CABIMER

 

 

Genome organization and function rely on the precise assembly and dynamics of chromatin. Histones H3 and H4 are first assembled into a core (H3/H4)2 tetramer that is the stable entity at physiological ionic strength. An H2A/H2B dimer associates on each side of the tetramer to form the histone octamer that is stabilized by the nucleosomal DNA. ATP-dependent remodeling complexes, histone post-translational modifications and replacement of canonical with variant histones can later modify nucleosomes thus altering the function of specific chromatin regions. We are interested in understanding how chromatin dynamics controls genome integrity. Using yeast genetics and molecular and cellular biology we are addressing this overall objective by analyzing different aspects of chromatin dynamics.

Chromatin assembly and cell cycle progression.

During DNA replication, the advance of the replication fork requires the disruption of the chromatin fiber in front of the fork and the assembly of the nascent DNA strands onto nucleosomes. DNA replication and chromatin assembly are tightly coupled by genetic and physical interactions between components of the replisome with histone deposition factors. Detailed molecular analysis in yeast has recently deciphered part of the mechanism of H3/H4 deposition during DNA replication. Thus, the histone chaperone Asf1 binds to newly synthesized H3/H4 dimers and presents them for acetylation of H3K56 by the histone acetyltransferase Rtt109. This histone modification enhances the binding of the chromatin assembly factors CAF1 and Rtt106 to both H3 and the replisome component PCNA, thus promoting histone deposition at the proximity of the fork.

Others and we have shown that defective histone H3/H4 deposition causes genetic instability, supporting the idea that proper chromatin assembly is required for the maintenance of genome integrity. We have recently uncovered a new function for chromatin assembly in the maintenance of genome integrity by keeping the stability of replication forks. We are currently trying to decipher the genetic requirements and molecular mechanisms by which this genetic instability takes place. It is also noted that defective chromatin assembly causes mitotic arrest. Our results point to a connection between proper nucleosome deposition and chromosome segregation that we are studying by time-lapse microscopy as well as genetic and molecular approaches.

Histone H2A-by-H2A.Z (Htz1) replacement and genome integrity.

Variant histones alter the physicochemical properties of nucleosomes and thereby not only the interactions of nucleosomes with other factors but also their stability and DNA accessibility. One such variant, H2A.Z – Htz1 in yeast –, is an evolutionary conserved histone with roles in transcription, silencing, genome integrity and cell cycle progression. H2A.Z/Htz1 is incorporated into chromatin by the Swi2/Snf2-related SWR complex (SWR.com), a 14-subunit complex that replaces the canonical histone H2A with Htz1 by not-yet defined mechanisms. In order to understand how histone replacement controls genome integrity we are currently analyzing the effect of mutations in the process of H2A-by-Htz1 exchange. Our results have shown that SWR/Htz1 maintains genome integrity by different mechanisms. As we get deeper insights in these processes, we understand better the molecular mechanisms of histone replacement and their importance in the maintenance of the structural and functional integrity of chromatin.

 

Role of chromatin in DNA damage repair.

Packaging of DNA into chromatin makes necessary the function of chromatin remodelling activities to facilitate the accessibility of repair proteins to DNA, as well as chromatin assembly factors to reset the original chromatin structure. A number of chromatin remodelling complexes have been shown to be recruited to double-strand breaks (DSBs), suggesting that DNA repair is associated with chromatin alterations that facilitate –and likely regulate – the repair of the break. We are addressing this interesting role of chromatin by following nucleosome dynamics during DSB repair.

Morillo-Huesca M, Clemente-Ruiz M, Andujar E, Prado F (2010) The SWR1 Histone Replacement Complex Causes Genetic Instability and Genome-Wide transcription Misregulation in the Absence of H2A.Z. PLoS ONE 5(8): e12143. doi:10.1371/journal.pone.0012143

Clemente-Ruiz and Félix Prado (2009) Chromatin assembly controls replication fork stability. EMBO reports (in press)

Félix Prado and Andrés Aguilera (2005) Impairment of replication fork progresión mediates RNA polII transcription-associated recombination. EMBO J 24: 1267-1276

Félix Prado and Andrés Aguilera (2005) Partial depletion of histone H4 increases homologous recombination-mediated genetic instability. Molecular and Cellular Biology 25:1526-1536

Félix Prado, Felipe Cortés-Ledesma and Andrés Aguilera (2004) The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange. EMBO reports 5: 497-502

Félix Prado, and Andrés Aguilera (2003) Control of crossing over by single strand DNA resection. Trends in Genetics 19:428-431

Mitotic recombination in Saccharomyces cerevisiae (2003) Félix Prado, Felipe Cortés-Ledesma, Pablo Huertas and Andrés Aguilera. Current Genetics 42; 185-198