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Drosophila Xpd regulates Cdk7 localization, mitotic kinase activity, spindle dynamics, and chromosome segregation.

Li X, Urwyler O, Suter B - PLoS Genet. (2010)

Bottom Line: This work proves that the multitask protein Xpd also plays an essential role in cell cycle regulation that appears to be independent of transcription or NER.Possibly through this activity, xpd controls spindle dynamics and chromosome segregation in our model system.This novel role of xpd should also lead to new insights into the understanding of the neurological and cancer aspects of the human XPD disease phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland.

ABSTRACT
The trimeric CAK complex functions in cell cycle control by phosphorylating and activating Cdks while TFIIH-linked CAK functions in transcription. CAK also associates into a tetramer with Xpd, and our analysis of young Drosophila embryos that do not require transcription now suggests a cell cycle function for this interaction. xpd is essential for the coordination and rapid progression of the mitotic divisions during the late nuclear division cycles. Lack of Xpd also causes defects in the dynamics of the mitotic spindle and chromosomal instability as seen in the failure to segregate chromosomes properly during ana- and telophase. These defects appear to be also nucleotide excision repair (NER)-independent. In the absence of Xpd, misrouted spindle microtubules attach to chromosomes of neighboring mitotic figures, removing them from their normal location and causing multipolar spindles and aneuploidy. Lack of Xpd also causes changes in the dynamics of subcellular and temporal distribution of the CAK component Cdk7 and local mitotic kinase activity. xpd thus functions normally to re-localize Cdk7(CAK) to different subcellular compartments, apparently removing it from its cell cycle substrate, the mitotic Cdk. This work proves that the multitask protein Xpd also plays an essential role in cell cycle regulation that appears to be independent of transcription or NER. Xpd dynamically localizes Cdk7/CAK to and away from subcellular substrates, thereby controlling local mitotic kinase activity. Possibly through this activity, xpd controls spindle dynamics and chromosome segregation in our model system. This novel role of xpd should also lead to new insights into the understanding of the neurological and cancer aspects of the human XPD disease phenotypes.

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Production of Xpd-deprived embryos.(A) Expression of Xpd::V5 from the endogenous xpd promoter is readily seen in the germ line where the signal becomes localized to the large nurse cell nuclei. In contrast, tub-Gal4 - UAST driven Xpd::V5 expression is much lower in the germ line, but the Xpd::V5 signal is readily seen in the cytoplasm of stage 8 follicle cells (all pictures were taken with the same laser power and scanner settings). Ovaries expressing Xpd::V5 under the endogenous promoter are from w; xpdP; P[w+xpd-V5] flies, and ovaries expressing Xpd::V5 under UAST control are from w; xpdP; tub-Gal4/UAST-xpd-V5 flies. Anti-V5 antibody was used for staining and scale bars represent 10 µm. (B) Zygotic expression of Xpd from the transgene is only detectable starting in late cycle 14. Embryos laid from w; xpdP/CyO; tub-Gal4/UAST-xpd-V5 mothers expressing both endogenous Xpd and transgenic Xpd::V5 under UAST control were stained with Hoechst and staged. Extracts of embryos at different stages were separated by SDS-Page and probed with anti-V5 and anti-Xpd. “N.S.” stands for non-specific cross reactive band. (C) No Xpd can be detected in xpdeE embryos, while other maternal proteins were loaded normally into xpdeE embryos. Extracts of 35 0–30 min xpdeE embryos or control embryos from w; xpdP/CyO; tub-Gal4/UAST-xpd-V5 mothers were loaded on each lane of a Western blot and probed with different antibodies. 1 and 2 indicate two samples collected at different times for each type of embryo. “1/3” means that one third of amount of the extract was loaded. YP is yolk protein and the masses of the markers are indicated in KDa. (D) Quantification of Xpd levels (from xpdP) in xpdeE embryos mixed with wild type embryos at different ratios. Numbers above lanes indicate ratios of xpdeE embryos mixed with control embryos with a functional xpd+. Even at ratios of 100∶1 (120+1.2), a faint Xpd band is still visible. Because no band is seen in this position in xpdeE embryos, Xpd levels in this mutant must be below 1% of normal levels. (E) Quantification of Xpd::V5 levels in xpdeE. Anti-V5 antibody staining of a Western blot showed that no Xpd::V5 could be detected from 100 xpdeE embryos, while it could be detected from even one single xpd− embryo rescued with a xpd-V5 transgene expressed under its endogenous xpd promoter after the sample was diluted 50 times (1+50).
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pgen-1000876-g002: Production of Xpd-deprived embryos.(A) Expression of Xpd::V5 from the endogenous xpd promoter is readily seen in the germ line where the signal becomes localized to the large nurse cell nuclei. In contrast, tub-Gal4 - UAST driven Xpd::V5 expression is much lower in the germ line, but the Xpd::V5 signal is readily seen in the cytoplasm of stage 8 follicle cells (all pictures were taken with the same laser power and scanner settings). Ovaries expressing Xpd::V5 under the endogenous promoter are from w; xpdP; P[w+xpd-V5] flies, and ovaries expressing Xpd::V5 under UAST control are from w; xpdP; tub-Gal4/UAST-xpd-V5 flies. Anti-V5 antibody was used for staining and scale bars represent 10 µm. (B) Zygotic expression of Xpd from the transgene is only detectable starting in late cycle 14. Embryos laid from w; xpdP/CyO; tub-Gal4/UAST-xpd-V5 mothers expressing both endogenous Xpd and transgenic Xpd::V5 under UAST control were stained with Hoechst and staged. Extracts of embryos at different stages were separated by SDS-Page and probed with anti-V5 and anti-Xpd. “N.S.” stands for non-specific cross reactive band. (C) No Xpd can be detected in xpdeE embryos, while other maternal proteins were loaded normally into xpdeE embryos. Extracts of 35 0–30 min xpdeE embryos or control embryos from w; xpdP/CyO; tub-Gal4/UAST-xpd-V5 mothers were loaded on each lane of a Western blot and probed with different antibodies. 1 and 2 indicate two samples collected at different times for each type of embryo. “1/3” means that one third of amount of the extract was loaded. YP is yolk protein and the masses of the markers are indicated in KDa. (D) Quantification of Xpd levels (from xpdP) in xpdeE embryos mixed with wild type embryos at different ratios. Numbers above lanes indicate ratios of xpdeE embryos mixed with control embryos with a functional xpd+. Even at ratios of 100∶1 (120+1.2), a faint Xpd band is still visible. Because no band is seen in this position in xpdeE embryos, Xpd levels in this mutant must be below 1% of normal levels. (E) Quantification of Xpd::V5 levels in xpdeE. Anti-V5 antibody staining of a Western blot showed that no Xpd::V5 could be detected from 100 xpdeE embryos, while it could be detected from even one single xpd− embryo rescued with a xpd-V5 transgene expressed under its endogenous xpd promoter after the sample was diluted 50 times (1+50).

