Analysis of Genetically Diverse Macrophages Reveals Local and Domain-wide Mechanisms that Control Transcription Factor Binding and Function.

Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of ∼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.

From promoter motif to cardiac function: A single DPE motif affects transcription regulation and organ function in vivo.

Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7bp substitution mutation results in massive perturbation of the Tinman regulatory network orchestrating dorsal musculature, manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation.

Yeast derlin Dfm1 employs a chaperone-like function to resolve misfolded membrane protein stress.

Protein aggregates are a common feature of diseased and aged cells. Membrane proteins comprise a quarter of the proteome, and yet, it is not well understood how aggregation of membrane proteins is regulated and what effects these aggregates can have on cellular health. We have determined in yeast that the derlin Dfm1 has a chaperone-like activity that influences misfolded membrane protein aggregation. We establish that this function of Dfm1 does not require recruitment of the ATPase Cdc48 and it is distinct from Dfm1's previously identified function in dislocating misfolded membrane proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Additionally, we assess the cellular impacts of misfolded membrane proteins in the absence of Dfm1 and determine that misfolded membrane proteins are toxic to cells in the absence of Dfm1 and cause disruptions to proteasomal and ubiquitin homeostasis.

The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Membrane Proteins.

Endoplasmic reticulum (ER)-associated degradation (ERAD) removes misfolded proteins from the ER membrane and lumen by the ubiquitin-proteasome pathway. Retrotranslocation of ubiquitinated substrates to the cytosol is a universal feature of ERAD that requires the Cdc48 AAA-ATPase. Despite intense efforts, the mechanism of ER exit, particularly for integral membrane (ERAD-M) substrates, has remained unclear. Using a self-ubiquitinating substrate (SUS), which undergoes normal retrotranslocation independently of known ERAD factors, and the new SPOCK (single plate orf compendium kit) micro-library to query all yeast genes, we found the rhomboid derlin Dfm1 was required for retrotranslocation of both HRD and DOA ERAD pathway integral membrane substrates. Dfm1 recruited Cdc48 to the ER membrane with its unique SHP motifs, and it catalyzed substrate extraction through its conserved rhomboid motifs. Surprisingly, dfm1Δ can undergo rapid suppression, restoring wild-type ERAD-M. This unexpected suppression explained earlier studies ruling out Dfm1, and it revealed an ancillary ERAD-M retrotranslocation pathway requiring Hrd1.

ElemeNT 2023: an enhanced tool for detection and curation of core promoter elements.

MOTIVATION: Prediction and identification of core promoter elements and transcription factor binding sites is essential for understanding the mechanism of transcription initiation and deciphering the biological activity of a specific locus. Thus, there is a need for an up-to-date tool to detect and curate core promoter elements/motifs in any provided nucleotide sequences. RESULTS: Here, we introduce ElemeNT 2023-a new and enhanced version of the Elements Navigation Tool, which provides novel capabilities for assessing evolutionary conservation and for readily evaluating the quality of high-throughput transcription start site (TSS) datasets, leveraging preferential motif positioning. ElemeNT 2023 is accessible both as a fast web-based tool and via command line (no coding skills are required to run the tool). While this tool is focused on core promoter elements, it can also be used for searching any user-defined motif, including sequence-specific DNA binding sites. Furthermore, ElemeNT's CORE database, which contains predicted core promoter elements around annotated TSSs, is now expanded to cover 10 species, ranging from worms to human. In this applications note, we describe the new workflow and demonstrate a case study using ElemeNT 2023 for core promoter composition analysis of diverse species, revealing motif prevalence and highlighting evolutionary insights. We discuss how this tool facilitates the exploration of uncharted transcriptomic data, appraises TSS quality, and aids in designing synthetic promoters for gene expression optimization. Taken together, ElemeNT 2023 empowers researchers with comprehensive tools for meticulous analysis of sequence elements and gene expression strategies. AVAILABILITY AND IMPLEMENTATION: ElemeNT 2023 is freely available at https://www.juven-gershonlab.org/resources/element-v2023/. The source code and command line version of ElemeNT 2023 are available at https://github.com/OritAdato/ElemeNT. No coding skills are required to run the tool.

Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.

Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique reporter inserts, we found that short (153 bp) MPRA promoter inserts replicate the endogenous TSS patterns of ∼60% of promoters. Lentiviral reporter chromatinization did not improve fidelity of TSS-MPRA initiation patterns, and increasing insert size frequently led to activation of extraneous TSS in the MPRA that are not active in vivo. We discuss the implications of our findings, which highlight important caveats when using MPRAs to study transcription mechanisms. Finally, we illustrate how TSS-MPRA and WIP scoring can provide novel insights into the impact of transcription factor motif mutations and genetic variants on TSS patterns and transcription levels.

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.

Nascent transcription reveals regulatory changes in extremophile fishes inhabiting hydrogen sulfide-rich environments.

Regulating transcription allows organisms to respond to their environment, both within a single generation (plasticity) and across generations (adaptation). We examined transcriptional differences in gill tissues of fishes in the Poecilia mexicana species complex (family Poeciliidae), which have colonized toxic springs rich in hydrogen sulfide (H2S) in southern Mexico. There are gene expression differences between sulfidic and non-sulfidic populations, yet regulatory mechanisms mediating this gene expression variation remain poorly studied. We combined capped-small RNA sequencing (csRNA-seq), which captures actively transcribed (i.e. nascent) transcripts, and messenger RNA sequencing (mRNA-seq) to examine how variation in transcription, enhancer activity, and associated transcription factor binding sites may facilitate adaptation to extreme environments. csRNA-seq revealed thousands of differentially initiated transcripts between sulfidic and non-sulfidic populations, many of which are involved in H2S detoxification and response. Analyses of transcription factor binding sites in promoter and putative enhancer csRNA-seq peaks identified a suite of transcription factors likely involved in regulating H2S-specific shifts in gene expression, including several key transcription factors known to respond to hypoxia. Our findings uncover a complex interplay of regulatory processes that reflect the divergence of extremophile populations of P. mexicana from their non-sulfidic ancestors and suggest shared responses among evolutionarily independent lineages.

DNA methylation-linked chromatin accessibility affects genomic architecture in Arabidopsis.

DNA methylation is a major epigenetic modification found across species and has a profound impact on many biological processes. However, its influence on chromatin accessibility and higher-order genome organization remains unclear, particularly in plants. Here, we present genome-wide chromatin accessibility profiles of 18 Arabidopsis mutants that are deficient in CG, CHG, or CHH DNA methylation. We find that DNA methylation in all three sequence contexts impacts chromatin accessibility in heterochromatin. Many chromatin regions maintain inaccessibility when DNA methylation is lost in only one or two sequence contexts, and signatures of accessibility are particularly affected when DNA methylation is reduced in all contexts, suggesting an interplay between different types of DNA methylation. In addition, we found that increased chromatin accessibility was not always accompanied by increased transcription, suggesting that DNA methylation can directly impact chromatin structure by other mechanisms. We also observed that an increase in chromatin accessibility was accompanied by enhanced long-range chromatin interactions. Together, these results provide a valuable resource for chromatin architecture and DNA methylation analyses and uncover a pivotal role for methylation in the maintenance of heterochromatin inaccessibility.

Human promoters are intrinsically directional.

Divergent transcription, in which reverse-oriented transcripts occur upstream of eukaryotic promoters in regions devoid of annotated genes, has been suggested to be a general property of active promoters. Here we show that the human basal RNA polymerase II transcriptional machinery and core promoter are inherently unidirectional and that reverse-oriented transcripts originate from their own cognate reverse-directed core promoters. In vitro transcription analysis and mapping of nascent transcripts in HeLa cells revealed that sequences at reverse start sites are similar to those of their forward counterparts. The use of DNase I accessibility to define proximal promoter borders revealed that about half of promoters are unidirectional and that unidirectional promoters are depleted at their upstream edges of reverse core promoter sequences and their associated chromatin features. Divergent transcription is thus not an inherent property of the transcription process but rather the consequence of the presence of both forward- and reverse-directed core promoters.

