Yeast strains are described in Supplementary Tables S1. A total of 15 plasmids (named d5-d70 and dT₇) were derived from the plasmid pCS6 (Supplementary Figures S1A, B and Supplementary Table S2). Three of them, d25, d35, and d70 were not used for most of the experiments because of very low transcription from them. The histone H4 depletion strain UKY 403 and control strain MHY308 (gifts from Michael Grunstein) were grown till 0.8 OD_600nm in YEPGal and then in YEPD for 3 h before harvesting and RNA extraction as described earlier (Arimbasseri and Bhargava 2008).

Yeast Nhp6A and B were HA-tagged at the C-terminal using the PCR toolbox (Janke et al., 2004). Both strains were used to measure Nhp6 occupancy over SNR6 by using the ChIP and real-time PCR method (Arimbasseri and Bhargava 2008) as described earlier.

The recombinant Nhp6A protein, with the N-terminal 6XHis-tag, was purified using an overexpression clone (gift from David Stillman, United States). The chromatin was assembled using the well-established Drosophila embryonic S-190 extract system, which gives equally spaced nucleosomal arrays over plasmids (Shivaswamy et al., 2004). The in vitro transcription using lab stocks of pure proteins TFIIIC, pol III, and recombinant TFIIIB was carried out as described in detail earlier and the transcripts were visualized by the primer-extension method (Shivaswamy et al., 2004). All transcript yields were normalized with corresponding levels from pCS6 in each experiment. At least three or more independent experiments were performed for all the measurements. The p-values were calculated by two-tailed Student’s t-test.

A positioned nucleosome between boxes A and B of the SNR6 gene was shown to enable the binding of TFIIIC and high transcriptional activation in vitro (Shivaswamy et al., 2004). The nucleosome positioned upstream (US) of the TATA box is regulatory in nature (Arimbasseri and Bhargava 2008), where it also blocks the 5′end of a solo δ element (YLRCdelta5) (Figure 1A). The PIC assembly occurs in the NFR, which encompasses the TATA box, TSS, T₇ element, and box A (Figure 1A). We had earlier reported the loss of the overall chromatin organization at the SNR6 locus under histone depletion conditions (Arimbasseri and Bhargava 2008). We found that under this condition, U6 snRNA levels are significantly reduced whereas the upstream solo δ element (pol II transcribed) is activated (Figures 1B–D), confirming that SNR6 transcription requires a properly configured chromatin organization in vivo.

Nhp6, a protein belonging to the HMG1 class has two 89% identical isoforms in yeast Nhp6A and B (Stillman 2010). The Nhp6 presence has been reported earlier on the SNR6 and some tRNA genes in vivo (Braglia et al., 2007). Our Nhp6 occupancy measurements by the ChIP and real-time PCR method found a similar enrichment of Nhp6A and Nhp6B on the TATA box and A–B box region of the SNR6 gene locus (Figure 1E). Under starvation, the repression of pol III transcription (Moir and Willis 2013) is found to be accompanied by increased occupancy of specifically Nhp6A and a small loss of Nhp6B on the SNR6 gene (Figure 1E). This suggests a repressive role of Nhp6A and a differential, non-redundant role of the Nhp6 isoforms on the SNR6 gene.

