Results
Screening of mVenus+transformants by flow
cytometry
Electroporated cells with pOpt_mVenus_Paro linearized vector (Figure
S1) were allowed to recover in liquid media for 24 hours and plated on
Petri dishes containing TAP agar medium supplemented with paromomycin,
as antibiotic selection, for 5-7 days. Cells were transferred twice in
fresh media before fluorescence assessment in 6-day old liquid culture.
Flow cytometry was used for the initial detection and screening of
mVenus+ positive colonies transferred to liquid
medium. The initial gating strategy to screen mVenus+clones was as follow: >1% mVenus+ total
cells and MFI > 1.5-fold compared to the WT (Figure 1 B). A
total of 88 (22.9%) colonies over the 384 antibiotic-resistant were
mVenus+ at day 6 of culture (Figures 1A and S2). Among
these positive colonies, 23 were selected and tested again to assess
fluorescence stability 6 weeks later; 10 colonies (43.5%) were still
mVenus+, while the others lost detectable levels of
mVenus fluorescence signal (Figure 1C). We hypothesized that the loss in
mVenus expression, despite antibiotic resistance, was due to silencing
events.
mVenus expression levels is modulated by hydroxamate family
of HDAC
inhibitors
Five transformants (numbered here as 16, 18, 20, 21, 22) were selected
with different levels of fluorescence in order to assess if chemical
treatment with silencing inhibitors could increase mVenus expression.
C. reinhardtii WT was used
as negative control. We used
HDACi targeting HDACs classes I
to IV. Sirtinol, nicotinamide target HDAC class III/sirtuin, and
OSS-128167 is specific to sirtuin 6. SAHA inhibits HDAC class I, II &
IV; and sodium butyrate is known as a HDAC class I & IIA inhibitor
(Wang & al., 2019). HDACi were added at initiation of culture.
For most of the tested clones, treatment with SAHA (2.5 µM) for 24 hours
increased the frequency of cells
expressing mVenus and its level of expression per cell (Figures 2A and
2B).
The % of mVenus+ cells increased by 5.1-fold in clone
16 (mean: 13.3 to 68.1%) and there was a 4.6-fold increase in protein
fluorescence intensity (MFI), when cells were treated with SAHA (Figure
2A). The rise was less marked in other clones (1.1- to 1.8-fold in
frequency) with higher basal fluorescence, consistent with a baseline
decreased silencing. OSS-128167 and sodium butyrate had a slight effect
on the frequency and the mean fluorescence intensity of
mVenus+ cells, while
none of the other inhibitors
consistently improved expression levels of mVenus.
We further evaluated a 6-day treatment of SAHA and a new generation HDAC
inhibitors, i.e. belinostat, dacinostat and panobinostat of
hydroxamate-class like SAHA,
mocetinostat and entinostat, two
benzamide derivatives (HDAC class I inhibitor), and romidepsin, a
bacterial-derived cyclic tetrapeptide (HDAC class I inhibitor)
(Karagiannis et Rampias, 2021). All treatments were done with 5 µM of
inhibitors at initiation of culture (Figures 3A and 3B).
Clone 22 was replaced by clone 17 that exhibited less fluorescence at
basal levels, i.e mVenus expression was suspected to be more
silenced in clone 17 compared to 22 (Figure 1C). Relative copy number ofmVenus was assessed to verify if the differences between the
clones were not due to the number of mVenus copies integrated into the
genome. There were no differences in the amount of integrated mVenus in
between the tested clones, as measured in copy number or relatively to
endogenous gene (Figure S3).
Results showed that the hydroxamate class of HDACs inhibitors was the
most potent in boosting both the frequency of mVenus cells in silenced
clones (8.6-fold in clone 16 for SAHA (8,5 to 73,1%), 27-fold in clone
17 for SAHA (2,2 to 59,9%)) and its level inside each cell (MFI,
3-4-fold in clone 16 for SAHA, 2-5-fold in clone 17) at day 3
post-treatment (Figures 3A, 3B and S4A). Comparable results were
obtained with the other inhibitors from this class, but SAHA was usually
less toxic than dacinostat and panobinostat (growth curves and
chlorophyl levels, although not to statistically significant levels) and
was generally more potent than belinostat (Figure S4B and S4C).
Thus, we continued investigating the efficiency of this molecule (SAHA)
to reverse silencing in C. reinhardtii .
mVenus levels are modulated over time and
treatment
Expression of mVenus was followed using both a microplate reader and a
flow cytometer for 12 days after treatment with SAHA. In untreated, or
solvent-treated cells, overall fluorescence peaked around day 6 in all
samples (day 5-7), corresponding to the end of the exponential phase and
the start of the stationary phase (Figures 4 A-C, S5A and B).
