Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome.

Gene expression is in part controlled by cis-regulatory elements (CREs) such as enhancers and repressive elements. Anecdotal evidence has indicated that a CRE and a promoter need to be biochemically compatible for promoter regulation to occur, but this compatibility has remained poorly characterised in mammalian cells. We used high-throughput combinatorial reporter assays to test thousands of CRE - promoter pairs from three Mb-sized genomic regions in mouse cells. This revealed that CREs vary substantially in their promoter compatibility, ranging from striking specificity for a single promoter to quantitative differences in activation across a broad set of promoters. More than half of the tested CREs exhibit significant promoter selectivity. Housekeeping promoters tend to have similar CRE preferences, but other promoters exhibit a wide diversity of compatibilities. Higher-order TF motif combinations may account for compatibility. CRE-promoter selectivity does not correlate with looping interactions in the native genomic context, suggesting that chromatin folding and compatibility are two orthogonal mechanisms that confer specificity to gene regulation.

Use of transcriptional slippage for diverse gene expression

We present here an alternative for two-promoter systems ensuring highly diverse expression of several genes from a single promoter. This approach assumes an introduction of a deletion mutation into an A/T homopolymeric run in a gene’s proximal part, and employs the transcriptional slippage mechanism for insertion-dependent reinstatement of the proper reading frame by the T7 RNA polymerase.

Lentiviral vector optimization enhances the expression and cytotoxicity of chimeric antigen receptors

Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer T/NK cells for the efficient expression of CARs. This study reported for the first time the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. Our results demonstrated that the selected CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered cells. This research will help to optimize the production of CAR-NK cells and improve their functional activity, to provide CAR-NK cell products with better and more uniform quality.

Targeting Epigenetics to Cure HIV-1: Lessons From (and for) Cancer Treatment

The Human Immunodeficiency Virus type 1 (HIV-1) integrates in the host genome as a provirus resulting in a long-lived reservoir of infected CD4 cells. As a provirus, HIV-1 has several aspects in common with an oncogene. Both the HIV-1 provirus and oncogenes only cause disease when expressed. A successful cure of both cancer and HIV-1 includes elimination of all cells with potential to regenerate the disease. For over two decades, epigenetic drugs developed against cancer have been used in the HIV-1 field to modulate the state of the proviral chromatin. Cells with an intact HIV-1 provirus exist in three states of infection: productive, inducible latent, and non-inducible latent. Here focus is on HIV-1, transcription control and chromatin structure; how the inducible proviruses are maintained in a chromatin structure that allows reactivation of transcription; and how transcription switches between different stages to allow for an abundance of different transcripts from a single promoter. Recently it was shown that a functional cure of HIV can be achieved by encapsulating all intact HIV-1 proviruses in heterochromatin, giving hope that epigenetic interventions may be used to end the HIV-1 epidemic.

A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

The contractile properties of adult myofibers are shaped by their Myosin heavy chain (MYH) isoform content. We identify by snATAC-seq a 42kb super-enhancer (SE) at the locus regrouping the fast Myh (fMyh) genes. By 4C-seq we show that active fMyh promoters interact with the SE by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the SE recapitulates the endogenous spatio-temporal expression of adult fMyh genes. In situ deletion of the SE by CRISPR/Cas9 editing demonstrates its major role in the control of associated fMyh genes, and deletion of two fMyh genes at the locus reveals an active competition of the promoters for the shared SE. Last, by disrupting the organization of fMyh, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

Independent control of mean and noise by convolution of gene expression distributions

Gene expression noise can reduce cellular fitness or facilitate processes such as alternative metabolism, antibiotic resistance, and differentiation. Unfortunately, efforts to study the impacts of noise have been hampered by a scaling relationship between noise and expression level from a single promoter. Here, we use theory to demonstrate that mean and noise can be controlled independently by expressing two copies of a gene from separate inducible promoters in the same cell. We engineer high- and low-noise inducible promoters to validate this result in Escherichia coli, and develop a model that predicts the experimental distributions. Finally, we use our method to reveal that the response of a promoter to a repressor is less sensitive with higher repressor noise, and explain this result using a law from probability theory. Our approach can be applied to investigate the effects of noise on diverse biological pathways or program cellular heterogeneity for synthetic biology applications.

Prediction and Recognition of Gram-Negative Bacterial Promoter Sequences: An Analysis of Available Web Tools

Many computational methods aim to improve the prediction and recognition of transcription elements in prokaryotes. Despite this, the natural features of those elements make their prediction and recognition remain as an open field of research. In this paper, we compared the open-access tools BacPP, BPROM, bTSSfinder, CNNPromoter_b, iPro70-PseZNC, NNPP2, PePPer, and PromPredict. First, we listed the overall functionalities of each tool and the resources available on their web pages. Later, we carried out a comparison of prediction results using 206 intergenic regions. When evaluating the prediction using intergenic regions containing a single promoter within each, NNPP2 and BacPP obtained >90% correct predictions, with NNPP2 obtaining the highest values of match between predicted promoter location and location indicated by RegulonDB. Overall, many discrepancies were observed among the results. They may be explained by the differences in the methodologies that each tool applies for promoter prediction, not excluding the natural features of promoters as a factor as well. In any case, the results highlight the necessity to continue the efforts to improve promoter prediction, perhaps combining multiple approaches. Through said efforts, some of the challenges of the postgenomic era may be tackled as well.

RNA Binding Motif Protein RBM45 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19 through Binding to Novel Intron Splicing Enhancers

ABSTRACT During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5′-GUA AAG CUA CGG GAC GGU-3′), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [KD] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA. IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.

A single promoter system co-expressing RNA sensor with fluorescent proteins for quantitative mRNA imaging in living tumor cells

Genetically encoded light-up RNA aptamers afford a valuable platform for developing RNA sensors toward live cell imaging.