Publications

2016

F*, Zhang, Ren C*, Zhao H, Yang L, Su F, Zhou MM, Han J, Sobie EA, and Walsh MJ. 2016. “Identification of novel prognostic indicators for triple-negative breast cancer patients through integrative analysis of cancer genomics data and protein interactome data”. Oncotarget 7 (44): 71620-34.
Publisher's Version
Triple negative breast cancers (TNBCs) are highly heterogeneous and aggressive without targeted treatment. Here, we aim to systematically dissect TNBCs from a prognosis point of view by building a subnetwork atlas for TNBC prognosis through integrating multi-dimensional cancer genomics data from The Cancer Genome Atlas (TCGA) project and the interactome data from three different interaction networks. The subnetworks are represented as the protein-protein interaction modules perturbed by multiple genetic and epigenetic interacting mechanisms contributing to patient survival. Predictive power of these subnetwork-derived prognostic models is evaluated using Monte Carlo cross-validation and the concordance index (C-index). We uncover subnetwork biomarkers of low oncogenic GTPase activity, low ubiquitin/proteasome degradation, effective protection from oxidative damage, and tightly immune response are linked to better prognosis. Such a systematic approach to integrate massive amount of cancer genomics data into clinical practice for TNBC prognosis can effectively dissect the molecular mechanisms underlying TNBC patient outcomes and provide potential opportunities to optimize treatment and develop therapeutics.
H, Wu, Ren C, Yang F, Qin Y, Zhang Y, and Liu J. 2016. “Extraction and identification of collagen-derived peptides with hematopoietic activity from Colla Corii Asini”. Journal of Ethnopharmacology 182: 129-36.
Publisher's Version

Ethnopharmacological relevance

Colla Corii Asini is a widely used traditional Chinese medicine to treat anemia with a long history due to its stimulating effect in hematopoiesis, but the components contributing to this effect are still unknown. In this study, we aimed to establish a methodology to isolate the bioactive components and provide pharmacological basis for its usage in treating anemia.

Methods

5-FU and γ-ray radiation induced anemic mice models were generated by treating with 5-FU at 150 mg/kg body weight and γ-rays by a 4 MV linear accelerator by total body irradiation using female ICR mice respectively. Oral administration of fraction A was performed by gastric lavage at 1 g/kg and 2 g/kg body weight for 12 days and 25 days and peripheral blood sample was collected from ocular sinus red blood cell (RBC) and white blood cell (WBC) counts every 3 days and 5 days for 5-FU and radiation induced models, respectively. Next, fraction A was separated to A1 and A2 using cation exchange chromatography (IEC) based on ionic strength. Fraction A1 was further separated using reverse phase chromatography (RPC) based on the hydrophobicity first with 0–10% linear gradient, then 20%, 30%, 50% constant gradient of 60% acetonitrile in neutral Na2HPO4 buffer. Peak fractions were pooled, evaporatively dried, and dissolved in ultrapure water. Finally, fraction A11 was analyzed combining tandem mass spectrometry and proteomic tools and two peptides (peptide 11 and 16) were identified. The hematopoietic effects of multiple fractions and the two peptides were measured using colony-forming units-erythroid (CFU-E), an indication of late erythroid progenitor cells and colony-forming units granulocyte-monocyte (CFU-GM), an indication of granulocyte and monocyte progenitor cells respectively on hematopoietic progenitor cells prepared from bone marrow (Till and Mcculloch 1961).

Results

Fraction A at 1 g/kg and 2 g/kg could increase RBC and WBC counts in 5-FU and radiation induced anemic mice models. Fraction A1 at 0.1 mg/ml and 0.5 mg/ml, exhibited stronger hematopoietic activity than fraction A2, both of which were subfractions from fraction A using IEX, by elevated CFU-E and CFU-GM of mouse bone marrow cells. Furthermore, fraction A11 at 0.1 mg/ml showed stronger CFU-E and CFU-GM than fractions A12 to A14 from RPC separation. Finally, peptide 11 and peptide 16 were identified from tandem mass spectrometry and peptide 11 increased CFU-E and CFU-GM in a dose dependent manner.

Conclusions

We combined multiple approaches including chromatography, mass spectrometry, cell-based assays, as well as animal studies to identify and demonstrate that the hematopoietic effect of Colla Corii Asini is at least in part from the peptidic components identified using our methodology. This is the first time to isolate peptidic components from Colla Corii Asini, and to provide molecular basis for its usage in treating anemia, which may particularly have the potential to benefit cancer patients suffering from myelosuppression due to radiotherapy or chemotherapy.

