BIOMAPP::CHIP: large-scale motif analysis.

BACKGROUND: Discovery biological motifs plays a fundamental role in understanding regulatory mechanisms. Computationally, they can be efficiently represented as kmers, making the counting of these elements a critical aspect for ensuring not only the accuracy but also the efficiency of the analytical process. This is particularly useful in scenarios involving large data volumes, such as those generated by the ChIP-seq protocol. Against this backdrop, we introduce BIOMAPP::CHIP, a tool specifically designed to optimize the discovery of biological motifs in large data volumes. RESULTS: We conducted a comprehensive set of comparative tests with state-of-the-art algorithms. Our analyses revealed that BIOMAPP::CHIP outperforms existing approaches in various metrics, excelling both in terms of performance and accuracy. The tests demonstrated a higher detection rate of significant motifs and also greater agility in the execution of the algorithm. Furthermore, the SMT component played a vital role in the system's efficiency, proving to be both agile and accurate in kmer counting, which in turn improved the overall efficacy of our tool. CONCLUSION: BIOMAPP::CHIP represent real advancements in the discovery of biological motifs, particularly in large data volume scenarios, offering a relevant alternative for the analysis of ChIP-seq data and have the potential to boost future research in the field. This software can be found at the following address: (https://github.com/jadermcg/biomapp-chip).

Chromatin Immunoprecipitation (ChiP) Protocol for the Analysis of Gene Regulation by Histone Modifications in Agave angustifolia Haw.

Chromatin is a dynamic entity that regulates different biological processes crucial for the proper functioning of the cell. Chromatin regulation depends largely on the interactions that occur between DNA with histones and nonhistone proteins. The chromatin immunoprecipitation assay (ChiP) is a widely used technique for the study of these DNA-histone and DNA-nonhistone interactions and their biological repercussions. Here we describe a ChiP protocol that allows in vivo analysis of the associations of histone modifications with genomic DNA in Agave angustifolia Haw. Although this protocol is established for A. angustifolia, it can be used in other species to obtain similar results. We also propose a strategy to shorten the times in some steps of the standard protocol.

Chromatin Immunoprecipitation in Early Mouse Embryos.

Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.

Regulators of genetic risk of breast cancer identified by integrative network analysis.

Genetic risk for breast cancer is conferred by a combination of multiple variants of small effect. To better understand how risk loci might combine, we examined whether risk-associated genes share regulatory mechanisms. We created a breast cancer gene regulatory network comprising transcription factors and groups of putative target genes (regulons) and asked whether specific regulons are enriched for genes associated with risk loci via expression quantitative trait loci (eQTLs). We identified 36 overlapping regulons that were enriched for risk loci and formed a distinct cluster within the network, suggesting shared biology. The risk transcription factors driving these regulons are frequently mutated in cancer and lie in two opposing subgroups, which relate to estrogen receptor (ER)(+) luminal A or luminal B and ER(-) basal-like cancers and to different luminal epithelial cell populations in the adult mammary gland. Our network approach provides a foundation for determining the regulatory circuits governing breast cancer, to identify targets for intervention, and is transferable to other disease settings.

Towards an understanding of the epigenetics of schistosomes: a comparative epigenomic study.

As in perhaps all eukaryotes, schistosomes use a supplementary information transmitting system, the epigenetic inheritance system, to shape genetic information and to produce different phenotypes. In contrast to other important parasites, the study of epigenetic phenomena in schistosomes is still in its infancy. Nevertheless, we are beginning to grasp what goes on behind the epigenetic scene in this parasite. We have developed techniques of native chromatin immunoprecipitation (N-ChIP) and associated the necessary bioinformatics tools that allow us to run genome-wide comparative chromatin studies on Schistosoma mansoni at different stages of its life cycle, on different strains and on different sexes. We present here an application of such an approach to study the genetic and epigenetic basis for a phenotypic trait, the compatibility of S. mansoni with its invertebrate host Biomphalaria glabrata. We have applied the ChIP procedure to two strains that are either compatible or incompatible with their intermediate host. The precipitated DNA was sequenced and aligned to a reference genome and this information was used to determine regions in which both strands differ in their genomic sequence and/or chromatin structure. This procedure allowed us to identify candidate genes that display either genetic or epigenetic difference between the two strains.

