15 research outputs found
RACS: Rapid Analysis of ChIP-Seq data for contig based genomes
Background: Chromatin immunoprecipitation coupled to next generation
sequencing (ChIP-Seq) is a widely used technique to investigate the function of
chromatin-related proteins in a genome-wide manner. ChIP-Seq generates large
quantities of data which can be difficult to process and analyse, particularly
for organisms with contig based genomes. Contig-based genomes often have poor
annotations for cis-elements, for example enhancers, that are important for
gene expression. Poorly annotated genomes make a comprehensive analysis of
ChIP-Seq data difficult and as such standardized analysis pipelines are
lacking. Methods: We report a computational pipeline that utilizes traditional
High-Performance Computing techniques and open source tools for processing and
analysing data obtained from ChIP-Seq. We applied our computational pipeline
"Rapid Analysis of ChIP-Seq data" (RACS) to ChIP-Seq data that was generated in
the model organism Tetrahymena thermophila, an example of an organism with a
genome that is available in contigs. Results: To test the performance and
efficiency of RACs, we performed control ChIP-Seq experiments allowing us to
rapidly eliminate false positives when analyzing our previously published data
set. Our pipeline segregates the found read accumulations between genic and
intergenic regions and is highly efficient for rapid downstream analyses.
Conclusions: Altogether, the computational pipeline presented in this report is
an efficient and highly reliable tool to analyze genome-wide ChIP-Seq data
generated in model organisms with contig-based genomes.
RACS is an open source computational pipeline available to download from:
https://bitbucket.org/mjponce/racs --or--
https://gitrepos.scinet.utoronto.ca/public/?a=summary&p=RACSComment: Submitted to BMC Bioinformatics. Computational pipeline available at
https://bitbucket.org/mjponce/rac
Nucleus-specific linker histones Hho1 and Mlh1 form distinct protein interactions during growth, starvation and development in Tetrahymena thermophila
Chromatin organization influences most aspects of gene expression regulation. The linker histone H1, along with the core histones, is a key component of eukaryotic chromatin. Despite its critical roles in chromatin structure and function and gene regulation, studies regarding the H1 protein-protein interaction networks, particularly outside of Opisthokonts, are limited. The nuclear dimorphic ciliate protozoan Tetrahymena thermophila encodes two distinct nucleus-specific linker histones, macronuclear Hho1 and micronuclear Mlh1. We used a comparative proteomics approach to identify the Hho1 and Mlh1 protein-protein interaction networks in Tetrahymena during growth, starvation, and sexual development. Affinity purification followed by mass spectrometry analysis of the Hho1 and Mlh1 proteins revealed a non-overlapping set of co-purifying proteins suggesting that Tetrahymena nucleus-specific linker histones are subject to distinct regulatory pathways. Furthermore, we found that linker histones interact with distinct proteins under the different stages of the Tetrahymena life cycle. Hho1 and Mlh1 co-purified with several Tetrahymena-specific as well as conserved interacting partners involved in chromatin structure and function and other important cellular pathways. Our results suggest that nucleus-specific linker histones might be subject to nucleus-specific regulatory pathways and are dynamically regulated under different stages of the Tetrahymena life cycle.York University Librarie
The bromodomain-containing protein Ibd1 links multiple chromatin related protein complexes to highly expressed genes in Tetrahymena thermophila
Background: The chromatin remodelers of the SWI/SNF family are critical
transcriptional regulators. Recognition of lysine acetylation through a
bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes,
this function is attributed to SNF2/Brg1.
Results: Using affinity purification coupled to mass spectrometry (AP-MS) we
identified members of a SWI/SNF complex (SWI/SNFTt) in Tetrahymena thermophila.
