Proximal Promoter Fragment Research Articles

Nfix  Promotes Survival of Immature Hematopoietic Cells Via Regulation of C-Mpl

Hematopoietic stem and progenitor cells (HSPC) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently identified the transcription factor nuclear factor I/X (Nfix) as a critical regulator of hematopoietic stem and progenitor cell (HSPC) survival post-transplantation. Nfix -depleted HSPC are lost from the marrow between 7-10 days post-transplant and residual Nfix -depleted HSPC display elevated levels of apoptosis at these time-points. To further characterize the anti-apoptotic role of Nfix in HSPCs, we overexpressed Nfix in Lineage- Sca-1+ c-Kit+ (LSK) cells that were then maintained ex vivo under serum-free conditions with supportive cytokines and monitored the cultures for cell surface phenotype, growth, vector selection, and apoptosis. Ectopic expression of Nfix in primary mouse HSPCs had no significant effect on the expression of lineage markers compared to controls, but did appear to accelerate the loss of immunophenotypic LSK cells, as well as colony forming units (p = 0.023). Nfix -overexpressing HSPCs were extended in their ex vivo culture lifespan from about 20 days to 40 days, and were steadily selected for in culture. HSPCs overexpressing Nfix also displayed hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation compared to controls (p = 0.032). NFIX has been shown to potentially influence transcription of several known regulators of HSC biology. Here, we show that ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Consequently, Bcl-XL, an anti-apoptotic factor induced by STAT5, was also significantly upregulated in Nfix -overexpressing cells by two weeks of culture compared to controls (p = 0.0038). Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody significantly negated the anti-apoptotic effect of Nfix overexpression on cultured HSPCs (p = 0.045, p = 0.0098, respectively). Further, NFIX was capable of transcriptional activation of a proximal c-Mpl promoter fragment (p = 0.0056). In sum, these data suggest that NFIX-mediated up-regulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo . DisclosuresNo relevant conflicts of interest to declare.

Nfix Promotes Survival of Immature Hematopoietic Cells via Regulation of c-Mpl.

Hematopoietic stem and progenitor cells (HSPCs) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently reported that nuclear factor I/X (Nfix) promotes HSPC survival post-transplant. Here, we report that ectopic expression of Nfix in primary mouse HSPCs extends their ex vivo culture from about 20 to 40 days. HSPCs overexpressing Nfix display hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation relative to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody negated the antiapoptotic effect of Nfix overexpression on cultured HSPCs. Furthermore, NFIX was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated upregulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Stem Cells 2018;36:943-950.

Characterization of 5′ promoter and exon 1–3 polymorphism of the RAET1E gene

NKG2D is an activating receptor utilized by natural killer (NK) cells that recognizes upregulated ligands on infected, tumorigenic and damaged cells, leading to their cytolysis. However, the NKG2D ligand (NKG2DL) system is very complex with eight known gene loci encoding slightly different molecules. Furthermore, most NKG2DL gene loci such as MICA and MICB are highly polymorphic with potential for functional differences. NKG2DL expression on tumors varies depending on the malignancy and tumors can also release soluble NKG2DL that exert anergic effects on NK cells when engagement with NKG2D occurs, allowing escape from NK cell immunosurveillance.We carried out RAET1E typing of IHW cell line DNA, including a 580bp proximal promoter fragment and exons 1–3 identifying 13 of 15 known RAET1E alleles. We determined 7 polymorphisms within the promoter region, including 2 already known that contributed to 9 promoter types. RAET1E alleles with variability in the extracellular region also differed with respect to promoter type and one allele, RAET1E∗003, associated with 5 promoter types. We then identified putative transcription factor binding sites for RAET1E, and found 5 of the 7 promoter polymorphisms may disrupt these sites, abrogating binding of transcription factors and varying the potential level of expression.

