spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 29 August 2006
doi: 10.1242/jcs.03150

Journal of Cell Science 119, 3866-3875 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aguilar-Fuentes, J.
Right arrow Articles by Zurita, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aguilar-Fuentes, J.
Right arrow Articles by Zurita, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

TFIIH trafficking and its nuclear assembly during early Drosophila embryo development

Javier Aguilar-Fuentes, Viviana Valadez-Graham, Enrique Reynaud and Mario Zurita*

Department of Developmental Genetics and Molecular Physiology, Institute of Biotechnology, National Autonomous University of México, Av. Universidad 2001, Cuernavaca Morelos 62250, Mexico


Figure 1
View larger version (87K):

[in a new window]
  		Fig. 1. XPB subcellular distribution in early Drosophila embryogenesis. XPB was localized using a polyclonal antibody. XPB signal is in red; DNA is in green after Sytox green staining. (a) Syncytial blastoderm embryo after the first nuclear division (two nuclei, stage 2). (b) Syncytial blastoderm embryo at stage 8; the nucleus starts to move towards the embryo periphery and XPB remains cytoplasmic. (c,c') A similar embryo at stage 8 of development, but only the surface staining shown. Note that XPB surrounds the nucleus in cytoplasmic domains or energids. (d) Syncytial blastoderm embryo at stage 10. XPB enters into the nuclei, which are now located in the embryo periphery. (e) Cellular blastoderm embryo. At this stage, most of XPB signal is located inside the nucleus. (f) Gastrulated embryo. XPB is preferentially nuclear in the interphase nuclei, but excluded from mitotic chromosomes. (g) Amplification of the periphery of a stage 10 embryo. The arrows indicate XPB signal inside the nucleus. (h) Amplification of a cellular blastoderm embryo. (i) Amplification of a gastrulated embryo. The arrow indicates a group of mitotic chromosomes. Bar, 100 µm.

 

Figure 2
View larger version (68K):

[in a new window]
  		Fig. 2. CDK7 and MAT1 subcellular distribution in early Drosophila embryogenesis. Cellular distribution of CDK7 (red signal) at different embryo developmental stages from syncytial blastoderm to gastrulation (a-d). DNA is shown in green. Note that the CDK7 signal is preferentially located in the cytoplasm at all stages. Distribution of the CAK complex protein MAT1 in early Drosophila development (e-h). The red signal is MAT1 after immunostaining with a polyclonal antibody (see Materials and Methods) and the green signal is DNA. (a,e) Early syncytial blastoderm embryos. (b,f) Syncytial blastoderm embryos at nuclear division 10. (c,g) Cellular blastoderm embryos. (d,h) Embryo during gastrulation.

 

Figure 3
View larger version (79K):

[in a new window]
  		Fig. 3. Co-immunostaining of CDK7 and XPD during early fly embryogenesis from syncytial blastoderm to gastrulation. (a) XPD (green signal) is preferentially nuclear after mitosis number 10. CDK7 (red signal) is preferentially located in the cytoplasm at these developmental stages. (b) Amplification of a cellular blastoderm co-stained with CDK7 (red) and XPD (green). (c) Amplification of a gastrulated embryo co-stained with CDK7 (red) and XPD (green).

 

Figure 4
View larger version (79K):

[in a new window]
  		Fig. 4. Subcellular and chromosomal distribution of the CAK and the core complexes of TFIIH in third-instar larvae salivary gland and polytene chromosomes. (a) Immunolocalization of CDK7 shown in red and DNA staining in green. (b) Immunolocalization of XPD (red) and DNA (green) in third-instar salivary glands. (c) Co-immunostaining of CDK7 (red) and XPD (green) in polytene chromosomes. Most of the two proteins colocalize in a specific euchromatin banding pattern and the signal is enriched in the puffs. An amplified view of the CDK7/XPD immunolocalization in polythene chromosomes is shown. The arrows indicate sites where CDK7 does not colocalize with XPD in the chromatin. (d) Chromosomal distribution of MAT1 and XPD in polytene chromosomes. The red signal is MAT 1 and the green signal is XPD. The arrows indicate an example of a site where MAT1 is localized without XPD in an amplified image.