Identification and dynamic quantification of regulatory elements using total RNA.

The spatial and temporal regulation of transcription initiation is pivotal for controlling gene expression. Here, we introduce capped-small RNA-seq (csRNA-seq), which uses total RNA as starting material to detect transcription start sites (TSSs) of both stable and unstable RNAs at single-nucleotide resolution. csRNA-seq is highly sensitive to acute changes in transcription and identifies an order of magnitude more regulated transcripts than does RNA-seq. Interrogating tissues from species across the eukaryotic kingdoms identified unstable transcripts resembling enhancer RNAs, pri-miRNAs, antisense transcripts, and promoter upstream transcripts in multicellular animals, plants, and fungi spanning 1.6 billion years of evolution. Integration of epigenomic data from these organisms revealed that histone H3 trimethylation (H3K4me3) was largely confined to TSSs of stable transcripts, whereas H3K27ac marked nucleosomes downstream from all active TSSs, suggesting an ancient role for posttranslational histone modifications in transcription. Our findings show that total RNA is sufficient to identify transcribed regulatory elements and capture the dynamics of initiated stable and unstable transcripts at single-nucleotide resolution in eukaryotes.

TRF2 and the evolution of the bilateria.

The development of a complex body plan requires a diversity of regulatory networks. Here we consider the concept of TATA-box-binding protein (TBP) family proteins as "system factors" that each supports a distinct set of transcriptional programs. For instance, TBP activates TATA-box-dependent core promoters, whereas TBP-related factor 2 (TRF2) activates TATA-less core promoters that are dependent on a TCT or downstream core promoter element (DPE) motif. These findings led us to investigate the evolution of TRF2. TBP occurs in Archaea and eukaryotes, but TRF2 evolved prior to the emergence of the bilateria and subsequent to the evolutionary split between bilaterians and nonbilaterian animals. Unlike TBP, TRF2 does not bind to the TATA box and could thus function as a new system factor that is largely independent of TBP. We postulate that this TRF2-based system served as the foundation for new transcriptional programs, such as those involved in triploblasty and body plan development, that facilitated the evolution of bilateria.

TRF2, but not TBP, mediates the transcription of ribosomal protein genes.

The TCT core promoter element is present in most ribosomal protein (RP) genes in Drosophila and humans. Here we show that TBP (TATA box-binding protein)-related factor TRF2, but not TBP, is required for transcription of the TCT-dependent RP genes. In cells, TCT-dependent transcription, but not TATA-dependent transcription, increases or decreases upon overexpression or depletion of TRF2. In vitro, purified TRF2 activates TCT but not TATA promoters. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) experiments revealed the preferential localization of TRF2 at TCT versus TATA promoters. Hence, a specialized TRF2-based RNA polymerase II system functions in the synthesis of RPs and complements the RNA polymerase I and III systems.

The Role of Auxin in the Pattern Formation of the Asteraceae Flower Head (Capitulum).

Nature often creates complex structures by rearranging pre-existing units. One such example is the flower head (capitulum) in daisies, where a group of flowers (florets) and phyllaries (modified bracts) are arranged to superficially mimic a single flower. The capitulum is a key taxonomical innovation that defines the daisy family (Asteraceae), the largest flowering plant group. However, patterning mechanisms underlying its structure remain elusive. Here, we show that auxin, a plant hormone, provides a developmental patterning cue for the capitulum. During capitulum development, a temporal auxin gradient occurs, regulating the successive and centripetal formation of distinct florets and phyllaries. Disruption of the endogenous auxin gradient led to homeotic conversions of florets and phyllaries in the capitulum. Furthermore, auxin regulates floral meristem identity genes, such as Matricaria inodora RAY2 and M inodora LEAFY, which determine floret and phyllary identity. This study reveals the mechanism of capitulum patterning and highlights how common developmental tools, such as hormone gradients, have independently evolved in plants and animals.

Evolution and diversification of the basal transcription machinery.

