Genomic discovery of potent chromatin insulators for human gene therapy.

Insertional mutagenesis and genotoxicity, which usually manifest as hematopoietic malignancy, represent major barriers to realizing the promise of gene therapy. Although insulator sequences that block transcriptional enhancers could mitigate or eliminate these risks, so far no human insulators with high functional potency have been identified. Here we describe a genomic approach for the identification of compact sequence elements that function as insulators. These elements are highly occupied by the insulator protein CTCF, are DNase I hypersensitive and represent only a small minority of the CTCF recognition sequences in the human genome. We show that the elements identified acted as potent enhancer blockers and substantially decreased the risk of tumor formation in a cancer-prone animal model. The elements are small, can be efficiently accommodated by viral vectors and have no detrimental effects on viral titers. The insulators we describe here are expected to increase the safety of gene therapy for genetic diseases.

Transcriptional environment and chromatin architecture interplay dictates globin expression patterns of heterospecific hybrids derived from undifferentiated human embryonic stem cells or from their erythroid progeny.

To explore the response of ß globin locus with established chromatin domains upon their exposure to new transcriptional environments, we transferred the chromatin-packaged ß globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroblasts into an adult transcriptional environment. Distinct globin expression patterns were observed. In hESC-derived erythroblasts where both ε and γ globin were active and marked by similar chromatin modifications, ε globin was immediately silenced upon transfer, whereas γ globin continued to be expressed for months, implying that different transcriptional environments were required for their continuing expression. Whereas ß globin was silent both in hESCs and in hESC-derived erythroblasts, ß globin was only activated upon transfer from hESCs, but not in the presence of dominant γ globin transferred from hESC-derived erythroblasts, confirming the competing nature of γ versus ß globin expression. With time, however, silencing of γ globin occurred in the adult transcriptional environment with concurrent activation of ß-globin, accompanied by a drastic change in the epigenetic landscape of γ and ß globin gene regions without apparent changes in the transcriptional environment. This switching process could be manipulated by overexpression or downregulation of certain transcription factors. Our studies provide important insights into the interplay between the transcription environment and existing chromatin domains, and we offer an experimental system to study the time-dependent human globin switching.

KLF11 mediates PPARγ cerebrovascular protection in ischaemic stroke.

Peroxisome proliferator-activated receptor gamma (PPARγ) is emerging as a major regulator in neurological diseases. However, the role of (PPARγ) and its co-regulators in cerebrovascular endothelial dysfunction after stroke is unclear. Here, we have demonstrated that (PPARγ) activation by pioglitazone significantly inhibited both oxygen-glucose deprivation-induced cerebral vascular endothelial cell death and middle cerebral artery occlusion-triggered cerebrovascular damage. Consistent with this finding, selective (PPARγ) genetic deletion in vascular endothelial cells resulted in increased cerebrovascular permeability and brain infarction in mice after focal ischaemia. Moreover, we screened for (PPARγ) co-regulators using a genome-wide and high-throughput co-activation system and revealed KLF11 as a novel (PPARγ) co-regulator, which interacted with (PPARγ) and regulated its function in mouse cerebral vascular endothelial cell cultures. Interestingly, KLF11 was also found as a direct transcriptional target of (PPARγ). Furthermore, KLF11 genetic deficiency effectively abolished pioglitazone cytoprotection in mouse cerebral vascular endothelial cell cultures after oxygen-glucose deprivation, as well as pioglitazone-mediated cerebrovascular protection in a mouse middle cerebral artery occlusion model. Mechanistically, we demonstrated that KLF11 enhanced (PPARγ) transcriptional suppression of the pro-apoptotic microRNA-15a (miR-15a) gene, resulting in endothelial protection in cerebral vascular endothelial cell cultures and cerebral microvasculature after ischaemic stimuli. Taken together, our data demonstrate that recruitment of KLF11 as a novel (PPARγ) co-regulator plays a critical role in the cerebrovascular protection after ischaemic insults. It is anticipated that elucidating the coordinated actions of KLF11 and (PPARγ) will provide new insights into understanding the molecular mechanisms underlying (PPARγ) function in the cerebral vasculature and help to develop a novel therapeutic strategy for the treatment of stroke.

Krüppel-like factor-11, a transcription factor involved in diabetes mellitus, suppresses endothelial cell activation via the nuclear factor-κB signaling pathway.