Identifying an optimal promoter sequence of goat β-lactoglobulin gene for constructing high-expression vectors in mammary epithelial cells

This study aimed to identify the highest efficiency promoter sequence of β-lactoglobulin (BLG) gene and construct a mammary-specific expression vector for expressing high level of transgene in goat mammary epithelial cells. We amplified 13 BLG proximal promoter fragments, 2 introns, and a 3′ untranslated region from goat genome. The dual-luciferase assay showed that the promoter activities of these BLG proximal promoter fragments were significantly higher in HC11 than those in 293T or GEF cells, and that BLG promoter region BLG11 (431bp, −391/+40) was an optimal sequence to drive transgenic expression. Western blot analysis confirmed that the promoter sequence BLG11 had a higher promoter activity in goat mammary epithelial cells than other promoter sequences. Moreover, the expression levels of GFP were significantly increased by combining promoter region BLG11 and native intron 1 in transfected goat mammary epithelial cells, and increased further with introns 1 and 2. This study demonstrated that a short BLG promoter region BLG11 combined with introns 1 and 2 could promote transgenic expression in goat mammary epithelial cells. These mammary regulated elements may be useful for production of mammary gland bioreactor.

Transcriptional inhibition of etv2 expression is essential for embryonic cardiac development

E-twenty six variant 2 (Etv2) transcription factor participates in cardiac, vascular-endothelial and blood cell lineage specification decisions during embryonic development. Previous studies have identified genomic elements in the etv2 locus responsible for vascular endothelial cell specification. Using transgenic analysis in zebrafish, we report here an etv2 proximal promoter fragment that prevents transgene misexpression in myocardial progenitor cells. This inhibition of etv2 expression in the cardiac progenitor population is partly mediated by Scl and Nkx2.5, likely through direct binding to the etv2 promoter, and cis-regulatory elements located in the first and second introns. The results identify an etv2 cis-regulatory mechanism controlling cardiovascular fate choice implying that etv2 participates in a transcriptional network mediating developmental plasticity of endothelial progenitor cells during embryonic development.

Monitoring bovine fetal fibroblast reprogramming utilizing a bovine NANOG promoter‐driven EGFP reporter system

NANOG is an essential transcription factor involved in the proliferation and maintenance of embryonic stem cells (ESC) and reprogramming of somatic cells to a pluripotent state. Oct4 and Nanog promoter-driven enhanced green fluorescent protein (EGFP) reporters have been employed for establishing lines of induced pluripotent stem cells (iPSC) from mouse, human, and pig. In ruminants, including cattle, in which no fully validated ESC lines have been established, iPSC generated by reprogramming somatic cells to an ESC-like state may prove useful in the production of genetically modified livestock. In this study, utility of the bovine NANOG reporter was tested for use with cattle. Seven proximal bovine NANOG promoter fragments of different size were fused to the LUC gene, and were tested in mouse ESC lines using a dual-luciferase assay. Three of the bovine NANOG promoters, 315 bp (-134/+181), 446 bp (-265/+181), and 1,100 bp (-919/+181), were fused to a nuclear localized signal EGFP reporter gene. The fidelity of these constructs was analyzed by transfection into mouse ESC and bovine fetal fibroblasts (bFFs), and subsequent reprogramming of the bFF. Fusion of the transgenic bFF with human teratocarcinoma (NTERA2) cells induced nuclear expression of the EGFP reporter. Similarly, bFF-derived somatic cell nuclear transfer (SCNT) embryos expressed EGFP in a stage- and location-appropriate manner. Following reprogramming of transgenic bFFs for 10 days with an Oct4-Sox2-Klf4-cMyc vector, iPSC expressed EGFP and alkaline phosphatase. These results indicate that NANOG reporters can be used to monitor nuclear reprogramming of bFFs and to distinguish cell allocation in SCNT-derived embryos.

Cyclic AMP Enhances TGFβ Responses of Breast Cancer Cells by Upregulating TGFβ Receptor I Expression

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein) and TβRI (TGFβ receptor 1), were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected.