 

Figure 5
View larger version (35K):

[in a new window]
  		Fig. 5. CDK7, XPB and XPD are positioned at active promoters at the onset of transcription in early Drosophila embryos. Chromatin of embryos at 30-180 minutes of development was precipitated using antisera against CDK7, XPD, XPB and RNA pol II. Two neutral, unrelated IgG (Mock 1) and IgM (Mock 2) antisera were used as controls. The immunoprecipitated regions were amplified by PCR using oligonucleotides that cover the zygotic hb promoter, hb second exon, the sgs5 promoter, the atrx second exon and the H3 promoter; each amplified region is indicated in the figure. Input amplification is also shown for each PCR. The fraction of the input for each ChIP is indicated below each band. The sequences of the different promoters analyzed in this work are derived form the Drosophila core promoter database (http://www-biology.ucsd.edu/labs/Kadonaga/DCPD.html).

 

Figure 6
View larger version (33K):

[in a new window]
  		Fig. 6. CDK7 and XPB co-immunoprecipitations (CoIP) of cytoplasmic and nuclear fractions from embryos at mitotic stages 8-14. The cytoplasmic fraction was immunoprecipitated with either an anti-CDK7 or anti-XPB antibody. The nuclear fraction was co-immunoprecipitated with the anti-CDK7 antibody. The precipitated material and total proteins from cytoplasmic fraction before CoIP were analyzed by SDS-PAGE. The proteins were transferred to a membrane and analyzed by western blot experiments for the presence of CDK7, MAT1, XPB and XPD using specific antibodies as indicated. The same blot was reused for each antibody. The antibody used for CoIP is indicated in the figure. Lanes labeled P show the CoIP material and those labeled I show the input from the original cytoplasmic or nuclear fraction, equal amounts of protein were loaded in each lane.

 

Figure 7
View larger version (41K):

[in a new window]
  		Fig. 7. XPB and XPD need to be assembled in the core of TFIIH for nuclear entry at the onset of transcription. Embryos at 0-30 minutes of development were injected with antibodies that specifically recognize XPB, XPD, GFP and CDK7. After injection, the embryos were allowed to develop for 2 hours, then were fixed and immunostained against either XPB or XPD as well as against TBP. The specific injected antibodies are indicated at the left of each panel. DNA staining with Sytox Green as well as the antibodies used for immunostaining are indicated at the top of each panel. Note that in the first panel (Ab-XPD), one half of the gastrulated embryo XPB is nuclear and in the other half is cytoplasmic. This effect is possible due to a gradient of neutralization of XPD by the antibody. Also note that the localization of XPD is cytoplasmic in a gastrulated embryo injected with the XPB antibody (Ab-XPB, middle panel). The injection of GFP antibody does not have any effect on the correct localization of XPB in a gastrulated embryo. Note that neither XPB nor XPD antibodies affect the TBP nuclear localization. Injection of the CDK7 antibody (Ab-CDK7 panel) arrests mitotic division and aberrant mitotic chromosomes are observed. The bottom right panel (Ab-XPB), shows a cellular blastoderm embryo stained with XPB after injection of its antibody. Note that a gradient of the nuclear XPB is observed in one half of the embryo and no signal is detected in the other half owing to the neutralization of XPB.

 

Figure 8
View larger version (69K):

[in a new window]
  		Fig. 8. XPB and XPD inactivation affects expression of the ftz gene. (a) Typical ftz expression pattern in a wild-type embryo. (b) Embryo microinjected with the XPB antibody. (c) Embryos microinjected with the XPD antibody. Arrows indicate examples of regions lacking the ftz mRNA.

 

Figure 9
View larger version (29K):

[in a new window]
  		Fig. 9. Nuclear translocation and assembly model of TFIIH at the onset of transcription in the early Drosophila embryo.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006