Yeast strains, plasmids, and growth conditions

S. cerevisiae yeast strains were grown in YPD (2% peptone, 1% yeast extract, 2% glucose) or yeast nitrogen base minus uracil (AA-Ura) media, to prevent plasmids loss at 30 °C [19]. Yeast strains used in this study are listed in Supplemental Table 1. Plasmids were transformed into yeast strains using the standard lithium acetate transformation method [20]. AA-Ura or YPD plates containing 2% glucose were supplemented with 0.2% w/v 2-DG. To prevent plasmid loss and ensure that only vector-containing cells can grow, yeast strain transformants are grown on selective media (AA-URA).

Mammalian cell culture

Human prostate cancer cell line, PC-3 cells were cultured in F-12 K Medium (Kaighn’s Modification of Ham’s F-12 Medium) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. All cells were cultured in 37 °C, 5% CO2 incubator.

HXK cloning and yeast strain construction

To amplify yHXK1 and yHXK2 from yeast genomic DNA, we employed specific primer pairs (Kpn1-HXK1/Xba1-HXK1 and Kpn1-HXK2/Xba1-HXK2; primer sequences provided in Supplemental Table 2). Following amplification, the resulting DNA fragments were gel-purified using a Qiagen gel purification kit and subsequently cleaved with Kpn1 and Xba1 restriction enzymes. The resulting fragments were inserted into the yeast expression vector PSF-TEF1-URA3 (OGS534, Milipore-Sigma) at the corresponding restriction enzyme sites.

The yeast-codon optimized human HK genes hHK1 and hHK2 were synthesized directly, with the sequences described in the Supplemental Data. These gene fragments were enzymatically cut using the restriction enzymes EcoRV and XhoI and integrated into the PSF-TEF1-URA3 vector, utilizing the matching restriction enzyme sites.

To introduce the HXK-containing vectors and vector controls into yeast, we followed the standard lithium acetate transformation protocol. Colonies were selected on yeast synthetic complete supplement mixture minus uracil (SC-Ura) plates.

ATP measurements

Yeast cells (2 OD units at 600 nm) were collected in a 1.5 ml microcentrifuge tube by centrifugation at maximum speed for 1 min and washed once with cold distilled water. The supernatant was removed, and 0.75 ml of 90% acetone was added to the pellet. The resuspended cells were then heated at 90 °C for 15 min to allow the acetone to evaporate, leaving approximately 50 µl of solution. Assay buffer (10 mM Tris, pH 8.0, and 1 mM EDTA) was added to bring the final volume to 500 µl. ATP concentration was measured using the ATP Determination Kit (A22066, Thermo Scientific Inc.) according to the manufacturer’s instructions, with a luminescence microplate reader (BioTek U.S.).

Immunoblotting assay

To monitor the levels of histone marks in yeast strains, we performed whole cell extraction using tris-buffered saline (20 mM Tris pH 7.5, 150 mM NaCl) with protease inhibitor (phenylmethylsulfonyl fluoride PMSF). Briefly, log-phase cells (5 OD600) were harvested by spinning cultures at 5000 rpm (revolutions per minute) then washing the pellets with water, heating them at 95° C for 3 min, and suspending them in 50 ul tris-buffered saline buffer with 2 mM PMSF. Cells were physically broken by glass bead–beating them for 2 cycles (30 s per cycle). Proteins were then solubilized in 50 ul 2x SDS sample buffer [Tris-Cl (pH 6.8), 4% (w/v) sodium dodecyl sulfate (SDS; electrophoresis grade), 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol]. Insoluble debris was separated from supernatant and removed by spinning the samples at the 15,000 g speed. To prepare mammalian cell lysates, cells were washed with 1x PBS and cells were resuspended in RIPA buffer (50 mM Tris-Cl (pH 8.0), 150 mM sodium chloride, 1% NP-40, 0.5% deoxycholate, 0.1% SDS). Protease inhibitors were added and kept on ice for 10 min. Soluble proteins were extracted by high-speed centrifugation. Soluble proteins (the supernatant) were separated by SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad 1620115) using Tris-Glycine buffer and probed with different primary antibodies [anti-γ-H2A antibody (ab15083 Abcam), tubulin (6A204 Santa Cruz), human hexokinase 2 (C64G5 Cellsignaling), H3K4me3 (ab8580 Abcam), Anti FLAG (F1804 Sigma), H3K36me3 (ab9050 Abcam), H3K56Ac (mc1681 provided by Dr. Zhiguo Zhang [21]), Sir2 (provided by Dr. Zhiguo Zhang) [22], H3K27Ac (07-360 Millipore), and H3K5,8,12Ac (C15410021 Diogenode), Hexokinase 2 (NBP2-44234, Novusbio), PGK1 (22C5D8 Abcam)]. The stain-free gel or Sir2, PGK1 is used as the loading control. The second antibody is either goat anti rabbit IgG HRP (AB_2337913, Jackson Immnoresearch) or goat anti mouse IgG HRP (AB_10015289, Jackson Immnoresearch). Immunoblots were developed by using Chemiluminescent Substrate (PI37069, Thermo-Scientific)and images were acquired using Bio-Rad Chemidoc (12003154, Bio-Rad).

Cell cycle analysis

Yeast strains were grown in YPD medium at 30° C until the cells reached mid-log phase (∼ 0.6 OD at 600 nm), at which time they were treated with 2-DG (0.2% w/v). A total of ∼ 1 × 107 cells were harvested at various time points by centrifugation, washed with water, and fixed with 95% ethanol overnight at 4° C. Fixed cells were collected by centrifugation, washed with 50 mM sodium citrate buffer (pH 7.5), and resuspended in 500 µL sodium citrate buffer (pH 7.5) with RNAase A (0.025 mg/mL). After cells were incubated for 1 h at 50° C, 25 µL proteinase K (20 mg/mL) was added to the cell suspension and the mixture was incubated at 50° C for another hour. Cells were subsequently stained overnight at 4° C using propidium iodide at a final concentration of 0.02 mg/mL. Finally, samples were analyzed in a fluorescence-activated cell sorting (FACS) flow cytometer (BD Fortessa cytometer). A minimum of 50,000 cells per sample were acquired.