Fluorescence levels of mVenus progressively decreased over the following
6 days of culture. When cells were treated with SAHA, the maximum
detection of mVenus was also measured at the end of the exponential
phase (day 4-7), reaching more than 74% of producing cells in all the
clones (Figure 4C), and a maximum of fluorescence intensity overall and
per cell (Figure 4A and 4B). Consistently with the low number of cells
impacting on the flow cytometer sensitivity in the first 3 days, the
analysis of the % of mVenus+ cells varied at early
time point (Figure 4C).
Nonetheless, SAHA treatment at initiation of culture steadily increased
fluorescence around day 4-5 in all clones, but did not prevent the
progressive decline in production when transformants advanced into
stationary phase around day 7-8. At day 12, most clones declined to
<10% of mVenus+ cells (except 16 and 21)
but had still more frequent population of mVenus expressing cells and
more mVenus fluorescence signal per cells compared to untreated or DMSO
treated cells (Figure 4A, 4B and 4C). SAHA did not seem to impact on
overall growth of microalgae (Figure S5A) but a significant decline in
chlorophyll fluorescence was noted (Figure S5B, One-way Anova, Dunett’s
multiple comparison test), suggesting that some metabolic pathways
related to photosynthesis are altered. This prompted more investigation
on SAHA’s impact on cell fitness.
SAHA’s effect on viability, cell growth, palmelloid and
chlorophyll
levels
We treated cells at initiation of culture with increasing concentrations
of the inhibitors and incubated them for 6 days. Viability was analyzed
through membrane integrity confirmation using propidium iodide (PI)
staining at day 6 on a flow cytometer. SAHA’s addition at concentration
of 2.5, 5 and 10 µM seemed to slightly increase the % of
PI+ cells, but ~90% of the
transformant cells remained intact and there was no significant increase
compared to background levels in WT cells (Figure 5A). Different stress
conditions have been reported to transiently induce palmelloid colonies
in C. reinhardtii without impacting viability (de Carpentier et
al., 2019). We observed that 6 days of treatment with 5 and 10 µM of
SAHA lead to the accumulation of small palmelloid colonies in C.
reinhardtii (Figure 5C and D compared to Figure 5B), while motility was
also progressively lost above 2.5 µM. We quantified palmelloid cells
formation by flow cytometry when transformants were treated with 5 µM of
SAHA and showed that in most cases palmelloid colonies increased at day
3 and 6 post-treatment (Figure 5E and S6A). These results suggest that
SAHA modifies chlorophyll content and colony morphology but does not
alter cell growth kinetic nor viability at concentrations of 10 µM and
lower.
SAHA’s addition at higher concentration of 20, 40 and 80 µM was also
tested to see at which degree it could affect the growth kinetic and
viability. Larger palmelloid colonies (or aggregates) were observed
(Figure S6B) with increasing concentrations, while growth kinetic was
inhibited partially with 20 and 40 µM SAHA, and totally with 80 µM SAHA
(data not shown). Chlorophyll content was also strongly modified, as
observed by the progressive loss of the usual dark green color ofC. reinhardtii healthy culture in favor of a more yellow tint (20
and 40 µM SAHA) or even red tint (80 µM SAHA).
SAHA upregulates mRNA transgene levels by inhibiting histone
deacetylation.
Then, we measured the impact of SAHA treatment on transgene mRNA levels.
SAHA being a HDAC inhibitor, its addition should increase the
availability of the chromatin to transcription factors, and hence the
mRNA levels. The relative mVenus mRNA expression over the housekeeping
gene h3 transcript expression increased 5.7- and 6.6-fold in
clone 16 and 17 respectively at day 6 of cultures, when cells were
treated with SAHA compared to the DMSO control (Figure 6A); the rise was
also observed in other clones, although less marked (1.2- to 2.8-fold).
Finally, we verified by western-blot that histones acetylation was
restored upon SAHA treatment (Figure 6B), leading to brighter band using
the H3K9Ac antibody in all transformants, while the amount of histone 3
remained consistent. In addition, we could detect an increase in mVenus
protein accumulation in clones 16, 17 and 18 at day 6 of cultures, but
not in 20 and 21 the two transformants with the highest level of mVenus
basal expression. Without treatment, mVenus was barely detectable in
clones 16, 17, and 18, consistently with the low fluorescence signal
detected by plate reader and FACS.