Prostaglandin E2 induces stromal cell-derived factor-1 expression in prostate stromal cells by activating protein kinase A and transcription factor Sp1

Recent reports indicate prostaglandin E2 (PGE2) can modulate tumor environment and promote angiogenesis through induction of stromal cell-derived factor 1 (SDF-1) production. We investigated the mechanism of PGE2-induced SDF-1 regulation in human prostate stromal cell and analyzed the effects in a stromal–epithelial interaction model. PGE2 stimulation increased SDF-1 expression in the prostate stromal cell lines WPMY-1 and NAF. We revealed signaling through the PGE2 receptor EP3 and activation of protein kinase A (PKA) are required. The EP3 agonist sulprostone and the cAMP analog forskolin mimicked and the EP3 siRNA, antagonist L798106 and the PKA inhibitor H89 abrogated the effect of PGE2 on SDF-1 expression. SDF-1 promoter truncation experiments demonstrated a 254bp (from nt −219 to nt +34) SDF-1 proximal promoter fragment containing 5 putative transcription factor Sp1 binding motifs is sufficient for PGE2 induction. CHIP assays confirmed binding and PGE2 induced recruitment of Sp1 to the SDF-1 promoter. Sp1 motif mutation identified Sp1 motifs −140/−133 and −9/+1 as the crucial elements responsible for PGE2 induction. Moreover, SDF-1 was up- or down-regulated by Sp1 over-expression or knock-down. We also demonstrate stimulation of migration of prostate cancer cell lines PC3 and DU145 with conditioned media collected from WPMY-1 or NAF cells stimulated with PGE2 and blockade of enhanced migration by a SDF-1 neutralizing antibody. In conclusion, we provide evidence for a paracrine prostate stromal–epithelial interaction induced by upregulation of expression of SDF-1 by PGE2. Our research provides new insights into the mechanism promoting metastasis of prostate carcinoma via stromal–epithelial interaction.

Selection of a common multipotent cardiovascular stem cell using the 3.4-kb MesP1 promoter fragment

Common cardiovascular progenitor cells are characterized and induced by expression of the transcription factor MesP1. To characterize this population we used a 3.4-kb promoter fragment previously described by our group. This served to isolate MesP1-positive cells from differentiating ES stem cells via magnetic cell sorting based on a truncated CD4 surface marker. As this proximal promoter fragment omits a distal non-cardiovasculogenic enhancer region, we were able to achieve a synchronized fraction of highly enriched cardiovascular progenitors. These led to about 90% of cells representing the three cardiovascular lineages: cardiomyocytes, endothelial cells and smooth muscle cells as evident from protein and mRNA analyses. In addition, electrophysiological and pharmacological parameters of the cardiomyocytic fraction show that almost all correspond to the multipotent early/intermediate cardiomyocyte subtype at day 18 of differentiation. Further differentiation of these cells was not impaired as evident from strong and synchronous beating at later stages. Our work contributes to the understanding of the earliest cardiovasculogenic events and may become an important prerequisite for cell therapy, tissue engineering and pharmacological testing in the culture dish using pluripotent stem cell-derived as well as directly reprogrammed cardiovascular cell types. Likewise, these cells provide an ideal source for large-scale transcriptome and proteome analyses.

Cloning of the human activated leukocyte cell adhesion molecule promoter and identification of its tissue-independent transcriptional activation by Sp1

Activated leukocyte cell adhesion molecule (ALCAM) belongs to the immunoglobulin cell adhesion molecule super family. ALCAM is implicated in tumor progression, inflammation, and the differentiation of hematopoietic stem cells. Hitherto, the identity of regulatory DNA elements and cognate transcription factors responsible for ALCAM gene expression remained unknown. In this report, the human ALCAM promoter was cloned and its transcriptional mechanisms elucidated. The promoter is TATA-less and contains multiple GC-boxes. A proximal 650-bp promoter fragment conferred tissue-independent activation, whereas two contiguous regions upstream of this region negatively influenced promoter activity in a tissue-specific manner. The positive regulatory promoter region was mapped to a core 50 base pair sequence containing a conical Sp1 element. Mutation analysis revealed that this element alone or in tandem with elements immediately upstream was required for maximal promoter activity. Chromatin analysis revealed that Sp1 binds exclusively to the canonical binding sequence in vivo, but not to DNA sequence immediately upstream. Finally, we showed that over-expression of Sp1 significantly increased the basal promoter activity. Thus, Sp1 activated the ALCAM promoter in most cells. These findings have important ramifications for unraveling the roles of ALCAM in inflammation and tumorigenesis.

