Caroline Schluth-Bolard

Identification of a perinuclear positioning element in human subtelomeres that requires A-type lamins and CTCF

The localization of genes within the nuclear space is of paramount importance for proper genome functions. However, very little is known on the cis‐acting elements determining subnuclear positioning of chromosome segments. We show here that the D4Z4 human subtelomeric repeat localizes a telomere at the nuclear periphery. This perinuclear activity lies within an 80 bp sequence included within a region known to interact with CTCF and A‐type Lamins. We further show that a reduced level of either CTCF or A‐type Lamins suppresses the perinuclear activities of D4Z4 and that an array of multimerized D4Z4 sequence, which has lost its ability to bind CTCF and A‐type Lamins, is not localized at the periphery. Overall, these findings reveal the existence of an 80 bp D4Z4 sequence that is sufficient to position an adjacent telomere to the nuclear periphery in a CTCF and A‐type lamins‐dependent manner. Strikingly, this sequence includes a 30 bp GA‐rich motif, which binds CTCF and is present at several locations in the human genome.

Replication timing of human telomeres is chromosome arm-specific, influenced by subtelomeric structures and connected to nuclear localization

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of β-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4–associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm–specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.

Telomere protection and TRF2 expression are enhanced by the canonical Wnt signalling pathway

The DNA‐binding protein TRF2 is essential for telomere protection and chromosome stability in mammals. We show here that TRF2 expression is activated by the Wnt/β‐catenin signalling pathway in human cancer and normal cells as well as in mouse intestinal tissues. Furthermore, β‐catenin binds to TRF2 gene regulatory regions that are functional in a luciferase transactivating assay. Reduced β‐catenin expression in cancer cells triggers a marked increase in telomere dysfunction, which can be reversed by TRF2 overexpression. We conclude that the Wnt/β‐catenin signalling pathway maintains a level of TRF2 critical for telomere protection. This is expected to have an important role during development, adult stem cell function and oncogenesis.

D4Z4 as a prototype of CTCF and lamins-dependent insulator in human cells

Using cellular models that mimic the organization of the subtelomeric 4q35 locus found in patients affected with Facio-Scapulo-Humeral Dystrophy (FSHD) and in healthy individuals, we recently investigated the biological function of the D4Z4 macrosatellite within this domain. We demonstrated that D4Z4 acts as a CTCF and A-type Lamins dependent insulator element exhibiting both enhancer-blocking and barrier activities, and displaces a telomere towards the nuclear periphery. This peripheral positioning activity lies within a short sequence that interacts with CTCF and A-type Lamins. Depletion in either of these two proteins suppresses these perinuclear activities, revealing the existence of a subtelomeric sequence that is sufficient to position an adjacent telomere to the nuclear periphery. We discuss here the biological implications of these results in the light of our current knowledge in related fields and the potential implication of other CTCF and A- type lamins insulators in the light of human pathologies.

A Distinct Class of Chromoanagenesis Events Characterized by Focal Copy Number Gains.

Chromoanagenesis is the process by which a single catastrophic event creates complex rearrangements confined to a single or a few chromosomes. It is usually characterized by the presence of multiple deletions and/or duplications, as well as by copy neutral rearrangements. In contrast, an array CGH screen of patients with developmental anomalies revealed three patients in which a single chromosome carries from 8 to 11 large copy number gains confined to a single chromosome or chromosomal arm, but the absence of deletions. Subsequent fluorescence in situ hybiridization and massive parallel sequencing revealed the duplicons to be clustered together in distinct locations across the altered chromosomes. Breakpoint junction sequences showed both microhomology and non‐templated insertions of up to 40 bp. Hence, these patients each demonstrate a single altered chromosome of clustered insertional duplications, no deletions, and breakpoint junction sequences showing microhomology and/or non‐templated insertions. These observations are difficult to reconcile with current mechanistic descriptions of chromothripsis and chromoanasynthesis. Therefore, we hypothesize those rearrangements to be of a mechanistically different origin. In addition, we suggest that large untemplated insertional sequences observed at breakpoints are driven by a non‐canonical non‐homologous end joining mechanism.

Characterization of a de novo Supernumerary Neocentric Ring Chromosome Derived from Chromosome 7

Supernumerary ring chromosomes (SRC) are usually derived from regions adjacent to the centromere. Their identification may be challenging, particularly in case of low mosaicism. Here, we report on a patient who was referred for major in utero growth retardation, severe developmental delay, facial dysmorphism, cleft palate, and hypospadias. The karyotype showed a small SRC in mosaic. The combination of FISH, M-FISH and array-CGH was necessary for a complete characterization of this SRC. M-FISH revealed that the SRC originated from chromosome 7. Array-CGH performed with a 400K oligonucleotide array showed a gain in region 7q22.1q31.1 present in low mosaic. This result was confirmed by FISH using BAC probes specific for chromosome 7. The SRC was a neocentric ring derived from 7q22.1q31.1 and was found in only 8% of the cells. This is the first patient carrying a mosaic neocentric SRC derived from the long arm of chromosome 7. Our study emphasizes the need to combine different techniques and to use adapted bioinformatic tools for low-mosaicism marker identification. It also contributes to the delineation of the partial trisomy 7q phenotype.