Mentions: Direct testing of potential cell cycle functions of xpd in the absence of a transcriptional requirement should be possible if we can make young preblastoderm embryos that lack Xpd. During the rapid nuclear divisions of the preblastoderm stage, embryonic transcription is not required for development and survival. Towards producing such embryos we constructed mothers that express xpd in the soma only. For this we first made transgenic flies that express Xpd under UAST control [21]. UAS (“upstream activating sequence”) is a transcriptional enhancer element from yeast that is normally inactive in Drosophila. It can be activated in a controlled manner by introducing into the fly the transcription factor Gal4 under control of a promoter of choice. The UAST system contains a hsp70 basal promoter to direct transcription initiation downstream of the UAS enhancer. This basal promoter is not active on its own, but needs to be combined with enhancer elements. In this combination the system allows good expression in the somatic tissue, but does not support expression in the germ line during the vitellarial stages of oogenesis, probably due to poor activation of the hsp70 basal promoter in this tissue [22]. To find out whether it was possible to produce embryos that lack Xpd, we studied the expression of UAST-xpd-V5 in the xpdP/P mutant background. Consistent with the expectation, we found that V5-tagged Xpd expressed under UAST control with a tubulin-Gal4 driver accumulated in the female germ line to a much lower degree than Xpd-V5 expressed under its endogenous promoter (w; xpdP; tub-Gal4/P[w+ UAST-xpd-V5] and w; xpdP; P[w+xpd-V5] ovaries, Figure 2A). In the somatic follicle cells, however, UAST-xpd-V5 is highly expressed from stage 8 onward. To monitor maternal deposition and zygotic accumulation of Xpd in the embryo, we analyzed embryos from mothers containing the transgenic UAST-xpd-V5 and one copy of endogenous xpd+ (required for development, see below; w; xpdP/CyO; tub-Gal4/UAST-xpd-V5). Early embryonic stages from nuclear cycle 1–13 do not show any Xpd-V5 expression and zygotic expression becomes detectable just prior to the start of mitosis in nuclear cycle 14 (Figure 2B).