SWI/SNFTt is composed of 11 proteins, Snf5Tt, Swi1Tt, Swi3Tt, Snf12Tt, Brg1Tt,
two proteins with potential chromatin interacting domains and four proteins
without orthologs to SWI/SNF proteins in yeast or mammals. SWI/SNFTt subunits
localize exclusively to the transcriptionally active macronucleus (MAC) during
growth and development, consistent with a role in transcription. While
Tetrahymena Brg1 does not contain a BRD, our AP-MS results identified a
BRD-containing SWI/SNFTt component, Ibd1 that associates with SWI/SNFTt during
growth but not development. AP-MS analysis of epitope-tagged Ibd1 revealed it
to be a subunit of several additional protein complexes, including putative
SWRTt, and SAGATt complexes as well as a putative H3K4-specific histone methyl
transferase complex. Recombinant Ibd1 recognizes acetyl-lysine marks on
histones correlated with active transcription. Consistent with our AP-MS and
histone array data suggesting a role in regulation of gene expression, ChIP-Seq
analysis of Ibd1 indicated that it primarily binds near promoters and within
gene bodies of highly expressed genes during growth.
Conclusions: Our results suggest that through recognizing specific histones
marks, Ibd1 targets active chromatin regions of highly expressed genes in
Tetrahymena where it subsequently might coordinate the recruitment of several
chromatin remodeling complexes to regulate the transcriptional landscape of
vegetatively growing Tetrahymena cells.Comment: Published on BMC Epigenetics & Chromati
Functional Proteomics of Nuclear Proteins in Tetrahymena thermophila: A Review
Identification and characterization of protein complexes and interactomes has been essential to the understanding of fundamental nuclear processes including transcription, replication, recombination, and maintenance of genome stability. Despite significant progress in elucidation of nuclear proteomes and interactomes of organisms such as yeast and mammalian systems, progress in other models has lagged. Protists, including the alveolate ciliate protozoa with Tetrahymena thermophila as one of the most studied members of this group, have a unique nuclear biology, and nuclear dimorphism, with structurally and functionally distinct nuclei in a common cytoplasm. These features have been important in providing important insights about numerous fundamental nuclear processes. Here, we review the proteomic approaches that were historically used as well as those currently employed to take advantage of the unique biology of the ciliates, focusing on Tetrahymena, to address important questions and better understand nuclear processes including chromatin biology of eukaryotes
Functional Proteomics of Nuclear Proteins in Tetrahymena thermophila: A Review
Identification and characterization of protein complexes and interactomes has been essential to the understanding of fundamental nuclear processes including transcription, replication, recombination, and maintenance of genome stability. Despite significant progress in elucidation of nuclear proteomes and interactomes of organisms such as yeast and mammalian systems, progress in other models has lagged. Protists, including the alveolate ciliate protozoa with Tetrahymena thermophila as one of the most studied members of this group, have a unique nuclear biology, and nuclear dimorphism, with structurally and functionally distinct nuclei in a common cytoplasm. These features have been important in providing important insights about numerous fundamental nuclear processes. Here, we review the proteomic approaches that were historically used as well as those currently employed to take advantage of the unique biology of the ciliates, focusing on Tetrahymena, to address important questions and better understand nuclear processes including chromatin biology of eukaryotes
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Sig. A4Segons consta en: Catálogo de Villancicos y Oratorios en la Biblioteca Nacional Siglos XVIII-XIX, nº 1264, l'autor de la lletra és José Vicente Ortí y MayorTotes les p. orlades. - Reclam
MOESM9 of The bromodomain-containing protein Ibd1 links multiple chromatin-related protein complexes to highly expressed genes in Tetrahymena thermophila
Additional file 9. Primers
MOESM4 of The bromodomain-containing protein Ibd1 links multiple chromatin-related protein complexes to highly expressed genes in Tetrahymena thermophila
Additional file 4. Peptide_array
MOESM5 of The bromodomain-containing protein Ibd1 links multiple chromatin-related protein complexes to highly expressed genes in Tetrahymena thermophila
Additional file 5. Ibd1_MS_5hConj
MOESM6 of The bromodomain-containing protein Ibd1 links multiple chromatin-related protein complexes to highly expressed genes in Tetrahymena thermophila
Additional file 6. ChIP_seq_ORF