Fig. 3

Analysis of enzymatic activity, gene transcription level, and histone methylation patterns in WT and PoSet2 mutants. a Activity assay of xylanase, arabinofuranosidase, pNPCase, and CMCase. Enzyme activities were normalized to the ratio of corresponding biomass (IU/mg). b Expression level assay of genes encoding four transcriptional factors (clr-2/clrB, xyr1/xlnR, amyR, and cre1/creA) via qRT-PCR analysis. Actin gene was used for data normalization. c Analysis of H3K4 and H3K36 methylation patterns through Western blot. Histone H3 was used as the loading control. The peak area and the data were obtained by the software Image J, representing the relative signal strength of the corresponding Western blot bands. d Transcript abundance of genes encoding eight subunits (i.e., Swd1, Swd2, Swd3, Bre2, Sdc1, Spp1, Shg1, and Set1) of COMPASS. e Analysis of the methylation modification level of H3K36 on the specific regions of three COMPASS subunits encoding genes (swd2, swd3, and set1). The levels of H3K36me1 and H3K36me3 of the target genes were assayed by ChIP-qPCR. Equal amounts of extracted chromatin (1 mg/IP) of each sample were used for IP reactions, and 0.1 mg chromatin DNA was used as input (without IP) for each sample. The purified IP products and input DNA were subjected to quantitative PCR (qPCR). The transcription start site (TSS) was designated as + 1. Six specific regions (R1 to R6) of each gene were designed for qPCR assay (as was shown at the top of the subgraph). Region 1 (R1) and region 2 (R2) were located upstream of the TSS. Region 3 (R3) covered the TATA-box and the initiator (Inr). Region 4 (R4) was located in the 5′ region of the CDS. Region 5 (R5) was located at the middle of the CDS, and region 6 (R6) was located in the 3′ region of the CDS. The relative enrichment of IP DNA was calculated by % of input. The values showed the means of the three biological replicates, and the error bar indicated standard deviation. Statistical significance tests were done by one tailed, unequal variance t-test. *P < 0.05, **P < 0.01, ***P < 0.001