Materials and Methods
C. reinhardtii cultivation and
transformation
C. reinhardtii strain CC-1690 (wild-type strain; WT) was
purchased from the Chlamydomonas Resource Center (The University
of Minnesota, USA). Growth of C. reinhardtii was performed under
mixotrophic conditions with Tris-acetate-phosphate (TAP) medium (Gorman
et Levine, 1965) on agar plates or liquid in 6 well plates, 96 well
plates or 125 mL shake flasks under 50±10 µmol photons
m−2 s−1 light intensity and a
photoperiod of 16h light:8h dark cycles at 21±0.5oC,
and 130 rpm agitation for liquid cultures.
Nuclear transformation was carried out by electroporation following
GeneArtTM Chlamydomonas Protein Expression
Vector protocol (Invitrogen, Life technologies, Thermo Fisher
Scientifics) using 0.5 µg of linearized pOpt_mVenus_Paro vector
(Laursen et al., 2015) by restriction endonucleases Xba I andKpn I. Transformants were selected on TAP agar plates supplemented
with paromomycin (10 mg. L−1) for 5-7 days.
Treatment with histone deacetylase
inhibitors
For all assays, C. reinhardtii WT and transformed colonies were
initially transferred from agar plates to a liquid preculture in 6 well
plates for 4-6 days, under culture conditions mentioned in section 2.1.
Inoculums of 105 cells. mL-1 in late
exponential phase of growth were used in this study.
For initial experiments, 200 µL of C. reinhardtii cells
grown to exponential phase were transferred into a 96-well plate (flat
bottom; Corning) and incubated for 24 hours with individual HDACi, 50 µM
sirtinol, 2.5 µM SAHA, 100 µM OSS-128167, 1 mM nicotinamide (all from
MedChem Express) and 5 mM sodium pyruvate (Fishers Bioreagents). A mix
of all inhibitors was also tested. As a negative control, samples were
incubated with DMSO.
SAHA analogs and other HDACi such as belinostat, dacinostat,
panobinostat, mocetinostat, entinostat, and romidepsin (all purchased
from MedChem Express) were also tested. Culture inoculums were
transferred in 200 µL of TAP medium in 96 well plates, with 5 µM of each
HDACi followed over a 6 days growth period. Other experiments were done
to follow the impact of SAHA on growth and fluorescence levels, inoculum
was transferred into 50 mL of TAP media in 125 mL shaking flasks and
treated with SAHA at final concentrations of 2.5, 5 and 10 µM for 6 to
12 days.
Growth curve, chlorophyll and mVenus fluorescence detection
using plate
reader
Growth curves were obtained using a Synergy H1 plate reader (Biotek,
Agilent), from three biological replicates for each C.reinhardtii strain studied as in (Molino et al., 2022). For SAHA
treatments, 250 µL of C. reinhardtii cells from 50 mL
cultures were transferred, in triplicate, into 96 well flat bottom
microplates (Corning Costar 96-Well, Cell Culture-Treated, Fisher
Scientifics). Microalgae growth was done by tracking the optical density
at 750 nm (OD750, 750/8 nm). Area scan mode was used to
measure the mean fluorescence intensity of mVenus (excitation 500/18 nm,
emission 541/18 nm) and chlorophyll (excitation 475/18 nm, emission
650/18 nm). C. reinhardtii WT was used as negative control for
mVenus fluorescence. Fluorescence intensity of mVenus was normalized to
the OD750.
Flow cytometry
A Cytomics FC500 cytometer equipped with Argon (488 nm) and HeNe (633
nm) lasers were used to measure mVenus emission on the FL1 channel
(525/15 nm), and chloroplast fluorescence on the FL4 channel (675/15 nm)
(Beckman Coulter Life Sciences). At least 10,000 events were acquired. A
homogeneous cloud of intact cells was first gated based on the size
(forward scatter, FSC) and the granulosity (side scatter, SSC). Then a
daughter gate was selected on the cloud of cells with homogenous
chlorophyll autofluorescence. The percentage (%) of
mVenus+ cells was measured on this daughter
population, with a gate in the FL1 channel that (filter at 525/10 nm)
excluded cells with autofluorescence levels similar to the negative
controls. Cells were considered mVenus+ when both the
% of gated mVenus+ cells (and >0.1%)
and mVenus mean fluorescence intensity (MFI) were higher than the values
of the wildtype (WT) cells used as a control. Propidium iodide (Thermo
Fisher, 7 µM) was used to verify viability (Cheloni et al., 2014) and
acquired on the FL3 channel (620/20 nm). To select palmelloid cells, a
second gate that included events with a minimum of 2-fold increase in
FSC mean intensity was drawn, similarly to (Cheloni and Slaveykova,
2021). In that case, cells were acquired on a Beckman Cytoflex S
equipped with violet (405nm), blue (488nm), yellow-green (561 nm) and
red (638 nm) lasers. Chlorophyll autofluorescence was detected in the
PerCP channel (690/50 nm), mVenus on the FITC (525/40 BP). Statistics
were obtained using BD FlowJo version 10 software (BD Biosciences, La
Jolla, CA, USA, 2020).
