Tuning transcriptional regulation through signaling: A predictive theory of allosteric induction

Allosteric regulation is found across all domains of life, yet we still lack simple, predictive theories that directly link the experimentally tunable parameters of a system to its input-output response. To that end, we present a general theory of allosteric transcriptional regulation using the Monod-Wyman-Changeux model. We rigorously test this model using the ubiquitous simple repression motif in bacteria by first predicting the behavior of strains that span a large range of repressor copy numbers and DNA binding strengths and then constructing and measuring their response. Our model not only accurately captures the induction profiles of these strains but also enables us to derive analytic expressions for key properties such as the dynamic range and $[EC_{50}]$. Finally, we derive an expression for the free energy of allosteric repressors which enables us to collapse our experimental data onto a single master curve that captures the diverse phenomenology of the induction profiles.Comment: Substantial revisions for resubmission (3 new figures, significantly elaborated discussion); added Professor Mitchell Lewis as another author for his continuing contributions to the projec

The use of primary dermal fibroblast cultures to evaluate type I collagen expression in the oim model mouse

Abstract only availableOsteogenesis imperfecta type III is a heritable disorder leading to impaired connective tissue function in type I collagen containing tissues including bone fragility, blue-grey sclera, short stature, and hearing loss. Normal type I collagen is a heterotrimeric molecule containing two pro1(I) collagen chains and a similar but genetically distinct pro2(I) collagen chain. The osteogenesis imperfecta murine (oim) model mouse produces only homotrimeric type I collagen due to a single nucleotide deletion in the COL1A2 gene resulting in a non-functional pro2(I) collagen chain. The result is expression of an abnormal type I collagen molecule which leads to the above phenotype. This study is aimed at developing a methodology whereby dermal fibroblast cultures can be utilized for a variety of assays, including type I collagen RNA and protein quantification. For genotype identification, primers flanking the site of the single nucleotide deletion allow for differentiation of wild type, heterozygous and homozygous animals at the genomic level. Upon confirmation, skin was removed from both oim and wildtype mice and the dermal layer harvested. Fibroblasts originating from the dermal layer of each genotype were then cultured and grown to confluence as separate cultures and the RNA and/or protein harvested from both cell types. Total RNA was harvested using the Qiagen RNeasy kit and used to make cDNA, which was then used in conjunction with specific PCR primers to differentiate between wildtype and oim transcripts. For protein studies, the cells were treated with ascorbic acid to maximize the production of collagen prior to harvesting. Type I collagen expression was then confirmed via a western blot using a type I collagen-specific antibody. Initial results indicate the presence of both pro1(I) and pro2(I) collagen chains from harvested wildtype dermal fibroblasts, while media harvested from oim dermal fibroblasts indicated the presence of only the pro1(I) collagen chains. These results confirm the in vivo protein expression profile seen in skin of both oim and wildtype mice is exhibited in vitro in the respective dermal fibroblast cultures. These results will allow us to quantitate pro1(I) and pro2(I) collagen mRNA and protein expression levels. Future experiments will include the quantitation of type I collagen mRNA levels using RT-PCR, as well as the use of densitometry to quantitate type I collagen protein levels by western blot analysis. This in turn promises to provide insight into the mechanism of formation of abnormal type I collagen in the oim model mouse.Life Sciences Undergraduate Research Opportunity Progra

The Energetics of Molecular Adaptation in Transcriptional Regulation

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only a subset of the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.Comment: 11 pages, 6 figures, supplemental info. available via http://rpgroup.caltech.edu/mwc_mutant

Integrating genomics into the care of people with palliative needs: A global scoping review of policy recommendations

BACKGROUND: Genomics has growing relevance to palliative care, where testing largely benefits relatives. Integration of genomics into the care of patients with palliative care needs has not received the critical attention it requires, and health professionals report a lack of policy guidance to support them to overcome practice barriers. SUMMARY: To identify policy recommendations related to: (1) integrating genomics into the care of patients with palliative care needs and their families, and (2) care of the family unit, we performed a scoping review of palliative care and genomic policies. Two of 78 policies recommended integrating genomics into palliative care. Six palliative care policies mentioned genomics in background information but were without relevant recommendations. No genomics policies mentioned palliative care in the background information. Across all policies, guidance related to “Delivering Family-Centred Care” was the most frequent recommendation related to care of the family unit, (n=62/78, 79.5%). KEY MESSAGES: We identified a policy gap related to integrating genomics into palliative care. Without policy guidance, health services are less likely to commit funding towards supporting health professionals, reducing the personal and clinical benefits of genomics to patients and relatives. Framing genomic information as family-centred care enables policy makers to communicate the value of genomics to palliative care that will resonate with genomic and palliative care stakeholders. These findings increase awareness among policy makers of the benefits of genomic information to patients with palliative care needs and their families and call for incorporation of appropriate recommendations into palliative care and genomic policy