Discussion
In this study, we propose a new
tool to successfully trigger the expression of silenced heterologous
proteins in the green microalgae C. reinhardtii . Integration of
exogenous DNA into the microalgae nuclear genome predominantly occurs
randomly, via non-homologous-end-joining (NHEJ), leading to a large and
heterogeneous population of transformed cells with varied expression
levels (Zhang et al., 2014; Nouemssi et al., 2020). Despite antibiotic
selection, not all transformed cells express the targeted transgene at a
desired level. This loss of expression occurred even though the
resistance marker was downstream of the reporter expression cassette in
the transformation vector. Hence, screening of mVenus+transformants by plate reader or flow cytometry becomes useful to target
the transformants with the highest expressing level of transgene. As
usually observed with C. reinhardtii nuclear transformants, many
(97.4%) early positive transformants expressing the transgene of
interest, which was not essential for survival, underwent
transcriptional and/or post-transcriptional gene silencing mechanisms
(Tran et Kaldenhoff, 2020). Thus, the number of positive transformants
falls overtime after rounds of subcultures despite antibiotic selection.
Our report demonstrated for the first time, to our knowledge, the use of
HDAC inhibitors of hydroxamate-class like SAHA in C. reinhardtiito trigger protein expression.
Upon SAHA treatment, the expression level of the mVenus reporter protein
can be recovered at doses that do not affect the cell growth. Treatment
at initiation of cultures lead to a maximal production of mVenus at the
end of the exponential phase (around day 6). However, SAHA did not
prevent the progressive decline in mVenus production when transformants
advanced into stationary phase, although cultures treated with SAHA
still expressed more transgene.
The overall growth pattern of microalgae was not impacted upon treatment
with 5 µM SAHA, but a significant decline in chlorophyll fluorescence
was observed. In addition, cell motility was progressively lost ≥2.5 µM
SAHA, while palmelloid formation was induced.
Some studies have shown that abiotic stress could transiently induce
palmelloid in C. reinhardtii without impacting viability (Cheloni
et Slaveykova, 2021; de Carpentier et al., 2022).
Cheloni et Slaveykova examined palmelloid colony formation upon
micropollutants (MPs) exposure. The number of palmelloid and their size
were dependent on MP concentration and exposure duration. Cells kept
growing and dividing within the palmelloid and reverted to their
unicellular lifestyle when colonies were harvested and inoculated in
fresh medium, indicating that palmelloid formation is a common (and not
specific) plastic response to different micropollutants. In mixed
populations cultures, the unicellular population exhibited chlorophyll
bleaching, membrane damage and oxidative stress, whereas palmelloids
were unaffected. In a different study, Carpentier et al. reported
on the characterization of an abiotic stress response that the algae can
trigger, forming massive multicellular structures called aggregates,
which are different from palmelloids. Aggregates are formed by a few
tens, to several thousand cells, held together in an extracellular
matrix, whereas palmelloids are composed of 4 to 16 cells surrounded by
a cell wall. These aggregates constitute an effective bulwark within
which the cells are efficiently protected from the toxic environment.
Aggregation is not the result of passive agglutination, but rather of
genetic reprogramming and substantial modification of the algae’s
secretome. Hence, the induction of aggregates and/or palmelloid
following SAHA treatment, could help concentrate proteins (and even
metabolites) within fewer cells linked together in these multicellular
structures, which would not hinder significantly usual cellular
metabolism.
High doses of SAHA (20, 40 and 80 µM) strongly increased the number and
promoted the larger size of the palmelloids and/or aggregations
formation and inhibited the cell growth. There have been some studies
using HDAC inhibitors of hydroxamate-class like SAHA in plant cell
cultures (Medicago truncatula and Bambusa multiplex ) to
boost protein expression (Santos et al., 2017; Nomura et al., 2021),
which reported cell toxicity induced by the tested HDACi. Toxicity was
lower when the cell culture was treated at day 3, at the onset of
exponential phase. In a different study, Nomura et al. boosted
the production of two endogenous specialized metabolic compounds
(3-O -p -coumaroylquinic acid and 3-O -feruloylquinic
acid in a cell line culture of B. multiplex treated with Suberoyl
bis‑hydroxamic acid (SBHA), an analog of SAHA. Production of both
compounds was induced by SBHA at concentrations between 2 µM and 100 µM,
but production decreased at concentrations ≥ 50 µM, mainly because of
the cytotoxicity of SBHA. Interestingly, in their study, smaller initial
cell density of 5% SCV (sedimented cell volume) lead to the strongest
induction of both metabolites. This suggests that inoculum size might be
an important contributing factor to the success of silencing reversal.
