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The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin unlike most human telomeres.

Tam R, Smith KP, Lawrence JB - J. Cell Biol. (2004)

Bottom Line: Studies of hybrid and translocation cell lines indicate this localization is inherent to the distal tip of 4q.However, consistent association of the pathogenic D4Z4 locus with the heterochromatic compartment supports a potential role in regulating the heterochromatic state and makes a telomere positioning effect more likely.Furthermore, D4Z4 repeats on other chromosomes also frequently organize with the heterochromatic compartment at the nuclear or nucleolar periphery, demonstrating a commonality among chromosomes harboring this subtelomere repeat family.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

ABSTRACT
This paper investigates the nuclear localization of human telomeres and, specifically, the 4q35 subtelomere mutated in facioscapulohumeral dystrophy (FSHD). FSHD is a common muscular dystrophy that has been linked to contraction of D4Z4 tandem repeats, widely postulated to affect distant gene expression. Most human telomeres, such as 17q and 17p, avoid the nuclear periphery to reside within the internal, euchromatic compartment. In contrast, 4q35 localizes at the peripheral heterochromatin with 4p more internal, generating a reproducible chromosome orientation that we relate to gene expression profiles. Studies of hybrid and translocation cell lines indicate this localization is inherent to the distal tip of 4q. Investigation of heterozygous FSHD myoblasts demonstrated no significant displacement of the mutant allele from the nuclear periphery. However, consistent association of the pathogenic D4Z4 locus with the heterochromatic compartment supports a potential role in regulating the heterochromatic state and makes a telomere positioning effect more likely. Furthermore, D4Z4 repeats on other chromosomes also frequently organize with the heterochromatic compartment at the nuclear or nucleolar periphery, demonstrating a commonality among chromosomes harboring this subtelomere repeat family.

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In FSHD patient muscle, truncation of D4Z4 repeats does not significantly alter 4q35 localization to the heterochromatic rim. (A) Cytogenetic preparation of FSHD lymphoblastoid cells demonstrating different intensities of D4Z4 signals (green) at each 4q35 allele (red), permitting the mutant and wild-type alleles to be distinguished. (B) Both 4q35 alleles (green) in a mutant FSHD myoblast remain at the nuclear periphery depleted of hnRNA (red). (C) Localization of wild-type (arrow) and mutant (arrowhead) alleles in a FSHD myoblast using the same probes as in A. DAPI (blue) delineates the nucleus. (D) Quantitation of the localization of mutant (mut) versus wild-type (wt) allele in three FSHD myoblast cell lines (GM 17731, GM17899, and GM17869A) and in a normal myoblast line (50MB-1) before and after differentiation. 100 cells analyzed per sample. Localization of 4q35 (red) in a normal (E) and a FSHD myotube (F) with more intense D4Z4 signal (green) demarcating wild type versus weaker mutant 4q35 allele. Red and green signals from a z-stack were projected onto a single plane of blue signal. In the FSHD myotube, lamin A and fibrillarin were simultaneously detected by Alexa 350. (G) The number of cells (of 100 analyzed) with both 4q35 alleles dissociated from the periphery is rare regardless of D4Z4 copy number and may correlate with the age of the patient (see online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200403128/DC1). Typically, both alleles (red) lie just below the nuclear envelope stained for nucleopores (green) in normal muscle (H; blue, nucleoli) and FSHD muscle (I; blue, sc35).
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fig5: In FSHD patient muscle, truncation of D4Z4 repeats does not significantly alter 4q35 localization to the heterochromatic rim. (A) Cytogenetic preparation of FSHD lymphoblastoid cells demonstrating different intensities of D4Z4 signals (green) at each 4q35 allele (red), permitting the mutant and wild-type alleles to be distinguished. (B) Both 4q35 alleles (green) in a mutant FSHD myoblast remain at the nuclear periphery depleted of hnRNA (red). (C) Localization of wild-type (arrow) and mutant (arrowhead) alleles in a FSHD myoblast using the same probes as in A. DAPI (blue) delineates the nucleus. (D) Quantitation of the localization of mutant (mut) versus wild-type (wt) allele in three FSHD myoblast cell lines (GM 17731, GM17899, and GM17869A) and in a normal myoblast line (50MB-1) before and after differentiation. 100 cells analyzed per sample. Localization of 4q35 (red) in a normal (E) and a FSHD myotube (F) with more intense D4Z4 signal (green) demarcating wild type versus weaker mutant 4q35 allele. Red and green signals from a z-stack were projected onto a single plane of blue signal. In the FSHD myotube, lamin A and fibrillarin were simultaneously detected by Alexa 350. (G) The number of cells (of 100 analyzed) with both 4q35 alleles dissociated from the periphery is rare regardless of D4Z4 copy number and may correlate with the age of the patient (see online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200403128/DC1). Typically, both alleles (red) lie just below the nuclear envelope stained for nucleopores (green) in normal muscle (H; blue, nucleoli) and FSHD muscle (I; blue, sc35).

