When Mcp is replaced by Fab-7 or Fab-8, they direct the iab-4 domain (which regulates abd-A) to inappropriately activate Abd-B in abdominal segment A4. For the Fab-8 replacement, ectopic induction was only observed when it was inserted in the same orientation as the endogenous Fab-8 boundary. A similar orientation dependence for bypass activity was observed when Fab-7 was replaced by Fab-8. Thus, boundaries perform two opposite functions in the context of BX-C-they block crosstalk between neighboring regulatory domains, but at the same time actively facilitate long distance communication between the regulatory domains and their respective target genes.

  • boundaries support specific long distance interactions between enhancers and promoters in drosophila Bithorax Complex
    bioRxiv, 2018
    Co-Authors: P. G. Georgiev, Paul Schedl, Martin Muller, Nikolay Postika, Mario Metzler, Markus Affolter, O. V. Kyrchanova
    Abstract:

    Drosophila Bithorax Complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously. Paradoxically, seven of the BX-C regulatory domains are separated from their gene target by at least one boundary, and must “jump over” the intervening boundaries. To understand the jumping mechanism, the Mcp boundary was replaced with Fab-7 and Fab-8. Mcp is located between the iab-4 and iab-5 domains, and defines the border between the set of regulatory domains controlling abd-A and Abd-B. When Mcp is replaced by Fab-7 or Fab-8, they direct the iab-4 domain (which regulates abd-A) to inappropriately activate Abd-B in abdominal segment A4. For the Fab-8 replacement, ectopic induction was only observed when it was inserted in the same orientation as the endogenous Fab-8 boundary. A similar orientation dependence for bypass activity was observed when Fab-7 was replaced by Fab-8. Thus, boundaries perform two opposite functions in the context of BX-C – they block crosstalk between neighboring regulatory domains, but at the same time actively facilitate long distance communication between the regulatory domains and their respective target genes.

  • Architectural protein Pita cooperates with dCTCF in organization of functional boundaries in Bithorax Complex.
    Development (Cambridge England), 2017
    Co-Authors: O. V. Kyrchanova, Vladic Mogila, Paul Schedl, Oksana Maksimenko, Nikolay Zolotarev, Pavel Georgiev
    Abstract:

    Boundaries in the Bithorax Complex (BX-C) of Drosophila delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two crucial functions: they must block crosstalk between adjacent regulatory domains and at the same time facilitate boundary bypass. The C2H2 zinc-finger protein Pita binds to several BX-C boundaries, including Fab-7 and Mcp To study Pita functions, we have used a boundary replacement strategy by substituting modified DNAs for the Fab-7 boundary, which is located between the iab-6 and iab-7 regulatory domains. Multimerized Pita sites block iab-6↔iab-7 crosstalk but fail to support iab-6 regulation of Abd-B (bypass). In the case of Fab-7, we used a novel sensitized background to show that the two Pita-binding sites contribute to its boundary function. Although Mcp is from BX-C, it does not function appropriately when substituted for Fab-7: it blocks crosstalk but does not support bypass. Mutation of the Mcp Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the Mcp dCTCF site does not affect Pita binding, and this mutant boundary retains partial function.

  • Different Evolutionary Strategies To Conserve Chromatin Boundary Function in the Bithorax Complex.
    Genetics, 2017
    Co-Authors: Fabienne Cléard, François Karch, Daniel Wolle, Tsutomu Aoki, Girish Deshpande, Andrew M Taverner, Peter Andolfatto, Paul Schedl
    Abstract:

    Chromatin boundary elements subdivide chromosomes in multicellular organisms into physically independent domains. In addition to this architectural function, these elements also play a critical role in gene regulation. Here we investigated the evolution of a Drosophila Bithorax Complex boundary element called Fab-7 , which is required for the proper parasegment specific expression of the homeotic Abd-B gene. Using a “gene” replacement strategy, we show that Fab-7 boundaries from two closely related species, D . erecta and D. yakuba , and a more distant species, D. pseudoobscura , are able to substitute for the melanogaster boundary. Consistent with this functional conservation, the two known Fab-7 boundary factors, Elba and LBC, have recognition sequences in the boundaries from all species. However, the strategies used for maintaining binding and function in the face of sequence divergence is different. The first is conventional, and depends upon conservation of the 8 bp Elba recognition sequence. The second is unconventional, and takes advantage of the unusually large and flexible sequence recognition properties of the LBC boundary factor, and the deployment of multiple LBC recognition elements in each boundary. In the former case, binding is lost when the recognition sequence is altered. In the latter case, sequence divergence is accompanied by changes in the number, relative affinity, and location of the LBC recognition elements.