Upon SAHA-treatment, the relative mVenus mRNA expression and protein
levels, together with fluorescence intensity increased in most clones.
These results are consistent with SAHA modus operandi at the DNA
levels, inducing gene expression. Transcriptional gene silencing is
believed to be the main cause for transgene expression inhibition inC. reinhardtii , which is largely mediated by protein factors that
place specific histone modifications onto nucleosomes at the transgene
loci to trigger the formation of a repressive chromatin structure, a
mechanism that may have evolved to protect the genome from invading DNA
(Schroda, 2019). H3K4 and K9 monomethylated are some of the histone
marks known to occur on nucleosomes in promoter regions of silent genes
in C. reinhardtii , while H3K4 trimethylated and H3K9 acetylated
appear in promoter regions of active genes (Yamasaki et al., 2008;
Shaver et al., 2010; Strenker et al., 2013; Barahimipour et al., 2015;
Schroda, 2019). SAHA triggered an increase in histone acetylation level
of H3K9 (determined by western-blot) in all clones, while the level of
H3 remained consistent. This further confirms that SAHA treatment
restored expression by increasing histone acetylation levels, or by
preventing histone deacetylation. In addition, Kaginkar et al.,using antibiotic resistance as readout for transgene expression inC. reinhardtii suggested that the use of some metal ions, light,
curcumin, cinnamic acid, quercetin sodium butyrate, decitabine
(5-aza-2’-deoxycytidine) could reverse stress-induced silencing, through
inhibition of DNA methylation or histone deacetylation (Kaginkar et al.,
2021). By contrast Neupert et al. could not induce expression
using sirtuin inhibitors and other HDACs inhibitors, despite increasing
histone acetylation levels (Neupert et al., 2020). In our hands,
inhibition of methylation did not yield to an increase in mVenus
expression, while hydroxamate-family HDAC inhibitors were very
efficient. Multiple and distinct mechanisms responsible for silencing
could occur in different clones. It might depend on the promoter,
implying that triggering expression with some inhibitors might work for
some clones and not others.
In this study, mVenus gene expression was driven under the hybridHSP70A-RBCS2 fusion promoter. Strenkert et al. (2011,
2013) demonstrated that the transgenic HSP70A promoters harbor
lower levels of active chromatin marks than the native HSP70A but
more than transgenic RBCS2 promoters (Strenkert et al., 2013;
Strenkert et al., 2011). The authors found that, first, heat shock
transcription factor 1 (HSF1) binds to the promoter, second histone
acetylation occurred, then nucleosomes were remodeled, and transcript
accumulated. This suggested that the HSF1 recruits histone
acetyltransferase (and other histone-modifying enzyme activities) to
target promoters. HSF1 could constitutively form a scaffold at the
transgenic HSP70A promoter, presumably containing mediator and
TFIID, from which local chromatin remodeling and polymerase II
recruitment to downstream promoters is realized. However, the authors
also observed HSF1-independent histone H3/4 deacetylation at theRBCS2 promoter after heat shock, suggesting interplay of specific
and presumably more generally acting factors to adapt gene expression to
the new requirements of a changing environment. Interestingly, in the
case of the HSP70A-RBCS2 fusion promoter, the chromatin state at
the HSP70A promoter was dominantly transferred to RBCS2 by
HSF1, to recruit all the machinery necessary for transcription. Here, we
show that HDAC inhibitors of hydroxamate-class like SAHA can further
help maintain active chromatin marks at the HSP70A-RBCS2 fusion
promoters.
In summary, we uncovered a new tool to successfully trigger the
expression of heterologous proteins in C. reinhardtii . This
method could also be useful and applicable for recombinant production in
other microalgae species and open the field to new studies.