Mentions: It would be valuable to discriminate the mutant from the normal 4q35 locus in single cells because this distinction was not explored in earlier localization studies (Winokur et al., 1996; Stout et al., 1999). The intensity of FISH signal is proportional to the DNA target size, and direct comparison of normal and mutant alleles in the same cell minimizes technical differences (Johnson et al., 2000). Therefore, we tested a strategy of hybridizing a 4q35 marker with the D4Z4 probe in two different colors to discern the normal from the mutant FHSD alleles in single cells. In the heterozygous SKFSHD5 lymphoblast cell line first tested, the mutant allele carries one D4Z4 repeat, whereas the other allele was within normal range. Both alleles showed a strong 4q35 marker signal, whereas only one associated with a normal bright D4Z4 signal, as seen in both interphase nuclei and metaphase spreads (Fig. 5 A). As shown in the following section, we were able to discern this for other FSHD patient cells that retained more than one copy of D4Z4 (see online supplemental material). Although not our primary purpose here, we suggest that this molecular cytogenetic assay may prove useful in diagnostic assays for FSHD, which are complicated by D4Z4 and 4q homologous sequences on other chromosomes (Kissel, 1999).


The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin unlike most human telomeres.

Tam R, Smith KP, Lawrence JB - J. Cell Biol. (2004)