Transcriptional Repression of the α7 Nicotinic Acetylcholine Receptor Subunit Gene (CHRNA7) by Activating Protein-2α (AP-2α)

The CHRNA7 gene, which encodes the α7 nicotinic acetylcholine receptor (α7*nAChR), has been implicated as a candidate gene in schizophrenia. Expression of the α7*nAChR mRNA and protein are reduced in multiple regions of post-mortem brain from patients diagnosed with schizophrenia. Transcriptional regulation may therefore be an important mechanism for the regulation of this gene. A 230-bp proximal promoter fragment, necessary for transcription in cultured neuroblastoma cells, was used to study a putative AP-2α binding site. Mutation of the site indicates that AP-2α plays a negative role in regulating CHRNA7 transcription. This was confirmed through knockdown and overexpression of AP-2α. Electrophoretic mobility shift assays (EMSAs) identified positive DNA-protein interaction at this same site, and supershift assays indicate that the complex includes AP-2α. The interaction was confirmed in cells using chromatin immunoprecipitation (ChIP). DNA methylation was discovered as an anomalous mechanism for CHRNA7 regulation in one cell line. These studies suggest a role for AP-2α regulation of CHRNA7 mRNA expression in multiple tissues during development.

Molecular Mechanism Underlying 1,25-Dihydroxyvitamin D Regulation of Nephrin Gene Expression

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.

Transcriptional analysis of the titin cap gene

Mutations in titin cap (Tcap), also known as telethonin, cause limb-girdle muscular dystrophy type 2G (LGMD2G). Tcap is one of the titin interacting Z-disc proteins involved in the regulation and development of normal sarcomeric structure. Given the essential role of Tcap in establishing and maintaining normal skeletal muscle architecture, we were interested in determining the regulatory elements required for expression of this gene in myoblasts. We have defined a highly conserved 421bp promoter proximal promoter fragment that contains two E boxes and multiple putative Mef2 binding sequences. This promoter can be activated by MyoD and myogenin in NIH3T3 fibroblast cells, and maintains the differentiated cell-specific expression pattern of the endogenous Tcap in C2C12 cells. We find that while both E boxes are required for full activation by MyoD or myogenin in NIH3T3 cells, the promoter proximal E box has a greater contribution to activation of this promoter in C2C12 cells and to activation by MyoD in NIH3T3 cells. Together, the data suggest an important role for MyoD in activating Tcap expression through the promoter proximal E box. We also show that myogenin is required for normal expression in vivo and physically binds to the Tcap promoter during embryogenesis.

Activation of Gene Transcription by Heat Shock Protein 27 May Contribute to Its Neuronal Protection

Heat shock proteins are up-regulated as a physiological response to stressful stimuli and generally function as molecular chaperones for improperly folded protein substrates. The small heat shock protein HSP27 (or HSPB1) has multiple cytoplasmic roles. HSP27 also can translocate to the nucleus in response to stress, but the functional significance of this nuclear distribution has not been elucidated. We have previously implicated HSP27 as a genetic modifier of spinocerebellar ataxia 17 (SCA17), a neurological disease caused by a polyglutamine expansion in the TATA-binding protein (TBP). Altered expression of HSP27 is also found in cell models of other polyglutamine diseases, including Huntington disease as well as SCA3 and SCA7. Here, we show that Hsp27, unlike Hsp70, is not detected in mutant TBP aggregates in primary cerebellar granule neurons from transgenic SCA17 mice. Although HSP27 overexpression does not reduce the aggregation of cotransfected mutant TBP containing 105 glutamines, it potentiates activated transcription from both TATA-containing and TATA-lacking promoters. Neither HSP40 nor HSP70 elicits the same transcriptional effect. Moreover, HSP27 interacts with the transcription factor SP1, and coexpression of SP1 and nuclear localization signal-tagged HSP27 synergistically activates reporter constructs for the SP1-responsive neurotrophic receptor genes Ngfr(p75) and TRKA. Overexpression of nuclear localization signal-tagged HSP27 also rescues mutant TBP-mediated down-regulation of TrkA in a PC12 cell model of SCA17. These results indicate that nuclear HSP27 can modulate SP1-dependent transcriptional activity to promote neuronal protection.

Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors

AbstractThe Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.

Characterization of promoter elements required for cardiac chamber-specific expression

Salmon cardiac natriuretic peptide (sCP, an A-type natriuretic peptide) is an excellent model for the study of cardiac chamber-specific gene expression because it is uniquely specific to the heart and its promoter drives gene expression effectively in mammalian cardiac atrial but not in ventricular cells. We have now prepared hybrid luciferase constructs containing specific sequences from both sCP and BNP 5′ promoters. According to our results the simple addition of a short rat BNP proximal promoter fragment to the inert 846 nucleotide sCP proximal promoter increases 100 times the basal activity of the sCP promoter in rat ventricular cardiomyocytes, and also conveys inducibility by mechanical load and endothelin-1. Thus, a small rBNP promoter fragment can transform the prototypical A-type natriuretic peptide regulation of sCP to B-type regulation, a result which argues against a major role of repressors causing the low expression level of A-type peptides in ventricular cardiomyocytes.

Induction and mechanism of growth arrest DNA damage-inducible gene 45 β in human hepatoma HepG2 cells by S-adenusylmethionine

Objective To identify the active region of proximal promoter growth arrest DNA damage-inducible gene 45 β (GADD45β) and to evaluate the influence of S-adenosylmethionine on HepG2. Methods The proximal promoter fragments (-618/-520) were synthesized in vitro by ev-ery 30-50 nucleotides, cloned into pGL3 basic luciferase expression plasmid respectively, then trans-fected into HepO2 cells by electroporation. The promoter regions were identified with reference to TRANSFAC database. Following S-adenosylmethionine administration, quantitative real-time PCR was employed to validate the expression of GADD45β in HepG2. The effect of S-adenosylmethionine to promoter activity was further focused on. Results Using the luciferase assay, several transcription factor binding sites were identified in the proximal promoter of GADD45β, which included three NF-kBs (- 602/- 593, - 581/- 572, and - 537/- 528) according to the consensus sequences in TRANSFAC database. After S-adenosylmethionine administration, the increasing expression of GADD45β was marked in a dose-dependent manner. The luciferase assay also demonstrated the pro-moter activity of fragment constructed the first NF-kB binding site could be induced by S-adenosylme-thionine. Conclusion S-adenosylmethionine can induce the expression of GADD45β, which is specific-ally down-regulated in HCC. The possible mechanism may be involved in the regulation of transcrip-tion factor NF-kB in GADD45β proximal promoter. Key words: Carcinoma,hepatocellular; S-adenosylmethionine; GADD45β; Promoter

Lineage-specific silencing of human IL-10 gene expression by promoter methylation in cervical cancer cells

Epigenetic analysis was performed to demonstrate that the normal and neoplastic epithelial cells do not serve as the source of the locally elevated IL-10 production during cervical carcinogenesis. Bisulfite sequencing was used to correlate promoter CpG methylation with the transcription of the gene. Lack of IL-10 transcription in HeLa, SiHa, Caski, HT-3, C33-A, HaCaT cell lines and in primary human keratinocytes correlated consistently with the methylated state of the proximal CpG residues, particularly with the two most proximal CpGs at positions –185 and –110. These two sites were also highly methylated in normal and malignant cervical cells directly isolated from patient material. On the other hand, IL-10 producing peripheral blood mononuclear cells had unmethylated CpG residues in the proximal promoter associated with acetylated H3 and H4 histones as determined by chromatin immunoprecipitation. In HeLa carrying epigenetically silenced endogeneous IL-10 promoter, the transfected non-CpG methylated 1 kb and 0.6 kb proximal promoter fragments could drive reporter gene expression, which was reversed by cassette methylation of these promoter fragments. In conclusion, the CpG methylation pattern of the proximal promoter is implicated as a major determinant of transcriptional silencing of human IL-10 expression in cells of cervical epithelial origin.