Drosophila Xpd regulates Cdk7 localization, mitotic kinase activity, spindle dynamics, and chromosome segregation.

Li X, Urwyler O, Suter B - PLoS Genet. (2010)

Production of Xpd-deprived embryos.(A) Expression of Xpd::V5 from the endogenous xpd promoter is readily seen in the germ line where the signal becomes localized to the large nurse cell nuclei. In contrast, tub-Gal4 - UAST driven Xpd::V5 expression is much lower in the germ line, but the Xpd::V5 signal is readily seen in the cytoplasm of stage 8 follicle cells (all pictures were taken with the same laser power and scanner settings). Ovaries expressing Xpd::V5 under the endogenous promoter are from w; xpdP; P[w+xpd-V5] flies, and ovaries expressing Xpd::V5 under UAST control are from w; xpdP; tub-Gal4/UAST-xpd-V5 flies. Anti-V5 antibody was used for staining and scale bars represent 10 µm. (B) Zygotic expression of Xpd from the transgene is only detectable starting in late cycle 14. Embryos laid from w; xpdP/CyO; tub-Gal4/UAST-xpd-V5 mothers expressing both endogenous Xpd and transgenic Xpd::V5 under UAST control were stained with Hoechst and staged. Extracts of embryos at different stages were separated by SDS-Page and probed with anti-V5 and anti-Xpd. “N.S.” stands for non-specific cross reactive band. (C) No Xpd can be detected in xpdeE embryos, while other maternal proteins were loaded normally into xpdeE embryos. Extracts of 35 0–30 min xpdeE embryos or control embryos from w; xpdP/CyO; tub-Gal4/UAST-xpd-V5 mothers were loaded on each lane of a Western blot and probed with different antibodies. 1 and 2 indicate two samples collected at different times for each type of embryo. “1/3” means that one third of amount of the extract was loaded. YP is yolk protein and the masses of the markers are indicated in KDa. (D) Quantification of Xpd levels (from xpdP) in xpdeE embryos mixed with wild type embryos at different ratios. Numbers above lanes indicate ratios of xpdeE embryos mixed with control embryos with a functional xpd+. Even at ratios of 100∶1 (120+1.2), a faint Xpd band is still visible. Because no band is seen in this position in xpdeE embryos, Xpd levels in this mutant must be below 1% of normal levels. (E) Quantification of Xpd::V5 levels in xpdeE. Anti-V5 antibody staining of a Western blot showed that no Xpd::V5 could be detected from 100 xpdeE embryos, while it could be detected from even one single xpd− embryo rescued with a xpd-V5 transgene expressed under its endogenous xpd promoter after the sample was diluted 50 times (1+50).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2837399&req=5

