Involvement of Mitochondrial Dysfunction in FOXG1 Syndrome.

FOXG1 (Forkhead box g1) syndrome is a neurodevelopmental disorder caused by a defective transcription factor, FOXG1, important for normal brain development and function. As FOXG1 syndrome and mitochondrial disorders have shared symptoms and FOXG1 regulates mitochondrial function, we investigated whether defective FOXG1 leads to mitochondrial dysfunction in five individuals with FOXG1 variants compared to controls (n = 6). We observed a significant decrease in mitochondrial content and adenosine triphosphate (ATP) levels and morphological changes in mitochondrial network in the fibroblasts of affected individuals, indicating involvement of mitochondrial dysfunction in FOXG1 syndrome pathogenesis. Further investigations are warranted to elucidate how FOXG1 deficiency impairs mitochondrial homeostasis.

Folate stress induces SLX1- and RAD51-dependent mitotic DNA synthesis at the fragile X locus in human cells.

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.

Folate deficiency drives mitotic missegregation of the human FRAXA locus.

The instability of chromosome fragile sites is implicated as a causative factor in several human diseases, including cancer [for common fragile sites (CFSs)] and neurological disorders [for rare fragile sites (RFSs)]. Previous studies have indicated that problems arising during DNA replication are the underlying source of this instability. Although the role of replication stress in promoting instability at CFSs is well documented, much less is known about how the fragility of RFSs arises. Many RFSs, as exemplified by expansion of a CGG trinucleotide repeat sequence in the fragile X syndrome-associated FRAXA locus, exhibit fragility in response to folate deficiency or other forms of "folate stress." We hypothesized that such folate stress, through disturbing the replication program within the pathologically expanded repeats within FRAXA, would lead to mitotic abnormalities that exacerbate locus instability. Here, we show that folate stress leads to a dramatic increase in missegregation of FRAXA coupled with the formation of single-stranded DNA bridges in anaphase and micronuclei that contain the FRAXA locus. Moreover, chromosome X aneuploidy is seen when these cells are exposed to folate deficiency for an extended period. We propose that problematic FRAXA replication during interphase leads to a failure to disjoin the sister chromatids during anaphase. This generates further instability not only at FRAXA itself but also of chromosome X. These data have wider implications for the effects of folate deficiency on chromosome instability in human cells.

The Detection and Analysis of Chromosome Fragile Sites.

A fragile site is a chromosomal locus that is prone to form a gap or constriction visible within a condensed metaphase chromosome, particularly following exposure of cells to DNA replication stress. Based on their frequency, fragile sites are classified as either common (CFSs; present in all individuals) or rare (RFSs; present in only a few individuals). Interest in fragile sites has remained high since their discovery in 1965, because of their association with human disease. CFSs are recognized as drivers of oncogene activation and genome instability in cancer cells, while some RFSs are associated with neurodegenerative diseases. This review summaries our current understanding of the nature and causes of fragile site "expression", including the recently characterized phenomenon of telomere fragility. In particular, we focus on a description of the methodologies and technologies for detection and analysis of chromosome fragile sites.

PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis.

PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.

Replication stress activates DNA repair synthesis in mitosis.

Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress. The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.