Experimental System to Study Instability of (CGG)n Repeats in Cultured Mammalian Cells.

Expansions of simple trinucleotide repeats, such as (CGG)n, (CAG)n or (GAA)n, are responsible for more than 40 hereditary disorders in humans including fragile X syndrome, Huntington's disease, myotonic dystrophy, and Friedreich's ataxia. While the mechanisms of repeat expansions were intensively studied for over two decades, the final picture has yet to emerge. It was important, therefore, to develop a mammalian experimental system for studying repeat instability, which would recapitulate repeat instability observed in human pedigrees. Here, we describe a genetically tractable experimental system to study the instability of (CGG)n repeats in cultured mammalian cells (Kononenko et al., Nat Struct Mol Biol 25:669-676, 2018). It is based on a selectable cassette carrying the HyTK gene under the control of the FMR1 promoter with carrier-size (CGG)n repeats in its 5' UTR, which was integrated into the unique RL5 site in murine erythroid leukemia cells. Expansions of these repeats and/or repeat-induced mutagenesis shut down the reporter, which results in the accumulation of ganciclovir-resistance cells. This system is useful for understanding the genetic controls of repeat instability in mammalian cells. In the long run, it can be adjusted to screen for drugs that either alleviate repeat expansions or reactivate the FMR1 promoter.

Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system.

We developed an experimental system for studying genome instability caused by fragile X (CGG)n repeats in mammalian cells. Our method uses a selectable cassette carrying the HyTK gene under the control of the FMR1 promoter with (CGG)n repeats in its 5' UTR, which is integrated into the unique RL5 site in murine erythroid leukemia cells. Carrier-size (CGG)n repeats markedly elevated the frequency of reporter inactivation, making cells ganciclovir resistant. These resistant clones had a unique mutational signature: a change in repeat length concurrent with mutagenesis in the reporter gene. Inactivation of genes implicated in break-induced replication, including Pold3, Pold4, Rad52, Rad51, and Smarcal1, reduced the frequency of ganciclovir-resistant clones to the baseline level that was observed in the absence of (CGG)n repeats. We propose that replication fork collapse at carrier-size (CGG)n repeats can trigger break-induced replication, which results in simultaneous repeat length changes and mutagenesis at a distance.

tRNA genes protect a reporter gene from epigenetic silencing in mouse cells.

It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.

Human gamma-satellite DNA maintains open chromatin structure and protects a transgene from epigenetic silencing.

The role of repetitive DNA sequences in pericentromeric regions with respect to kinetochore/heterochromatin structure and function is poorly understood. Here, we use a mouse erythroleukemia cell (MEL) system for studying how repetitive DNA assumes or is assembled into different chromatin structures. We show that human gamma-satellite DNA arrays allow a transcriptionally permissive chromatin conformation in an adjacent transgene and efficiently protect it from epigenetic silencing. These arrays contain CTCF and Ikaros binding sites. In MEL cells, this gamma-satellite DNA activity depends on binding of Ikaros proteins involved in differentiation along the hematopoietic pathway. Given our discovery of gamma-satellite DNA in pericentromeric regions of most human chromosomes and a dynamic chromatin state of gamma-satellite arrays in their natural location, we suggest that gamma-satellite DNA represents a unique region of the functional centromere with a possible role in preventing heterochromatin spreading beyond the pericentromeric region.

Co-localization of centromere activity, proteins and topoisomerase II within a subdomain of the major human X alpha-satellite array.

Dissection of human centromeres is difficult because of the lack of landmarks within highly repeated DNA. We have systematically manipulated a single human X centromere generating a large series of deletion derivatives, which have been examined at four levels: linear DNA structure; the distribution of constitutive centromere proteins; topoisomerase IIalpha cleavage activity; and mitotic stability. We have determined that the human X major alpha-satellite locus, DXZ1, is asymmetrically organized with an active subdomain anchored approximately 150 kb in from the Xp-edge. We demonstrate a major site of topoisomerase II cleavage within this domain that can shift if juxtaposed with a telomere, suggesting that this enzyme recognizes an epigenetic determinant within the DXZ1 chromatin. The observation that the only part of the DXZ1 locus shared by all deletion derivatives is a highly restricted region of <50 kb, which coincides with the topo isomerase II cleavage site, together with the high levels of cleavage detected, identify topoisomerase II as a major player in centromere biology.

Expression of Hqk encoding a KH RNA binding protein is altered in human glioma.

The quaking gene family encodes single KH domain RNA-binding proteins that play vital roles in cell differentiation, proliferation, and apoptotic processes. The human quaking gene, Hqk, maps to 6q25-q26, where cytogenetic alterations associated with a variety of human malignancies, including gliomas have been reported. To assess possible relationships of Hqk with human diseases such as glial tumors, we first isolated the Hqk gene, characterized its structure and expression pattern, and carried out mutational analysis of Hqk in primary tumor samples. The Hqk gene contains 8 exons spanning a approximately 200 kb genomic region, and generating at least four alternatively spliced transcripts, Hqk-5, Hqk-6, Hqk-7 and Hqk-7B, of which Hqk-7 is abundantly expressed in brain. Analysis of primary tumors demonstrated a high incidence of expression alterations of Hqk in gliomas (30%; 6/20), but not in other tumors such as schwannomas (0/3), or meningiomas (0/8). Among the tumor samples showing expression alterations, two were devoid of all three major transcripts, one was missing only the Hqk-5 message, and only the Hqk-7 message was absent in two cases. Our results thus imply the involvement of Hqk in human glial tumor progression.