Towards an understanding of the epigenetics of schistosomes: a comparative epigenomic study

As in perhaps all eukaryotes, schistosomes use a supplementary information transmitting system, the epigenetic inheritance system, to shape genetic information and to produce different phenotypes. In contrast to other important parasites, the study of epigenetic phenomena in schistosomes is still in its infancy. Nevertheless, we are beginning to grasp what goes on behind the epigenetic scene in this parasite. We have developed techniques of native chromatin immunoprecipitation (N-ChIP) and associated the necessary bioinformatics tools that allow us to run genome-wide comparative chromatin studies on Schistosoma mansoni at different stages of its life cycle, on different strains and on different sexes. We present here an application of such an approach to study the genetic and epigenetic basis for a phenotypic trait, the compatibility of S. mansoni with its invertebrate host Biomphalaria glabrata. We have applied the ChIP procedure to two strains that are either compatible or incompatible with their intermediate host. The precipitated DNA was sequenced and aligned to a reference genome and this information was used to determine regions in which both strands differ in their genomic sequence and/or chromatin structure. This procedure allowed us to identify candidate genes that display either genetic or epigenetic difference between the two strains.

Protein tagging for chromatin immunoprecipitation from Arabidopsis.

A powerful method to identify binding sites in target genes is chromatin immunoprecipitation (ChIP), which allows the purification of in vivo formed complexes of a DNA-binding protein and associated DNA. Briefly, the method involves the fixation of plant tissue and the isolation of the total protein-DNA mixture, followed by an immunoprecipitation step with an antibody directed against the protein of interest and, subsequently, the DNA can be purified. Finally, the DNA can be analyzed by PCR for the enrichment of specific regions. A drawback of ChIP is that for each protein another antibody is needed. To overcome this, a generic strategy is possible using tags fused to the protein of interest. In this case, only antibody is needed against the tag. This protocol describes the tagging of proteins and how to perform ChIP.

Toxoplasma H2A variants reveal novel insights into nucleosome composition and functions for this histone family.

Toxoplasma gondii is an obligate intracellular parasite. Toxoplasmosis is incurable because of its ability to differentiate from the rapidly replicating tachyzoite stage into a latent cyst form (bradyzoite stage). Gene regulation pertinent to Toxoplasma differentiation involves histone modification, but very little is known about the histone proteins in this early branching eukaryote. Here, we report the characterization of three H2A histones, variants H2AX and H2AZ, and a canonical H2A1. H2AZ is the minor parasite H2A member. H2A1 and H2AX both have an SQ motif, but only H2AX has a complete SQ(E/D)varphi (where varphi denotes a hydrophobic residue) known to be phosphorylated in response to DNA damage. We show that a novel H2B variant interacts with H2AZ and H2A1 but not with H2AX. Chromatin immunoprecipitation (ChIP) revealed that H2AZ and H2Bv are enriched at active genes while H2AX is enriched at repressed genes as well as the silent TgIRE repeat element. During DNA damage, we detected an increase in H2AX phosphorylation as well as increases in h2a1 and h2ax transcription. We found that expression of h2ax, but not h2a1 or h2az, increases in bradyzoites generated in vitro. Similar analysis performed on mature bradyzoites generated in vivo, which are arrested in G0, showed that h2az and h2ax are expressed but h2a1 is not, consistent with the idea that h2a1 is the canonical histone orthologue in the parasite. The increase of H2AX, which localizes to silenced areas during bradyzoite differentiation, is consistent with the quiescent nature of this stage of the life cycle. Our results indicate that the early-branching eukaryotic parasite Toxoplasma contains nucleosomes of novel composition, which is likely to impact multiple facets of parasite biology, including the clinically important process of bradyzoite differentiation.

Sequential chromatin immunoprecipitation protocol: ChIP-reChIP.

Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene transcription. Therefore, it has become relevant to determine the cohabitation of several proteins in a particular developmental stage and cell type. Furthermore, multiple post-translational histone modifications can be analyzed on the same genomic location with the aim of deciphering the combinatorial pattern of histone modifications associated to specific transcriptional stages during cell commitment. Here we describe the ChIP-reChIP assay that represents a direct strategy to determine the in vivo colocalization of proteins interacting or in close contact in a chromatinized template on the basis of double and independent rounds of immunoprecipitations with high-quality ChIP grade antibodies.