ChIP for Hox Proteins from Drosophila Imaginal Discs

Agrawal, Pavan; Shashidhara, L. S.

doi:10.1007/978-1-4939-1242-1_15

Cited by 5 publications

(3 citation statements)

References 19 publications

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“…We used a modified protocol that was used earlier for Drosophila wing discs738. All ChIP-Seq and RNA-Seq experiments (described below) and data analyses conform to The Functional Genomics Data Society (http://fged.org/) guidelines.…”

Section: Experimental Methodsmentioning

confidence: 99%

A comparative genomic analysis of targets of Hox protein Ultrabithorax amongst distant insect species

Prasad

Tarikere

Khanale

et al. 2016

Sci Rep

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In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is dependent on the function of the Hox protein Ultrabithorax (Ubx). Here we compare Ubx-mediated regulation of wing patterning genes between the honeybee, Apis mellifera, the silkmoth, Bombyx mori and Drosophila. Orthologues of Ubx are expressed in the third thoracic segment of Apis and Bombyx, although they make functional hindwings. When over-expressed in transgenic Drosophila, Ubx derived from Apis or Bombyx could suppress wing development, suggesting evolutionary changes at the level of co-factors and/or targets of Ubx. To gain further insights into such events, we identified direct targets of Ubx from Apis and Bombyx by ChIP-seq and compared them with those of Drosophila. While majority of the putative targets of Ubx are species-specific, a considerable number of wing-patterning genes are retained, over the past 300 millions years, as targets in all the three species. Interestingly, many of these are differentially expressed only between wing and haltere in Drosophila but not between forewing and hindwing in Apis or Bombyx. Detailed bioinformatics and experimental validation of enhancer sequences suggest that, perhaps along with other factors, changes in the cis-regulatory sequences of earlier targets contribute to diversity in Ubx function.

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Section: Experimental Methodsmentioning

confidence: 99%

A comparative genomic analysis of targets of Hox protein Ultrabithorax amongst distant insect species

Prasad

Tarikere

Khanale

et al. 2016

Sci Rep

Self Cite

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“…Chromatin immunoprecipitation was performed on 60 wing imaginal discs from Ore R wandering third instar larvae per biological sample as described in 57 with minor changes. IgA- and IgG-coupled Dynabeads (Invitrogen), were used in a 1:1 ratio for chromatin immunoprecipitation in place of protein A or G agarose/salmon sperm DNA beads.…”

Section: Methodsmentioning

confidence: 99%

TnaA, a trithorax group protein, modulates wingless expression in different regions of the Drosophila wing imaginal disc

Rosales-Vega,

Reséndez-Pérez,

Zurita

et al. 2023

Sci Rep

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Abstractwingless expression is exquisitely regulated by different factors and enhancers in the imaginal wing discs of Drosophila melanogaster in four domains: the dorsal band, the dorso-ventral boundary, and the inner and outer ring domains. tonalli is a trithorax group gene that encodes a putative SUMO E3 ligase that binds to chromatin to regulate the expression of its targets, including the Hox genes. However, its role in modulating gene expression is barely known. Here, we show that TnaA modulates the wingless expression at two domains of the wing disc, the dorso-ventral boundary and the inner ring. At first, tonalli interacts genetically with Notch to form the wing margin. In the inner ring domain, TnaA modulates wingless transcription. When the dosage of TnaA increases in or near the inner ring since early larval stages, this domain expands with a rapid increase in wingless expression. TnaA occupies the wingless Inner Ring Enhancer at the wing disc, meanwhile it does not affect wingless expression directed by the Ventral Disc Enhancer in leg discs, suggesting that TnaA acts as a wingless enhancer-specific factor. We describe for the first time the presence of TnaA at the Inner Ring Enhancer as a specific regulator of wingless in the development of wing boundaries.

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“…The protocol was essentially as reported by (Agrawal and Shashidhara 2014). Third instar wandering larvae were cut, inverted and fixed with 1.5% PFA (ThermoFisher scientific) for 20 minutes at room temperature and subsequently quenched (125mM Glycine solution, 1XPBS).…”

Section: Chip Sequencingmentioning

confidence: 99%

A Micro-evolutionary Change in Target Binding Sites as a Key Determinant of Ultrabithorax Function in Drosophila

et al. 2023

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All Hox proteins are known to recognize, in vitro, similar DNA-binding sites containing a TAAT core sequence. This poor DNA-binding specificity is in sharp contrast with their specific functions in vivo. Here we report a new binding motif with TAAAT core sequence to which the Hox protein Ultrabithorax (Ubx) binds with higher affinity and specificity. Using transgenic and luciferase assays, we show that this new motif is critical for Ubx-mediated regulation of a target gene in Drosophila melanogaster. Interestingly, this new motif with TAAAT core sequences is not associated with the targets of Ubx in the honeybee, Apis mellifera, wherein hindwings are nearly identical to the forewings. We show that introduction of TAAAT motif in the place of TAAT motif is sufficient to bring an enhancer of a wing-promoting gene of A. mellifera under the regulation of Ubx. Our results, thus, suggest that binding motifs with a TAAAT core sequence may help identify functionally relevant direct targets of Ubx in D. melanogaster and the emergence of these binding sites may be crucial for Hox-mediated morphological changes during insect evolution.

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ChIP for Hox Proteins from Drosophila Imaginal Discs

Cited by 5 publications

References 19 publications

A comparative genomic analysis of targets of Hox protein Ultrabithorax amongst distant insect species

A comparative genomic analysis of targets of Hox protein Ultrabithorax amongst distant insect species

TnaA, a trithorax group protein, modulates wingless expression in different regions of the Drosophila wing imaginal disc

A Micro-evolutionary Change in Target Binding Sites as a Key Determinant of Ultrabithorax Function in Drosophila

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