OBJECTIVE: Endothelial cell (EC) inflammatory status is critical to many vascular diseases. Emerging data demonstrate that mutations of Krüppel-like factor-11 (KLF11), a gene coding maturity-onset diabetes mellitus of the young type 7 (MODY7), contribute to the development of neonatal diabetes mellitus. However, the function of KLF11 in the cardiovascular system still remains to be uncovered. In this study, we aimed to investigate the role of KLF11 in vascular endothelial inflammation. METHODS AND RESULTS: KLF11 is highly expressed in vascular ECs and induced by proinflammatory stimuli. Adenovirus-mediated KLF11 overexpression inhibits expression of tumor necrosis factors-α-induced adhesion molecules. Moreover, small interfering RNA-mediated KLF11 knockdown augments the proinflammatory status in ECs. KLF11 inhibits promoter activity of adhesion molecules induced by tumor necrosis factor-α and nuclear factor-κB p65 overexpression. Mechanistically, KLF11 potently inhibits nuclear factor-κB signaling pathway via physical interaction with p65. Furthermore, KLF11 knockdown results in increased binding of p65 to vascular cell adhesion molecule-1 and E-selectin promoters. At the whole organism level, KLF11(-/-) mice exhibit a significant increase in leukocyte recruitment to ECs after lipopolysaccharide administration. CONCLUSIONS: Taken together, our data demonstrate for the first time that KLF11 is a suppressor of EC inflammatory activation, suggesting that KLF11 constitutes a novel potential molecular target for inhibition of vascular inflammatory diseases.

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling.

Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1beta (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.

The use of in vitro transcription to probe regulatory functions of viral protein domains.

Adenoviruses (Ads), like other DNA tumor viruses, have evolved specific regulatory genes that facilitate virus replication by controlling the transcription of other viral genes as well as that of key cellular genes. In this regard, the E1A transcription unit contains multiple protein domains that can transcriptionally activate or repress cellular genes involved in the regulation of cell proliferation and cell differentiation. Studies using in vitro transcription have provided a basis for a molecular understanding of the interaction of viral regulatory proteins with the transcriptional machinery of the cell and continue to inform our understanding of transcription regulation. This chapter provides examples of the use of in vitro transcription to analyze transcriptional activation and transcriptional repression by purified, recombinant Ad E1A protein domains and single amino acid substitution mutants as well as the use of protein-affinity chromatography to identify host cell transcription factors involved in viral transcriptional regulation. A detailed description is provided of the methodology to prepare nuclear transcription extract, to prepare biologically active protein domains, to prepare affinity depleted transcription extracts, and to analyze transcription by primer extension and by run-off assay using naked DNA templates.

MicroRNA expression dynamics during murine and human erythroid differentiation.

OBJECTIVE: MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that regulate diverse cellular functions by sequence-specific inhibition of gene expression. We determined miRNA expression profile during erythroid differentiation and putative roles in erythroid differentiation. METHODS: The expression profile of 295 miRNAs before and after their erythroid differentiation induction was analyzed using microarray. Fluorescein-activated cell sorting analysis was used to isolate mouse spleen erythroblasts at different differentiation stages. Human cord blood CD34+ progenitors were differentiated in vitro. Real-time reverse transcriptase polymerase chain reaction was used to confirm the results of miRNA microarray. Synthetic oligonucleotides for miR-451 overexpression or knockdown were transfected into MEL cells. RESULTS: More than 100 miRNAs were found to be expressed in erythroid cells. The majority of them showed changes in their expression levels with progression of erythroid differentiation. Further analysis revealed that overall miRNA expression levels are increased upon erythroid differentiation. Of the miRNAs analyzed, miR-451 was most significantly upregulated during erythroid maturation. Functional studies using gain of function and loss of function approaches showed that miR-451 is associated with erythroid maturation. CONCLUSIONS: Dynamic changes in miRNA expression occurred during erythroid differentiation, with an overall increase in the levels of miRNAs upon terminal differentiation of erythroid cells. MiR-451 may play a role in promoting erythroid differentiation.

Systematic RNAi studies on the role of Sp/KLF factors in globin gene expression and erythroid differentiation.

Sp/KLF family of factors regulates gene expression by binding to the CACCC/GC/GT boxes in the DNA through their highly conserved three zinc finger domains. To investigate the role of this family of factors in erythroid differentiation and globin gene expression, we first measured the expression levels of selected Sp/KLF factors in primary cells of fetal and adult stages of erythroid development. This quantitative analysis revealed that their expression levels vary significantly in cells of either stages of the erythroid development. Significant difference in their expression levels was observed between fetal and adult erythroid cells for some Sp/KLF factors. Functional studies using RNA interference revealed that the silencing of Sp1 and KLF8 resulted in elevated level of gamma globin expression in K562 cells. In addition, K562 cells become visibly red after Sp1 knockdown. Benzidine staining revealed significant hemoglobinization of these cells, indicating erythroid differentiation. Moreover, the expression of PU.1, ETS1 and Notch1 is significantly down-regulated in the cells that underwent erythroid differentiation following Sp1 knockdown. Overexpression of PU.1 or ETS1 efficiently blocked the erythroid differentiation caused by Sp1 knockdown in K562 cells. The expression of c-Kit, however, was significantly up-regulated. These data indicate that Sp1 may play an important role in erythroid differentiation.

Functional study of transcription factor KLF11 by targeted gene inactivation.

