Transcription factors (TFs) are key players in evolution. Changes affecting their function can yield novel life forms but may also have deleterious effects. Consequently, gene duplication events that release one gene copy from selective pressure are thought to be the common mechanism by which TFs acquire new activities. Here, we show that LEAFY, a major regulator of flower development and cell division in land plants, underwent changes to its DNA binding specificity, even though plant genomes generally contain a single copy of the LEAFY gene. We examined how these changes occurred at the structural level and identify an intermediate LEAFY form in hornworts that appears to adopt all different specificities. This promiscuous intermediate could have smoothed the evolutionary transitions, thereby allowing LEAFY to evolve new binding specificities while remaining a single-copy gene.

Deciphering the mechanisms directing transcription factors (TFs) to specific genome regions is essential to understand and predict transcriptional regulation. TFs recognize short DNA motifs primarily through their DNA-binding domain. Some TFs also possess an oligomerization domain suspected to potentiate DNA binding but for which the genome-wide influence remains poorly understood. Here we focus on the LEAFY transcription factor, a master regulator of flower development in angiosperms. We have determined the crystal structure of its conserved amino-terminal domain, revealing an unanticipated Sterile Alpha Motif oligomerization domain. We show that this domain is essential to LEAFY floral function. Moreover, combined biochemical and genome-wide assays suggest that oligomerization is required for LEAFY to access regions with low-affinity binding sites or closed chromatin. This finding shows that domains that do not directly contact DNA can nevertheless have a profound impact on the DNA binding landscape of a TF.

RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5 ′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential "housekeeping" roles. Many tRNA genes were found to generate long, 3′ -extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3 ′ -extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5 ′ -exonuclease Rat1.[Supplemental material is available for this article.]Transcription of nuclear DNA in eukaryotes is carried out by at least three different RNA polymerases. RNA polymerase III (RNAPIII) is specialized for high-level synthesis of small noncoding RNAs. The most abundant products of RNAPIII-dependent transcription are the 5S rRNA and the many pre-tRNA species. In addition, RNAPIII synthesizes numerous, less abundant small RNAs that are involved in diverse cellular processes, including protein translocation and the processing of pre-rRNA and pre-tRNAs (Dieci et al. 2013). The nuclear genome of Saccharomyces cerevisiae contains 275 actively transcribed tRNA genes (including a tRNA of undetermined specificity [tX(XXX)D]). These are grouped into 20 isotypes, each charged with a single amino acid, which are subdivided into 41 isoacceptors that each recognize the same anticodon sequence(s) (Hani and Feldmann 1998; Chan and Lowe 2009). The reported lengths of the primary transcripts vary between 72 and 133 nucleotides (nt), and ∼25% of pre-tRNAs include introns. Primary pre-tRNA transcripts undergo 5 ′ and 3 ′ maturation and intron excision to generate the mature tRNAs. In tRNA genes, the transcription machinery recognizes conserved promoter elements, termed box A and box B, which are located within the transcribed region and form a bipartite binding site for the six-subunit basal transcription factor TFIIIC (Acker et al. 2013). Box A starts at position +8 of the mature tRNA, and the transcription start site is most frequently located 18-20 nt upstream (Dieci et al. 2013). Within yeast tRNA genes, boxes A and B are localized 31-93 nt apart and correspond to the universally conserved D-and T-loops in the tRNA structure. Internally located A and B boxes are...

SUMMARYThe transition to flowering in Arabidopsis is characterized by the sharp and localized upregulation of APETALA1 (AP1) transcription in the newly formed floral primordia. Both the flower meristem-identity gene LEAFY (LFY) and the photoperiod pathway involving the FLOWERING LOCUS T (FT) and FD genes contribute to this upregulation. These pathways have been proposed to act independently but their respective contributions and mode of interaction have remained elusive. To address these questions, we studied the AP1 regulatory region. Combining in vitro and in vivo approaches, we identified which of the three putative LFY binding sites present in the AP1 promoter is essential for its activation by LFY. Interestingly, we found that this site is also important for the correct photoperiodic-dependent upregulation of AP1. In contrast, a previously proposed putative FD-binding site appears dispensable and unable to bind FD and we found no evidence for FD binding to other sites in the AP1 promoter, suggesting that the FT/FD-dependent activation of AP1 might be indirect. Altogether, our data give new insight into the interaction between the FT and LFY pathways in the upregulation of AP1 transcription under long-day conditions.

SUMMARYIn indeterminate inflorescences, floral meristems develop on the flanks of the shoot apical meristem, at positions determined by auxin maxima. The floral identity of these meristems is conferred by a handful of genes called floral meristem identity genes, among which the LEAFY (LFY) transcription factor plays a prominent role. However, the molecular mechanism controlling the early emergence of floral meristems remains unknown. A body of evidence indicates that LFY may contribute to this developmental shift, but a direct effect of LFY on meristem emergence has not been demonstrated. We have generated a LFY allele with reduced floral function and revealed its ability to stimulate axillary meristem growth. This role is barely detectable in the lfy single mutant but becomes obvious in several double mutant backgrounds and plants ectopically expressing LFY. We show that this role requires the ability of LFY to bind DNA, and is mediated by direct induction of REGULATOR OF AXILLARY MERISTEMS1 (RAX1) by LFY. We propose that this function unifies the diverse roles described for LFY in multiple angiosperm species, ranging from monocot inflorescence identity to legume leaf development, and that it probably pre-dates the origin of angiosperms.

Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo. High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional posttranscriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity.

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Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In particular, pre-mRNA splicing was reported to be associated with slowed RNAPII elongation. Here, we identify a site of ubiquitination (K1246) in the catalytic subunit of RNAPII close to the DNA entry path. Ubiquitination was increased in the absence of the Bre5-Ubp3 ubiquitin protease complex. Bre5 binds RNA in vivo, with a preference for exon 2 regions of intron-containing pre-mRNAs and poly(A) proximal sites. Ubiquitinated RNAPII showed similar enrichment. The absence of Bre5 led to impaired splicing and defects in RNAPII elongation in vivo on a splicing reporter construct. Strains expressing RNAPII with a K1246R mutation showed reduced co-transcriptional splicing. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.