Zhi-Chen Guo

Inter-MAR Association Contributes to Transcriptionally Active Looping Events in Human β-globin Gene Cluster

Matrix attachment regions (MARs) are important in chromatin organization and gene regulation. Although it is known that there are a number of MAR elements in the β-globin gene cluster, it is unclear that how these MAR elements are involved in regulating β-globin genes expression. Here, we report the identification of a new MAR element at the LCR(locus control region) of human β-globin gene cluster and the detection of the inter-MAR association within the β-globin gene cluster. Also, we demonstrate that SATB1, a protein factor that has been implicated in the formation of network like higher order chromatin structures at some gene loci, takes part in β-globin specific inter-MAR association through binding the specific MARs. Knocking down of SATB1 obviously reduces the binding of SATB1 to the MARs and diminishes the frequency of the inter-MAR association. As a result, the ACH establishment and the α-like globin genes and β-like globin genes expressions are affected either. In summary, our results suggest that SATB1 is a regulatory factor of hemoglobin genes, especially the early differentiation genes at least through affecting the higher order chromatin structure.

Endothelium-specific SIRT1 overexpression inhibits hyperglycemia-induced upregulation of vascular cell senescence.

The rapidly increasing prevalence of diabetes mellitus worldwide is one of the most serious and challenging health problems in the 21st century. Mammalian sirtuin 1 (SIRT1) has been shown to decrease high-glucose-induced endothelial cell senescence in vitro and prevent hyperglycemia-induced vascular dysfunction. However, a role for SIRT1 in prevention of hyperglycemia-induced vascular cell senescence in vivo remains unclear. We used endothelium-specific SIRT1 transgenic (SIRT1-Tg) mice and wild-type (WT) mice to construct a 40-week streptozotocin (STZ)-induced diabetic mouse model. In this mode, 42.9% of wild-type (WT) mice and 38.5% of SIRT1-Tg mice were successfully established as diabetic. Forty weeks of hyperglycemia induced significant vascular cell senescence in aortas of mice, as indicated by upregulation of expression of senescence-associated markers including p53, p21 and plasminogen activator inhibitor-1 (PAI-1). However, SIRT1-Tg diabetic mice displayed dramatically decreased expression of p53, p21 and PAI-1 compared with diabetic WT mice. Moreover, manganese superoxide dismutase expression (MnSOD) was significantly downregulated in the aortas of diabetic WT mice, but was preserved in diabetic SIRT1-Tg mice. Furthermore, expression of the oxidative stress adaptor p66Shc was significantly decreased in aortas of SIRT1-Tg diabetic mice compared with WT diabetic mice. Overall, these findings suggest that SIRT1-mediated inhibition of hyperglycemia-induced vascular cell senescence is mediated at least partly through the reduction of oxidative stress.

The AT-rich DNA-binding Protein SATB2 Promotes Expression and Physical Association of Human Gγ- and Aγ-Globin Genes

Background: Matrix attachment region-binding proteins regulate gene expression through anchoring chromatin loops to nuclear scaffold. Results: Matrix attachment region-binding protein SATB2 promotes G/Aγ-globin genes transcription, facilitates histone acetylase recruitment, and mediates physical association of G/Aγ-globin promoters. Conclusion: SATB2 activates γ-globin gene expression as an active chromatin organizer. Significance: The role of SATB2 as a novel γ-globin gene regulator is explored. Matrix attachment region (MAR)-binding protein (MARBP) has profound influence on gene transcriptional control by tethering genes to the nuclear scaffold. MARBP SATB2 is recently known as a versatile regulator functioning in the differentiation of multiple cell types including embryonic stem cells, osteoblasts and immunocytes. Roles of SATB2 in erythroid cells and its working mechanism in orchestrating target gene expression are largely unexplored. We show here that SATB2 is expressed in erythroid cells and activates γ-globin genes by binding to MARs in their promoters and recruiting histone acetylase PCAF. Further analysis in higher-order chromatin structure shows that SATB2 affects physical proximity of human Gγ- and Aγ-globin promoters via self-association. We also found that SATB2 interacts with SATB1, which specifically activates ϵ-globin gene expression. Our results establish SATB2 as a novel γ-globin gene regulator and provide a glimpse of the differential and cooperative roles of SATB family proteins in modulating clustered genes transcription and mediating higher-order chromatin structures.

Inversion and transposition of Tc1 transposon of C. elegans in mammalian cells.

Tc1/mariner transposons are widespread in the eukaryotes. In vitro transposition test indicated that the transposase is the only protein that is needed in transpositions. It was shown later that the reconstructed Tc1-like transposon, “sleeping beauty” in fish, and the Tc1 transposon in C. elegans jumps in human cells. This discovery indicates that the Tc1/mariner transposon may be engineered as a somatic gene therapy vector if coupled with an efficient gene delivery system. We introduced the Tc1 transposon from C. elegans into different mammalian cell lines and detected the transposition events, indicating that Tc1 transposon functions in different mammalian cells. Interestingly, a high inversion frequency of the transposon was also detected, suggesting that this type of transposon may add variations to host genome when it is horizontally transferred into a new species.

