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

ABSTRACT: This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

ORGANISM(S): Bombyx mori

SUBMITTER: Shreeharsha Tarikere

PROVIDER: E-GEOD-71992 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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

Prasad Naveen N Tarikere Shreeharsha S Khanale Dhanashree D Habib Farhat F Shashidhara L S LS

Scientific reports 20160614

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 B ...[more]

PMID: 27296678

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Project description:Amongst the various different insect groups, there is remarkable diversity in the number and size of wings. However the development of the basic body plan in insects is similar to a large extent. The genes of the hox complex regulate various pathways to bring about the development or modification of different organs. Ubx, a gene of the bithorax hox complex is expressed in the third thoracic segment of insects and is known to specify the fate of wing appendage in that segment.To understand the role of Ubx and how its regulatory mechanism has evolved through the course of evolution we have compared its genome wide targets in different insect orders. The identification of regulatory pathways and the key players Ubx regulates is crucial to understand how it has controlled wing development across insect orders. Our lab has previously identified direct targets of Ubx in Drosophila using ChIP-chip (Agrawal et al, 2011). To further our knowledge on the role of regulation in development and modification of hind wing appendage we have studied the targets in the hind wings of other insects (silk moth; Lepidoptera and honeybee; Hymenoptera) and performed a comparative analysis. We have employed ChIP followed by illumina sequencing to identify the targets of Ubx in developing hind and fore wing buds of Bombyx larvae. This is a first next generation sequencing study in Lepidoptera in an attempt to understand wing development. Chromatin Immunoprecipitation (ChIP) was used to identify genome wide targets bound by Ubx in Bombyx larval wing buds. The experiment to enrich Ubx bound regions was carried out using a Bombyx N terminal-Ubx specific poylclonal antibody raised in Rabbit and purified against a Protein A column to obtain IgG fraction. An Immunoprecipitation (IP) with Normal Rabbit IgG was used as a negative control to eliminate the regions that pertained to non specific binding to an Immunogloubulin. The normalization of both ChIP and IgG was done against sequenced input chromatin. Two replicates of single end 36 bp reads were sequenced using Ilumina for all the three conditions and for both fore and hind wing tissue samples.The peaks common to both the replicates were considered after applying a FDR cutoff.The fore wing target set was used for comparison with the hind wing targets.

Project description:Amongst the various different insect groups, there is remarkable diversity in the number and size of wings. However the development of the basic body plan in insects is similar to a large extent. The genes of the hox complex regulate various pathways to bring about the development or modification of different organs. Ubx, a gene of the bithorax hox complex is expressed in the third thoracic segment of insects and is known to specify the fate of wing appendage in that segment.To understand the role of Ubx and how its regulatory mechanism has evolved through the course of evolution we have compared its genome wide targets in different insect orders. The identification of regulatory pathways and the key players Ubx regulates is crucial to understand how it has controlled wing development across insect orders. Our lab has previously identified direct targets of Ubx in Drosophila using ChIP-chip (Agrawal et al, 2011). To further our knowledge on the role of regulation in development and modification of hind wing appendage we have studied the targets in the hind wings of other insects (silk moth; Lepidoptera and honeybee; Hymenoptera) and performed a comparative analysis.To understand the differential development of wing appendages in insects we intend to compare the differential expression of fore and hind wing appendages in Diptera and Lepidoptera. We have employed RNA-seq using by illumina sequencing to identify the genes that are differentially expressed between fore and the hind wing bud of the Bombyx larvae. Only one replicate was performed as the intended study was to validate the differential targets in comparison to ChIP studies and other methods of select candidates. Total RNA was extarcted from wing buds of IV instar Bombyx larvae and sequenced on an illumina sequencer. Wing buds were collected directly in liquid nitrogen and 80 such buds each for fore and hind wing were collected from IV instar larvae. RNA was isolated from them and processed for Library preperation and illumina sequencing. One replicate was done as a supplement to ChIP studies.

Project description:It has widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes, whereas the other forms, such as N6-methyladenine, primarily exist in prokaryotes and only a few eukaryotes. Herein, we demonstrated the surprising presence of N6-methyladenine in mammalian genomes, especially, mouse embryonic stem cells. This modification is enriched at histone variant H2A.X-deposited genomic regions in wild type embryonic stem cells. Our work also showed that a previously unknown DNA demethylase, Alkbh1, is the major demethylase for N6-methyladenine in embryonic stem cells. Increase of N6-methyladenine levels in Alkbh1 deficient cells leads to silencing of genes that regulate embryonic development. Surprisingly, genes located on the X-chromosome, but not the Y-chromosome or autosomes are preferentially silenced by N6-methyladenine. Strikingly, N6-methyladenine in Alkbh1 deficient cells are specifically deposition at young, full-length subfamilies of LINE1 transposons that are strongly enriched on the X chromosome. Furthermore, N6-methyladenine deposition on LINE1s pattern is inversely correlated with their evolutionary age. The deposition of N6-methyladenine results in epigenetic silencing of such L1s, which are otherwise actively transcribed in wild type embryonic stem cells, and the neighboring enhancers and genes. Furthermore, N6-methyladenine induced-silencing resists gene activation signals during embryonic stem cell differentiation. Thus, N6-methyladenine adopts a new function in epigenetic silencing in evolution, distinct from its role in gene activation in other organisms. In summary, our results demonstrate that N6-methyladenine unexpectedly constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes. First, we used different histone antibodies to enrich for DNA molecules with histone modification or specific variant in mouse ESCs as previously described Native-ChIP methods. Then, co-purified DNA molecules from WT or KO ESCs were subject to HiSeq2000 sequencing and data analysis for histone modification or variant peaks. Native ChIP coupling with HiSeq sequencing

Dataset Information

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

Publications

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

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