RESEARCH

What are our areas of interest?

FUNDAMENTAL RESEARCH

Molecular biophysics of DNA recognition

Transcription is the process by which genetic information encoded in the DNA is copied as messenger RNA (mRNA). Proteins that are eventually synthesized by the ribosome require this mRNA as templates. The intricate business of executing and regulating transcription is carried out by a large suite of DNA-binding proteins. Our group is interested in the principles that govern the interactions between transcription factors, a major functional class of DNA-binding proteins that direct transcriptional initiation, and DNA. Our studies are focused on the role of conformational dynamics and molecular hydration on DNA recognition and self-regulation by the transcription factors. A major thrust of our effects is aimed at understanding how these interactions occur in the cellular environment, which is crowded with biological polymers and metabolites. Such an environment has the tendency of realizing weak interactions and being highly sensitive to the behavior of water molecules. Our studies are aimed at understanding the physicochemical bases of these actions and establishing their biological effects in live cells.

TRANSLATIONAL RESEARCH

Transcription factor pharmacology

Molecular definition of DNA binding by transcription factors also helps reveal novel routes to targeting specific members within transcription factor families, for which strong structural homology has frustrated their translational potential as drug targets. A strategic extension of our interest in transcription factor biophysics is the development of chemical control of specific transcription factors in vivo. Factor-specific reagents represent much-needed additions to the chemical biology toolbox with respect to targeted transcriptional control and have strong potential to impact multiple areas of bioengineering and therapeutics. A particular target of interest is PU.1, a member of the ETS family and a key regulator of hematopoiesis. PU.1-specific compounds are attractive drug discovery leads in a growing list of diseases characterized by de-regulated PU.1 activity. We are currently engaged in collaborative preclinical studies to evaluate the effect of targeting PU.1 in model systems such as acute myeloid leukemia. In addition to pharmacology, we also work collaboratively on the medicinal chemistry of DNA-targeting drugs, with a particular interest in how the physicochemical properties of these compounds impact their cellular uptake and trafficking in cells.

What systems do we study?

Over the past several years, we have been interested in transcription factors involved in hematopoiesis, the process by which all the different lineages of blood cells are stepwise programmed from a single progenitor that resides in the bone marrow. Hematopoiesis is essential to life, and malfunctioning transcription factors are involved in blood-borne cancers (leukemias, lymphomas, and myeloma) and autoimmune diseases. Hematopoiesis is also a model for understanding stem cell programming, which is a major area of interest in bioengineering and regenerative medicine. Experimentally, hematopoietic phenotypes are readily observed and quantified by cell biology techniques. Finally, as adaptive immunity and merging of the marrow with bone are major evolutionary milestones, hematopoietic factors embody many molecular properties we associate with the vertebrate evolution. Nevertheless, the principles we draw from studying hematopoietic factors are applicable to transcriptional regulators in general to the extent that the crowded, osmotically labile environment is a common feature in virtually all cells.

What techniques do we use?

 To enable our efforts to draw the broadest principles, we combine three orthogonal approaches to our study of protein/DNA interactions

Quantitative model analysis

Experimental biophysics

We use a range of spectroscopic (fluorescence, circular dichroism, NMR, surface plasmon resonance), volumetric, and calorimetric techniques to extract thermodynamic, kinetic, and structural information about DNA binding and protein-protein interactions by a wide range of recombinant protein constructs.

Leveraging data science

Computational biophysics

To complement our experimental data, which reflect the average behavior of ensembles of molecules, we perform molecular dynamics simulations to observe structural and dynamic changes at atomistic resolution. The huge amount of stochastic data generated is analyzed using various statistical and probabilistic programming techniques in order to generate physical insight into these fluctuating systems.

Functional relevance

Cell biology

We design reporter systems that incorporate native and non-native features to test biological hypothesis prompted by our biophysical studies. These reporters respond to factor-specific transactivation or de-repression to generate signals that can be spatio-temporally quantified by microscopy or flow cytometry. We also follow native cellular targets using standard molecular biology techniques such as RT-PCR, immunoblotting, and immunofluorescence.

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