Sp1/Krüppel-like factor (KLF) family of transcription factors regulates diverse biological processes including cell growth, differentiation, and development through modulation of gene expression. This family of factors regulates transcription positively and negatively by binding to the GC and GT/CACCC boxes in the promoter through their highly conserved three zinc finger domains. Although the molecular mechanism of gene regulation by this family of proteins has been well studied, their exact role in growth and development in vivo remains largely unknown. KLF11 has been implicated in the regulation of cell growth and gene expression. To determine the physiological function of KLF11, we generated KLF11-null mice by gene-targeting technology. Homologous KLF11(-/-) mice were bred normally and were fertile. Hematopoiesis at all stages of development was normal in the KLF11(-/-) mice. There was no effect on globin gene expression. These mice lived as long as the wild-type mice without evident pathological defects. Thus, despite its cell growth inhibition and transcriptional regulation functions observed when transiently or stably expressed in cultured cells in vitro, the results from genetic knockout suggest that KLF11 is not absolutely required for hematopoiesis, growth, and development.

Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro.

RNA interference (RNAi) is a powerful tool for studying gene function. Here, we describe an inducible small interfering RNA expression system that allows a tight control of the specific gene silencing by RNAi. Using this system, we demonstrated the inducible RNAi effect on the gene expression in mammalian cells. We further showed that inducible knockdown of endogenous CXC chemokine receptor-4 (CXCR4) gene expression in breast cancer cells resulted in significant inhibition of breast cancer cell migration in vitro. This system should be useful for both basic researches on gene function and therapeutic applications of RNAi.

Functional interplay between CBP and PCAF in acetylation and regulation of transcription factor KLF13 activity.

The transcriptional co-activators CBP/p300 and PCAF participate in transcriptional activation by many factors. We have shown that both CBP/p300 and PCAF stimulate the transcriptional activation by KLF13, a member of the KLF/Sp1 family, either individually or cooperatively. Here we further investigated how CBP and PCAF acetylation regulate KLF13 activity, and how these two co-activators functionally interplay in the regulation of KLF13 activity. We found that CBP and PCAF acetylated KLF13 at specific lysine residues in the zinc finger domain of KLF13. The acetylation by CBP, however, resulted in disruption of KLF13 DNA binding. Although the acetyltransferase activity of CBP is not required for stimulating the DNA binding activity of all of the transcription factors that we have examined, the disruption of factor DNA binding by CBP acetylation is factor-specific. We further showed that PCAF and CBP act synergistically and antagonistically to regulate KLF13 DNA binding depending on the status of acetylation. PCAF blocked CBP acetylation and disruption of KLF13 DNA binding. Conversely, acetylation of KLF13 by CBP prevented PCAF stimulation of KLF13 DNA binding. PCAF blocked CBP disruption of KLF13 DNA binding by preventing CBP acetylation of KLF13. These results demonstrate that acetylation by CBP has distinct effects on transcription factor DNA binding, and that CBP and PCAF regulate each other functionally in their regulation of transcription factor DNA binding.

Transcription coactivator CBP has direct DNA binding activity and stimulates transcription factor DNA binding through small domains.

CBP and p300 are transcriptional coactivators that physically interact with diverse sequence-specific DNA-binding factors through conserved domains. To further investigate the functional roles of these protein-interaction domains in CBP/p300 regulation, we have identified multiple domains of CBP that interact with FKLF2 and the CH2 domain as a new p53 interacting domain of CBP. Functional studies demonstrate that several domains of CBP are capable of stimulating FKLF2 and p53 DNA binding. In addition, we found that CBP through distinct domain is able to bind DNA directly with no specificity. We identified a 51-residue domain in CBP that is capable of interacting with both transcription factors and DNA. We named this domain PDBD for protein and DNA binding domain. These results unveiled two novel activities of CBP. First, these highly conserved domains of CBP not only function to recruit CBP to the target promoter through interaction with DNA-bound transcription factors, but they also actively regulate the DNA binding activity of their interacting factors. Second, by directly interacting with DNA, CBP may orchestrate the formation of stable and promoter-committed transcriptional complexes through interactions with both proteins and promoter DNA.

Functional interaction between coactivators CBP/p300, PCAF, and transcription factor FKLF2.

The Sp1/KLF family of factors regulates diverse cellular processes, including growth and development. Fetal Krüppel-like factor (FKLF2) is a new member of this family. In this study, we characterized the coactivators involved in FKLF2 transcriptional activation. Our results show that both CBP/p300 and p300/CBP-associated factor (PCAF) enhance FKLF2 transcriptional activity. We demonstrate that the acetyltransferase activity of PCAF but not that of CBP/p300 is required for stimulating FKLF2 transcription activity. We further show that p300 and PCAF act cooperatively in stimulating FKLF2 transcriptional activation. FKLF2 interacts with both CBP and PCAF through specific domains, and CBP and PCAF acetylate FKLF2. Both CBP/p300 and PCAF stimulate FKLF2 DNA binding activity. The integrity of the acetyltransferase domain of PCAF but not that of CBP/p300 is required for stimulating FKLF2 DNA binding activity. These results demonstrate that CBP/p300 and PCAF stimulate FKLF2 transcriptional activity at least by enhancing its DNA binding. The acetyltransferase activities of CBP/p300 and PCAF play a distinct role in stimulating FKLF2 transcription and DNA binding.