Intragenic transcription from the ASP3 ORF is detected in both ammonium-containing medium and under nitrogen starvation. (A) The wild-type strain was pre-grown in ammonium-containing medium. Cells were then washed with phosphate buffer and transferred to a 3% glucose solution for nitrogen starvation for 2 h. Northern analysis of the two different transcripts of ASP3 was conducted using an ASP3 probe. The blot was subsequently probed against ACT1 as a loading control. The sizes of transcripts are labeled at right. (B,C) Intragenic transcription is independent of GAT1 and GLN3. (B) The wild-type, gat1Δ, gln3Δ, and gat1Δ gln3Δ mutant strains were treated as described in A, and then analyzed by Northern blotting. (C) Strains described in B were pre-grown in ammonium-containing medium, treated with rapamycin, and then analyzed by Northern blotting. In B and C, ASP3 and ncASP3 signals were normalized to ACT1 signals. Values are displayed below the blots.

RNAPII-independent transcription of the short form of ASP3. Northern analysis of total RNA was performed under RNAPII inactivation. The rpb1Δ single copy–ASP3 cells containing either wild-type or rpb1-1 mutant plasmid were pre-grown at 23°C, and then shifted to 37°C for the indicated times. ACT1 is an RNAPII-dependent transcript, and scR1 is an RNAPIII-dependent transcript.

Intragenic transcription promotes full-length ASP3 gene expression in cis but not in trans. (A) Northern analysis of the single copy–ASP3 wild-type and iceΔ strains in ammonium-containing medium and under nitrogen starvation. The ASP3 and ncASP3 signals were normalized to that of ACT1 and are shown below as the average of three independent experiments. (B) Total RNAs were extracted from the wild-type strain containing the empty vector or ncASP3-expressing plasmid under the control of the ADH1 promoter in ammonium-containing medium and under nitrogen starvation. Following Northern blotting, the ASP3 and ncASP3 signals were normalized to the signal of ACT1. Values are displayed below the blots.

Model for the chromatin status at the ASP3 promoter in the presence and absence of intragenic transcription. The chromatin status in wild-type (left) and iceΔ (right) strains is shown under nitrogen-rich conditions (upper) and under nitrogen starvation (bottom). With the help of intragenic transcription (light-blue lines), the promoter region of ASP3 is immediately accessible for RNAPII upon nitrogen starvation. In the absence of intragenic transcription, the promoter region is more condensed, thus requiring chromatin-remodeling ATPase to enhance the accessibility of this region to RNAPII for ASP3 transcription (red lines) after nitrogen depletion.

5′ and 3′ RACE analyses of transcripts obtained in ammonium-containing medium or under nitrogen starvation. A schematic diagram shows the results of 5′ (upper arrows) and 3′ (lower bars) RACE from sequencing analyses. The intragenic transcript is initiated at position 658 nt of the ASP3 coding sequence and polyadenylated under both conditions.

Expression of the short transcript is regulated by an intragenic control element but not the TATA element of ASP3. (A) Reporter assays of the wild-type strain with an empty vector (pLG669Z-UASΔ) or a plasmid containing the ASP3(306-657) coding region upstream of the LacZ reporter gene (pLG669Z-P_ncASP3) were conducted under ammonium-containing or nitrogen starvation conditions. The β-galactosidase activities shown are means of three independent measurements. (B) Total RNAs were extracted from wild-type and asp3Δ mutant strains containing an empty vector (Vec), wild-type (ASP3), TATA box-deleted (tataΔ), or intragenic control element-deleted (iceΔ) expressing plasmid (pRS316 backbone) in ammonium-containing medium and under nitrogen starvation. The ASP3 and ncASP3 signals determined by Northern blotting were normalized to ACT1 and are displayed below the blots.

Disruption of the intragenic transcription hampers chromatin remodeling and transcriptional initiation of ASP3. (A) Schematic diagram of the ASP3 locus. The three regions used for PCR amplification in the ChIP assay are marked below. (B–D) ChIP analyses performed at the ASP3 locus. Samples were collected at the indicated time points after nitrogen starvation. Regions 1–3 used for the PCR amplification are indicated. (B) ChIP analyses of histone H3 occupancy. (C) ChIP analyses of H3K4me3 at the ASP3 locus. (D) Rate of RNAPII-mediated initiation of transcription at the TATA-box of ASP3 was assayed by ChIP, using antibody against Ser5-phosphorylated CTD. Data are shown as relative levels compared to signals measured at time 0. (E) Nuclear run-on analysis was conducted to detect the transcription rate of ASP3 in the wild-type and iceΔ strains after nitrogen starvation. Fragments of ACT1 and the multiple cloning sites of the pBSKS plasmid were used as positive and negative controls, respectively. The ASP3 signals were normalized to that of ACT1 and are shown below.