C, Ren, Zeng L, and Zhou MM. 2016. “Preparation, Biochemical Analysis, and Structure Determination of the Bromodomain, an Acetyl-Lysine Binding Domain”. Methods in Enzymology 573: 321–343.
Publisher's Version
The bromodomain (BrD) represents an evolutionarily conserved protein domain whose function mostly is to recognize acetylated lysine residues in histones and nuclear proteins in regulation of gene transcription in chromatin. The highly conserved BrD structure features an unusual left-handed, antiparallel four-helix bundle and a hydrophobic pocket between the interhelical ZA and BC loops important for acetyl-lysine binding. Many proteins, particularly transcriptional activators, contain BrDs, and mutation or deletion of the BrDs impairs the protein function, implying their critical role in human biology and disease. In this chapter, we provide general protocols of the preparation, biochemical analysis, and structure determination of BrDs, aiming to offer a general guideline for structural and biochemical functional characterization of BrD-containing proteins.
C*, Ren, Smith SG*, Yap KL, Li S, Li J, Mezei M, Vincek AS, Aguilo F, Walsh MJ, and Zhou MM. 2016. “Structure-Guided Discovery of Selective Antagonists for the Chromodomain of Polycomb Repressive Protein CBX7”. ACS Medicinal Chemistry Letters 7 (6): 601-5.
Publisher's Version
The chromobox 7 (CBX7) protein of the polycomb repressive complex 1 (PRC1) functions to repress transcription of tumor suppressor p16INK4a through long noncoding RNA, ANRIL (antisense noncoding RNA in the INK4 locus) directed chromodomain (ChD) binding to trimethylated lysine 27 of histone H3 (H3K27me3), resulting in chromatin compaction at the INK4a/ARF locus. In this study, we report structure-guided discovery of two distinct classes of small-molecule antagonists for the CBX7ChD. Our Class A compounds, a series including analogues of the previously reported MS452, inhibit CBX7ChD/methyl-lysine binding by occupying the H3K27me3 peptide binding site, whereas our Class B compound, the newly discovered MS351, appears to inhibit H3K27me3 binding when CBX7ChD is bound to RNA. Our crystal structure of the CBX7ChD/MS351 complex reveals the molecular details of ligand recognition by the aromatic cage residues that typically engage in methyl-lysine binding. We further demonstrate that MS351 effectively induces transcriptional derepression of CBX7 target genes, including p16INK4a in mouse embryonic stem cells and human prostate cancer PC3 cells. Thus, MS351 represents a new class of ChD antagonists that selectively targets the biologically active form of CBX7 of the PRC1 in long noncoding RNA- and H3K27me3-directed gene transcriptional repression.

2015

C, Ren, Morohashi K, Plotnikov AN, Jakoncic J, Smith SG, Li J, Zeng L, et al. 2015. “Small-molecule modulators of methyl-lysine binding for the CBX7 chromodomain”. Chemistry & Biology 2 (22): 161-68.
Publisher's Version
Chromobox homolog 7 (CBX7) plays an important role in gene transcription in a wide array of cellular processes, ranging from stem cell self-renewal and differentiation to tumor progression. CBX7 functions through its N-terminal chromodomain (ChD), which recognizes trimethylated lysine 27 of histone 3 (H3K27me3), a conserved epigenetic mark that signifies gene transcriptional repression. In this study, we report the discovery of small molecules that inhibit CBX7ChD binding to H3K27me3. Our crystal structures reveal the binding modes of these molecules that compete against H3K27me3 binding through interactions with key residues in the methyl-lysine binding pocket of CBX7ChD. We further show that a lead compound, MS37452, derepresses transcription of Polycomb repressive complex target gene p16/CDKN2A by displacing CBX7 binding to the INK4A/ARF locus in prostate cancer cells. These small molecules have the potential to be developed into high-potency chemical modulators that target CBX7 functions in gene transcription in different disease pathways.

2013

K, Jiang, Ren C, and Nair VD. 2013. “MicroRNA-137 represses Klf4 and Tbx3 during differentiation of mouse embryonic stem cells”. Stem Cell Research 11 (3): 1299-1313.
Publisher's Version

MicroRNA-137 (miR-137) has been shown to play an important role in the differentiation of neural stem cells. Embryonic stem (ES) cells have the potential to differentiate into different cell types including neurons; however, the contribution of miR-137 in the maintenance and differentiation of ES cells remains unknown. Here, we show that miR-137 is mainly expressed in ES cells at the mitotic phase of the cell cycle and highly upregulated during differentiation. We identify that ES cell transcription factors, Klf4 and Tbx3, are downstream targets of miR-137, and we show that endogenous miR-137 represses the 3′ untranslated regions of Klf4 and Tbx3. Transfection of ES cells with mature miR-137 RNA duplexes led to a significant reduction in cell proliferation and the expression of Klf4, Tbx3, and other self-renewal genes. Furthermore, we demonstrate that increased miR-137 expression accelerates differentiation of ES cells in vitro. Loss of miR-137 during ES cell differentiation significantly impeded neuronal gene expression and morphogenesis. Taken together, our results suggest that miR-137 regulates ES cell proliferation and differentiation by repressing the expression of downstream targets, including Klf4 and Tbx3.