Sgt1 has co-chaperone properties and is up-regulated by heat shock

The Sgt1 protein is a binding partner of heat shock proteins such as Hsp90, Hsp70 or Hsc70. In this work we show that the level of Sgt1 is increased in HEp-2 cells exposed to heat shock or radicicol. The citrate synthase aggregation assay shows that Sgt1 attenuates aggregation of the enzyme induced by increased temperature as efficiently as p23, a known co-chaperone of Hsp90. We have cloned two fragments of the human Sgt1 gene promoter (−708/+98 and −351/+98) into pGL3-luciferase vector and found that both fragments generated a 2-fold increase in luciferase activity upon heat shock. Furthermore, electrophoretic mobility shift assay revealed binding of the HSF-1 transcription factor to the heat shock element in the proximal (−42/−2) Sgt1 gene promoter fragment. These results indicate that Sgt1 is a co-chaperone protein with an expression pattern matching that of the well known heat shock proteins.

HTERT Transcription Is Repressed by Cbfa1 in Human Mesenchymal Stem Cell Populations

Human BMSSCs lose telomerase activity in vitro, which leads to chromosomal instability and cellular senescence. We observed an inverse expression pattern between the osteogenic master regulatory gene, CBFA1, and the stem cell-associated gene, hTERT. We showed that Cbfa1 acts as a partial repressor of TERT, which may facilitate cellular differentiation. The absence of telomerase activity by cultured human bone marrow stromal stem cells (BMSSCs) causes critical shortening of chromosomal telomeres, leading eventually to cellular senescence. Ex vivo expansion of BMSSCs correlates to an increase in osteogenic lineage associated markers such as alkaline phosphatase, bone sialoprotein, and osteocalcin that are regulated by the master regulatory transcription factor, Cbfa1 (Runx2). This study examined whether Cbfa1 was capable of regulating the promoter of the early stem cell-associated gene, telomerase reverse transcriptase (TERT). Human BMSSCs were isolated by fluorescence-activated cell sorting. Telomerase activity was determined using the telometric repeat amplification protocol. CBFA1 and TERT gene expression was assessed by real-time PCR. The functional capacity of Cbfa1 to bind to the hTERT promoter was performed using a modified electrophoretic mobility shift assay (EMSA). Chromatin immunoprecipitation (ChIP) analysis was used to examine Cbfa1 binding to the hTERT promoter in vivo. Functional analysis of CBFA-1 wildtype and mutant DNA binding sites on TERT promoter fragments was assessed using the promoterless green fluorescence protein (GFP) reporter vector, pEGFP-1, after transfection into HOS cells. This study showed an inverse expression pattern between the osteogenic master regulatory gene, CBFA1, and the stem cell-associated gene, hTERT. The data showed that BMSSCs undergo osteogenic commitment after the loss of hTERT expression, with concomitant elevated levels of CBFA1 transcripts. In addition, two unique Cbfa1 DNA binding sites were identified on the hTERT proximal promoter by EMSA supershift assay. Mutated forms of the putative Cbfa1 binding sites, created by site-directed mutagenesis, were able to abolish this interaction. ChIP analysis showed that Cbfa1 interacted directly with the hTERT promoter in vivo. Functional studies using GFP reporter constructs, driven by 2- and 3-kbp hTERT proximal promoter fragments, showed significantly lower levels of transcriptional activity compared with corresponding constructs with mutated Cbfa1 binding site Oligo 2. These studies suggest that Cbfa1 may act as a repressor of early stem cell markers such as hTERT as one possible mechanism for facilitating cellular differentiation.