H3k9me3

ChIP assays were performed as previously described24 (link). Briefly, protein-DNA complexes were cross-linked by 1% formaldehyde for 10 min at 37 °C. Cells were resuspended in 200 μl of SDS lysis buffer and subjected to three cycles of sonication on ice with 10-S pulses. Then, sonicated samples were centrifuged to spin down cell debris, and the soluble chromatin solutions were immunoprecipitated using antibodies specific for p65(Bethyl Lab., Montgomery, TX, USA), HP1(GeneTex, San Antonio, TX, USA), SUV39H1, H3K9me3, and RNA polymerase II(Millipore, Billerica, MA, USA). The final immunoprecipitated DNA(IP DNA) were resolved in nuclease-free H₂O and the DNA solutions were quantified by quantitative PCR. Primers amplifying irrelevant segments of DNA in the IL-8 gene, the 3′ UTR sites, were used as site-specific binding controls.

ChIP analysis was performed as previously described [41 (link)]. Briefly, 4 × 10⁶ cells were used for each experimental point. Chromatin fragments were incubated, overnight at 4 °C on a rotating platform with different antibodies: H3 (Abcam), H3K9me3 (Millipore), H4 (Abcam), H4K20me3 (Millipore) and the preimmune serum (Jackson Immuno Research Laboratories, Inc. Baltimore Pike, PA, USA). DNA was then recovered by phenol-chloroform extraction and ethanol precipitation, slot-blotted into a Hybond N⁺ membrane (Amersham Pharmacia Biotech) and hybridized with a telomeric probe (kindly provided by M. Blasco, Spanish National Cancer Research Centre-CNIO) obtained from a plasmid containing 1.6 kb of TTAGGG repeats labelled with α-32P. The signal was quantified using the ImageJ software. For total DNA samples, aliquots corresponding to a 1:10 dilution of the amount of lysate used in the immunoprecipitations were processed along with the rest of the samples during the crosslink reversal step. Data were normalized on the telomeric H3 and H4 signal, respectively. We represented the ChIP values as a percentage of the total input telomeric DNA, thus correcting for differences in the number of telomere repeats [42 (link)]. Experiments were performed at least two times.

Chromatin from synchronous-rings-stage parasites of 3D7 clone G7 was prepared and 3*10⁸ cells per ChIP used for the previously described protocol (Lopez-Rubio et al., 2013 (link)). Briefly, chromatin was crosslinked in 1% formaldehyde for 10 min (Sigma-Aldrich, #SZBD1830V), sheared to an average length of 300 bp using the BioRuptor Pico, and individual histone modifications were pulled down using 0.5 μg of antibody for H3K4me3 (Diagenode, cat # K2921004), H3K9me3 (Millipore, cat # 257833), and homemade rabbit polyclonal anti-PfHP1. 5 μl rabbit polyclonal anti-H4K31me1 and 15 μl anti-H4K31ac were used for each experiment. To generate Illumina-compatible sequencing libraries, the immunoprecipitated DNA and input was processed using the MicroPlex Library Preparation Kit (Diagenode C05010014) according to manufacturer’s instructions. The optimized library amplification step used KAPA Biosystems HIFI polymerase (KAPA Biosystems KK2101). Pooled, multiplexed libraries were sequenced on an Illumina NextSeq 500/550 system as a 150-nucleotide single-end run. The raw data were demultiplexed using bcl2fastq2 (Illumina) and converted to fastq format files for downstream analysis. Two biological replicates were analyzed for each antibody.

Cells were grown for on cover slips either in normoxic or hypoxic incubation. Immunostaining was performed on 4% PFA-PBS fixed cells. Cells were permeabilized with 1% TritonX-PBS for 10 minutes and washed with PBS, subsequent incubation with primary antibody was performed overnight, at 4°C. The following antibodies were used: Ki-67 (clone TEC-3, M-7249, Dako), H3K9me3 (07-523, Millipore) and γH2AX (05-636, Millipore).
Cover slips containing cells were washed and incubated with secondary antibody (AlexaFluor488 goat anti-rat, AlexaFluor488 goat anti-rabbit and AlexaFluor488 rabbit anti-mouse, (Invitrogen)) in 3% BSA in PBS/0.05% Tween for one hour at 37°C in the dark. Slides were washed, and for nuclei counterstaining briefly incubated in PBS containing DAPI, mounted and analyzed.