DNA extraction and qPCR for transgene relative
quantity
Genomic DNA (gDNA) was extracted using the protocol from theChlamydomonas Resource Center (The University of Minnesota, USA)
with minor modifications. Two mL of a 6 days C. reinhardtii cell
cultures were harvested and centrifuged in 1.7 tubes for 5 min at 4000
g. The cell pellet was resuspended in 500 µL of CTAB-buffer (2% (w/v)
CTAB, 100 mM Tris-HCl pH 8.0, 1,4 M NaCl, 20 mM EDTA pH 8.0 and 2%
(v/v) 2-mercaptoethanol) and incubated at 65ºC for 1h. The DNA was
extracted with 500 µL of chloroform/isoamyl alcohol (24:1). The upper
phase was transferred and 0.7 volumes of isopropanol was added for 15
min at 4ºC. The DNA was spin down at 4ºC for 20 min at 12000 rpm. The
pellet was washed twice with 1 mL of cold 70% ethanol and centrifuged
at 4ºC for 5 min at 12000 rpm. The supernatant was discarded and the
pellet was air dry under the hood until the pellet was completely dried.
The gDNA was dissolved in 50 µL of TE-buffer (1 mM Tris-HCl pH 8.0 and
0,1 mM EDTA pH 8.0). gDNA yield and quality were determined by measuring
the 260/280 ratios using a Nanodrop instrument (Thermo Fisher
Scientific).
gDNA samples were subjected to qPCR amplification using Luna® Universal
qPCR Master Mix (New England Biolabs). Briefly, 250 ng of gDNA were used
as template in 20 μL reactions according to manufacturer instructions.
Initial denaturation was 2 min at 95°C followed by 45 cycles of
denaturation for 15 seconds at 95°C and extension for 30 seconds at
60°C. Primers used to detect mVenus and histone 3(h3 ) (reference gene) were designed using
PrimerQuestTM Tool (Integrated DNA Technologies, IDT)
(primer sequences are listed in Table S1). The oligonucleotides were
validated by performing a standard curve and through dissociation curves
analysis (60-95°C for the melt curves). SYBR Green fluorescence was
recorded in the FAM channel of a CFX connect real time system (Bio-Rad).
PCR runs were analyzed with CFX Manager software (Bio-Rad).
pOpt_mVenus_Paro was used to determine the copy numbers. Dilutions
ranging from 5 to 1000 pg of vector were used to perform the standard
curve. Plasmid copy number was estimated following this formula:
(pOpt_mVenus (ng) * 6.0221.1023) / (pOpt_mVenus (bp)
* 660 *1.109)
(Integrated DNA Technologies,
https://www.idtdna.com/pages/education/decoded/
article/calculations-converting-from-nanograms-to-copy-number).
Relative mVenus gene copy numbers for each strain were determined using
the equation of the standard curve log(y) = ax + b; where y = plasmid
copy number and x= Ct. Each sample copy number was then determined with
the formula 10((Ctsample−b)/a), as in (Masroori et
al., 2016), normalized to the gDNA weight (ng). Relative mVenus gene
detection was also calculated, using h3 as an endogenous control
(Veillette et al., 2013). Experiments were performed with three
biological replicates.
RNA extraction and RT-qPCR for mRNA relative
quantification
Four mL of a 6 days C. reinhardtii grown culture were harvested
and centrifuged in 1.7 mL tube for 5 min at 4000 g. Cells were lysed by
immersion in liquid nitrogen for 1 min. Total RNA was extracted using
InvitrogenTM TRIzolTM reagent (Life
technologies, Thermo Fisher Scientifics) according to the manufacturer
protocol with minor modifications, including the addition of NaCl (final
concentration of 100 mM) in isopropanol to improve nucleic acids
precipitation. Samples were further treated with Turbo DNase
(Invitrogen™ TURBO DNA-free ™ Kit, Thermo Fisher Scientifics)
according to the manufacturer’s instructions. RNA yield and quality were
determined by measuring the 260/280 ratios using a Nanodrop instrument.
