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Silencing is noisy: population and cell level noise in telomere-adjacent genes is dependent on telomere position and sir2.

Anderson MZ, Gerstein AC, Wigen L, Baller JA, Berman J - PLoS Genet. (2014)

Bottom Line: Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes.This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise.Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota - Twin Cities, Minneapolis, Minnesota, United States of America.

ABSTRACT
Cell-to-cell gene expression noise is thought to be an important mechanism for generating phenotypic diversity. Furthermore, telomeric regions are major sites for gene amplification, which is thought to drive genetic diversity. Here we found that individual subtelomeric TLO genes exhibit increased variation in transcript and protein levels at both the cell-to-cell level as well as at the population-level. The cell-to-cell variation, termed Telomere-Adjacent Gene Expression Noise (TAGEN) was largely intrinsic noise and was dependent upon genome position: noise was reduced when a TLO gene was expressed at an ectopic internal locus and noise was elevated when a non-telomeric gene was expressed at a telomere-adjacent locus. This position-dependent TAGEN also was dependent on Sir2p, an NAD+-dependent histone deacetylase. Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes. This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise. Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

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Related in: MedlinePlus

TPE produces TLO expression plasticity.(A) A cartoon represents URA3 inserted into TLO-adjacent subtelomeres in a head-to-head orientation to test the effect of regulating URA3 expression on TLO expression variability. (B). A diagram depicts the effect on URA3 expression under growth in different conditions and the effect on TLO TAGEN. (C). qRT-PCR measured transcript abundance of TLOα9 and TLOα12 when URA3 was either unselected, selected on media lacking uracil, or selected on 5-FOA. Selection of URA3 expression significantly reduced expression plasticity of the adjacent TLO at either locus but not at the unlinked TLO gene. (D) Subtelomeric loci transition between active and inactive chromatin states. This transcriptional toggling results in a population of cells expressing subtelomeric loci over a wide range. Cells locked into a repressive transcriptional state have lower expression and reduced noise from transcriptional bursting at both the single cell and population level. Conversely, increased transcriptional activity, potentially due to loss of SIR2, increases expression and reduces noise due to increased transcriptional bursting.
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pgen-1004436-g008: TPE produces TLO expression plasticity.(A) A cartoon represents URA3 inserted into TLO-adjacent subtelomeres in a head-to-head orientation to test the effect of regulating URA3 expression on TLO expression variability. (B). A diagram depicts the effect on URA3 expression under growth in different conditions and the effect on TLO TAGEN. (C). qRT-PCR measured transcript abundance of TLOα9 and TLOα12 when URA3 was either unselected, selected on media lacking uracil, or selected on 5-FOA. Selection of URA3 expression significantly reduced expression plasticity of the adjacent TLO at either locus but not at the unlinked TLO gene. (D) Subtelomeric loci transition between active and inactive chromatin states. This transcriptional toggling results in a population of cells expressing subtelomeric loci over a wide range. Cells locked into a repressive transcriptional state have lower expression and reduced noise from transcriptional bursting at both the single cell and population level. Conversely, increased transcriptional activity, potentially due to loss of SIR2, increases expression and reduces noise due to increased transcriptional bursting.

Mentions: We next asked if TAGEN and TPE are functionally related by measuring TLO expression variability in cells selected for constant expression or constant silencing of a TLO-adjacent selectable marker, URA3. We measured levels of the adjacent TLO (in cis) as well as an unlinked TLO (in trans), when cells were selected for expression of URA3 (ON state selected on medium lacking uridine) or when cells were selected for repression of URA3 (OFF state selected on medium containing 5-FOA) vs cells being free to ‘toggle’ between the two states (ON and OFF states, no selection on YPAD medium). We first constructed two strains, each with URA3 inserted head-to-head at a TLO-adjacent position (adjacent to TLOα9 or TLOα12; Fig. 8A) in the subtelomeres. These strains enabled the selection of cells expressing URA3 (by growth in media lacking uracil (“-ura”)), or to select for silencing of URA3 (by growth in the presence of 5-floroorotic acid (“5-FOA”)). Growth of TLO-adjacent URA3 strains on media lacking uracil or with 5-FOA reduced or increased transcript abundance of URA3, respectively (data not shown). We then asked if selection in –ura or 5-FOA influenced TLO expression plasticity (Fig. 8B). Importantly, in both strains, selection either for or against URA3 expression significantly reduced variability of the URA3-adjacent TLO transcript levels, yet it did not affect the transcript variability at an unlinked TLO (Fig. 8C; presence of selection: F1, 20 = 40.4, p<0.0001, gene: F1, 20 = 0.28, p = 0.60, interaction: F1, 20 = 0.174, p = 0.69). This occurred without a significant effect on expression levels (Fig. S11; presence of selection: F1, 20 = 0.03, = 0.87, gene: F1, 20 = 2.48, p = 0.13, interaction: F1, 20 = 0.145, p = 0.71). Thus, TAGEN at a specific TLO locus requires that cells toggle between the ON and OFF states and is lost if expression of an adjacent gene is constitutively ON or OFF. Furthermore, the effect of telomeric silencing on TAGEN occurs in cis and does not affect silencing or TLO expression at other subtelomeres.


