The FOXO Transcription Factor DAF-16 Bypasses ire-1 Requirement to Promote Endoplasmic Reticulum Homeostasis

SummaryThe unfolded protein response (UPR) allows cells to adjust the capacity of the endoplasmic reticulum (ER) to the load of ER-associated tasks. We show that activation of the Caenorhabditis elegans transcription factor DAF-16 and its human homolog FOXO3 restore secretory protein metabolism when the UPR is dysfunctional. We show that DAF-16 establishes alternative ER-associated degradation systems that degrade misfolded proteins independently of the ER stress sensor ire-1 and the ER-associated E3 ubiquitin ligase complex sel-11/sel-1. This is achieved by enabling autophagy-mediated degradation and by increasing the levels of skr-5, a component of an ER-associated ubiquitin ligase complex. These degradation systems can act together with the conserved UPR to improve ER homeostasis and ER stress resistance, beyond wild-type levels. Because there is no sensor in the ER that activates DAF-16 in response to intrinsic ER stress, natural or artificial interventions that activate DAF-16 may be useful therapeutic approaches to maintain ER homeostasis

Integration of multiple epigenomic marks improves prediction of variant impact in saturation mutagenesis reporter assay

The integrative analysis of highâ throughput reporter assays, machine learning, and profiles of epigenomic chromatin state in a broad array of cells and tissues has the potential to significantly improve our understanding of noncoding regulatory element function and its contribution to human disease. Here, we report results from the CAGI 5 regulation saturation challenge where participants were asked to predict the impact of nucleotide substitution at every base pair within five diseaseâ associated human enhancers and nine diseaseâ associated promoters. A library of mutations covering all bases was generated by saturation mutagenesis and altered activity was assessed in a massively parallel reporter assay (MPRA) in relevant cell lines. Reporter expression was measured relative to plasmid DNA to determine the impact of variants. The challenge was to predict the functional effects of variants on reporter expression. Comparative analysis of the full range of submitted prediction results identifies the most successful models of transcription factor binding sites, machine learning algorithms, and ways to choose among or incorporate diverse datatypes and cellâ types for training computational models. These results have the potential to improve the design of future studies on more diverse sets of regulatory elements and aid the interpretation of diseaseâ associated genetic variation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151884/1/humu23797_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151884/2/humu23797.pd

Caudal, a key developmental regulator, is a DPE-specific transcriptional factor

The regulation of gene transcription is critical for the proper development and growth of an organism. The transcription of protein-coding genes initiates at the RNA polymerase II core promoter, which is a diverse module that can be controlled by many different elements such as the TATA box and downstream core promoter element (DPE). To understand the basis for core promoter diversity, we explored potential biological functions of the DPE. We found that nearly all of the Drosophila homeotic (Hox) gene promoters, which lack TATA-box elements, contain functionally important DPE motifs that are conserved from Drosophila melanogaster to Drosophila virilis. We then discovered that Caudal, a sequence-specific transcription factor and key regulator of the Hox gene network, activates transcription with a distinct preference for the DPE relative to the TATA box. The specificity of Caudal activation for the DPE is particularly striking when a BREu core promoter motif is associated with the TATA box. These findings show that Caudal is a DPE-specific activator and exemplify how core promoter diversity can be used to establish complex regulatory networks

Quantitative Analysis of Differential Expression of HOX Genes in Multiple Cancers

Transcription factors encoded by Homeobox (HOX) genes play numerous key functions during early embryonic development and differentiation. Multiple reports have shown that mis-regulation of HOX gene expression plays key roles in the development of cancers. Their expression levels in cancers tend to differ based on tissue and tumor type. Here, we performed a comprehensive analysis comparing HOX gene expression in different cancer types, obtained from The Cancer Genome Atlas (TCGA), with matched healthy tissues, obtained from Genotype-Tissue Expression (GTEx). We identified and quantified differential expression patterns that confirmed previously identified expression changes and highlighted new differential expression signatures. We discovered differential expression patterns that are in line with patient survival data. This comprehensive and quantitative analysis provides a global picture of HOX genes’ differential expression patterns in different cancer types

Computational identification and experimental characterization of preferred downstream positions in human core promoters