  • functional requirements for fab 7 boundary activity in the Bithorax Complex
    Molecular and Cellular Biology, 2015
    Co-Authors: Daniel Wolle, Paul Schedl, Fabienne Cléard, Tsutomu Aoki, Girish Deshpande, François Karch
    Abstract:

    Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila Bithorax Complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa Complex, the late boundary Complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

E. B. Lewis - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of the intergenic RNA profile at abdominal-A and Abdominal-B in the Drosophila Bithorax Complex.
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Esther Bae, E. B. Lewis, Michael S. Levine, Vincent C. Calhoun, Robert A. Drewell
    Abstract:

    The correct spatial expression of two Drosophila Bithorax Complex (BX-C) genes, abdominal-A (abdA) and Abdominal-B (AbdB), is dependent on the 100-kb intergenic infraabdominal (iab) region. The iab region is known to contain a number of different domains (iab2 through iab8) that harbor cis-regulatory elements responsible for directing expression of abdA and AbdB in the second through eighth abdominal segments. Here, we use in situ hybridization to perform high-resolution mapping of the transcriptional activity in the iab control regions. We show that transcription of the control regions themselves is abundant and precedes activation of the abdA and AbdB genes. As with the homeotic genes of the BX-C, the transcription patterns of the RNAs from the iab control regions demonstrate colinearity with the sequence of the iab regions along the chromosome and the domains in the embryo under the control of the specific iab regions. These observations suggest that the intergenic RNAs may play a role in initiating cis regulation at the BX-C early in development.

  • Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila Bithorax Complex.
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Robert A. Drewell, Esther Bae, John G. Burr, E. B. Lewis
    Abstract:

    The extensive infraabdominal (iab) region contains a number of cis-regulatory elements, including enhancers, silencers, and insulators responsible for directing the developmental expression of the abdominal-A and Abdominal-B homeotic genes at the Drosophila Bithorax Complex. It is unclear how these regulatory elements are primed for activity early in embryogenesis, but the 100-kb intergenic region is subject to a Complex transcriptional program. Here, we use molecular and genetic methods to examine the functional activity of the RNAs produced from this region and their role in cis regulation. We show that a subset of these transcripts demonstrates a distinct pattern of cellular localization. Furthermore, the transcripts from each iab region are discrete and the transcripts do not spread across the insulator elements that delineate the iab regions. In embryos carrying a Mcp deletion, the intergenic transcription pattern is disrupted in the iab4 region and the fourth abdominal segment is transformed into the fifth. We propose that intergenic transcription is required early in embryogenesis to initiate the activation of the Drosophila Bithorax Complex and define the domains of activity for the iab cis-regulatory elements. We also discuss a possible mechanism by which this may occur.

  • Complete sequence of the Bithorax Complex of Drosophila
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: Christopher Martin, E. B. Lewis, Susan E. Celniker, Carol A. Mayeda, Cheryl A. Davis, Cheryl L. Ericsson, John D. Knafels, David R. Mathog, Michael J. Palazzolo
    Abstract:

    The Bithorax Complex (BX-C) of Drosophila, one of two Complexes that act as master regulators of the body plan of the fly, is included within a sequence of 338,234 bp (SEQ89E). This paper presents the strategy used in sequencing SEQ89E and an analysis of its open reading frames. The BX-C sequence (BXCALL) contains 314,895 bp obtained by deletion of putative genes that are located at each end of SEQ89E and appear to be functionally unrelated to the BX-C. Only 1.4% of BXCALL codes for the three homeodomain-containing proteins of the Complex. Principal findings include a putative ABD-A protein (ABD-AII) larger than a previously known ABD-A protein and a putative glucose transporter-like gene (1521 bp) located at or near the Bithoraxoid (bxd), infra-abdominal-2 (iab-2) boundary on the opposite strand relative to that of the homeobox-containing genes.

  • Sequence analysis of the cis-regulatory regions of the Bithorax Complex of Drosophila
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: E. B. Lewis, John D. Knafels, David R. Mathog, Susan E. Celniker
    Abstract:

    The Bithorax Complex (BX-C) of Drosophila, one of two Complexes that act as master regulators of the body plan of the fly, has now been entirely sequenced and comprises ≈315,000 bp, only 1.4% of which codes for protein. Analysis of this sequence reveals significantly overrepresented DNA motifs of unknown, as well as known, functions in the non-protein-coding portion of the sequence. The following types of motifs in that portion are analyzed: (i) concatamers of mono-, di-, and trinucleotides; (ii) tightly clustered hexanucleotides (spaced ≤5 bases apart); (iii) direct and reverse repeats longer than 20 bp; and (iv) a number of motifs known from biochemical studies to play a role in the regulation of the BX-C. The hexanucleotide AGATAC is remarkably overrepresented and is surmised to play a role in chromosome pairing. The positions of sites of highly overrepresented motifs are plotted for those that occur at more than five sites in the sequence, when

  • sequence analysis of the cis regulatory regions of the Bithorax Complex of drosophila
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: E. B. Lewis, John D. Knafels, David R. Mathog, Susan E. Celniker
    Abstract:

    The Bithorax Complex (BX-C) of Drosophila, one of two Complexes that act as master regulators of the body plan of the fly, has now been entirely sequenced and comprises ≈315,000 bp, only 1.4% of which codes for protein. Analysis of this sequence reveals significantly overrepresented DNA motifs of unknown, as well as known, functions in the non-protein-coding portion of the sequence. The following types of motifs in that portion are analyzed: (i) concatamers of mono-, di-, and trinucleotides; (ii) tightly clustered hexanucleotides (spaced ≤5 bases apart); (iii) direct and reverse repeats longer than 20 bp; and (iv) a number of motifs known from biochemical studies to play a role in the regulation of the BX-C. The hexanucleotide AGATAC is remarkably overrepresented and is surmised to play a role in chromosome pairing. The positions of sites of highly overrepresented motifs are plotted for those that occur at more than five sites in the sequence, when <0.5 case is expected. Expected values are based on a third-order Markov chain, which is the optimal order for representing the BXCALL sequence.