A specialized TFIIB is required for transcription of transposon-targeting noncoding RNAs

Abstract

Transposable elements (TEs) pose threats to genome stability. Therefore, small RNA-mediated heterochromatinization suppresses the transcription and hence the mobility of TEs. Paradoxically, transcription of noncoding RNA (ncRNA) from TEs is needed for the production of TE-targeting small RNAs and/or recruiting the silencing machinery to TEs. Hence, specialized RNA polymerase II (Pol II) regulators are required for such unconventional transcription in different organisms, including the developmental stage-specific Mediator complex (Med)-associated proteins in the ncRNA transcription from TE-related sequences in Tetrahymena. Yet it remains unclear how the Pol II transcriptional machinery is assembled at TE-related sequences for the ncRNA transcription. Here, we report that Pol II is regulated by Emit3, a stage-specific TFIIB-like protein specialized in TE transcription. Emit3 interacts with the TFIIH complex and localizes to TE-dense regions, especially at sites enriched with a G-rich sequence motif. Deletion of Emit3 globally abolishes Pol II-chromatin association in the meiotic nucleus, disrupts the chromatin binding of Med, and impairs the TE-biased localization of TFIIH. Conversely, Emit3's preferential localization to TE-rich loci relies in part on Med-associated proteins. These findings suggest that Emit3, TFIIH, and Med-associated proteins work together to initiate Pol II ncRNA transcription from TE-dense regions, possibly in a sequence-dependent manner.

Graphical Abstract

Figure 1.

Tetrahymena life cycle and the identification of Emit3. (A) Tetrahymena cells contain two distinct types of nuclei: the somatic macronucleus (MAC) and the germline micronucleus (MIC). The MAC has continuous transcriptional activity, while the MIC remains transcriptionally inactive until meiosis begins. TEs (depicted in magenta) are predominantly retained in the MIC but eliminated from the new MAC. During conjugation, when two cells of different mating types pair, TE-targeting ncRNAs are synthesized in the meiotic MIC. Meiotic prophase is categorized based on the extent of nuclear elongation. In stages II–IV, centromeres (Cen.) and telomeres (Tel.) are clustered at opposite poles of the elongating MIC. By stage V, these structures detach from the MIC termini, although telomeres remain clustered. The ncRNA transcription (indicated by dots) is initially concentrated in IES-rich telomeric and pericentromeric regions during stages II and III. In stages IV and V, transcription is restricted to a narrow telomeric region. (B) Current knowledge about the MIC ncRNA transcription activation mechanism. See the text for details. (C) MIC ncRNA transcription activators and meiosis regulators were grouped into different clusters with respect to the timing of gene expression. Hpm: Hours post mixing. (D) Upper panel: Sequence alignment shows that Emit3 and Tfiibl1 have a conserved zinc-ribbon motif. Tthe: Tetrahymena thermophila; Tmal: Tetrahymena malaccensis; Tell: Tetrahymena elliotti; Tbor: Tetrahymena borealis; Tvor: Tetrahymena vorax; Tsp: Tetrahymena sp.; Tepi: Tetrahymena empidokyrea; Tsha: Tetrahymena shanghaiensis; Tpar: Tetrahymena paravorax; Hasp: Homo sapiens; Atha: Arabidopsis thaliana; Scer: Saccharomyces cerevisiae. Lower panel: The N-terminal region contains a putative zinc-ribbon motif, with the positions of the zinc-binding cysteines indicated. The zinc-ribbon structure of Emit3 is similar to that of Pyrococcus furiosus TFIIB (PfTFIIB), as retrieved from the Protein Data Bank (PDB code: 1pft). Homology modeling of the C-terminal regions is presented in Supplementary Fig. S1. (E) DNA FISH analysis of REP2 IESs (magenta) in emit3Δ and WT cells harvested at 32 h after the induction of conjugation. REP2 IESs are retained in progeny MACs of the mutant. Scale bars: 10 μm. (F) The scnRNA (indicated by an arrowhead) was detected in WT cells but not in emit3Δ cells.

Figure 2.

Emit3 localizes to meiotic MIC and is essential for MIC ncRNA transcription. (A) Strand-specific Northern blotting was used to investigate transcripts from (+) and (−) strands of an MIC-specific DNA locus, known as the M-element. Transcripts were detected in WT and rib1Δ cells, and were accumulated in dcl1Δ cells due to the lack of ncRNA cleavage. In contrast, transcripts were not detected in emit1Δ, emit2Δ, or emit3Δ mutants. The expression of the conjugation-specific PDD1 gene was analyzed as a control for loading and developmental stages. (B) Immunostaining of dsRNA (and Rpb3) in WT and emit3Δ cells. Scale bar: 10 μm. (C) Upper panel: Costaining of endogenously expressed Emit3-HA (by immunostaining) and telomeric repeats (by FISH) in a stage IV meiotic cell. Lower panel: Immunostaining of endogenously expressed Tfiibl1-HA in a stage IV meiotic cell. (D) Costaining of Emit3-HA and Rpb3 (an RNA Pol II-specific subunit) was performed to assess their localization. Emit3 and Rpb3 colocalize in the meiotic MIC of Tetrahymena cells, fixed under a mild condition (i.e. conventional fixation). (E) Costaining of Emit3-HA and Rpb3 was performed to assess their localization. Emit3 and Rpb3 colocalize in the meiotic MIC, fixed under a harsh condition (i.e. pre-fixation detergent treatment, which removes nucleoplasmic proteins not tightly associated with chromatin). For technical details, see “Materials and methods” section. (F) Investigation of Emit3 localization in rib1Δ, emit1Δ, and emit2Δ mutants, which exhibit disrupted MIC ncRNA biogenesis. Scale bars: 10 μm.

Figure 3.