Dynamics and plasticity of chromosome ends: consequences in human pathologies

In eukaryotic cells, chromosome ends of linear chro mosomes are particular regions of the genome formed by telomeres at the very end and subtelomeres, complex sequences that separate telomeres from chromosome-specific r egions. These two regions are highly dynamic are contribute to th e stability and integrity of the human genome. Furt hermore, in human cells, dysregulation of these regions are imp licated in a wide range of physiological events and pathological manifestations. Due to the amount of information av ailable on the biology of telomeres, we will give a n overview and discuss here what is currently known of the regulat ion of telomere length and homeostasis and describe the complex organization of subtelomeric regions and their impl ication in multiple pathologies.

Chromosomal Position Effects and Gene Variegation: Impact in Pathologies

One of the most intriguing aspects of epigenetics is the dynamic nature of chromatin and the transmission of various chromatin conformations, sometimes over a large region of the genome. This is likely to involve long-range chromatin interactions, which is well illustrated by the capacity of chromatin to propagate into neighboring loci. Chromatin regulation involves finely tuned processes and it becomes more and more clear that the tri-dimensional organization of genomic domains within the nuclear space also contributes to genome regulation. In eukaryotic genomes, beside non-coding regions, regulatory elements can extend their influence outside of a transcription unit and reside within an independently regulated locus. Thus, sequences with different functions or spatio-temporal activities are juxtaposed next to another. Affecting this equilibrium by changing their relative positioning represents a risk in the maintenance of chromatin architecture, gene regulation and in turn, the fate of a cell. Position effect mechanisms, first discovered and described in model organisms, remain poorly investigated in human pathology. However, common themes are emerging from the review of fundamental research and clinical observations of patients. In this chapter, we try to outline and discuss different examples of diseases linked to position effects and bring into focus potential conserved mechanisms underlying their pathogenesis.Publisher Summary This chapter discusses the chromosomal position effects (CPE) and gene variegation with its impact in pathologies. CPE was originally discovered in flies in a study of X-ray-induced chromosomal rearrangements or P element insertions that placed euchromatic genes into heterochromatic regions and rearrangements that positioned euchromatin domains into heterochromatin or vice versa. Mechanisms through which CPE can cause human diseases are diverse—separation of the transcription unit from an essential distant regulatory element, juxtaposition of the gene with the enhancer element of another gene, competition for the same regulatory element, and classical position effect variegation in which a gene is moved to a new chromatin environment. CPE-associated pathogenesis has been described in cancers as well as in constitutional pathologies, essentially in the context of chromosomal rearrangements (translocations, deletions, inversions). For instance, malignant hemopathies are characterized by acquired chromosomal rearrangements, mainly translocations, which are clonal, nonrandom, recurrent, and often specific for a tumor type. These translocations have two main consequences—the formation of a chimerical gene encoding a new fusion protein or the combination between the coding region of a gene and the promoter/enhancer region of another one leading to inappropriate overexpression of the former gene.

Genetic Counselling Pitfall: Co-Occurrence of an 11.8-Mb Xp22 Duplication and an Xp21.2 Duplication Disrupting IL1RAPL1

We report a 3-generation family in which 2 Xp copy number variations (CNVs) co-segregate. The proband presented with syndromic intellectual disability. The CNV had been revealed by conventional karyotyping, identifying a large Xp22 duplication causing an Xp functional disomy. Family studies found that this duplication was inherited from the probands mother and was also present in one of his sisters. This sister had conventional karyotyping performed during pregnancy with a normal result. Postnatally, her child, the probands nephew, presented with autism spectrum disorders. aCGH revealed a 339-kb IL1RAPL1 duplication. Overall, the proband, his mother, and one of his sisters all harboured both CNVs, while his other sister and the 2 sons of each sister only carried the IL1RAPL1 intragenic duplication. As seen in this family, we emphasise the importance of small CNV detection, the pathogenicity of IL1RAPL1 exonic duplications in male carriers, and the difficulties for genetic counselling with the risk of double diagnosis in a single patient.

Prenatal Diagnosis of Trisomy 2p due to Terminal 2p Duplication including Interstitial Telomeric Sequences

We report on a prenatally diagnosed unusual case of inverted terminal duplication of the short arm of chromosome 2, leading to interstitial telomeric sequences (ITSs) and partial trisomy 2p. To our knowledge, there are only 4 further cases of pure partial trisomy 2p reported prenatally. Here, the mother was referred at 22 weeks of gestation for isolated fetal congenital heart malformation at ultrasound. The karyotype of amniotic fluid cells displayed a large duplication of the short arm of chromosome 2 that was further investigated by array-CGH, which detected a 1-copy gain of 43.75 Mb in chromosome 2 at 2p21p25.3. FISH confirmed the presence of an inverted duplication in the short arm of chromosome 2 involving the region 2p21pter and revealed the presence of ITSs at the breakpoint in chromosome 2p21. This report contributes to the prenatal description of the syndrome. We also discuss the possible mechanisms leading to this duplication and the formation of ITSs which are rarely described in constitutional rearrangements.