The deletion of 30–45 bp resulted in partial removal whereas longer deletions of 50 bp upwards in the complete removal of the pseudoA boxes (Supplementary Table S2). As expected, reducing the distance between the binding sites of TFIIIC resulted in somewhat periodic up and down levels of transcription (Supplementary Figure S1C), which reflect the changing helical phase of the DNA with deletions in 5 bp steps. TFIIIC- in dependent transcription was lowest for d15, d30, d60, and d65, while d10 and d55 were higher than pCS6 with/without TFIIIC (Figure 2A). The rest of the deletion clone NDs could be similarly transcribed with/without TFIIIC addition, staying below the pCS6 level (Figure 2A). This is not surprising since the transcription of SNR6 ND is TFIIIC-independent. TFIIIC is known to slightly inhibit the naked pCS6 transcription. However, deleting one helical turn immediately next to the pseudoA boxes in the d10 plasmid gives ∼1.5-fold gain of transcription, whereas deletions of 5 bp or more than 15 bp up to 50 bp deletion, return only ∼60–90% of pCS6 transcription levels (Figure 2A). Interestingly, the transcription of d55–d65 increases to more than pCS6 levels with TFIIIC (Figure 2B, Supplementary Figure S1C, D). More than 50 bp deletions may reduce the linear distance between A and B boxes, but also constrain the steric flexibility of the intervening DNA, turning them out of phase on looping. Accordingly, d40–d55 are similarly transcribed with/without TFIIIC, and TFIIIC-dependent transcription increases for d55 whereas TFIIIC-independent transcription decreases for d60 and d65 with respect to the pCS6 level (Figures 2A,B, Supplementary Figures S1C, D). The increase on longer deletions with the deletion of the pseudoA boxes (Supplementary Figure S1A; Supplementary Table S2), suggests that the reduced TFIIIC-dependent transcription of pCS6 could be due to the sequestration of TFIIIC by the pseudoA boxes.

The chromatin transcription of the deletion clones with and without TFIIIC showed an undulating pattern (Figures 2B–D, Supplementary Figures S1E, F) with a gradual decrease of TFIIIC-dependent transcriptional activation (Figure 2D, Supplementary Figure S1E). A decrease in transcription was seen followed by an increase with every 5 bp deletion in the next step up to 50 bp deletions. As a 5 bp deletion reduces the distance from optimal to less optimal, boxes A and B also fall out of phase with each other. With the next 5bp deletion, the boxes may again come in phase, resulting in a gain of transcription, although not to the original level. Therefore, an alternating decrease and increase suggests a change in the phase as the reason behind the pattern, which could directly influence the simultaneous binding of the multi-subunit TFIIIC to its two widely separated binding sites.

Earlier studies reported that a 42-bp deletion between the terminator and B box (Δ42) reduces transcription from SNR6 more than an 84-bp deletion (Δ84) could (Eschenlauer et al., 1993). In agreement with this, transcription was found at very low levels when 20–40 bp were deleted (Figures 2C,D), with the lowest observed levels from the d40 plasmid (Supplementary Figure S1E). Moreover, although TFIIIC-independent transcription increased with further deletions, the TFIIIC-dependent chromatin transcription remained lower than the pCS6 level (Figures 2B,D, Supplementary Figure S1F). In the absence of TFIIIC, the highest transcription was seen from d50, on both the chromatin and ND, but the highest activation was on the d45 chromatin (Figure 2D, Supplementary Figure S1E). Surprisingly, transcription from the d20, d30, and d50–d65 chromatin remains repressed with TFIIIC addition, suggesting severe compromise of TFIIIC binding to these templates (Figures 2B–D, Supplementary Figure S1E). The aforementioned results show a very subtle effect of the intervening DNA in the transcription of SNR6 according to the gap length, DNA phase, and hence, the orientation of TFIIIC binding sites as discussed later. The results agree with earlier studies suggesting TFIIIC-dependence of transcriptional activation by Nhp6.

Nhp6 showed a dose-dependent effect on the in vitro chromatin transcription of a tRNA gene (Mahapatra et al., 2011). We found that the addition of 60 ng Nhp6 activated two of the templates, d40 and d45, more than two-fold (Supplementary Figure S2A). As the Nhp6 amount is increased further, chromatin activation in the presence of TFIIIC is reduced (Supplementary Figure S2A), suggesting that Nhp6 influences SNR6 transcription in a dose-dependent manner. Nhp6 is reported to work through the stabilization of the TFIIIC-DNA complex (Kassavetis and Steiner 2006), which is essential for chromatin transcription. Much of the non-specific transcription from naked pCS6 is suppressed in the presence of TFIIIC or Nhp6, which together increased the initiation from the +1 site (Figure 3A, lanes 1–5). Similar to previous reports (Lopez et al., 2001), Nhp6 gave a 1.5- to 2-fold increase of the naked pCS6 transcription but inhibited d5-d15 ND or chromatin transcription with/without TFIIIC (Figures 3A,B, Supplementary Figure S2B). ND transcription of SNR6 with further deletions could not be enhanced by Nhp6 without/with TFIIIC addition (Figure 3C, Supplementary Figures S2C, D). Surprisingly, Nhp6 activated d50 and d65 ND transcription by ∼2- to 2.5-fold in the presence of TFIIIC (Figure 3C, Supplementary Figure S2C); their chromatin form is not activated by TFIIIC (Figure 2D).

