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Actin-dependent intranuclear repositioning of an active gene locus in vivo.

Dundr M, Ospina JK, Sung MH, John S, Upender M, Ried T, Hager GL, Matera AG - J. Cell Biol. (2007)

Bottom Line: However, the mechanism responsible for this association is uncertain.In the presence of a dominant-negative mutant of beta-actin, the repositioning of activated U2 genes is markedly inhibited.This supports a model in which nuclear actin is required for these rapid, long-range chromosomal movements.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA. mirek.dundr@rosalindfranklin.edu

ABSTRACT
Although bulk chromatin is thought to have limited mobility within the interphase eukaryotic nucleus, directed long-distance chromosome movements are not unknown. Cajal bodies (CBs) are nuclear suborganelles that nonrandomly associate with small nuclear RNA (snRNA) and histone gene loci in human cells during interphase. However, the mechanism responsible for this association is uncertain. In this study, we present an experimental system to probe the dynamic interplay of CBs with a U2 snRNA target gene locus during transcriptional activation in living cells. Simultaneous four-dimensional tracking of CBs and U2 genes reveals that target loci are recruited toward relatively stably positioned CBs by long-range chromosomal motion. In the presence of a dominant-negative mutant of beta-actin, the repositioning of activated U2 genes is markedly inhibited. This supports a model in which nuclear actin is required for these rapid, long-range chromosomal movements.

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The association between U2 genes and CBs was disrupted by the expression of a β-actin mutant. TetU2-45 cells were transfected with mCherry-lac repressor, reverse Tet-activator, and wild-type (wt) or dominant-negative (dn) YFP-actin constructs. (A) In the presence of wt YFP-actin, association of CBs and U2 arrays was not disrupted (arrow). However, in the presence of the nonpolymerizable dn YFP-actin, the association between CBs and the U2 array was significantly diminished. (B) Quantification of the relative repositioning of the U2 array within the chromosome 7 territory. Cells were scored (n > 85 cells per time point) on 3D z-axis projections as in Fig. 4 B in the presence of wt actin or dn actin. (C) Quantification of the effect of wt and dn YFP-actin on association of the U2 array with CBs. n > 250 cells. (D) The effect of dn YFP-actin on transcription of the U2 array as analyzed by quantitative PCR. The levels of 18S ribosomal RNA were monitored as a control. Bar, 2 μm.
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fig5: The association between U2 genes and CBs was disrupted by the expression of a β-actin mutant. TetU2-45 cells were transfected with mCherry-lac repressor, reverse Tet-activator, and wild-type (wt) or dominant-negative (dn) YFP-actin constructs. (A) In the presence of wt YFP-actin, association of CBs and U2 arrays was not disrupted (arrow). However, in the presence of the nonpolymerizable dn YFP-actin, the association between CBs and the U2 array was significantly diminished. (B) Quantification of the relative repositioning of the U2 array within the chromosome 7 territory. Cells were scored (n > 85 cells per time point) on 3D z-axis projections as in Fig. 4 B in the presence of wt actin or dn actin. (C) Quantification of the effect of wt and dn YFP-actin on association of the U2 array with CBs. n > 250 cells. (D) The effect of dn YFP-actin on transcription of the U2 array as analyzed by quantitative PCR. The levels of 18S ribosomal RNA were monitored as a control. Bar, 2 μm.

Mentions: To investigate this mechanism, we tested whether the association between U2 genes and CBs is dependent on nuclear actin. Chuang et al. (2006) recently showed that intranuclear repositioning of a chromosomal locus after targeting a transcriptional activator to a promoterless cassette was perturbed by overexpression of a dn actin mutant. Therefore, we transiently overexpressed wild-type (wt) YFP-actin or a nonpolymerizable mutant, YFP-actin(G13R), along with the mCherry-NLS-lac repressor in TetU2-45 cells. The dn actin mutant not only abolished the association between CBs and the U2 array (Fig. 5 C) but also inhibited the repositioning of the locus within the chromosome 7 territory (Fig. 5 B). In the presence of wt YFP-actin, the U2 array was repositioned normally (compare Figs. 4 B with 5 B); however, in the presence of the mutant actin, the intranuclear repositioning of the U2 array was significantly reduced. To test the effect of actin overexpression on transcription of the U2 array, we transfected with wt or mutant YFP-actin constructs and sorted cells by FACS (gating on YFP) such that all cells analyzed were expressing the desired constructs. After sorting, total RNA was isolated, and array-specific U2 RNA transcription was analyzed by quantitative PCR. No appreciable difference was observed (Fig. 5 D). Thus, the overexpression of dn actin has little overall effect on transcription of the U2 array but has a significant effect on intranuclear repositioning and CB association.


