Yanqun Song

Converting a solvatochromic fluorophore into a protein-based pH indicator for extreme acidity.

Live-cell pH measurements: An environment-sensitive fluorophore (green) was site-specifically introduced on HdeA, an acid-resistant chaperone showing pH-mediated conformational changes under low pH conditions. A survey of the attachment sites led to the discovery of one position on HdeA at which the attached fluorophore showed a strong fluorescence increase upon acidification.

A Highly Selective Fluorescent Probe for Visualization of Organic Hydroperoxides in Living Cells

The transcriptional regulatory protein OhrR is converted into a fluorescent bioprobe capable of detecting organic hydroperoxides in living cells with high sensitivity and selectivity.

A Genetically Encoded FRET Sensor for Intracellular Heme

Heme plays pivotal roles in various cellular processes as well as in iron homeostasis in living systems. Here, we report a genetically encoded fluorescence resonance energy transfer (FRET) sensor for selective heme imaging by employing a pair of bacterial heme transfer chaperones as the sensory components. This heme-specific probe allows spatial-temporal visualization of intracellular heme distribution within living cells.

5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers

DNA modifications such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are epigenetic marks known to affect global gene expression in mammals. Given their prevalence in the human genome, close correlation with gene expression and high chemical stability, these DNA epigenetic marks could serve as ideal biomarkers for cancer diagnosis. Taking advantage of a highly sensitive and selective chemical labeling technology, we report here the genome-wide profiling of 5hmC in circulating cell-free DNA (cfDNA) and in genomic DNA (gDNA) of paired tumor and adjacent tissues collected from a cohort of 260 patients recently diagnosed with colorectal, gastric, pancreatic, liver or thyroid cancer and normal tissues from 90 healthy individuals. 5hmC was mainly distributed in transcriptionally active regions coincident with open chromatin and permissive histone modifications. Robust cancer-associated 5hmC signatures were identified in cfDNA that were characteristic for specific cancer types. 5hmC-based biomarkers of circulating cfDNA were highly predictive of colorectal and gastric cancers and were superior to conventional biomarkers and comparable to 5hmC biomarkers from tissue biopsies. Thus, this new strategy could lead to the development of effective, minimally invasive methods for diagnosis and prognosis of cancer from the analyses of blood samples.

Structural characterization of the DNA-binding mechanism underlying the copper(II)-sensing MarR transcriptional regulator.

Multiple antibiotic resistance regulator (MarR) family proteins are widely conserved transcription factors that control bacterial resistance to antibiotics, environmental stresses, as well as the regulation of virulence determinants. Escherichia coli MarR, the prototype member of this family, has recently been shown to undergo copper(II)-catalyzed inter-dimer disulfide bond formation via a unique cysteine residue (Cys80) residing in its DNA-binding domain. However, despite extensive structural characterization of the MarR family proteins, the structural mechanism for DNA binding of this copper(II)-sensing MarR factor remains elusive. Here, we report the crystal structures of DNA-bound forms of MarR, which revealed a unique, concerted generation of two new helix–loop–helix motifs that facilitated MarR’s DNA binding. Structural analysis and electrophoretic mobility shift assays (EMSA) show that the flexibility of Gly116 in the center of helix α5 and the extensive hydrogen-bonding interactions at the N-terminus of helix α1 together assist the reorientation of the wHTH domains and stabilize MarR’s DNA-bound conformation.

Allosteric histidine switch for regulation of intracellular zinc(II) fluctuation

Significance Metal homeostasis is critical to numerous biological processes, and metalloregulators play key roles in its regulation. In transcriptional regulation, which is allosterically controlled by metalloregulators, reorganization of their metal-binding residues and/or related hydrogen bonding networks is usually utilized, while the coordination atoms on the same metal-binding residues remain seldom changed. Our study shows an example whereby the zinc-induced transcriptional regulator ZitR switches one of its histidine nitrogen atoms for zinc coordination in response to zinc fluctuation. This histidine-switch process facilitates conformational change of ZitR protein, allowing allosteric and fine-tuned control of DNA binding and transcriptional regulation. Metalloregulators allosterically control transcriptional activity through metal binding-induced reorganization of ligand residues and/or hydrogen bonding networks, while the coordination atoms on the same ligand residues remain seldom changed. Here we show that the MarR-type zinc transcriptional regulator ZitR switches one of its histidine nitrogen atoms for zinc coordination during the allosteric control of DNA binding. The Zn(II)-coordination nitrogen on histidine 42 within ZitR’s high-affinity zinc site (site 1) switches from Nε2 to Nδ1 upon Zn(II) binding to its low-affinity zinc site (site 2), which facilitates ZitR’s conversion from the nonoptimal to the optimal DNA-binding conformation. This histidine switch-mediated cooperation between site 1 and site 2 enables ZitR to adjust its DNA-binding affinity in response to a broad range of zinc fluctuation, which may allow the fine tuning of transcriptional regulation.

DNA 5-Hydroxymethylcytosines from Cell-free Circulating DNA as Diagnostic Biomarkers for Human Cancers

DNA modifications such as 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) are epigenetic marks known to affect global gene expression in mammals(1, 2). Given their prevalence in the human genome, close correlation with gene expression, and high chemical stability, these DNA epigenetic marks could serve as ideal biomarkers for cancer diagnosis. Taking advantage of a highly sensitive and selective chemical labeling technology(3), we report here genome-wide 5hmC profiling in circulating cell-free DNA (cfDNA) and in genomic DNA of paired tumor/adjacent tissues collected from a cohort of 90 healthy individuals and 260 patients recently diagnosed with colorectal, gastric, pancreatic, liver, or thyroid cancer. 5hmC was mainly distributed in transcriptionally active regions coincident with open chromatin and permissive histone modifications. Robust cancer-associated 5hmC signatures in cfDNA were identified with specificity for different cancers. 5hmC-based biomarkers of circulating cfDNA demonstrated highly accurate predictive value for patients with colorectal and gastric cancers versus healthy controls, superior to conventional biomarkers, and comparable to 5hmC biomarkers from tissue biopsies. This new strategy could lead to the development of effective blood-based, minimally-invasive cancer diagnosis and prognosis approaches.

Physiological distribution and regulation of heme

Heme is an essential cofactor in almost all organisms. Proteins that utilize heme and heme itself play diverse but fundamental roles in cellular process, even in physiological process. However, due to its inherent toxicity to cell, the concentration of heme is strictly regulated to avoid cell damage. Several separate pathways are involved to maintain the heme homeostasis, dysfunction of which may lead to serious even deadly disease such as porphyrias.