No activation of chromatin by Nhp6 could be seen in the absence of TFIIIC (Figures 3A–C). TFIIIC binding to the repressed SNR6 chromatin results in its high activation (Shivaswamy et al., 2004). On pCS6, ∼10-fold TFIIIC-dependent activation of transcription is inhibited to ∼2.4-fold with Nhp6 addition (Figure 3D, lanes 5 and 6). As compared with the pCS6 chromatin, comparatively lower activation with TFIIIC (Figure 2) is further reduced on shorter deletion clones by Nhp6 (Figures 3A,B). While on longer deletion clones, Nhp6 addition to the d40, d45, and d60 chromatin reduced the TFIIIC-dependent activation to almost pCS6 level, and d30, d50, and d65 were unaffected (Figures 3B–D). One reason for the observed differences in the Nhp6 effect on the longer deletion clones (Figure 3B) could be the differential effects of Nhp6 on their ND transcription (Supplementary Figure S2C).

We also noticed that chromatin formation on the deletion plasmids suppressed the +1 transcription, giving a downstream initiated transcript instead, which is seen in all the conditions (Figure 3D, chromatin lanes). On the d50 plasmid, which showed the lowest (of all longer deletion clones) activation of chromatin transcription with TFIIIC (Figures 2B, 3C, Supplementary Figure S2E), Nhp6 addition could not restore the transcription from the right TSS (+1 transcript). The persistence of downstream initiation of transcription from the +7 bp position suggests altered TFIIIC and hence, TFIIIB placement upstream, which has been earlier suggested as the cause of different TSS selections in TATA box–A box double mutants (Eschenlauer et al., 1993).

The aforementioned results demonstrate that the Nhp6 effect is stronger on longer deletion clones where the pseudoA boxes are deleted and it generally represses the chromatin transcription in a TFIIIC-dependent manner. It appears that the pseudoA boxes may be serving as a guide to TFIIIC for binding to the upstream, right A boxes. Therefore, with a perturbation in TFIIIC binding in their absence, chromatin activation and right +1 site selection are both compromised on the plasmids d45–d65.

The T₇ promoter element, positioned between the TATA box and TSS is reported to co-operate with Nhp6 in the transcriptional activation of yeast SNR6 (Martin et al., 2001). The chromatin transcription shows higher sensitivity to Nhp6 levels (Figure 3). Nhp6 clearly showed stronger inhibition of pCS6 than dT₇ transcription in a dose-dependent manner (Supplementary Figure S3), suggesting that the T₇ sequence may not be required for normal transcription but enhances the effects of Nhp6 on SNR6. In the presence of TFIIIC, Nhp6 suppresses the downstream transcription initiation from the pCS6 chromatin and dT₇ ND templates (Figure 4A, lanes 6 vs. 10 and 11 vs. 15). Consistent with the previously reported role of Nhp6 in increasing the transcriptional fidelity of PoI III on tRNA genes (Kassavetis and Steiner 2006; Mahapatra et al., 2011), Nhp6 could abolish downstream initiation of the pCS6 ND and chromatin. In contrast, transcription was completely inhibited by Nhp6 on the dT₇ chromatin (Figure 4A), suggesting a role for T₇ deletion in the chromatin repression. Thus, apart from the reported roles of TATA and A boxes (Gerlach et al., 1995), the T₇ stretch promoter element may also have a role in the TSS selection and TFIIIC binding.

As compared with ∼1.9-fold Nhp6-dependent activation of ND transcription on pCS6, T₇ disruption returned only ∼1.25-fold (p < 0.1) activation in the presence of TFIIIC (Figure 4B). With respect to pCS6+TFIIIC, ∼0.7-fold (p = 0.0082) activation for dT₇ -TFIIIC resulted in repression (Figure 4B). TFIIIC absence and T₇ disruption influence Nhp6 similarly. Additive effects of the three components demonstrate that both Nhp6 and the T₇ element co-operate with TFIIIC to activate transcription on ND.