Silencing is noisy: population and cell level noise in telomere-adjacent genes is dependent on telomere position and sir2.

Anderson MZ, Gerstein AC, Wigen L, Baller JA, Berman J - PLoS Genet. (2014)

TPE produces TLO expression plasticity.(A) A cartoon represents URA3 inserted into TLO-adjacent subtelomeres in a head-to-head orientation to test the effect of regulating URA3 expression on TLO expression variability. (B). A diagram depicts the effect on URA3 expression under growth in different conditions and the effect on TLO TAGEN. (C). qRT-PCR measured transcript abundance of TLOα9 and TLOα12 when URA3 was either unselected, selected on media lacking uracil, or selected on 5-FOA. Selection of URA3 expression significantly reduced expression plasticity of the adjacent TLO at either locus but not at the unlinked TLO gene. (D) Subtelomeric loci transition between active and inactive chromatin states. This transcriptional toggling results in a population of cells expressing subtelomeric loci over a wide range. Cells locked into a repressive transcriptional state have lower expression and reduced noise from transcriptional bursting at both the single cell and population level. Conversely, increased transcriptional activity, potentially due to loss of SIR2, increases expression and reduces noise due to increased transcriptional bursting.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109849&req=5

pgen-1004436-g008: TPE produces TLO expression plasticity.(A) A cartoon represents URA3 inserted into TLO-adjacent subtelomeres in a head-to-head orientation to test the effect of regulating URA3 expression on TLO expression variability. (B). A diagram depicts the effect on URA3 expression under growth in different conditions and the effect on TLO TAGEN. (C). qRT-PCR measured transcript abundance of TLOα9 and TLOα12 when URA3 was either unselected, selected on media lacking uracil, or selected on 5-FOA. Selection of URA3 expression significantly reduced expression plasticity of the adjacent TLO at either locus but not at the unlinked TLO gene. (D) Subtelomeric loci transition between active and inactive chromatin states. This transcriptional toggling results in a population of cells expressing subtelomeric loci over a wide range. Cells locked into a repressive transcriptional state have lower expression and reduced noise from transcriptional bursting at both the single cell and population level. Conversely, increased transcriptional activity, potentially due to loss of SIR2, increases expression and reduces noise due to increased transcriptional bursting.
Mentions: We next asked if TAGEN and TPE are functionally related by measuring TLO expression variability in cells selected for constant expression or constant silencing of a TLO-adjacent selectable marker, URA3. We measured levels of the adjacent TLO (in cis) as well as an unlinked TLO (in trans), when cells were selected for expression of URA3 (ON state selected on medium lacking uridine) or when cells were selected for repression of URA3 (OFF state selected on medium containing 5-FOA) vs cells being free to ‘toggle’ between the two states (ON and OFF states, no selection on YPAD medium). We first constructed two strains, each with URA3 inserted head-to-head at a TLO-adjacent position (adjacent to TLOα9 or TLOα12; Fig. 8A) in the subtelomeres. These strains enabled the selection of cells expressing URA3 (by growth in media lacking uracil (“-ura”)), or to select for silencing of URA3 (by growth in the presence of 5-floroorotic acid (“5-FOA”)). Growth of TLO-adjacent URA3 strains on media lacking uracil or with 5-FOA reduced or increased transcript abundance of URA3, respectively (data not shown). We then asked if selection in –ura or 5-FOA influenced TLO expression plasticity (Fig. 8B). Importantly, in both strains, selection either for or against URA3 expression significantly reduced variability of the URA3-adjacent TLO transcript levels, yet it did not affect the transcript variability at an unlinked TLO (Fig. 8C; presence of selection: F1, 20 = 40.4, p<0.0001, gene: F1, 20 = 0.28, p = 0.60, interaction: F1, 20 = 0.174, p = 0.69). This occurred without a significant effect on expression levels (Fig. S11; presence of selection: F1, 20 = 0.03, = 0.87, gene: F1, 20 = 2.48, p = 0.13, interaction: F1, 20 = 0.145, p = 0.71). Thus, TAGEN at a specific TLO locus requires that cells toggle between the ON and OFF states and is lost if expression of an adjacent gene is constitutively ON or OFF. Furthermore, the effect of telomeric silencing on TAGEN occurs in cis and does not affect silencing or TLO expression at other subtelomeres.

Bottom Line: Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes.This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise.Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota - Twin Cities, Minneapolis, Minnesota, United States of America.

ABSTRACT
Cell-to-cell gene expression noise is thought to be an important mechanism for generating phenotypic diversity. Furthermore, telomeric regions are major sites for gene amplification, which is thought to drive genetic diversity. Here we found that individual subtelomeric TLO genes exhibit increased variation in transcript and protein levels at both the cell-to-cell level as well as at the population-level. The cell-to-cell variation, termed Telomere-Adjacent Gene Expression Noise (TAGEN) was largely intrinsic noise and was dependent upon genome position: noise was reduced when a TLO gene was expressed at an ectopic internal locus and noise was elevated when a non-telomeric gene was expressed at a telomere-adjacent locus. This position-dependent TAGEN also was dependent on Sir2p, an NAD+-dependent histone deacetylase. Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes. This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise. Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

Show MeSH
Related in: MedlinePlus