Biomedical subjects

M S Santisteban

Publications and source records attributed to M S Santisteban.

11 recordsLinked to original sources

PubMed2000

Histone H2A.Z regulats transcription and is partially redundant with nucleosome remodeling complexes.

Nucleosomes impose a block to transcription that can be overcome in vivo by remodeling complexes such as SNF/SWI and histone modification complexes such as SAGA. Mutations in the major core histones relieve transcriptional repression and bypass the requirement for SNF/SWI and SAGA. We have found that the variant histone H2A.Z regulates gene transcription, and deletion of the gene encoding H2A.Z strongly increases the requirement for SNF/SWI and SAGA. This synthetic genetic interaction is seen at the level of single genes and acts downstream of promoter nucleosome reorganization. H2A.Z is preferentially crosslinked in vivo to intergenic DNA at the PH05 and GAL1 loci, and this association changes with transcriptional activation. These results describe a novel pathway for regulating transcription using variant histones to modulate chromatin structure.

Adenosine Triphosphatases↗

PubMed2000

Histone-histone interactions and centromere function.

Cse4p is a structural component of the core centromere of Saccharomyces cerevisiae and is a member of the conserved CENP-A family of specialized histone H3 variants. The histone H4 allele hhf1-20 confers defects in core centromere chromatin structure and mitotic chromosome transmission. We have proposed that Cse4p and histone H4 interact through their respective histone fold domains to assemble a nucleosome-like structure at centromeric DNA. To test this model, we targeted random mutations to the Cse4p histone fold domain and isolated three temperature-sensitive cse4 alleles in an unbiased genetic screen. Two of the cse4 alleles contain mutations at the Cse4p-H4 interface. One of these requires two widely separated mutations demonstrating long-range cooperative interactions in the structure. The third cse4 allele is mutated at its helix 2-helix 3 interface, a region required for homotypic H3 fold dimerization. Overexpression of wild-type Cse4p and histone H4 confer reciprocal allele-specific suppression of cse4 and hhf1 mutations, providing strong evidence for Cse4p-H4 protein interaction. Overexpression of histone H3 is dosage lethal in cse4 mutants, suggesting that histone H3 competes with Cse4p for histone H4 binding. However, the relative resistance of the Cse4p-H4 pathway to H3 interference argues that centromere chromatin assembly must be highly regulated.

Centromere↗

PubMed1998

Genetic dissection of histone function.

Mutational analysis is an essential tool for understanding the functions of genes within a living organism. The budding yeast Saccharomyces cerevisiae provides an excellent model system for dissecting the genetics of histone function at the molecular and cellular levels. A simple gene organization, plus a wide variety of genetic strategies, makes it possible to directly manipulate a specific histone gene in vitro and then examine the expression of mutant alleles in vivo. Recent methods for manipulating the yeast histone genes have been designed to facilitate both side-directed analysis of structure/function relationships and unbiased screens targeted at specific functional pathways. The conservation of histone and nucleosome structure throughout evolution means that the principles discovered through genetic studies in yeast will be broadly applicable to the chromatin of more complex eukaryotes.

Cell Cycle↗

PubMed1997

Histone octamer function in vivo: mutations in the dimer-tetramer interfaces disrupt both gene activation and repression.

Within the core histone octamer each histone H4 interacts with each H2A-H2B dimer subunit through two binding surfaces. Tyrosines play a central role in these interactions with H4 tyrosines 72 and 88 contacting one H2A-H2B dimer subunit, and tyrosine 98 contacting the other. To investigate the roles of these interactions in vivo, we made site-directed amino acid substitutions at each of these tyrosine residues. Elimination of either set of interactions is lethal, suggesting that binding of the tetramer to both dimers is essential. Temperature-sensitive mutants were obtained through single amino acid substitutions at each of the tyrosines. The mutants show both strong positive and negative effects on transcription. Positive effects include Spt- and Sin-phenotypes resulting from mutations at each of the three tyrosines. One allele has a strong negative effect on the expression of genes essential for the G1 cell cycle transition. At restrictive temperature, mutant cells fail to express the CLN1, CLN2, SWI4 and SWI6 genes, and have reduced levels of CLN3 mRNA. These results demonstrate the critical role of histone dimer-tetramer interactions in vivo, and define their essential role in the expression of genes regulating G1 cell cycle progression.

Chromatin↗

PubMed1996

A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission.

The histone proteins are essential for the assembly and function of th e eukaryotic chromosome. Here we report the first isolation of a temperature-sensitive lethal histone H4 mutant defective in mitotic chromosome transmission Saccharomyces cerevisiae. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, was found to suppress the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation.

Chromatin↗

PubMed1995

Fluorescence image analysis of the MCF-7 cycle related changes in chromatin texture. Differences between AT- and GC-rich chromatin.

This paper reports on quantitative in situ changes in chromatin structure that occur throughout the cell cycle of the human breast cancer epithelial cell line, MCF-7. Texture parameters were measured by image cytometry on nuclei stained by DNA specific fluorochromes. These parameters calculated from the co-occurrence and run length matrices of grey level images were previously shown to be related to condensation, organization and distribution of DNA. In some experiments, cells were triple stained for DNA/Ki-67/PCNA, and compartmentalization in the cycle was ascertained from the Ki-67/PCNA pattern expression. In these experiments, Hoechst dye was used to stain DNA. Chromatin of cells traversing G1 phase progressively decondensed and became homogeneously distributed. In addition, these G1 cells had more condensed chromatin than cells in G0 phase (as determined by Ki-67 negative staining). During the S and G2 phases, chromatin condensation took place and an increasing reticulated organization was quantified. Similar profile of changes in chromatin texture was found in experiments done with cells double stained by AT-specific Hoechst dye and the GC-specific mithramycin dye. GC-rich chromatin texture-associated parameters greatly varied comparing to those of AT-rich chromatin during the G0/G1 phase as well as in the first mid-S phase. Conversely, variation of the AT-associated parameters was much greater in the second half of S phase as compared to the GC-associated parameters that barely varied during this period. This study well establishes the correlation between in situ chromatin texture and proliferation state because the latter is assessed by proliferation-associated antigens. Moreover, changes in chromatin texture are independently ascribed to the AT- and GC-rich regions suggesting that these 2 types of chromatin are involved to different extents in transcriptional and replicational tasks.