Emit3 is required for the localization of MIC ncRNA transcription regulators. (A) Immunostaining of endogenously expressed Emit1-HA in WT and emit3Δ cells that were fixed under different conditions. Arrowheads indicate eye-catching telomeric clustered regions in the MIC. Dashed lines indicate MIC termini. Scale bar: 10 μm. (B) Immunostaining of endogenously expressed Emit2-HA in WT and emit3Δ cells that were fixed under different conditions. Arrowheads indicate the telomeric clustered regions in the MIC. Dashed lines indicate MIC termini. Scale bar: 10 μm. (C) Immunostaining of endogenously expressed Rib1-HA in WT and emit3Δ cells. Telomeric repeats were stained using FISH. Arrowheads denote telomeric foci. Scale bar: 10 μm. (D) FISH staining of telomeric repeats in spo11Δ meiotic MIC. Scale bar: 10 μm. (E) The abundance profiles of scnRNAs on chromosome 2 of the meiotic MIC in WT and spo11Δ cells. BPM: Bins per million mapped reads.

Figure 4.

Emit3 is required for the association of the Pol II transcription machinery with meiotic chromatin. (A-B) Immunostaining of the endogenously expressed Pol II subunit, Rpb3, in WT and emit3Δ cells that were fixed under different conditions. Arrowheads indicate the telomeric clustered region in the MIC. Dashed lines indicate MIC termini. Scale bar: 10 μm. (C) A schematic diagram summarizing the Rpb3 immunostaining results. (D-E) Immunostaining of the endogenously expressed Med subunit, Med31, in WT and emit3Δ cells that were fixed under different conditions. Arrowheads indicate the eye-catching telomeric clustered region in the MIC. Dashed lines indicate MIC termini. Scale bar: 10 μm. (F) A schematic diagram summarizing the Med31 immunostaining results.

Figure 5.

Emit3 interacts with TFIIH through a zinc-ribbon and this interaction is required for recruiting Pol II to the MIC chromatin. (A) Interactions (depicted in blue) between Emit3 and TFIIH components were identified through immunoprecipitation. Components indicated by dashed lines are not present in Tetrahymena. The shape and arrangement of the complex subunits are modeled based on the human TFIIH core complex (PDB code: 6nmi). (B) Tetrahymena Rpb1 lacks both the conserved TFIIB-interacting dock domain and the canonical carboxy-terminal domain (CTD) that contains phosphorylatable heptad repeats. (C-D) Immunostaining of the endogenously expressed TFIIH complex subunit P34-HA in WT and emit3Δ cells that were fixed under different conditions. Arrowheads indicate the telomeric clustered region in the MIC. Dashed lines indicate MIC termini. Scale bars: 10 μm. (E) Costaining of P34-HA (using immunostaining) and telomeric repeats (using FISH) in a stage V meiotic cell. Arrowheads indicate the telomere-clustered region in the MIC. Scale bar: 10 μm. (F) Immunostaining of another endogenously expressed TFIIH complex subunit, Xpd1. Scale bar: 10 μm. (G) Immunostaining of endogenously expressed Emit3-WT-HA (i.e. unmodified Emit3) and Emit3-CS-HA (i.e. zinc ribbon mutated Emit3). Arrowheads indicate enriched Emit3-WT-HA at a pole of the elongated meiotic MIC. Scale bar: 10 μm. (H) Immunostaining of Rpb3 in Emit3-WT-HA and Emit3-CS-HA cells that were detergent-treated prior to fixation. Scale bar: 10 μm.

Figure 6.

Chromatin occupancy analysis revealed that Emit3 preferentially associates with meiotic scnRNA production regions. (A) Occupancy of Emit3 and the abundance profile of scnRNAs on the meiotic MIC chromosome 2 (left panel) and a representative region of chromosome 2 (right panel). Blue vertical lines indicate Emit3-HA peak summits, while magenta vertical lines mark scnRNA peak summits. BPM: Bins per million mapped reads. RPCG: Reads per genome coverage. (B) Profile visualization of Emit3 CUT&Tag reads distribution around scnRNA peak summits and an equivalent number of random sites in the MIC. (C) Chromatin occupancies of Emit3 (in WT and rib1Δ cells) and Emit3-CS (with a mutated zinc-ribbon) on the MIC chromosome 2. (D) Profile visualization of CUT&Tag reads distribution around the summit of scnRNA peaks, compared to an equivalent number of random sites in the MIC. (E) A consensus motif identified from the Emit3-bound sequences. (F) Profile visualization of CUT&Tag reads for Emit3 (in WT and rib1Δ cells), Emit3-CS, Rpb3 (in WT and emit3Δ cells), and Med31 (in WT and emit3Δ cells), alongside meiotic scnRNA reads. The data are centered around MIC sites containing the Emit3-bound AGGGSS motif. (G) Chromatin occupancies of Rpb3 (in WT and emit3Δ cells) on the MIC chromosome 2. (H) Profile visualization of Rpb3 CUT&Tag reads (in WT and emit3Δ cells) around scnRNA peak summits and an equivalent number of random sites in the MIC. (I) Chromatin occupancies of Med31 (in WT and emit3Δ cells) on the MIC chromosome 2. (J) Profile visualization of Med31 CUT&Tag reads (in WT and emit3Δ cells) around scnRNA peak summits and an equivalent number of random sites in the MIC. (K) Schematic summary of the MIC ncRNA transcription activation mechanism. Pol II, Med, and their regulators are imported to the MIC during meiosis. Subsequently, Emit3 mediates the recruitment of the Pol II transcription machinery to the G-rich regions of meiotic MIC chromatin. The engagement of Emit1, Emit2, Rib1, Med, and possibly also TFIIH stabilizes the transcription initiation complex and activates ncRNA transcription.