Genetic and Molecular Analysis of Chromatin Domains

Vázquez, Julio; Farkas, Gabriella; Gaszner, Miklos; Udvardy, Andor; Müller, Matthias A.; Hagstrom, Kirsten; Gyurkovics, Henrik; Sípos, László; Gausz, János; Galloni, Mireille; Hogga, Iiham; Karch, François; Schedl, Paul

doi:10.1101/sqb.1993.058.01.008

Cited by 41 publications

(32 citation statements)

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“…If the initial activation of the cis-regulatory elements in the iab regions is dependent on the intergenic transcripts, then it would be necessary not only to have unique transcription patterns within each region but also to ensure that transcription does not spread from one region to the next. Our study provides evidence that the insulators that organize the different iab regions into discrete chromatin domains (8,14,15) act to regulate the intergenic transcription program in this way.…”

Section: Discussionmentioning

confidence: 92%

“…Although not all insulators are thought to act in the same way (12,13), it has been suggested that the BX-C insulators organize the different iab regions into discrete chromatin domains ( Fig. 1 A) (8,14,15). In this way, cis-regulatory elements within the separate domains are able to direct expression of abdA and AbdB in specific abdominal segments of the Drosophila embryo.…”

Section: E Xpression Of the Abdominal-a (Abda) And Abdominal-b (Abdb)mentioning

confidence: 99%

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Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila bithorax complex

Drewell

Bae

Burr

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Discussionmentioning

confidence: 92%

Section: E Xpression Of the Abdominal-a (Abda) And Abdominal-b (Abdb)mentioning

confidence: 99%

Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila bithorax complex

Drewell

Bae

Burr

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…A class would be defined by conserved sequence elements and the proteins involved in establishing boundary activity, although there could be overlap between classes. Yet no notable sequence homology or common binding activity has been found for the few boundary elements described so far, such as the su(Hw) binding sites, scs, scsЈ, Mcp, and Fab7, although in all cases examined, nuclease-hypersensitive sites localize to these elements (6,7,12,18,34). This lack of common features, combined with the difficulty of identifying boundary elements despite their proposed importance, has called the functional-domain model into question.…”

Section: Discussionmentioning

confidence: 96%

“…Regulatory DNA elements often form structures in chromatin that are hypersensitive to nuclease digestion, and the scs and scsЈ special chromatin structures were originally identified as major nuclease-hypersensitive sites bracketing the heat shock puff (33). Indeed, it appears that boundary elements are generally associated with hypersensitive sites (4,6,18,34,35). It was of interest, therefore, to examine cBE76 for nuclease hypersensitivity.…”

Section: Resultsmentioning

confidence: 99%

“…Consequently, when the gene is integrated into fly strains that have white eyes, the transformed flies display a range of eye color phenotypes. The relative level of expression is conveniently assessed on the basis of eye color, which changes with increased expression from yellow through orange to dark red (20,34). When bracketed by boundary elements, the mini-white gene is insulated from these chromosomal position effects so that there is less variability in eye color between independent transgenic lines, and most lines have yellow eyes.…”

Section: Resultsmentioning

confidence: 99%

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Identification of a Class of Chromatin Boundary Elements

Cuvier

Hart

Laemmli

1998

Molecular and Cellular Biology

137

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Boundary elements are thought to define the ends of functionally independent domains of genetic activity. An assay for boundary activity based on this concept measures the ability to insulate a bracketed, chromosomally integrated reporter gene from position effects. Despite their presumed importance, the few examples identified to date apparently do not share sequence motifs or DNA binding proteins. The Drosophila protein BEAF binds the scs boundary element of the 87A7 hsp70 locus and roughly half of polytene chromosome interband loci. To see if these sites represent a class of boundary elements that have BEAF in common, we have isolated and studied several genomic BEAF binding sites as candidate boundary elements (cBEs). BEAF binds with high affinity to clustered, variably arranged CGATA motifs present in these cBEs. No other sequence homologies were found. Two cBEs were tested and found to confer position-independent expression on a miniwhite reporter gene in transgenic flies. Furthermore, point mutations in CGATA motifs that eliminate binding by BEAF also eliminate the ability to confer position-independent expression. Taken together, these findings suggest that clustered CGATA motifs are a hallmark of a BEAF-utilizing class of boundary elements found at many loci. This is the first example of a class of boundary elements that share a sequence motif and a binding protein.Chromatin appears to be partitioned into chromosomal domains that are operationally defined by bracketing DNA regions called boundary elements or insulators (10; see reference 34 for a review). Boundary elements are presumably necessary to curtail the potentially promiscuous behavior of enhancers, limiting their action to the domain in which they reside. The biological activity of a boundary element is experimentally measured by either position-independent expression or enhancerblocking assays. If this view of chromosomal organization is correct, boundary elements play a very important functional role. Yet only a few examples have been identified, and each is so far a unique case, as they do not appear to have notable sequence homologies or to have binding activities in common.The best-characterized boundary elements are the scs and scsЈ regions found to bracket the 87A7 hsp70 heat shock puff of Drosophila melanogaster polytene chromosomes (33) and a 340-bp fragment from the gypsy retrotransposon (11). The scs/scsЈ and the gypsy-derived elements have a boundary function in both of the assays mentioned above. They confer position-independent expression on a bracketed reporter gene by insulating the transgene from both activating and repressive effects at the site of chromosomal integration, and they block communication between a specific enhancer and promoter when interposed (20,21,31). It is important to note that boundary elements do not inactivate promoters or enhancers; they only block communication when interposed (2, 3, 21, 32). For instance, if an enhancer and boundary element are located between two divergently transcribed promoters, t...

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Sequences required for enhancer blocking activity of scs are located within two nuclease-hypersensitive regions.

Vázquez

Schedl

1994

The EMBO Journal

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The Drosophila 87A7 heat shock locus is bordered, on the proximal and distal sides, by two special chromatin structures, scs and scs‘. Each structure is characterized by two sets of nuclease‐hypersensitive sites, located within moderately G/C‐rich DNA, flanking an A/T‐rich nuclease‐resistant region. scs and scs’ have been shown to insulate a white reporter gene from position effects and to prevent enhancer‐promoter interactions. These and other properties suggest scs and scs’ might function as chromatin domain boundaries. To identify the DNA sequences which are essential for the insulating activity of scs we used an enhancer blocking assay based on the white gene. Sequences capable of suppressing activation of white by its upstream enhancer elements reside within a 900 bp DNA fragment corresponding to the scs chromatin structure. Within this region, DNA fragments associated with the two nuclease‐hypersensitive regions are essential for full enhancer blocking activity, while the central A/T‐rich region is dispensable. Deletions which remove part of the hypersensitive regions result in intermediate levels of white activity. Insulating activity can, however, be reconstituted by multimerizing DNA fragments from either hypersensitive region. Our results suggest that the scs boundary is assembled from a discrete number of functionally redundant DNA sequences located within both hypersensitive regions and that boundaries act by decreasing the frequency of enhancer‐promoter interactions. We also show that certain types of position effects, like those involved in dosage compensation, are not efficiently blocked by scs.

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Genetic and Molecular Analysis of Chromatin Domains

Cited by 41 publications

References 0 publications

Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila bithorax complex

Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila bithorax complex

Identification of a Class of Chromatin Boundary Elements

Sequences required for enhancer blocking activity of scs are located within two nuclease-hypersensitive regions.

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