Base Composition↗

PubMed1995

[Structure of chromatin. 2: levels of organization of DNA in the nucleus. Highly organized structures].

In the interphase nucleus, the 30 nm fiber, whose characteristics were discussed in the first part of this review, is condensed 10-15 fold to form loops or domains of chromatin with a DNA packing ratio of 600. In the metaphase chromosomes, the packing ratio is 12000. The existence of loops in the interphase nucleus has been suggested by experiments from electronic microscopy, sedimentation and nuclease digestion. These loops of 20-200Kb are attached at their bases to a non-histone protein network, that is the nuclear matrix or scaffold whose major component is the topoisomerase II. There is disagreement regarding the arrangement of the loops in metaphase chromosomes and several models have been proposed. Six of these models are presented. A number of data suggest that the sequences of attachment (SARs/MARs) define functional units containing related genes. Thus, the compartmentation of the genome in topographically independent domains might ensure the fidelity of epigenetic transmission of the chromatin structure, and it might help to control gene expression. In the interphase nucleus, the chromosomes coexist with other domains involved in major nuclear functions such as transcription, replication or post-transcriptional modifications and whose compartmentation is not always evident.

Centromere↗

PubMed1995

[Structure of chromatin and cell cycle. 3].

During the cell cycle, chromatin undergoes structural changes associated with cellular functions such as DNA replication during the S phase or the transcriptional inactivation during metaphase. All levels of chromatin organization from the nucleosome to the loops attached to the scaffold are concerned by these changes. Biochemical or biophysical studies from 70's, as well as more recent studies using nondestructive in situ methods, that allow to obtain information at the individual cell level, tend to demonstrate a correlation between the condensation state of the chromatin and the position of the cell in the cycle. The majority of these studies confirm the existence of one single cycle of condensation-decondensation for the chromatin. These studies are discussed in the first part of this chapter. In the second part, the role of the cdc (cell division cycle) gene's products in the control of the cell cycle and in the regulation of the chromatin structure are analyzed.

CDC2 Protein Kinase↗

PubMed1994

[Structure of chromatin. I: Levels of DNA organization in the nucleus; nucleosome and chromatin fibres].

This bibliographic review, the first of a series of three on chromatin structure, discusses the current knowledge and hypotheses concerning chromatin organization in nucleosomes and fibers. Presently, the structural features of the chromatin arrangement are better known for the low levels of organization, e.g. nucleosome, but there is much disagreement about levels of organization higher than the 10 nm fiber. The published reports are consistent with the model that, in the nucleosome, the DNA is wrapped around the histone octamer. The histone octamer whose structure and dimensions were investigated for years is viewed today as a tripartite particle with the (H3-H4)2 tetramer flanked by two H2A-H2B dimers. In the chromatosome, two turns of DNA around of the histone octamer are sealed by the histone H1 that binds DNA at the points of entry and exit. A chain of nucleosomes, each with about 200 bp associated DNA, forms a 10 nm fiber with a DNA packing ratio of 6-7. The 10 nm fiber is then condensed into a 30 nm fiber, likely of solenoid structure, that is stabilized by histone H1. All the different models proposed for the structure of the 30 nm fiber are consistent with a DNA packing ratio of 35-40.

Chromatin↗

PubMed1992

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytometry (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes fixed by the Boehm-Sprenger procedure optimal for preserving the chromatin pattern. The question was whether fluorochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2 c ratio was still equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the fixation conditions used. The intranuclear and the internuclear SD of the fluorescence intensities describing the fluorescence pattern of the 2c hepatocytes proved to vary according to both the basepair specificity and the binding mode of the fluorochromes. The results reported here argue in favor of an external binding of 7-AMD to DNA and an increased quantum yield of QM when bound to AT-rich DNA. For PI, EB, 7-AMD, and OM, the measured DNA content increased with the fluorescence distribution heterogeneity. This correlation was not observed with other fluorochromes and is suggested to result from decreased fluorochrome accessibility to DNA when the chromatin is condensed. This study demonstrates that under conditions that preserve chromatin organization, only HO for AT-rich DNA and MA for GC-rich DNA can be used, alone or in combination, to measure nuclear DNA content. With other fluorochromes, either the measured DNA content or the chromatin pattern is assessed in suboptimal conditions when fluorescent image cytometry is used.

Animals↗

PubMed1992

Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells.

The intranuclear distribution of newly replicated DNA and of the proliferating cell nuclear antigen (PCNA) was mapped by confocal laser scanning microscopy after simultaneous immunofluorescent labelling of incorporated bromodeoxyuridine (BrdUrd) and PCNA. A mild hydrolysis with HCl followed by an enzymic digestion of DNA was used to produce single-stranded DNA required for BrdUrd immunorevelation, since this procedure preserves PCNA antigenicity. Optical sections obtained with a laser scanning microscope clearly showed a similar distribution of PCNA and BrdUrd within the nuclei, thus confirming previous observations on parallel labelled synchronized cultures. The intranuclear distribution of PCNA and BrdUrd varies concomitantly during the S phase of MCF-7 cells.

Autoantigens↗