Actin-dependent intranuclear repositioning of an active gene locus in vivo.

Dundr M, Ospina JK, Sung MH, John S, Upender M, Ried T, Hager GL, Matera AG - J. Cell Biol. (2007)

The association between U2 genes and CBs was disrupted by the expression of a β-actin mutant. TetU2-45 cells were transfected with mCherry-lac repressor, reverse Tet-activator, and wild-type (wt) or dominant-negative (dn) YFP-actin constructs. (A) In the presence of wt YFP-actin, association of CBs and U2 arrays was not disrupted (arrow). However, in the presence of the nonpolymerizable dn YFP-actin, the association between CBs and the U2 array was significantly diminished. (B) Quantification of the relative repositioning of the U2 array within the chromosome 7 territory. Cells were scored (n > 85 cells per time point) on 3D z-axis projections as in Fig. 4 B in the presence of wt actin or dn actin. (C) Quantification of the effect of wt and dn YFP-actin on association of the U2 array with CBs. n > 250 cells. (D) The effect of dn YFP-actin on transcription of the U2 array as analyzed by quantitative PCR. The levels of 18S ribosomal RNA were monitored as a control. Bar, 2 μm.
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Related In: Results  -  Collection

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fig5: The association between U2 genes and CBs was disrupted by the expression of a β-actin mutant. TetU2-45 cells were transfected with mCherry-lac repressor, reverse Tet-activator, and wild-type (wt) or dominant-negative (dn) YFP-actin constructs. (A) In the presence of wt YFP-actin, association of CBs and U2 arrays was not disrupted (arrow). However, in the presence of the nonpolymerizable dn YFP-actin, the association between CBs and the U2 array was significantly diminished. (B) Quantification of the relative repositioning of the U2 array within the chromosome 7 territory. Cells were scored (n > 85 cells per time point) on 3D z-axis projections as in Fig. 4 B in the presence of wt actin or dn actin. (C) Quantification of the effect of wt and dn YFP-actin on association of the U2 array with CBs. n > 250 cells. (D) The effect of dn YFP-actin on transcription of the U2 array as analyzed by quantitative PCR. The levels of 18S ribosomal RNA were monitored as a control. Bar, 2 μm.
Mentions: To investigate this mechanism, we tested whether the association between U2 genes and CBs is dependent on nuclear actin. Chuang et al. (2006) recently showed that intranuclear repositioning of a chromosomal locus after targeting a transcriptional activator to a promoterless cassette was perturbed by overexpression of a dn actin mutant. Therefore, we transiently overexpressed wild-type (wt) YFP-actin or a nonpolymerizable mutant, YFP-actin(G13R), along with the mCherry-NLS-lac repressor in TetU2-45 cells. The dn actin mutant not only abolished the association between CBs and the U2 array (Fig. 5 C) but also inhibited the repositioning of the locus within the chromosome 7 territory (Fig. 5 B). In the presence of wt YFP-actin, the U2 array was repositioned normally (compare Figs. 4 B with 5 B); however, in the presence of the mutant actin, the intranuclear repositioning of the U2 array was significantly reduced. To test the effect of actin overexpression on transcription of the U2 array, we transfected with wt or mutant YFP-actin constructs and sorted cells by FACS (gating on YFP) such that all cells analyzed were expressing the desired constructs. After sorting, total RNA was isolated, and array-specific U2 RNA transcription was analyzed by quantitative PCR. No appreciable difference was observed (Fig. 5 D). Thus, the overexpression of dn actin has little overall effect on transcription of the U2 array but has a significant effect on intranuclear repositioning and CB association.

Bottom Line: However, the mechanism responsible for this association is uncertain.In the presence of a dominant-negative mutant of beta-actin, the repositioning of activated U2 genes is markedly inhibited.This supports a model in which nuclear actin is required for these rapid, long-range chromosomal movements.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA. mirek.dundr@rosalindfranklin.edu

ABSTRACT
Although bulk chromatin is thought to have limited mobility within the interphase eukaryotic nucleus, directed long-distance chromosome movements are not unknown. Cajal bodies (CBs) are nuclear suborganelles that nonrandomly associate with small nuclear RNA (snRNA) and histone gene loci in human cells during interphase. However, the mechanism responsible for this association is uncertain. In this study, we present an experimental system to probe the dynamic interplay of CBs with a U2 snRNA target gene locus during transcriptional activation in living cells. Simultaneous four-dimensional tracking of CBs and U2 genes reveals that target loci are recruited toward relatively stably positioned CBs by long-range chromosomal motion. In the presence of a dominant-negative mutant of beta-actin, the repositioning of activated U2 genes is markedly inhibited. This supports a model in which nuclear actin is required for these rapid, long-range chromosomal movements.

Show MeSH