The Nhp6 effects on the TFIIIC-dependent dT₇ chromatin and ND transcription activation were opposite. The T₇ disruption gave ∼2.7-fold gain (p = 0.006) of transcription in the absence of Nhp6 (Figure 4C), whereas Nhp6 addition significantly reduced this gain (cf. pCS6 and dT₇, Nhp6+ condition, Figure 4C) to only ∼1.6-fold (p = 0.028), indicating a reduced TFIIIC binding to the dT₇ chromatin. Consistent with an earlier report (Martin et al., 2001), the results show that Nhp6 requires the T₇ element to manifest its influence fully on the transcription of SNR6.

The aforementioned results show that the T₇ sequence regulates the dose-dependent effects of Nhp6 on the TFIIIC-dependent chromatin transcription of the SNR6 gene. Taken together, this study has demonstrated that reducing the distance by short 5–40 bp deletions between the terminator and box B does not improve transcription; a longer deletion including extragenic pseudoA boxes does. Chromatin transcription from yeast SNR6 is activated at lower and repressed at higher Nhp6 levels. Nhp6 increases transcription fidelity by abolishing non-canonical initiations in favor of +1 transcription. This transcriptional activation depends on TFIIIC and the cis promoter element T₇ stretch. Occupancy of specifically the Nhp6A isoform on the gene goes up under repression, attributing a repressive role to Nhp6 in keeping the highly active SNR6 gene expression under check in vivo.

Reducing the distance between the terminator and box B may constrain the TFIIIC binding whenever the A–B boxes do not fall in phase. For a particular DNA sequence wound over the nucleosome surface, rotational positioning decides the DNA phase accessible for a DNA-binding factor (Albert et al., 2007), while proximity of the two far apart binding sites may become possible by the looping out of intervening naked DNA (Bhargava and Chaterji, 1992) and winding of nucleosomal DNA (Pusarla et al., 2007). The SNR6 gene sequence directs the assembly of nucleosomes with unique rotational settings on the whole gene (Vinayachandran et al., 2009). The nucleosome between boxes A and B, which is both rotationally and translationally placed on the gene body, gives a clear 145-bp nucleosomal footprint (Shivaswamy and Bhargava 2006). Considering the possibility of change in this position with reduced spacing, the nucleosome may or may not support TFIIIC binding and interaction with the TFIIIB upstream. Our earlier measurements on a template with multiple operator sites for the binding of a lac repressor found that for a nucleosome to translationally and symmetrically position between two protein-binding sites, a minimum of 165 bp should be freely available such that a 145-bp core DNA length leaves 10 bp free DNA room from the protein binding sites at both ends (Pusarla et al., 2007). Therefore, we predict that the nucleosome position between A and B boxes may remain unaltered till 35 bp deletions, while on d40, d45, and d50 it may be difficult to fit in, which may hamper the juxtaposing of the boxes. This nay result in inefficient TFIIIC binding and loss of transcription, as observed in this study.

Further deletions may either include the A/B boxes in the core DNA wound over the nucleosome making TFIIIC/B binding non-productive, or the TFIIIC binding may exclude the nucleosome, alleviating the chromatin repression. The increase in TFIIIC-dependent ND transcription on d55, d60, and d65 plasmids may be explained by the absence of the interfering pseudoA boxes, whereas the opposite results on the chromatin may be the outcome of two effects. First, the TFIIIC binding may lead to nucleosome exclusion but a steric obstruction of the gene body may reduce transcription. Alternatively, in the TFIIIC absence, the nucleosome may be found only downstream of the +85 bp position, as seen earlier in vivo (Marsolier et al., 1995). This would enable the gene to be transcribed as naked DNA, without chromatin repression.

