Mediator promotes CENP-a incorporation at fission yeast centromeres
Authors: Marcela Davila Lopez
Karl Ekwall
Beidong Liu
Ingela Djupedal
Jonas O Carlsten
Erzs√©bet Sz√°szi
Claes M Gustafsson
Thomas Nystr√∂m
Xuefeng Zhu
Zsolt Szilagyi
Journal: Molecular and Cellular Biology (2012)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Schizosaccharomyces pombe; centromeres; heterochromatin formation; histone H3 variant; CENP-A(Cnp1)
Affiliations: Institute of Biomedicine, University of Gothenburg, G√∂teborg, Sweden, Department of Cell and Molecular Biology, University of Gothenburg, G√∂teborg, Sweden, Department of Life Science and Engineering, Harbin Institute of Technology, Harbin, China, Department of Biosciences and Nutrition, Center for Biosciences, Karolinska Institutet, Huddinge, Sweden

Abstract:  At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A(Cnp1), which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-A(Cnp1) from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Œî cells appears to directly block CENP-A(Cnp1) incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function. 

Retracted: No

PubPeer comments:
 This paper has an erratum concerning author contributions, but could the authors also please check Figure 1F? Two dg panels appear to be unexpectedly similar. Shown with red boxes.  , 
 Dear Elisabeth, Thank you for your questions about our paper. We have attached the raw, high-resolution image of the experiment presented in figure 1F (Blot 1). All the bands come from one single Northern blot, hybridized with two different probes, (one against the dg transcript and one against a U6 RNA control) From looking carefully at the individual bands in the Northern and comparing them with figure 1F, it is clear that we present the same band twice. The dg band from the med20 deletion strain (blot 1, lane 5, upper panel) is presented both in itsu2019 correct place and as the dg band in the wt strain. This is a mistake, since the correct dg band for the wt strain should be the one seen in blot 1, lane 6. We have compared the other bands in the figure with the raw image, and they all come from the correct lanes. Please note that the quantification is correct, so the fold changes above the individual lanes should not be altered. In the cases where we have run duplicates of the samples (like for the med20 deletion strain in lanes 4 and 5), we have averaged the ratios obtained from the two lanes. The experiment itself has been performed multiple times and by different co-authors. As you will see from the unpublished Northern blot (Blot 2), the results are consistent between different experiments. The levels of the dg transcript in the med20 deletion strain compared to the wt strain are similar. In contrast, the dg band cannot be clearly distinguished in the dcr1 deletion strain. We will contact the journal about the possibility to write an erratum. Xuefeng Zhu (Corresponding author).  , 
 Dear Dr. Zhu, it has been more than a year after you promised to issue a correction - can I ask you for an update, please? ,