Engineered endosymbionts capable of directing mammalian cell gene expression

SummaryModular methods for directing mammalian gene expression would enable advances in tissue regeneration, enhance cell-based therapeutics and improve modulation of immune responses. To address this challenge, engineered endosymbionts (EES) that escape endosomal destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to control host cell gene expression were developed. Microscopy confirmed that EES escape phagosomes, replicate within the cytoplasm, and can secrete reporter proteins into the cytoplasm that were then transported to the nucleus. Synthetic operons encoding the mammalian transcription factors, Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the EES genome. Using controlled induction, these EES were shown to direct gene expression in J774A.1 macrophage/monocyte cells and modulate the host cell fates. Expressing mammalian transcription factors from engineered intracellular bacteria as endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes.Graphical abstract

Topological isolation of developmental regulators in mammalian genomes

Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression.

MiR-378b Modulates Chlamydia-Induced Upper Genital Tract Pathology

Genital Chlamydia trachomatis infection causes severe reproductive pathologies such as salpingitis and pelvic inflammatory disease that can lead to tubal factor infertility. MicroRNAs (miRNAs) are evolutionarily conserved regulators of mammalian gene expression in development, immunity and pathophysiologic processes during inflammation and infection, including Chlamydia infection. Among the miRNAs involved in regulating host responses and pathologic outcome of Chlamydia infection, we have shown that miR-378b was significantly differentially expressed during primary infection and reinfection. In this study, we tested the hypothesis that miR-378b is involved in the pathological outcome of Chlamydia infection. We developed miR-378b knockout mice (miR-378b−/−) using Crispr/Cas and infected them along with their wild-type (WT) control with Chlamydia to compare the infectivity and reproductive pathologies. The results showed that miR-378b−/− mice were unable to clear the infection compared to WT mice; also, miR-378b−/− mice exhibited a relatively higher Chlamydia burden throughout the duration of infection. However, gross pathology results showed that miR-378b−/− mice had significantly reduced uterine dilatations and pathologic lesions after two infections compared to WT mice. In addition, the pregnancy and fertility rates for infected miR-378b−/− mice showed protection from Chlamydia-induced infertility with fertility rate that was comparable to uninfected WT mice. These results are intriguing as they suggest that miR-378b is important in regulating host immune responses that control Chlamydial replication and drive the inflammation that causes complications such as infertility. The finding has important implications for biomarkers of Chlamydial complications and targets for prevention of disease.

The Arg/N-degron pathway targets transcription factors and regulates specific genes

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degrons. Our previous work produced double-knockout (2-KO) HEK293T human cell lines that lacked the functionally overlapping UBR1 and UBR2 E3 ubiquitin ligases of the Arg/N-degron pathway. Here, we studied these cells in conjunction with RNA-sequencing, mass spectrometry (MS), and split-ubiquitin binding assays. 1) Some mRNAs, such as those encoding lactate transporter MCT2 and β-adrenergic receptor ADRB2, are strongly (∼20-fold) up-regulated in 2-KO cells, whereas other mRNAs, including those encoding MAGEA6 (a regulator of ubiquitin ligases) and LCP1 (an actin-binding protein), are completely repressed in 2-KO cells, in contrast to wild-type cells. 2) Glucocorticoid receptor (GR), an immunity-modulating transcription factor (TF), is up-regulated in 2-KO cells and also physically binds to UBR1, strongly suggesting that GR is a physiological substrate of the Arg/N-degron pathway. 3) PREP1, another TF, was also found to bind to UBR1. 4) MS-based analyses identified ∼160 proteins whose levels were increased or decreased by more than 2-fold in 2-KO cells. For example, the homeodomain TF DACH1 and the neurofilament subunits NF-L (NFEL) and NF-M (NFEM) were expressed in wild-type cells but were virtually absent in 2-KO cells. 5) The disappearance of some proteins in 2-KO cells took place despite up-regulation of their mRNAs, strongly suggesting that the Arg/N-degron pathway can also modulate translation of specific mRNAs. In sum, this multifunctional proteolytic system has emerged as a regulator of mammalian gene expression, in part through conditional targeting of TFs that include ATF3, GR, and PREP1.

Identification of putative calorie restriction mimetics using mammalian gene expression profiles

Obesity is a risk factor for cardiovascular diseases, diabetes and cancer. In theory, the obesity problem could be solved by the adherence to a calorie-restricted diet, but that is not generally achieved in practice. An alternative is a pharmacological approach, using compounds that trigger the same metabolic changes associated with calorie restriction. Here, I expand in the pharmacological direction by identifying compounds that induce liver gene signature profiles that mimic those induced by calorie restriction. Using gene expression profiles from mice and rat, I identify corticosteroids, PPAR agonists and some antibacterial/antifungal as candidate compounds mimicking the response to calorie restriction in the liver gene signatures.