Author summary Transcription of genes by the RNA polymerase II enzyme initiates at a genomic region termed the core promoter. The core promoter is a regulatory region that may contain diverse short DNA sequence motifs/elements that confer specific properties to it. Interestingly, core promoter motifs can be located both upstream and downstream of the transcription start site. Variable compositions of core promoter elements were identified. The initiator (Inr) motif and the downstream core promoter element (DPE) is a combination of elements that has been identified and extensively characterized in fruit flies. Although a few Inr+DPE -containing human promoters were identified, the presence of transcriptionally important downstream core promoter positions within human promoters has been a matter of controversy in the literature. Here, using a newly-designed motif discovery strategy, we discovered preferred downstream positions in human promoters that resemble fruit fly DPE. Clustering of the corresponding sequence motifs in eight additional species indicated that such promoters could be common to multicellular non-plant organisms. Importantly, functional characterization of the newly discovered preferred downstream positions supports the existence of Inr+DPE-containing promoters in human genes. Metazoan core promoters, which direct the initiation of transcription by RNA polymerase II (Pol II), may contain short sequence motifs termed core promoter elements/motifs (e.g. the TATA box, initiator (Inr) and downstream core promoter element (DPE)), which recruit Pol II via the general transcription machinery. The DPE was discovered and extensively characterized in Drosophila, where it is strictly dependent on both the presence of an Inr and the precise spacing from it. Since the Drosophila DPE is recognized by the human transcription machinery, it is most likely that some human promoters contain a downstream element that is similar, though not necessarily identical, to the Drosophila DPE. However, only a couple of human promoters were shown to contain a functional DPE, and attempts to computationally detect human DPE-containing promoters have mostly been unsuccessful. Using a newly-designed motif discovery strategy based on Expectation-Maximization probabilistic partitioning algorithms, we discovered preferred downstream positions (PDP) in human promoters that resemble the Drosophila DPE. Available chromatin accessibility footprints revealed that Drosophila and human Inr+DPE promoter classes are not only highly structured, but also similar to each other, particularly in the proximal downstream region. Clustering of the corresponding sequence motifs using a neighbor-joining algorithm strongly suggests that canonical Inr+DPE promoters could be common to metazoan species. Using reporter assays we demonstrate the contribution of the identified downstream positions to the function of multiple human promoters. Furthermore, we show that alteration of the spacing between the Inr and PDP by two nucleotides results in reduced promoter activity, suggesting a spacing dependency of the newly discovered human PDP on the Inr. Taken together, our strategy identified novel functional downstream positions within human core promoters, supporting the existence of DPE-like motifs in human promoters

TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription

The RNA polymerase II core promoter is a structurally and functionally diverse transcriptional module. RNAi depletion and overexpression experiments revealed a genetic circuit that controls the balance of transcription from two core promoter motifs, the TATA box and the downstream core promoter element (DPE). In this circuit, TBP activates TATA-dependent transcription and represses DPE-dependent transcription, whereas Mot1 and NC2 block TBP function and thus repress TATA-dependent transcription and activate DPE-dependent transcription. This regulatory circuit is likely to be one means by which biological networks can transmit transcriptional signals, such as those from DPE-specific and TATA-specific enhancers, via distinct pathways

Engineered Promoters for Potent Transient Overexpression.

The core promoter, which is generally defined as the region to which RNA Polymerase II is recruited to initiate transcription, plays a pivotal role in the regulation of gene expression. The core promoter consists of different combinations of several short DNA sequences, termed core promoter elements or motifs, which confer specific functional properties to each promoter. Earlier studies that examined the ability to modulate gene expression levels via the core promoter, led to the design of strong synthetic core promoters, which combine different core elements into a single core promoter. Here, we designed a new core promoter, termed super core promoter 3 (SCP3), which combines four core promoter elements (the TATA box, Inr, MTE and DPE) into a single promoter that drives prolonged and potent gene expression. We analyzed the effect of core promoter architecture on the temporal dynamics of reporter gene expression by engineering EGFP expression vectors that are driven by distinct core promoters. We used live cell imaging and flow cytometric analyses in different human cell lines to demonstrate that SCPs, particularly the novel SCP3, drive unusually strong long-term EGFP expression. Importantly, this is the first demonstration of long-term expression in transiently transfected mammalian cells, indicating that engineered core promoters can provide a novel non-viral strategy for biotechnological as well as gene-therapy-related applications that require potent expression for extended time periods