Transcription initiation was once thought to be regulated primarily by sequence-specific transcription factors with the basal transcription machinery being largely invariant. Gradually it became apparent that the basal transcription machinery greatly diversified during evolution and new studies now demonstrate that diversification of the TATA-binding protein (TBP) family yielded specialized and largely independent transcription systems.

Nascent RNA sequencing reveals distinct features in plant transcription.

Transcriptional regulation of gene expression is a major mechanism used by plants to confer phenotypic plasticity, and yet compared with other eukaryotes or bacteria, little is known about the design principles. We generated an extensive catalog of nascent and steady-state transcripts in Arabidopsis thaliana seedlings using global nuclear run-on sequencing (GRO-seq), 5'GRO-seq, and RNA-seq and reanalyzed published maize data to capture characteristics of plant transcription. De novo annotation of nascent transcripts accurately mapped start sites and unstable transcripts. Examining the promoters of coding and noncoding transcripts identified comparable chromatin signatures, a conserved "TGT" core promoter motif and unreported transcription factor-binding sites. Mapping of engaged RNA polymerases showed a lack of enhancer RNAs, promoter-proximal pausing, and divergent transcription in Arabidopsis seedlings and maize, which are commonly present in yeast and humans. In contrast, Arabidopsis and maize genes accumulate RNA polymerases in proximity of the polyadenylation site, a trend that coincided with longer genes and CpG hypomethylation. Lack of promoter-proximal pausing and a higher correlation of nascent and steady-state transcripts indicate Arabidopsis may regulate transcription predominantly at the level of initiation. Our findings provide insight into plant transcription and eukaryotic gene expression as a whole.

RNA polymerase III accurately initiates transcription from RNA polymerase II promoters in vitro.

In eukaryotes, there are three major RNA polymerases (Pol) in the nucleus, which are commonly described as transcribing non-overlapping subsets of genes. Structural studies have highlighted a conserved core shared among all three transcription systems. Initiation of human Pol III from TATA box-containing Pol II promoters under conditions with impaired Pol II transcription activity have been described previously. RNA polymerase III and Pol II were found to co-localize at the promoters of the c-myc gene and the RPPH1 sRNA in vivo. Here, I report that Pol III can, like Pol II, initiate transcription from most tested Pol II core promoters when assayed with crude human nuclear extracts (HSK, SNF, or Dignam). Both polymerases often initiate from the same transcription start site, and depend on a TATA box or AT-rich region but not the downstream promoter element (DPE) or the motif ten element (MTE). Moderate (∼2-fold) changes in the ratio of DNA template to nuclear extract were sufficient to change Pol II-mediated transcription to a mixture of Pol II- and Pol III-, or to a solely Pol III-dependent initiation of transcription from Pol II promoters. Polymerase specificity is thus not fixed but a variable that depends on the properties of the promoter and the transcription conditions. These findings provide functional evidence for a close similarity between the Pol II and Pol III transcription complexes, and additionally explain previous controversies in the literature.

Efficient small fragment sequencing of human, cow, and bison miRNA, small RNA or csRNA-seq libraries using AVITI.

Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been essential for NGS but emerging technologies like Element Biosciences' sequencing by avidity (AVITI) represent a novel approach. It has been reported that AVITI offers improved signal-to-noise ratios and cost reductions. However, the method relies on rolling circle amplification which can be impacted by polymer size, raising questions about its efficacy sequencing small RNAs (sRNA) molecules including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and others that are crucial regulators of gene expression and involved in various biological processes. In addition, capturing capped small RNAs (csRNA-seq) has emerged as a powerful method to map active or "nascent" RNA polymerase II transcription initiation in tissues and clinical samples. Here, we report a new protocol for seamlessly sequencing short DNA fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle (Bos taurus) and the bison (Bison bison) sRNA or csRNA sequencing libraries, augmenting the confidence in both approaches. Additionally, analysis of generated nascent transcription start sites (TSSs) data for cattle and bison revealed inaccuracies in their current genome annotations and highlighted the possibility and need to translate small RNA sequencing methodologies to livestock. Our accelerated and optimized protocol therefore bridges the advantages of AVITI sequencing and critical methods that rely on sequencing short DNA fragments.