Balancing acts of SRI and an auto-inhibitory domain specify Set2 function at transcribed chromatin

Set2-mediated H3K36 methylation ubiquitously functions in coding regions in all eukaryotes. It has been linked to the regulation of acetylation states, histone exchange, alternative splicing, DNA repair and recombination. Set2 is recruited to transcribed chromatin through its SRI domain's direct association with phosphorylated Pol II. However, regulatory mechanisms for histone modifying enzymes like Set2 that travel with elongating Pol II remain largely unknown beyond their initial recruitment events. Here, by fusing Set2 to RNA Pol II, we found that the SRI domain can also recognize linker DNA of chromatin, thereby controlling Set2 substrate specificity. We also discovered that an auto-inhibitory domain (AID) of Set2 primarily restricts Set2 activity to transcribed chromatin and fine-tunes several functions of SRI. Finally, we demonstrated that AID mutations caused hyperactive Set2 in vivo and displayed a synthetic interaction with the histone chaperone FACT. Our data suggest that Set2 is intrinsically regulated through multiple mechanisms and emphasize the importance of a precise temporal control of H3K36 methylation during the dynamic transcription elongation process.

To construct Rpb1-Set2 fusion plasmids, an intermediate vector, pBL875, was first constructed. The KpnI/SnaBI fragment at the RPB1 region was subcloned into a modified pBluescript vector, pBL766. A linker containing two restriction sites (SmaI and XhoI) and the sequence for an HA tag was inserted between the last codon of the heptad repeat sequence and the 3' UTR of RPB1. The coding sequences of SET2 and its derivatives were then subcloned into the XhoI site of pBL875 to form pWY041 (full length Set2; FL), pWY042 (coding for Set2 residues 1-618) and pBL876 (SET domain). The KpnI/SnaBI fragments from these intermediate vectors were subsequently transferred into pBL855 (pY1A) to generate pWY043 (CTD-Set2 potassium acetate for about 1 week due to inefficient sporulation of these mutants. Spores were dissected and the presence of HIS and KANMX cassettes was screened by PCR. Lastly, western blotting with anti-HA antibody was performed to confirm proper expression of HA-tagged Set2 in dextrose-and galactose-containing media.

Preparation of whole-cell extracts for immunoblotting:
Cells were grown in 3 ml YPD or synthetic drop-out media supplemented with 2% dextrose or 2% galactose at 30 o C overnight until the OD 600 reached 1.0-1.2. Cell pellets were resuspended in 45 µl of STE buffer (500 mM NaCl, 10 mM Tris HCl pH8.0 and 1 mM EDTA) and 40 µl of 3xSDS buffer. 100 µl of 0.5 mm glass beads were then added and boiled at 95 o C for 5 min. Samples were then vigorously vortexed to break the cells, and cell suspensions were clarified by spinning at 14,000 rpm for 5 min. Anti-H3K36me3 (Abcam, 9050), anti-H3K36me2 (Abcam, 9049), anti-H4 (Abcam, 10158) and anti-FLAG HRP (Sigma, A8592) antibodies were used according to the manufacturers' instructions. For anti-H3K36me2 antibody, 5% BSA was used for blocking and 1% BSA was present in all antibody incubations.

Recombinant Histone Purification and Nucleosome Reconstitution
Xenopus recombinant histones (H3, H4, H2A and H2B) were individually expressed in BL21CodonPlus-RIL (Stratagene) cells and purified as described (3). Histone octamers were assembled and fractionated through gel-filtration column Superdex 200. To generate mononucleosomes with different linker DNA lengths, plasmids that carry 16 copies of 216 bp (pBL645), 153 bp (pBL647) or 147 bp (pBL648) DNA fragments were digested by EcoRV, and the template DNA were purified using the 491 Prep-cell (Bio-Rad) (4). These DNA fragments were then mixed with histone octamers at a pre-determined ratio in high salt buffer and dialyzed against a buffer with serial dilutions of salt concentrations (4). The reconstituted nucleosomes were finally purified through a 4% native gel using 491 Prep-cell (Bio-Rad) and concentrated to about 1 µg/µl (4).

Yeast native nucleosome purification
Yeast nuclei were prepared as previously described (5)  Thomas Pestle Tissue Grinder (Size C). The resulting suspension was laid over 10 ml GB buffer (20 mM PIPES.NaOH pH6.5, 0.5 mM MgCl 2 , 7% Ficoll 400 and 20% glycerol) and centrifuged at 11,500 rpm using a HB6 swinging-bucket rotor. The pellets were resuspended in 20 ml of FB buffer, and spun at 4,500 rpm to remove cell debris. The nuclei were finally collected by centrifugation at 11,500 rpm for 30 min, and extracted three times with 120 ml of EBX buffer (50 mM HEPES.NaOH pH7.5, 2.5 mM MgCl 2 , 400 mM KCl and 0.25% Triton X-100). The remaining chromatin pellets were resuspended in 10 ml MNase digestion buffer (10 mM HEPES.NaOH pH7.5, 0.5 mM MgCl 2 and 0.05 CaCl 2 ) and sonicated briefly to break the clumps.
After a small-scale MNase titration test, the optimal concentration of Micrococcal nuclease (MNase) was applied to the rest of the chromatin suspension at 30 o C for 15 min. MNase digestion was stopped by addition of EDTA. The digested oligonucleosomes were then fractionated on a Sepharose CL-6B column using 0.1 M HB buffer (20 mM HEPES.NaOH pH7.5, 1 mM EDTA, 100 mM NaCl, 10% glycerol and 1 mM βME). Nucleosomes were pooled based on the size of DNA and concentrated using 30 kd cutoff concentrators (Amicon).

Figure S3. Full-length Set2 and SET domain have distinct substrate preferences. (A) An experimental strategy to test if SET activity is intrinsically inhibited. (B)
Coomassie staining of Set2 TEV proteins that were purified from insect cells as indicated in Figure 4A. (C) Set2 constructs for mapping minimal deletion that causes de-repression of SET activity. (D) Coomassie staining of purified Set2 proteins. The asterisk indicates the degraded protein. Note that Set2 ∆C mutant purified from insect cell expression system consistently displayed severe degradation. (E) Integrity of the AID domain is essential for regulating Set2 activity in vitro as measured by HMT assays using HeLa oligonucleosome substrates as described in Figure 5B.

Figure S4. AID mutations render Set2 more sensitive to degradation
Western blots of whole cell extracts from the indicated yeast strains. #1 and #2 represent two independent clones of the same strain that were tested. (A) Plasmids carrying WT or AIDmutated Set2 under the control of native SET2 promoters were transformed into YYW010 (∆SET2). (B) Deletion of RCO1 does not alleviate H3K36 status changes caused by AID mutations. pWY126 and pWY127 were transformed into YYW132(∆SET2/∆RCO1). Western blotting was performed with the indicated antibodies. (C). Protein levels of Set2 and H3K36 methylation status in strains in which SET2 is under the control of different promoters: pWY069 (GAL1) and pWY131 (SET2 native promoter). Two plasmids containing ADH1 promoter, pWY088 (Ura3) and pWY091 (Leu2), were transformed into ∆SET2. Their empty vector counterparts were transformed into WT strain. Western blotting was performed with anti-Set2 antibody (a generous gift from Dr. Strahl) to detect the expression levels of plasmid-borne Set2 compared to the endogenous source.