Papers by Valerio Orlando
ABSTRACT Immunoprecipitation using in vivo, formaldehyde-fixed chromatin technique (X-ChIP) has b... more ABSTRACT Immunoprecipitation using in vivo, formaldehyde-fixed chromatin technique (X-ChIP) has been providing a powerful tool to unravel basic mechanisms in gene expression. Wide-spread application of this method for the analysis of many chromosomal proteins has allowed substantial progress in the current understanding of gene regulation and chromosome domain structure. Major breakthroughs have been achieved, for example, in the sequence of events that follow mitosis and lead to resetting of transcriptional competence by chromatin remodelling complexes (1) and the replication machinery (2). Other important achievements in gene regulation were the dynamics of recruitment of TBP and associated factors at promoters (3) and changes in histone modification (4). Further, the structure and role of heterochromatin at telomeres, centromeres, and developmentally regulated complex loci in the epigenetic control of gene silencing (5–9) have been thoroughly investigated with this method (see also Ref. 10). The method relies, in the first place, on the availability of high-quality antibodies. Affinity-purified and monoclonal antibodies should be tested for their efficiency in immunoprecipitation and immunoistochemistry. The latter may be highly indicative of the ability of the antibody to recognize a given epitope in formaldehyde-fixed preparations. The efficiency of immunoprecipitation is normally quite low (10-50 micrograms). Assuming that a given factor may be associated with roughly 0.1% of the genome, to get enough material to be analyzed by PCR or ligation and cloning (0.5-5 ng of DNA), each chromatin immunoprecipitation has to contain the equivalent of 25-50 micrograms of DNA. Thus, tissue culture cells, yeast, and other homogeneous cell suspensions are routinely used. Drosophila and mouse embryos have been also successfully used. More detailed comments and technical tips on the procedure in various systems are discussed in Ref. 10. Indeed, by "visualizing" regulatory proteins in vivo at high resolution, new functional aspects of regulatory proteins in the context of living interphase and mitotic chromosomes may be anticipated. Nevertheless, the occupancy of specific genomic sites by a given protein does not always allow easy extrapolation of the functional relevance of those binding sites. Formaldehyde may freeze transient interactions occurring in vivo, which reflect regulated binding of factors acting on the same site. Thus, the presence of factors at specific DNA sequences may simply reflect the chance that a protein has to access a particular site. The relevance of "binding" of a protein for gene function has then to be evaluated by other means. To this concern, appropriate functional tests should be considered before embarking on X-ChIP analysis. Because immunopurified chromatin contains all binding sites in the genome for a given factor, the exciting possibility exists that this method will contribute to a genome-wide identification of target sites of transcription factors (11).Finally, the X-ChIP assay is a powerful tool to take high resolution "pictures" of proteins in the context of chromosome structures. By fixing, we take a snapshot of a largely dynamic situation. Thus, like the single frames in a movie, one has to wait until the end before writing about it.
Scientific Reports, Apr 26, 2016
PIWI-interacting RNAs (piRNAs) are responsible for maintaining the genome stability by silencing ... more PIWI-interacting RNAs (piRNAs) are responsible for maintaining the genome stability by silencing retrotransposons in germline tissues-where piRNAs were first discovered and thought to be restricted. Recently, novel functions were reported for piRNAs in germline and somatic cells. Using deep sequencing of small RNAs and CAGE of postnatal development of mouse brain, we identified piRNAs only in adult mouse brain. These piRNAs have similar sequence length as those of MILI-bound piRNAs. In addition, we predicted novel candidate regulators and putative targets of adult brain piRNAs.
Nature Genetics, Apr 28, 2006
Molecular Cell, 2001
vicinity (Cockell and Gasser, 1999). This phenomenon is known as position effect variegation (PEV... more vicinity (Cockell and Gasser, 1999). This phenomenon is known as position effect variegation (PEV) and is thought to occur by spreading of repressive chromatin protein complexes along the nucleosomal fiber into the translocated gene. Immunoprecipitation experiments of
Cell systems, Nov 1, 2019
Highlights d AGO1 enrichment on chromatin and transcriptional enhancers is mediated by eRNAs d AG... more Highlights d AGO1 enrichment on chromatin and transcriptional enhancers is mediated by eRNAs d AGO1 depletion alters global transcriptional output including eRNA transcripts d Enhancer-associated AGO1 contributes to the maintenance of 3D chromatin organization
PLOS Genetics, Apr 28, 2006
Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes a... more Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis-antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis-antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis-antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis-antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis-antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis-antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.
EMBO Reports, Aug 3, 2023
CSH Protocols, Sep 1, 2006
INTRODUCTIONThis protocol describes the use of chromatin immunoprecipitation technology (ChIP) to... more INTRODUCTIONThis protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. The following protocol has been established for the cultured cell line Schneider 2 (S2) from Drosophila melanogaster. If other tissue is used, certain steps of the protocol may need to be optimized; the main variation is likely to be in the cross-linking step.
Proceedings of the National Academy of Sciences of the United States of America, Oct 2, 2001
PLOS ONE, Jun 13, 2013
Background: Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two co... more Background: Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.
Nature Genetics, Sep 1, 2007
× 10-9 DSBs per bp are replicated, or about fourfold fewer than the estimate of about 0.8 × 10-8 ... more × 10-9 DSBs per bp are replicated, or about fourfold fewer than the estimate of about 0.8 × 10-8 DSBs per bp replicated in human somatic cells (or 50 DSBs per diploid human genome replication) from Vilenchik and Knudson (Proc. Natl. Acad. Sci. USA 100, 12871-12876; 2003). This would bring the number of DSBs per human genome replication down to approximately 13, if it were proportional to that in E. coli. Our error arose from calculating the human equivalent based on haploid, not diploid, human genome size. This error has been corrected in the HTML and PDF versions of the article.
Nature Structural & Molecular Biology, Dec 1, 2002
ABSTRACT Histone methylation regulates the transcriptional activity of genes in the chromatin fib... more ABSTRACT Histone methylation regulates the transcriptional activity of genes in the chromatin fiber and might provide a mechanistic basis for inheritable epigenetic patterns of gene transcription.
International review of cytology, 2007
Cellular phenotypes can be ascribed to different patterns of gene expression. Epigenetic mechanis... more Cellular phenotypes can be ascribed to different patterns of gene expression. Epigenetic mechanisms control the generation of different phenotypes from the same genotype. Thus differentiation is basically a process driven by changes in gene activity during development, often in response to transient factors or environmental stimuli. To keep the specific characteristics of cell types, tissue-specific gene expression patterns must be transmitted stably from one cell to the daughter cells, also in the absence of the early-acting determination factors. This heritability of patterns of active and inactive genes is enabled by epigenetic mechanisms that create a layer of information on top of the DNA sequence that ensures mitotic and sometimes also meiotic transmission of expression patterns. The proteins of the Polycomb and Trithorax group comprise such a cellular memory mechanism that preserves gene expression patterns through many rounds of cell division. This review provides an overview of the genetics and molecular biology of these maintenance proteins, concentrating mainly on mechanisms of Polycomb group-mediated repression.
Sample information (SDRF file) for human, including their details and details of their RNA extrac... more Sample information (SDRF file) for human, including their details and details of their RNA extractions
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Papers by Valerio Orlando