The human initiator is a distinct and abundant element that is precisely positioned in focused core promoters.

DNA sequence signals in the core promoter, such as the initiator (Inr), direct transcription initiation by RNA polymerase II. Here we show that the human Inr has the consensus of BBCA+1BW at focused promoters in which transcription initiates at a single site or a narrow cluster of sites. The analysis of 7678 focused transcription start sites revealed 40% with a perfect match to the Inr and 16% with a single mismatch outside of the CA+1 core. TATA-like sequences are underrepresented in Inr promoters. This consensus is a key component of the DNA sequence rules that specify transcription initiation in humans

RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.

Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide small interfering RNAs (siRNAs) that bind to ARGONAUTE 4 (AGO4) and target genomic regions for silencing. RdDM also requires non-coding RNAs transcribed by RNA polymerase V (Pol V) that probably serve as scaffolds for binding of AGO4-siRNA complexes. Here, we used a modified global nuclear run-on protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5' adenine found in many AGO4-bound 24-nucleotide siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10 U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expression of wild-type AGO4 in ago4/6/9 mutants was able to restore slicing of Pol V transcripts, but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), an elongation factor whose function is not well understood. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes

Diverse motif ensembles specify non-redundant DNA binding activities of AP-1 family members in macrophages

Mechanisms by which members of the AP-1 family of transcription factors play non-redundant biological roles despite recognizing the same DNA sequence remain poorly understood. To address this question, here we investigate the molecular functions and genome-wide DNA binding patterns of AP-1 family members in primary and immortalized mouse macrophages. ChIP-sequencing shows overlapping and distinct binding profiles for each factor that were remodeled following TLR4 ligation. Development of a machine learning approach that jointly weighs hundreds of DNA recognition elements yields dozens of motifs predicted to drive factor-specific binding profiles. Machine learning-based predictions are confirmed by analysis of the effects of mutations in genetically diverse mice and by loss of function experiments. These findings provide evidence that non-redundant genomic locations of different AP-1 family members in macrophages largely result from collaborative interactions with diverse, locus-specific ensembles of transcription factors and suggest a general mechanism for encoding functional specificities of their common recognition motif

Core promoter-specific gene regulation: TATA box selectivity and Initiator-dependent bi-directionality of serum response factor-activated transcription

Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter. Core promoters are diverse and may contain a variety of sequence elements such as the TATA box, the Initiator (INR), and the downstream promoter element (DPE) recognized, respectively, by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex. Core promoter elements contribute to the gene selectivity of enhancers, and INR/DPE-specific enhancers and activators have been identified. Here, we identify a TATA box-selective activating sequence upstream of the human β-actin (ACTB) gene that mediates serum response factor (SRF)-induced transcription from TATA-dependent but not INR-dependent promoters and requires the TATA-binding/bending activity of TBP, which is otherwise dispensable for transcription from a TATA-less promoter. The SRF-dependent ACTB sequence is stereospecific on TATA promoters but activates in an orientation-independent manner a composite TATA/INR-containing promoter. More generally, we show that SRF-regulated genes of the actin/cytoskeleton/contractile family tend to have a TATA box. These results suggest distinct TATA-dependent and INR-dependent mechanisms of TFIID-mediated transcription in mammalian cells that are compatible with only certain stereospecific combinations of activators, and that a TBP-TATA binding mechanism is important for SRF activation of the actin/cytoskeleton-related gene family

Evolution and Diversification of the Core Transcription Regulatory Network

The manifold species we marvel today reflect the diverse regulatory mechanisms facilitating differential gene expression and therefore cell differentiation. Transcription is the initial step in gene expression and three RNA polymerases (RNAPs) with reportedly non- overlapping functions reside in the human nucleus. All three RNAPs share one common ancestor, which may be reflected in the finding that RNAP III accurately initiates transcription from RNAP II promoters. These results propose a functional conservation among RNAP II & III and further imply polymerase specificity as not constant, but a variable that depends on promoter properties and transcription conditions. The regulation of gene expression was previously assumed to be primarily mediated by sequence-specific transcription factors. It has now become evident that also the basal transcription machinery greatly diversified during evolution. Independent duplications of the TATA-binding protein (TBP) yielded paralogs, termed TBP-related factors (TRFs) which support different transcription systems. For example, TRF2 but not TBP is required for TCT core promoter motif- dependent transcription in Drosophila melanogaster while TBP but not TRF2 is essential for the initiation of transcription from TATA box-dependent promoters. The observation that TBP family proteins each support distinct sets of transcriptional programs led to the proposal to refer to them as "system factors". Notably, subdivision of the transcription systems likely reduced constraints, thereby facilitating evolvability and ultimately specialization. One example for specialization was encountered by the functional characterization of the TCT motif that was found to be highly enriched at the promoters of genes involved in translation. These findings propose TCT-dependent transcription to constitute a conserved and specialized RNAP II system complementing RNAP I and III to ensure translation, thereby proving an example how distinct core promoter motifs and TRFs facilitates regulatory specialization. In addition, the directionality of the human transcription apparatus was analyzed which revealed core promoter and the basal transcription apparatus to be highly directional. Using DNase I accessibility defined promoter borders revealed that about half of the promoters are unidirectional. Reverse-oriented transcripts of bidirectional or divergent promoters originated from their own cognate reverse- directed core promoter. Unidirectional promoters are depleted of reverse core promoter sequences or associated chromatin features at their upstream edge