MBG*4110 Eden Kinzel

Toggle Involving cis-Interfering Noncoding RNAs Controls Variegated Gene Expression in

Yeast
Introduction
The FLO11 locus in S. Cerevisiae displays a binary variegated phenotype. FLO11 is
typically silenced when the yeast have sufficient nitrogen. FLO11 is expressed primarily when
the yeast are in a nitrogen-starved state as it encodes a cell surface flocculin allowing for the
formation of pseudohyphae which are advantageous in scavenging nutrients (Gancedo, 2001).
Long noncoding RNAs (ncRNAs) are RNA molecules longer than 200 nucleotides which
do not encode proteins. ncRNAs have many proposed functions including modulating protein
activity and localization, altering chromatin structure, and controlling transcriptional, post-
transcriptional, and epigenetic processes (Mercer, Dinger, & Mattick, 2009). The ncRNAs
explored in this paper are Promoting Watson RNA (PWR1) and Interfering Crick RNA (ICR1).
The purpose of this study was to find the mechanism regulating the variegation at the
FLO11 locus. Flo8, Rpd3L, and PWR1 were identified to be necessary for FLO11 expression and
Sfl1 and ICR1 were implicated in FLO11 repression via a promoter occlusion model.
Experimental Results
Rpd3L Activates FLO11 Transcription
Three experiments involving Rpd3L mutants (null mutations Cti6, Rxt2, or Pho23)
demonstrated silencing of the FLO11 promoter, indicating Rpd3L activates FLO11 expression.
An assay of promoter activity where the FLO11 promoter was fused to reporter genes in wild-type
and Rpd3L mutants demonstrated a lack of reporter gene expression in Rpd3L mutants. Northern
blotting observed less FLO11 mRNA in Rpd3L mutants than in wild-type and experiments
evaluating FLO11 function noted a loss of FLO11-dependent phenotypes in Rpd3L mutants
which was alleviated by transformation with a plasmid containing FLO11. Rpd3S mutants did
not show a reduction in FLO11 activity, reaffirming the role of Rpd3L in FLO11 activation.
The role of Rpd3L in activating FLO11 transcription was identified to be dependent upon
Sfl1 function, but does not involve Rpd3L repressing the transcription of SFL1. This was
determined in experiments that showed identical FLO11 silencing in Rpd3L sfl1 double mutants
and sfl1 mutants. Rpd3L mutants showed the same amount of SFL1 mRNA as the wild-type yeast
demonstrating no Rpd3L-dependent SFL1 repression.
Rpd3L Modulates Transcription Factor Binding and Chromatin Remodeling at FLO11 Promoter
Rpd3L localizes at two regions upstream of FLO11, close in proximity to Flo8 and Sfl1
binding sites as was detected by Genome Wide ChIP-chip. This study indicates that Rpd3L
regulates the binding of the transcription factors. In wild-type yeast, the absence of Sfl1 permits
Flo8 binding, however there was a reduction in Flo8 binding observed in Rpd3L sfl1 double
mutants from the typical wild-type levels. This indicates that Rpd3L may affect Flo8 binding.
Both TATA box-binding protein and H4 localization were decreased in Rpd3L mutants as
compared to sfl mutants, implying that Rpd3L also modulates chromatin remodelling.
MBG*4110 Eden Kinzel

Rpd3L, Sfl1, and Flo8 Regulate cis-Acting ncRNAs to Control FLO11 Variegation
Two ncRNAs, PWR1 and ICR1, were detected several kilobases upstream of the FLO11
locus. They were initially identified by strand-specific microarrays and were further quantified by
Northern blotting and cap-dependent RACE. ICR1 was determined to be 3.2 kb long and have
start sites ranging between 3,197-3,445 bp upstream of FLO11. ICR1 stop sites occurred either
shortly upstream or within the FLO11 open reading frame. The 1.2 kb long ncRNA PWR1 has
start sites found within a range of DNA located between 2,190-2,339 bp upstream of the FLO11
locus. PWR1 is almost entirely complementary to the 5 sequence of ICR1, terminating between
3,246-3,409 bp upstream of FLO11.
An inverse correlation between ICR1 and PWR1 transcription was discovered and
supported a promoter occlusion model. ICR1 experienced the highest expression in yeast not
expressing FLO11 which occurred in any mutants lacking Rpd3L or Flo8 and Rpd3L flo8 sfl1
triple mutants. This determined that Flo8 and Rpd3L repress ICR1. Sfl1 was shown to promote
ICR1 expression because there were extremely low levels in sfl1 mutants which retained Rpd3L
function. PWR1 transcription primarily occurred in yeast which silence FLO11. PWR1
expression is dependent upon Flo8, promoted by Rpd3L, and supressed by Sfl1.
ICR1 transcription was causative in silencing FLO11. FLO11 expression was rescued in
Rpd3L mutants when constructs terminating ICR1 were introduced upstream of the FLO11 locus.
The transcription of ICR1 and PWR1 is relatively mutually exclusive, in that the
transcription of one of the ncRNAs will interfere with the transcription of the other. The evidence
for interference was demonstrated by Northern blots displaying transcript sizes characteristic of
interference and by a reporter gene inserted to mimic ICR1 showing reduced expression with the
transcription of PWR1. Basal levels of ICR1 transcription in sfl1 mutants indicate that ICR1 may
be expressed constitutively, but modulated by PWR1 expression.
ICR1 and PWR1 were shown to only function in cis when overexpression of the ncRNAs
in trans did not increase FLO11 silencing or expression.
Discussion
When sufficient nitrogen is supplied, yeast do not express FLO11. Sfl1 outcompetes Flo8
in binding upstream of FLO11 and induces ICR1 expression. ICR1 restricts the access of
chromatin remodelers and transcription factors to FLO11 which inhibits its transcription via
promoter occlusion. When yeast lack nitrogen, they express FLO11. Flo8 associates with Rpd3L
and outcompetes Sfl1 in binding upstream of FLO11. PWR1 is transcribed and represses ICR1
transcription. FLO11 is expressed and yeast can form pseudohyphae. Rpd3L assists in FLO11
expression by repressing ICR1 transcription or by obstructing the Sfl1 binding site.
Take Home Message
This paper introduces a new mechanism of variegation involving the function of ncRNAs.
This may explain variegation at binary loci in other organisms.
MBG*4110 Eden Kinzel

References
Bumgarner, S. L., Dowell, R. D., Grisafi, P., Gifford, D. K., & Fink, G. R. (2009). Toggle
involving cis-interfering noncoding RNAs controls variegated gene expression in yeast.
Proceedings of the National Academy of the Sciences of the United States of America,
106(43), 18321-18326. 10.1073/pnas.0909641106
Gancedo, J. M. (2001). Control of pseudohyphae formation in Saccharomyces cerevisiae. FEMS
Microbiology Reviews, 25, 107-123.
Mercer, T. R., Dinger, M. E., & Mattick J. S. (2009). Long non-coding RNAs: insights into
functions. Nature Reviews Genetics, 10(3), 155-159. 10.1038/nrg2521