Papers by alejandra Loyola
Biochimica Et Biophysica Acta-gene Structure and Expression, 2004
In eukaryotic cells, highly basic histone proteins are associated with the DNA to form the nucleo... more In eukaryotic cells, highly basic histone proteins are associated with the DNA to form the nucleosome, the fundamental unit of chromatin. Histones are closely escorted by histone chaperones from their point of synthesis up to their delivery site. We will present an overview of the histone chaperones identified to date with their various roles, in an attempt to highlight their importance in cellular metabolism. Nucleoplasmin will illustrate a role in histone storage and Nap-1, a histone translocator. CAF-1 and Hira will provide examples of distinct histone deposition factors coupled to and uncoupled from DNA synthesis, respectively, while Asf1 could act as a histone donor. We then will illustrate with two examples how histone chaperones can be associated with chromatin remodeling activities. Finally, we will discuss how the RbAp46/48 proteins, as escort factors, are part of multiple complexes with various functions. Based on these examples, we will propose a scheme in which the diverse roles of histone chaperones are integrated within an assembly line for chromatin formation and regulation. Finally, we discuss how these chaperones may have more than a supporting role in a histone metabolic pathway. D
Trends in Biochemical Sciences, 2007
DNA in eukaryotic cells is compacted into chromatin, a regular repeated structure in which the nu... more DNA in eukaryotic cells is compacted into chromatin, a regular repeated structure in which the nucleosome represents the basic unit. The nucleosome not only serves to compact the genetic material but also provides information that affects nuclear functions including DNA replication, repair and transcription. This information is conveyed through numerous combinations of histone post-translational modifications (PTMs) and histone variants. A recent challenge has been to understand how and when these combinations of PTMs are imposed and to what extent they are determined by the choice of a specific histone variant. Here we focus on histone H3 variants and the PTMs that they carry before and after their assembly into chromatin. We review and discuss recent knowledge about how the choice and initial modifications of a specific variant might affect PTM states and eventually the final epigenetic state of a chromosomal domain.
Embo Reports, 2009
Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric ... more Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1a (HP1a)-chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non-nucleosomal histone H3. Therefore, the heterochromatic HP1a-CAF1-SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1a with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.
Molecular Cell, 2006
Extract preparation. We prepared cytosolic and nuclear extracts as previously described , and mon... more Extract preparation. We prepared cytosolic and nuclear extracts as previously described , and mononucleosomes and oligonucleosomes from nuclear pellets digested by micrococcal nuclease . Complex purification. We purified H3.1 and H3.3 histone complexes by immunoprecipitation on anti-Flag antibody-conjugated agarose beads in the presence of 0.1 µM of TSA and washed with 500 mM KCl/0.02% NP-40 for nonnucleosomal complexes and 200 mM KCl/0.02% NP-40 for mononucleosomes and oligonucleosomes. In order to remove DNA, mononucleosones and oligonucleosomes were further purified with hydroxyapatite resin, washed with 1M NaCl and eluted with 2 M NaCl. This washing condition removed not only remaining contaminants but also H2A/H2B.
Trends in Cell Biology, 2004
The bromodomain, a module of , 110 amino acids, is found in several chromatin-associated proteins... more The bromodomain, a module of , 110 amino acids, is found in several chromatin-associated proteins, including histone acetyltransferases and chromatin-remodeling factors, and can bind to acetylated lysines. Such post-translational modifications occur mainly in the N-terminal tail of the histone proteins and, in combination with other modifications, are thought to participate in defining a histone code. Recent findings provide a model for how bromodomain-containing proteins participate in the recognition of acetylated histones.
Journal of Biological Chemistry, 2000
We have previously reported the isolation and characterization of a nucleosome remodeling and spa... more We have previously reported the isolation and characterization of a nucleosome remodeling and spacing factor, RSF. One of the RSF subunits is hSNF2h, a SNF2 homologue. Here we set out to isolate and characterize other hSNF2h-containing complexes. We have identified a novel hSNF2h complex that facilitates ATP-dependent chromatin assembly with the histone chaperone NAP-1. The complex possesses ATPase activity that is DNA-dependent and nucleosome-stimulated. This complex is capable of facilitating ATP-dependent nucleosome remodeling and transcription initiation from chromatin templates. In addition to hSNF2h, this complex also contains a 190-kDa protein encoded by the BAZ1A gene. Since both subunits are homologues of the Drosophila ACF complex (ATP-utilizing chromatin assembly and remodeling factor), we have named this factor human ACF or hACF.
