[Chromatin dynamics during the repair of DNA lesions].
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Chromatin↗
Biomedical subjects
Sophie E Polo
Publications and source records attributed to Sophie E Polo.
New histone incorporation marks sites of UV repair in human cells.
Chromatin organization is compromised during the repair of DNA damage. It remains unknown how and to what extent epigenetic information is preserved in vivo. A central question is whether chromatin reorganization involves recycling of parental histones or new histone incorporation. Here, we devise an approach to follow new histone deposition upon UV irradiation in human cells. We show that new H3.1 histones get incorporated in vivo at repair sites. Remarkably we find that H3.1, which is deposited during S phase, is also incorporated outside of S phase. Histone deposition is dependent on nucleotide excision repair (NER), indicating that it occurs at a postrepair stage. The histone chaperone chromatin assembly factor 1 (CAF-1) is directly involved in the histone deposition process in vivo. We conclude that chromatin restoration after damage cannot rely simply on histone recycling. New histone incorporation at repair sites both challenges epigenetic stability and possibly contributes to damage memory.
Chromatin↗
Chromatin assembly: a basic recipe with various flavours.
Packaging of eukaryotic genomes into chromatin is a hierarchical mechanism, starting with histone deposition onto DNA to produce nucleosome arrays, which then further fold and ultimately form functional domains. Recent studies provide interesting insight into how nucleosome assembly is coordinated with histone and DNA metabolism and underline the combined contribution of histone chaperones and chromatin remodelers. How these factors operate at a molecular level is a matter of current investigation. New data highlight the importance of histone dimers as deposition entities for de novo nucleosome assembly and identify dedicated machineries involved in histone variant deposition.
Animals↗
Methods for studying chromatin assembly coupled to DNA repair.
In the eukaryotic nucleus, the DNA repair machinery operates on chromatin-embedded DNA substrates. Currently, a favored model for DNA repair into chromatin involves the transient disruption of chromatin organization to facilitate access of the repair machinery to DNA lesions. Importantly, this model implies that, in addition to DNA repair, a subsequent step is necessary to restore a proper chromatin structure. To study this latter step, we describe here methods for simultaneously analyzing chromatin assembly and DNA repair both in vitro and in vivo. Several cell-free systems have been developed that reproduce both DNA repair and nucleosome assembly. These in vitro systems are based on the use of defined damaged DNA. Two complementary assays are routinely used: (i) with circular DNA molecules, one can monitor in a combined analysis both repair synthesis and plasmid supercoiling; (ii) with immobilized damaged DNA, one follows specific protein interactions including histone deposition. In addition, in vivo assays have been designed to monitor the recruitment of chromatin assembly factors onto damaged chromatin either at a global level over the whole cell nucleus or locally at sites of DNA damage. Combination of these approaches provides powerful tools to gain insights into the mechanism by which chromatin organization can be restored after repair of DNA lesions.
Cell Line↗
Histone metabolic pathways and chromatin assembly factors as proliferation markers.
The structural organization of DNA into chromatin is of key importance to regulate genome function and stability. Maintenance of such an organization is thus crucial to preserve cellular identity. At each cell cycle, during S phase, this is achieved by duplication of chromatin structure in tight coordination with DNA replication. Such a coordinate process requires histone synthesis and their deposition onto DNA by chromatin assembly factors to be efficiently coupled to DNA synthesis. In this review, we highlight the intimate relationship between these chromatin-related events and DNA replication and we show how it is possible to take advantage of this coupling in order to identify cells with high replicative potential such as tumor cells. On the basis of recent data, we discuss the potential use of chromatin-associated factors as new proliferation markers of interest for cancer diagnosis and prognosis.
Biomarkers↗
Chromatin assembly factor-1, a marker of clinical value to distinguish quiescent from proliferating cells.
Histone synthesis and chromatin assembly are mainly associated with DNA replication and are thus intimately involved in cell cycle regulation. The expression of key components involved in these events in human cells was studied in relation to cell-proliferative status. Among several chromatin assembly factors, chromatin assembly factor (CAF)-1 stood out as the most discriminating marker of the proliferative state. We show, using both immunofluorescence and Western blot analysis, that the expression of both CAF-1 large subunits, p150 and p60, is massively down-regulated during quiescence in several cell lines. Upon exit from the quiescent state, the CAF-1 subunits are re-expressed early, before DNA replication. The amounts of either total or chromatin-associated pools of CAF-1 proteins correlate directly with cell proliferation. Regulation of CAF-1 expression is partly controlled at the RNA level, as shown by quantitative reverse transcription-PCR and Northern blot experiments. Biological material from benign and malignant human breast tumors analyzed by immunocytochemistry and immunohistochemistry exhibits a strong positive correlation between CAF-1 p60 expression and the following proliferation markers: S-phase fraction (r = 0.84, P < 0.0001); Ki-67 (r = 0.94, P < 0.0001); and proliferating cell nuclear antigen (r = 0.95, P = 0.0001). We discuss the advantages of using CAF-1 to assess cell proliferation. High CAF-1 p60 levels are also shown to be associated with various prognostic factors. Our data highlight the precise association of CAF-1 expression with the proliferative state and validate the use of this factor as a useful proliferation marker and prognostic indicator in malignant and benign breast lesions.
Biomarkers, Tumor↗