Samples of 100 ng total RNA were subjected to reverse transcription and
qPCR amplification in a single reaction using the
Luna® Universal One-Step RT-qPCR Kit Protocol (New
England Biolabs). Briefly, 2 μL of RNA were subjected to reverse
transcription performed at 55°C for 10 minutes. Initial denaturation was
1 min at 95°C followed by 45 cycles of denaturation for 10 seconds at
95°C and extension for 30 seconds at 60°C. A melt curve analysis was
performed from 60-95°C with an increment of 0.5°C each 5 seconds.
Primers used for mVenus transcript and h3 as a housekeeping gene
were designed using PrimerQuestTM Tool (IDT) (primer sequences are
listed in Table S1). SYBR Green fluorescence was recorded in the FAM
channel of a CFX connect real time system. PCR runs were analyzed with
Bio-Rad CFX Manager version 3.1 software. Relative mRNA expression
levels were determined according to the 2(-ΔΔCt)method (Livak et Schmittegen, 2001). Experiments were performed using
three technical replicates.
Protein extraction and
western-blot
Twenty-five mL of a 6 days C. reinhardtii cell cultures were
harvested and centrifuged in 50 mL tube at 4000 g for 10 min at 4°C. The
pellets were washed once with ice-cold PBS 1X supplemented with 5 mM
sodium butyrate, to retain levels of histone acetylation. Then, pellets
were weighed and resuspended with a ratio 0.5 g FW
mL-1 in Triton Extraction Buffer (TEB: PBS 1X
containing 0.5% Triton X 100 (v/v), 0.02% (w/v) NaN3).
PMSF, final concentration 2 mM, and protease inhibitor (32 µL g
FW-1) were subsequently added (both from Thermo Fisher
Scientific). Sonication was performed 6 times at 35% amplitude, 30 sec
on, 30 sec off for 3 min total using FisherbrandTMModel 505 Sonic Dismembrator (Thermo Fisher Scientific). Protein
extracts were centrifuged at 14000 g for 30 min at 4°C. Supernatants
containing the total soluble protein fractions were kept at -80°C to be
used for western blot. Proteins were quantified with the RC DC™ Protein
Assay Kit I (Bio-Rad).
To detect mVenus and Histone H3 Lysine 9 acetylated (H3K9ac), 50 µg and
25 µg of total proteins were loaded respectively, in 15% SDS-PAGEs.
Purified mVenus protein from colony 21 using GFP trap agarose
(Chromotek, Germany) was loaded as a positive control. Proteins were
then transferred to the 0.2 µm PVDF membrane (settings: 1 mA constant
and 25 V for 30 min). Primary antibodies for mVenus (27 kDa) and H3K9ac
(15.4 kDa) detection were purchased from Cedarlane and from Agrisera,
respectively. Both antibodies were diluted at 1:1000 in 3% BSA and were
incubated overnight at 4°C. Actin (40 kDa) and histone 3 (H3, 15.4 kDa)
detection was performed as internal standards of cytosolic and nuclear
proteins, respectively, respectively. Blots were incubated with
anti-actin-HRP solution, 1:10,000 in BSA 3%, from Abcam (Cambridge) and
anti-H3 (1:1,000 in BSA 3%, Agrisera). After three washes with
Tris-buffered saline, 0.1% Tween 20 (TBST) solution, blots were
incubated for 1 hour in a 1:20,000 dilution, in 5% milk, of Immun-Star
Goat Anti-Mouse (GAM)-HRP Conjugate from Bio-Rad to detect mVenus, and
Immun-Star Goat Anti-Rabbit (GAR)-HRP Conjugate from Bio-Rad to detect
H3K9ac. After three washes of the membrane with TBST solution, protein
detection was revealed using Clarity Max Western ECL Substrate-Luminol
solution from Bio-Rad. Chemiluminescence detection and Ponceau S stained
(Glacial Acetic Acid 5% v/v, Ponceau Red dye 0.1% m/v) of blots were
visualized using ChemiDoc Imaging System with Image Lab Touch Software
(Bio-Rad) and Image Lab™ Software (Bio-Rad). The molecular weight of the
protein corresponding to the detected band was confirmed with the
protein marker (Precision Plus Protein Dual Color Standards #1610374).
Statistical analysis
Statistical analyses were performed using GraphPad Prism (Version 9.4.1,
GraphPad Software, US). Data are expressed as means ± SD of three
biological replicated performed at least twice in independent
experiments. Parametric tests (ANOVA and Student’s t test) were
used when population followed normal distribution, and non-parametric
(ANOVA and Mann–Whitney test) were used when the population could not
be assumed to be normally distributed. A p value < 0.05
was considered to be significant.