Methods in Enzymology, 2003
Note that the protocol described here is for the use of templates with Gless cassette with transc... more Note that the protocol described here is for the use of templates with Gless cassette with transcript quantitation by incorporation of radiolabeled UTP. When primer extension is used to quantitate transcription, the 20Â nucleotide mixture in step 6 is replaced with a mixture giving 0.5 mM of each nucleoside triphosphates in the assay, step 7 is omitted, and transcripts are amplified by PCR using a 32 P-labeled probe and reverse transcriptase after RNA purification (step 10). In our experience, identical results are obtained with either transcription methodology.
Proceedings of The National Academy of Sciences, 2003
The functional capacity of genetically encoded histone proteins can be powerfully expanded by pos... more The functional capacity of genetically encoded histone proteins can be powerfully expanded by posttranslational modification. A growing body of biochemical and genetic evidence clearly links the unique combinatorial patterning of side chain acetylation, methylation, and phosphorylation mainly within the highly conserved N termini of histones H2A, H2B, H3, and H4 with the regulation of gene expression and chromatin assembly and remodeling, in effect constituting a ''histone code'' for epigenetic signaling. Deconvoluting this code has proved challenging given the inherent posttranslational heterogeneity of histone proteins isolated from biological sources. Here we describe the application of native chemical ligation to the preparation of full-length histone proteins containing site-specific acetylation and methylation modifications. Peptide thioesters corresponding to histone N termini were prepared by solid phase peptide synthesis using an acid labile Boc͞HF assembly strategy, then subsequently ligated to recombinantly produced histone C-terminal globular domains containing an engineered N-terminal cysteine residue. The ligation site is then rendered traceless by hydrogenolytic desulfurization, generating a native histone protein sequence. Synthetic histones generated by this method are fully functional, as evidenced by their self-assembly into a higher order H3͞H4 heterotetramer, their deposition into nucleosomes by human ISWI-containing (Imitation of Switch) factor RSF (Remodeling and Spacing Factor), and by enzymatic modification by human Sirt1 deacetylase and G9a methyltransferase. Site-specifically modified histone proteins generated by this method will prove invaluable as novel reagents for the evaluation of the histone code hypothesis and analysis of epigenetic signaling mechanisms. ‡ Present address:
Methods, 2003
It is becoming clear that the structure of cellular chromatin is dynamic and capable of undergoin... more It is becoming clear that the structure of cellular chromatin is dynamic and capable of undergoing rapid changes to respond to the metabolic requirements of the cell. These changes have a direct impact on gene expression and, therefore, the chromatin context must be considered when biochemical reactions that involve DNA are studied. Over the past several decades, a number of methods for assembling chromatin in vitro have been described. Some of them use chemical compounds to deposit histone octamers onto the DNA. Others take advantage of cellular protein complexes that have the ability to assemble chromatin. Some of these complexes have been identified and purified. This article focuses on one of these factors, RSF (remodeling and spacing factor), which was identified in our laboratory. We describe how the chromatin assembly reaction is performed and how it can be monitored to evaluate its efficiency.
Molecular and Cellular Biology, 2003
The human ISWI-containing factor RSF (for remodeling and spacing factor) is composed of two subun... more The human ISWI-containing factor RSF (for remodeling and spacing factor) is composed of two subunits: the ATPase hSNF2H and p325 (Rsf-1), a protein encoded by a novel human gene. We previously showed that RSF mediates nucleosome deposition and generates regularly spaced nucleosome arrays. Here we report the characterization of the largest subunit of RSF, Rsf-1. We found that Rsf-1 is a highly acidic protein containing a plant homology domain. The present study includes the cloning of Rsf-1, the preparation of recombinant RSF, and the dissection of the role of each subunit in the chromatin assembly reaction. The sequence of the gene for Rsf-1 includes a recently characterized cDNA, HBXAP; postulated to be involved in the transcriptional regulation of the hepatitis B virus. HBXAP actually contains a 252-amino-acid truncation of the amino terminus of Rsf-1. Finally, comparison of HBXAP and Rsf-1 properties shows that they are functionally different.
Aprueban Reglamento para la valorización de mercancías según el Acuerdo sobre Valoración en Aduan... more Aprueban Reglamento para la valorización de mercancías según el Acuerdo sobre Valoración en Aduana de la OMC DECRETO SUPREMO Nº 186-99-EF CONCORDANCIAS: R.M.Nº 256-99-EF-15 CIRCULAR N° INTA-CR.62
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Papers by alejandra Loyola