pgen-1000876-g002: Production of Xpd-deprived embryos.(A) Expression of Xpd::V5 from the endogenous xpd promoter is readily seen in the germ line where the signal becomes localized to the large nurse cell nuclei. In contrast, tub-Gal4 - UAST driven Xpd::V5 expression is much lower in the germ line, but the Xpd::V5 signal is readily seen in the cytoplasm of stage 8 follicle cells (all pictures were taken with the same laser power and scanner settings). Ovaries expressing Xpd::V5 under the endogenous promoter are from w; xpdP; P[w+xpd-V5] flies, and ovaries expressing Xpd::V5 under UAST control are from w; xpdP; tub-Gal4/UAST-xpd-V5 flies. Anti-V5 antibody was used for staining and scale bars represent 10 µm. (B) Zygotic expression of Xpd from the transgene is only detectable starting in late cycle 14. Embryos laid from w; xpdP/CyO; tub-Gal4/UAST-xpd-V5 mothers expressing both endogenous Xpd and transgenic Xpd::V5 under UAST control were stained with Hoechst and staged. Extracts of embryos at different stages were separated by SDS-Page and probed with anti-V5 and anti-Xpd. “N.S.” stands for non-specific cross reactive band. (C) No Xpd can be detected in xpdeE embryos, while other maternal proteins were loaded normally into xpdeE embryos. Extracts of 35 0–30 min xpdeE embryos or control embryos from w; xpdP/CyO; tub-Gal4/UAST-xpd-V5 mothers were loaded on each lane of a Western blot and probed with different antibodies. 1 and 2 indicate two samples collected at different times for each type of embryo. “1/3” means that one third of amount of the extract was loaded. YP is yolk protein and the masses of the markers are indicated in KDa. (D) Quantification of Xpd levels (from xpdP) in xpdeE embryos mixed with wild type embryos at different ratios. Numbers above lanes indicate ratios of xpdeE embryos mixed with control embryos with a functional xpd+. Even at ratios of 100∶1 (120+1.2), a faint Xpd band is still visible. Because no band is seen in this position in xpdeE embryos, Xpd levels in this mutant must be below 1% of normal levels. (E) Quantification of Xpd::V5 levels in xpdeE. Anti-V5 antibody staining of a Western blot showed that no Xpd::V5 could be detected from 100 xpdeE embryos, while it could be detected from even one single xpd− embryo rescued with a xpd-V5 transgene expressed under its endogenous xpd promoter after the sample was diluted 50 times (1+50).
Mentions: Direct testing of potential cell cycle functions of xpd in the absence of a transcriptional requirement should be possible if we can make young preblastoderm embryos that lack Xpd. During the rapid nuclear divisions of the preblastoderm stage, embryonic transcription is not required for development and survival. Towards producing such embryos we constructed mothers that express xpd in the soma only. For this we first made transgenic flies that express Xpd under UAST control [21]. UAS (“upstream activating sequence”) is a transcriptional enhancer element from yeast that is normally inactive in Drosophila. It can be activated in a controlled manner by introducing into the fly the transcription factor Gal4 under control of a promoter of choice. The UAST system contains a hsp70 basal promoter to direct transcription initiation downstream of the UAS enhancer. This basal promoter is not active on its own, but needs to be combined with enhancer elements. In this combination the system allows good expression in the somatic tissue, but does not support expression in the germ line during the vitellarial stages of oogenesis, probably due to poor activation of the hsp70 basal promoter in this tissue [22]. To find out whether it was possible to produce embryos that lack Xpd, we studied the expression of UAST-xpd-V5 in the xpdP/P mutant background. Consistent with the expectation, we found that V5-tagged Xpd expressed under UAST control with a tubulin-Gal4 driver accumulated in the female germ line to a much lower degree than Xpd-V5 expressed under its endogenous promoter (w; xpdP; tub-Gal4/P[w+ UAST-xpd-V5] and w; xpdP; P[w+xpd-V5] ovaries, Figure 2A). In the somatic follicle cells, however, UAST-xpd-V5 is highly expressed from stage 8 onward. To monitor maternal deposition and zygotic accumulation of Xpd in the embryo, we analyzed embryos from mothers containing the transgenic UAST-xpd-V5 and one copy of endogenous xpd+ (required for development, see below; w; xpdP/CyO; tub-Gal4/UAST-xpd-V5). Early embryonic stages from nuclear cycle 1–13 do not show any Xpd-V5 expression and zygotic expression becomes detectable just prior to the start of mitosis in nuclear cycle 14 (Figure 2B).

Bottom Line: This work proves that the multitask protein Xpd also plays an essential role in cell cycle regulation that appears to be independent of transcription or NER.Possibly through this activity, xpd controls spindle dynamics and chromosome segregation in our model system.This novel role of xpd should also lead to new insights into the understanding of the neurological and cancer aspects of the human XPD disease phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland.

ABSTRACT
The trimeric CAK complex functions in cell cycle control by phosphorylating and activating Cdks while TFIIH-linked CAK functions in transcription. CAK also associates into a tetramer with Xpd, and our analysis of young Drosophila embryos that do not require transcription now suggests a cell cycle function for this interaction. xpd is essential for the coordination and rapid progression of the mitotic divisions during the late nuclear division cycles. Lack of Xpd also causes defects in the dynamics of the mitotic spindle and chromosomal instability as seen in the failure to segregate chromosomes properly during ana- and telophase. These defects appear to be also nucleotide excision repair (NER)-independent. In the absence of Xpd, misrouted spindle microtubules attach to chromosomes of neighboring mitotic figures, removing them from their normal location and causing multipolar spindles and aneuploidy. Lack of Xpd also causes changes in the dynamics of subcellular and temporal distribution of the CAK component Cdk7 and local mitotic kinase activity. xpd thus functions normally to re-localize Cdk7(CAK) to different subcellular compartments, apparently removing it from its cell cycle substrate, the mitotic Cdk. This work proves that the multitask protein Xpd also plays an essential role in cell cycle regulation that appears to be independent of transcription or NER. Xpd dynamically localizes Cdk7/CAK to and away from subcellular substrates, thereby controlling local mitotic kinase activity. Possibly through this activity, xpd controls spindle dynamics and chromosome segregation in our model system. This novel role of xpd should also lead to new insights into the understanding of the neurological and cancer aspects of the human XPD disease phenotypes.

Show MeSH
Related in: MedlinePlus