Analysis of silencing-loss at the HML (HoMothallism Left) locus using the CRASH assay

The yeast strains WT (cyc1250), hxk2Δ (cyc1253), WT + PSF-yHXK2 (cyc1247), and hxk2Δ + PSF-yHXK2 (cyc1251) were used to measure the apparent silencing-loss rate at the HML locus. Briefly, 10 colonies of each strain were grown separately overnight in SC media, diluted to 0.01 OD at 600 nm in SC media, and grown for 5 h at 30° C. Cells were treated with 20 µM nicotinamide for the green fluorescent protein (GFP) + control; cells were grown in hygromycin (200 µg/ml) for the red fluorescent protein (RFP) + control. The apparent silencing-loss rate at the HML locus was calculated by dividing the number of RFP + GFP + cells (cells that have recently undergone Cre-mediated recombination express GFP but not RFP) by the total number of cells with the potential to lose silencing (RFP + GFP- and RFP + GFP+). For each colony, 50,000 events were analyzed using a BD Fortessa cytometer.

Enrichment and sequencing of protein-associated nascent (eSPAN)

The yeast strains WT (cyc1023), hxk2Δ (cyc1025), WT + PSF-HXK2 (cyc1022), and hxk2Δ + PSF-HXK2 (cyc1024) were used in this experiment. Briefly, yeast cells were grown in SC-URA- medium to exponential growth phase (∼ 0.5 OD at 600 nm). Two doses of α factor (5 µg/ml; EZBiolab) were added for 3 h at 25° C to arrest cell growth at the G1 phase. Cells were washed twice with cold water and then released into fresh YPD medium containing 400 mg/L Bromodeoxyuridine (BrdU) and 200 mM hydroxyurea for 45 min at 30° C. Hydroxyurea stalls the replication fork but does not interfere with assembly of newly synthesized and parental histones [23,24,25].

Cells were fixed by adding freshly prepared paraformaldehyde (final concentration 1%) at 25° C for 20 min, followed by quenching with 0.125 M glycine for 5 min at room temperature. After fixation, cells were washed twice with cold water and collected by centrifugation at 3000 rpm for 5 min. For chromatin immunoprecipitation (ChIP), cells were washed and lysed in 0.1 ml ChIP lysis buffer [50 mM HEPES (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate] with glass beads. Broken cells were collected and washed twice with NP buffer [1.6 M sorbitol, 2 mM CaCl2, 5mM MgCl, 50 mM NaCl, 14 mM b-mercaptoethanol, 10 mM Tris-HCl (pH 7.4), 0.075% NP-40, 5 mM spermidine]. Chromatin was digested primarily to di- and mononucleosomes using the proper amount of MNase (LS004797, Worthington, ∼ 1 unit) at 37° C for 20 min. The digestion was terminated with 5 µl 0.5 M EDTA and 90 µl 5X ChIP lysis buffer and kept on ice for 30 min. Next, cells were lightly sonicated for five cycles (Bioraptor Pico machine, 30 s ON/OFF) at 4 °C to release chromatin fragments into solution. Soluble chromatin was immunoprecipitated with anti-H3K4me3 antibody (ab8580 Abcam) [21]. Prewashed Protein G Sepharose beads (17-0618-02, GE Healthcare) were used to recover the immunoprecipitated chromatin. After washing the beads extensively, ChIP DNA was recovered using the Chelex-100 protocol [15, 26,27,28].

ChIP DNA was denatured by incubating it at 100° C for 5 min and then on ice for 5 min. DNA was diluted with BrdU IP buffer [1X PBS, 0.0625% Triton X-100 (v/v)]. BrdU antibody (0.17 µg/ml; 555627, BD Biosciences) was added, and samples were incubated at 4° C for 2 h. Next, 20 µl prewashed Protein G beads (17-0618-02, GE Healthcare) were added to each sample and incubated for an additional hour at 4° C. The beads were extensively washed, then DNA was eluted with 100 µl 1X TE buffer containing 1% SDS and purified using a QIAGEN MinElute PCR Purification kit. Single-stranded DNA libraries were prepared using an Accel-NGS 1 S Plus DNA library kit (10096, Swift Biosciences).

Sequence mapping and data analysis

Sequence mapping, nucleosome mapping, and eSPAN analysis were performed similarly to what has been previously described [23, 24]. Briefly, reads were mapped to the Saccharomyces Genome Database (http://www.yeastgenome.org/) reference genome with Bowtie2 software [29]. Only paired-end reads with both ends mapped correctly were selected for continued analysis. We determined nucleosome occupancy using 120–170 bp DNA fragments calculated from paired-reads, using Python programs we developed ourselves. To calculate the eSPAN bias pattern, we separated forward (Watson strand) and reverse (Crick strand) reads following the reference genome. Nucleosome positions around DNA replication origins were determined previously [30]. Total eSPAN sequence reads at ± 10 nucleosomes surrounding the DNA replication origins were counted. The log2 ratio of Watson over Crick strand reads at each nucleosome position was used to obtain the average eSPAN bias pattern.

Western blot to detect yeast Rad53-P and γH2AX

The sample collection and process with Rad53-P and γH2AX followed previous publication [31]. Blotting was then performed with anti- Rad53 antibody (ab104232, Abcam) and γH2A.X (phospho S139) (ab11174 Abcam).