Systematic interrogation of human promoters.

Despite much research, our understanding of the architecture and cis-regulatory elements of human promoters is still lacking. Here, we devised a high-throughput assay to quantify the activity of approximately 15,000 fully designed sequences that we integrated and expressed from a fixed location within the human genome. We used this method to investigate thousands of native promoters and preinitiation complex (PIC) binding regions followed by in-depth characterization of the sequence motifs underlying promoter activity, including core promoter elements and TF binding sites. We find that core promoters drive transcription mostly unidirectionally and that sequences originating from promoters exhibit stronger activity than those originating from enhancers. By testing multiple synthetic configurations of core promoter elements, we dissect the motifs that positively and negatively regulate transcription as well as the effect of their combinations and distances, including a 10-bp periodicity in the optimal distance between the TATA and the initiator. By comprehensively screening 133 TF binding sites, we find that in contrast to core promoters, TF binding sites maintain similar activity levels in both orientations, supporting a model by which divergent transcription is driven by two distinct unidirectional core promoters sharing bidirectional TF binding sites. Finally, we find a striking agreement between the effect of binding site multiplicity of individual TFs in our assay and their tendency to appear in homotypic clusters throughout the genome. Overall, our study systematically assays the elements that drive expression in core and proximal promoter regions and sheds light on organization principles of regulatory regions in the human genome.

Core promoter sequence in yeast is a major determinant of expression level.

The core promoter is the regulatory sequence to which RNA polymerase is recruited and where it acts to initiate transcription. Here, we present the first comprehensive study of yeast core promoters, providing massively parallel measurements of core promoter activity and of TSS locations and relative usage for thousands of native and designed sequences. We found core promoter activity to be highly correlated to the activity of the entire promoter and that sequence variation in different core promoter regions substantially tunes its activity in a predictable way. We also show that location, orientation, and flanking bases critically affect TATA element function, that transcription initiation in highly active core promoters is focused within a narrow region, that poly(dA:dT) orientation has a functional consequence at the 3' end of promoters, and that orthologous core promoters across yeast species have conserved activities. Our results demonstrate the importance of core promoters in the quantitative study of gene regulation.

Systematic dissection of the sequence determinants of gene 3' end mediated expression control.

The 3'end genomic region encodes a wide range of regulatory process including mRNA stability, 3' end processing and translation. Here, we systematically investigate the sequence determinants of 3' end mediated expression control by measuring the effect of 13,000 designed 3' end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3' end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3' end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3' UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3'UTRs, confirming the role for this element in 3' end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3' end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3' end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3' end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.

Molecular dissection of the genetic mechanisms that underlie expression conservation in orthologous yeast ribosomal promoters.

Recent studies have shown a surprising phenomenon, whereby orthologous regulatory regions from different species drive similar expression levels despite being highly diverged in sequence. Here, we investigated this phenomenon by genomically integrating hundreds of ribosomal protein (RP) promoters from nine different yeast species into S. cerevisiae and accurately measuring their activity. We found that orthologous RP promoters have extreme expression conservation even across evolutionarily distinct yeast species. Notably, our measurements reveal two distinct mechanisms that underlie this conservation and which act in different regions of the promoter. In the core promoter region, we found compensatory changes, whereby effects of sequence variations in one part of the core promoter were reversed by variations in another part. In contrast, we observed robustness in Rap1 transcription factor binding sites, whereby significant sequence variations had little effect on promoter activity. Finally, cases in which orthologous promoter activities were not conserved could largely be explained by the sequence variation within the core promoter. Together, our results provide novel insights into the mechanisms by which expression is conserved throughout evolution across diverged promoter sequences.

Sequence features of yeast and human core promoters that are predictive of maximal promoter activity.

The core promoter is the region in which RNA polymerase II is recruited to the DNA and acts to initiate transcription, but the extent to which the core promoter sequence determines promoter activity levels is largely unknown. Here, we identified several base content and k-mer sequence features of the yeast core promoter sequence that are highly predictive of maximal promoter activity. These features are mainly located in the region 75 bp upstream and 50 bp downstream of the main transcription start site, and their associations hold for both constitutively active promoters and promoters that are induced or repressed in specific conditions. Our results unravel several architectural features of yeast core promoters and suggest that the yeast core promoter sequence downstream of the TATA box (or of similar sequences involved in recruitment of the pre-initiation complex) is a major determinant of maximal promoter activity. We further show that human core promoters also contain features that are indicative of maximal promoter activity; thus, our results emphasize the important role of the core promoter sequence in transcriptional regulation.

Modeling interactions between adjacent nucleosomes improves genome-wide predictions of nucleosome occupancy.

MOTIVATION: Understanding the mechanisms that govern nucleosome positioning over genomes in vivo is essential for unraveling the role of chromatin organization in transcriptional regulation. Until now, models for predicting genome-wide nucleosome occupancy have assumed that the DNA associations of neighboring nucleosomes on the genome are independent. We present a new model that relaxes this independence assumption by modeling interactions between adjacent nucleosomes. RESULTS: We show that modeling interactions between adjacent nucleosomes improves genome-wide nucleosome occupancy predictions in an in vitro system that includes only nucleosomes and purified DNA, where the resulting model has a preference for short spacings (linkers) of less than 20 bp in length between neighboring nucleosomes. Since nucleosome occupancy in vitro depends only on properties intrinsic to nucleosomes, these results suggest that the interactions we find are intrinsic to nucleosomes and do not depend on other factors, such as transcription factors and chromatin remodelers. We also show that modeling these intrinsic interactions significantly improves genome-wide predictions of nucleosome occupancy in vivo. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A feature-based approach to modeling protein-DNA interactions.

Transcription factor (TF) binding to its DNA target site is a fundamental regulatory interaction. The most common model used to represent TF binding specificities is a position specific scoring matrix (PSSM), which assumes independence between binding positions. However, in many cases, this simplifying assumption does not hold. Here, we present feature motif models (FMMs), a novel probabilistic method for modeling TF-DNA interactions, based on log-linear models. Our approach uses sequence features to represent TF binding specificities, where each feature may span multiple positions. We develop the mathematical formulation of our model and devise an algorithm for learning its structural features from binding site data. We also developed a discriminative motif finder, which discovers de novo FMMs that are enriched in target sets of sequences compared to background sets. We evaluate our approach on synthetic data and on the widely used TF chromatin immunoprecipitation (ChIP) dataset of Harbison et al. We then apply our algorithm to high-throughput TF ChIP data from mouse and human, reveal sequence features that are present in the binding specificities of mouse and human TFs, and show that FMMs explain TF binding significantly better than PSSMs. Our FMM learning and motif finder software are available at http://genie.weizmann.ac.il/.