Evaluation of optimal expression cassette in retrovirus vector for β-thalassemia gene therapy

Trials of retroviral vector-mediated human β-globin gene transfer were hampered by low titers, unstable vector transmission, and low-level expression of transferred gene. With the goal of optimizing the retrovirally encoded human β-globin gene expression cassette for gene therapy of β-thalassemia, we generated 3 series of vector constructs (a total of 12 constructs) and investigated the effects of the proximal promoter, 3′-enhancer, and derivatives from the β-locus control region or α-major regulatory element on virus titer, vector transmission stability, and gene expression. The virus titers for 9 of the 12 vector constructs ranged between 2.8×104 cfu/mL and 1.0×106 cfu/mL. We found that proviral DNA was intact in most G418-resistant murine erythroleukemia (MEL) cell clones for 5 vector constructs, while obvious genetic instability was observed for 4 other vector constructs. MEL cells harboring the intact provirus were induced to differentiate, and human β-globin gene expression was analyzed with RNase protection assay. The percentage of human β-globin transcript relative to endogenous murine α-globin transcript were 101.8±64.3% (n=10), 40.1±28.7% (n=4), 31.1±31.9% (n=12), 52.4±11.2% (n=12), and 53.6±8.6% (n=12) for the 5 constructs, respectively, demonstrating the development of optimized retroviral vectors for β-globin gene therapy with murine erythroid cell lines as a model. Unexpectedly, we also documented that the point mutation 8700(C→T) in DNase I hypersensitive site 2 (HS2) core fragment might contribute to low-level expression of the human β-globin gene, based on a comparison of results from transfected and transduced MEL cells and sequence analysis of proviral DNA.

Comparison of two kinds of methods to determine the titer of recombinant retrovirus containing β-globin gene based on G418 selection

Four recombinant retrovirus (RV) vectors containing human β-globin gene and regulatory elements were constructed. To determine the titers of recombinant RV from corresponding producer cell lines, we compared two kinds of method (the simple and the conventional) based on G418 resistance, which is conferred by neo gene of RV vector. The results demonstrated that the simple method shortened the selection period to 3 d instead of 10–12 d with the conventional method and reduced the amount of work; importantly, the titers determined by the simple method were not different significantly from those measured by the conventional method. It can be concluded that the simple method can be used to determine the titers of recombinant RV containing not only cDNA but also genomic DNA with introns and complex regulatory elements instead of the conventional method.

Identification of long range regulatory elements of mouse α‐globin gene cluster by quantitative associated chromatin trap (QACT)

Chromatin from different regions of the genome frequently forms steady associations that play important roles in regulating gene expression. The widely used chromatin conformation capture (3C) assay allows determination of the in vivo structural organization of an active endogenous locus. However, unpredicted chromatin associations within a given genomic locus can not be identified by 3C. Here, we describe a new strategy, quantitative associated chromatin trap (QACT), which incorporates a modified 3C method and a quantitative assay tool, to capture and quantitatively analyzes all possible associated chromatin partners (ACPs) of a given chromatin fragment. Using QACT, we have analyzed the chromatin conformation of the mouse α‐globin gene cluster and proved the extensive interaction between HS26 and α‐globin genes. In addition, we have identified a candidate α1‐globin gene specific silencer 475A8 which shows the differentiation‐stage specific DNase I hypersensitivity. Functional analysis suggests that 475A8 may regulate the α1‐globin gene during terminal differentiation of committed erythroid progenitor cells. ChIP (chromatin immunoprecipitation) and cotransfection assays demonstrate that GATA‐1, a hemopoietic specific transcriptional factor, may increase α1‐globin gene expression by suppressing the function of 475A8 in terminally differentiated erythroid cells. J. Cell. Biochem. 105: 301–312, 2008.

Improvement of SSO-mediated gene repair efficiency by nonspecific oligonucleotides

Targeted gene repair mediated by single-stranded DNA oligonucleotides (SSOs) is a promising method to correct the mutant gene precisely in prokaryotic and eukaryotic systems. We used a HeLa cell line, which was stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome, to test the efficiency of SSO-mediated gene repair. We found that the mEGFP gene was successfully repaired by specific SSOs, but the efficiency was only approximately 0.1%. Then we synthesized a series of nonspecific oligonucleotides, which were single-stranded DNA with different lengths and no significant similarity with the SSOs. We found the efficiency of SSO-mediated gene repair was increased by 6-fold in nonspecific oligonucleotides-treated cells. And this improvement in repair frequency correlated with the doses of the nonspecific oligonucleotides, instead of the lengths. Our evidence suggested that this increased repair efficiency was achieved by the transient alterations of the cellular proteome. We also found the obvious strand bias that antisense SSOs were much more effective than sense SSOs in the repair experiments with nonspecific oligonucleotides. These results provide a fresh clue into the mechanism of SSO-mediated targeted gene repair in mammalian cells.