The chromatin footprint on SNR6 in a strain with deletion of 42 bp between boxes A and B, was found similar to that in a strain with a lethal point mutation on box B (Gerlach et al., 1995). The recognition of box A by TFIIIC in SNR6 is reported to be an inefficient step during transcription complex assembly in vitro (Gerlach et al., 1995) and Nhp6A is shown to stabilize the TFIIIC-box A interaction (Kassavetis and Steiner 2006). A positive effect of Nhp6 specifically on SNR6 transcription and synthetic lethality of Nhp6 with a 42 bp deletion between the terminator and box B, reducing the distance between boxes A and B to the near subnucleosomal size (Kruppa et al., 2001; Lopez et al., 2001; Martin et al., 2001), synthetic lethality with SNR6 TATA box mutations (Gerlach et al., 1995), and nhp6ΔΔ condition (Martin et al., 2001), all could be explained by increased TFIIIC binding and single-round transcription with Nhp6 addition to SNR6 in vitro (Kruppa et al., 2001). The in vivo chromatin structure altered around the TATA box region of SNR6 in the nhp6ΔΔ cells was taken as an indication of altered TFIIIB binding, which could be a reason for the transcriptional repression of SNR6 (Lopez et al., 2001). Therefore, the reduced transcription in deletion clones could be due to a loss or non-productive TFIIIC/TFIIIB binding to SNR6. This may be the reason that earlier a deletion of 42 bp between the terminator and box B showed synthetic lethality with several other promoter mutations in SNR6 (Gerlach et al., 1995).

The T₇ mutations do not abolish the TFIIIB footprint but show lethality in the absence of Nhp6 (Martin et al., 2001). While the TATA box and T₇ stretch are found near the exit point of DNA in the US nucleosome, box A sits close to the DNA entry spot in the A–B box nucleosome. Nhp6 is generally found in the NFR near the entry/exit points of nucleosomal DNA (Dowell et al., 2010) and Nhp6A/B can cause looping and bending of DNA by at least 90° (Paull and Johnson 1995). Together, these observations raise the possibility that Nhp6 might be recruited to the T₇ stretch, just upstream of TSS and stabilize the TFIIIC interaction with box A in turn. This is consistent with the highest association of Nhp6 with TFIIIC, out of all the components of the pol III transcription complex (Bhalla et al., 2019; Shukla et al., 2021). The inherent rigidity of a stretch of T’s confers inflexibility to DNA, which may allow their presence only at the entry/exit or dyad axis positions in the nucleosome. Thus, T₇ may interfere with the encroachment of NFR by the US regulatory nucleosome on SNR6 (Arimbasseri and Bhargava 2008).

Transcription was found refractory to ∼300 ng Nhp6, whereas after saturation at ∼100 ng, higher Nhp6 additions inhibited chromatin transcription (Mahapatra et al., 2011). No transcription inhibition of SNR6 ND was seen even up to 500-ng Nhp6 addition (Kruppa et al., 2001), whereas we found more than 180 ng Nhp6 as inhibitory for chromatin transcription in vitro. At lower levels, it caused even activation by enhancing the +1 transcription initiation. The requirement of both the upstream T₇ stretch and TFIIIC for transcription activation by Nhp6 implies a balancing role for the T₇ element in the dose-dependent effects of Nhp6. As Nhp6A is an abundant, non-sequence-specific DNA-binding protein, its effects may easily be dose-dependent in vivo. Increased Nhp6A occupancy on the SNR6 gene under repression is consistent with a role for Nhp6 in further establishing the repressed chromatin state of SNR6.

Yeast SNR6 is regulated by its unique chromatin organization and targeted by a plethora of epigenetic regulatory complexes (Bhargava 2013). This study shows that TFIIIC sequestration by pseudoA boxes, difficulty in chromatin formation, or TFIIIC binding due to distance/phase differences between A and B boxes also influence the SNR6 transcription. The effects are individually small but subtle and significant when together. Enhancing transcription activation on the SNR6 chromatin by Nhp6 is the outcome of a combined influence of TFIIIC and the T₇ element on chromatin transcription while T₇ stretch also affects TFIIIC binding. It appears that every part of the SNR6 gene sequence has evolved with a unique role in fine-tuning its chromatin expression levels, making SNR6 a specific target for Nhp6.