Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8,269 rationally designed IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. Using these data, we fit a statistical mechanics model that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three novel promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

Editing a γ-Globin Repressor Binding Site Restores Fetal Hemoglobin Synthesis and Corrects the Phenotype of Sickle Cell Disease Erythrocytes

Sickle cell disease (SCD) is a severe, life-threatening disorder caused by a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) β-chain that causes Hb polymerization with consequent red blood cell (RBC) rigidity, anemia, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) decreases sickling and significantly reduces the clinical severity of SCD. HPFH mutations cluster at several loci in the promoters of the two γ-globin genes, HBG1 and HBG2, and, disrupt binding sites for transcriptional repressors (e.g., BCL11A and LRF), leading to elevated levels of fetal hemoglobin (HbF) representing up to 40% of total hemoglobin tetramers. In addition, SNPs at position -158 of both γ-globin promoters are associated with enhanced γ-globin expression and may identify a putative transcriptional repressor binding site. Here, we used the CRISPR/Cas9 system to mimic HPFH mutations in the HBG promoters by generating insertion and deletions (InDels) leading to disruption of known and putative binding sites for transcriptional repressors via non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) DNA repair mechanisms. Efficient editing of the LRF binding site (≥ 3 γ-globin promoters/cell in >70% of cells) in SCD patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in a robust, virtually pancellular HbF reactivation and a concomitant reduction in βS-globin levels, recapitulating the phenotype of asymptomatic SCD-HPFH patients. Of note, in RBCs derived from edited HSPCs, HbF levels exceeded 40% of total Hb tetramers, suggesting that CRISPR/Cas9 mediated disruption of the LRF binding site is even more potent than naturally occurring HPFH point mutations in reactivating HbF expression. RBCs derived from edited HSPCs displayed HbF levels sufficient to substantially correct the SCD cell phenotype (~80% of non-sickling RBCs). Although larger deletions (in part generated likely by MMEJ) disrupt more efficiently the LRF binding site, shorter InDels mainly produced by NHEJ, the most active repair mechanism in bona fide hematopoietic stem cells (HSCs), were still able to induce a potent γ-globin reactivation and correct the SCD cell phenotype. Interestingly, upon targeting of the -158 site, we observed HBG de-repression, which, however, was mild, indicating that this region contains a sequence that moderately inhibits γ-globin expression in adult cells, consistently with the mild increase in HbF associated with the -158 SNP. To evaluate the efficiency and the safety of this approach in long-term repopulating HSCs, untreated and edited HSPCs were injected into NSG immunodeficient mice. 16 weeks after transplantation, the engraftment of control and HBG-edited cells was comparable, as analyzed in bone marrow, spleen, thymus and blood. Edited HSCs were capable to differentiate into multiple hematopoietic cell types, with no skewing towards any particular lineage. Importantly, xenotransplantation of HSPCs edited using the gRNAs targeting the LRF binding site demonstrated a high editing efficiency in long-term repopulating HSCs (up to 75%) and a modest reduction in the frequency of deletions likely generated by MMEJ. Overall, this study identifies the binding site for the LRF repressor as a novel, potent and safe target for a genome editing-based treatment of SCD. Disclosures El Nemer: Hemanext: Other: Other. Cavazzana:SmartImmune: Other: Founder.

Feedback Regulation of Lac Repressor Expression in Escherichia coli

ABSTRACT Negative feedback regulation, mediated through repressor binding site O3, which overlaps the lacI gene, could explain the robustness of the weak expression of Lac repressor. Significant autorepression of Lac repressor has never been ruled out. In the work presented here, the degree of autoregulation of Lac repressor was determined. It is negligible.

Phosphoinositides Are Involved in Control of the Glucose-Dependent Growth Resumption That Follows the Transition Phase in Streptomyces lividans

ABSTRACT The interruption of the sblA gene of Streptomyces lividans was previously shown to lead to relief of glucose repression of the normally strongly glucose-repressed α-amylase gene. In addition to this relief, an early entry into stationary phase was observed when cells were grown in a minimal medium containing glucose as the main carbon source. In this study, we established that this mutant does not resume growth after the transition phase when cultured in the complex glucose-rich liquid medium R2YE and sporulates much earlier than the wild-type strain when plated on solid R2YE. These phenotypic differences, which were abolished when glucose was omitted from the R2YE medium, correlated with a reduced glucose uptake ability of the sblA mutant strain. sblA was shown to encode a bifunctional enzyme possessing phospholipase C-like and phosphoinositide phosphatase activities. The cleavage of phosphoinositides by SblA seems necessary to trigger the glucose-dependent renewed growth that follows the transition phase. The transient expression of sblA that takes place just before the transition phase is consistent with a regulatory role for this gene during the late stages of growth. The tight temporal control of sblA expression was shown to depend on two operator sites. One, located just upstream of the −35 promoter region, likely constitutes a repressor binding site. The other, located 170 bp downstream of the GTG sblA translational start codon, may be involved in the regulation of the degradation of the sblA transcript. This study suggests that phosphoinositides constitute important regulatory molecules in Streptomyces, as they do in eukaryotes.

The Moloney Murine Leukemia Virus Repressor Binding Site Represses Expression in Murine and Human Hematopoietic Stem Cells

ABSTRACT The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34+ CD38− hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.

Genetics of Nitrogen Regulation in Methanococcus maripaludis

We have used genetic methods in Methanococcus maripaludis to study nitrogen metabolism and its regulation. We present evidence for a “nitrogen regulon” in Methanococcus and Methanobacterium species containing genes of nitrogen metabolism that are regulated coordinately at the transcriptional level via a common repressor binding site sequence, or operator. The implied mechanism for regulation resembles the general bacterial paradigm for repression, but contrasts with well-known mechanisms of nitrogen regulation in bacteria, which occur by activation. Genes in the nitrogen regulons include those for nitrogen fixation, glutamine synthetase, (methyl)ammonia transport, the regulatory protein GlnB, and ammonia-dependent NAD synthetase, as well as a gene of unknown function. We also studied the function of two novel GlnB homologues that are encoded within the nif gene cluster of diazotrophic methanogens. The phenotype resulting from a glnB null mutation in M. maripaludis provides direct evidence that glnB-like genes are involved in “ammonia switch-off,” the post-transcriptional inhibition of nitrogen fixation upon addition of ammonia. Finally, we show that the gene nifX is not required for nitrogen fixation, in agreement with findings in several bacteria. These studies illustrate the utility of genetic methods in M. maripaludis and show the enhanced perspective that studies in the Archaea can bring to known biological systems.