In FSHD patient muscle, truncation of D4Z4 repeats does not significantly alter 4q35 localization to the heterochromatic rim. (A) Cytogenetic preparation of FSHD lymphoblastoid cells demonstrating different intensities of D4Z4 signals (green) at each 4q35 allele (red), permitting the mutant and wild-type alleles to be distinguished. (B) Both 4q35 alleles (green) in a mutant FSHD myoblast remain at the nuclear periphery depleted of hnRNA (red). (C) Localization of wild-type (arrow) and mutant (arrowhead) alleles in a FSHD myoblast using the same probes as in A. DAPI (blue) delineates the nucleus. (D) Quantitation of the localization of mutant (mut) versus wild-type (wt) allele in three FSHD myoblast cell lines (GM 17731, GM17899, and GM17869A) and in a normal myoblast line (50MB-1) before and after differentiation. 100 cells analyzed per sample. Localization of 4q35 (red) in a normal (E) and a FSHD myotube (F) with more intense D4Z4 signal (green) demarcating wild type versus weaker mutant 4q35 allele. Red and green signals from a z-stack were projected onto a single plane of blue signal. In the FSHD myotube, lamin A and fibrillarin were simultaneously detected by Alexa 350. (G) The number of cells (of 100 analyzed) with both 4q35 alleles dissociated from the periphery is rare regardless of D4Z4 copy number and may correlate with the age of the patient (see online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200403128/DC1). Typically, both alleles (red) lie just below the nuclear envelope stained for nucleopores (green) in normal muscle (H; blue, nucleoli) and FSHD muscle (I; blue, sc35).
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fig5: In FSHD patient muscle, truncation of D4Z4 repeats does not significantly alter 4q35 localization to the heterochromatic rim. (A) Cytogenetic preparation of FSHD lymphoblastoid cells demonstrating different intensities of D4Z4 signals (green) at each 4q35 allele (red), permitting the mutant and wild-type alleles to be distinguished. (B) Both 4q35 alleles (green) in a mutant FSHD myoblast remain at the nuclear periphery depleted of hnRNA (red). (C) Localization of wild-type (arrow) and mutant (arrowhead) alleles in a FSHD myoblast using the same probes as in A. DAPI (blue) delineates the nucleus. (D) Quantitation of the localization of mutant (mut) versus wild-type (wt) allele in three FSHD myoblast cell lines (GM 17731, GM17899, and GM17869A) and in a normal myoblast line (50MB-1) before and after differentiation. 100 cells analyzed per sample. Localization of 4q35 (red) in a normal (E) and a FSHD myotube (F) with more intense D4Z4 signal (green) demarcating wild type versus weaker mutant 4q35 allele. Red and green signals from a z-stack were projected onto a single plane of blue signal. In the FSHD myotube, lamin A and fibrillarin were simultaneously detected by Alexa 350. (G) The number of cells (of 100 analyzed) with both 4q35 alleles dissociated from the periphery is rare regardless of D4Z4 copy number and may correlate with the age of the patient (see online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200403128/DC1). Typically, both alleles (red) lie just below the nuclear envelope stained for nucleopores (green) in normal muscle (H; blue, nucleoli) and FSHD muscle (I; blue, sc35).
Mentions: It would be valuable to discriminate the mutant from the normal 4q35 locus in single cells because this distinction was not explored in earlier localization studies (Winokur et al., 1996; Stout et al., 1999). The intensity of FISH signal is proportional to the DNA target size, and direct comparison of normal and mutant alleles in the same cell minimizes technical differences (Johnson et al., 2000). Therefore, we tested a strategy of hybridizing a 4q35 marker with the D4Z4 probe in two different colors to discern the normal from the mutant FHSD alleles in single cells. In the heterozygous SKFSHD5 lymphoblast cell line first tested, the mutant allele carries one D4Z4 repeat, whereas the other allele was within normal range. Both alleles showed a strong 4q35 marker signal, whereas only one associated with a normal bright D4Z4 signal, as seen in both interphase nuclei and metaphase spreads (Fig. 5 A). As shown in the following section, we were able to discern this for other FSHD patient cells that retained more than one copy of D4Z4 (see online supplemental material). Although not our primary purpose here, we suggest that this molecular cytogenetic assay may prove useful in diagnostic assays for FSHD, which are complicated by D4Z4 and 4q homologous sequences on other chromosomes (Kissel, 1999).

Bottom Line: Studies of hybrid and translocation cell lines indicate this localization is inherent to the distal tip of 4q.However, consistent association of the pathogenic D4Z4 locus with the heterochromatic compartment supports a potential role in regulating the heterochromatic state and makes a telomere positioning effect more likely.Furthermore, D4Z4 repeats on other chromosomes also frequently organize with the heterochromatic compartment at the nuclear or nucleolar periphery, demonstrating a commonality among chromosomes harboring this subtelomere repeat family.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

ABSTRACT
This paper investigates the nuclear localization of human telomeres and, specifically, the 4q35 subtelomere mutated in facioscapulohumeral dystrophy (FSHD). FSHD is a common muscular dystrophy that has been linked to contraction of D4Z4 tandem repeats, widely postulated to affect distant gene expression. Most human telomeres, such as 17q and 17p, avoid the nuclear periphery to reside within the internal, euchromatic compartment. In contrast, 4q35 localizes at the peripheral heterochromatin with 4p more internal, generating a reproducible chromosome orientation that we relate to gene expression profiles. Studies of hybrid and translocation cell lines indicate this localization is inherent to the distal tip of 4q. Investigation of heterozygous FSHD myoblasts demonstrated no significant displacement of the mutant allele from the nuclear periphery. However, consistent association of the pathogenic D4Z4 locus with the heterochromatic compartment supports a potential role in regulating the heterochromatic state and makes a telomere positioning effect more likely. Furthermore, D4Z4 repeats on other chromosomes also frequently organize with the heterochromatic compartment at the nuclear or nucleolar periphery, demonstrating a commonality among chromosomes harboring this subtelomere repeat family.

Show MeSH
Related in: MedlinePlus