BCL11B Expression Research Articles

A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis

The development of the dentate gyrus is characterized by distinct phases establishing a durable stem-cell pool required for postnatal and adult neurogenesis. Here, we report that Bcl11b/Ctip2, a zinc finger transcription factor expressed in postmitotic neurons, plays a critical role during postnatal development of the dentate gyrus. Forebrain-specific ablation of Bcl11b uncovers dual phase-specific functions of Bcl11b demonstrated by feedback control of the progenitor cell compartment as well as regulation of granule cell differentiation, leading to impaired spatial learning and memory in mutants. Surprisingly, we identified Desmoplakin as a direct transcriptional target of Bcl11b. Similarly to Bcl11b, postnatal neurogenesis and granule cell differentiation are impaired in Desmoplakin mutants. Re-expression of Desmoplakin in Bcl11b mutants rescues impaired neurogenesis, suggesting Desmoplakin to be an essential downstream effector of Bcl11b in hippocampal development. Together, our data define an important novel regulatory pathway in hippocampal development, by linking transcriptional functions of Bcl11b to Desmoplakin, a molecule known to act on cell adhesion.

Abstract 2204: BCL11B is a critical EWS/FLI up-regulated target in Ewing's sarcoma

Abstract Objective: Ewing's sarcoma is a highly aggressive tumor of the bone and soft tissue that affects the pediatric and young adult population, and is characterized by the EWS/FLI chromosomal translocation. This fusion protein acts as an oncogenic transcription factor to dysregulate the expression of a number of genes that contribute to the transformed phenotype. One challenge is to define which of these dysregulated genes are important for the oncogenic phenotype of Ewing's sarcoma. To address this issue, we used a proteomics approach to identify new protein-protein interactions among target genes of EWS/FLI. This screen identified BCL11B, which had previously been identified in genome-wide transcriptional profiling studies by our group, and others, as an up-regulated target of EWS/FLI. Methods: To define the function of BCL11B in Ewing's sarcoma, we used retroviral mediated shRNA knock-down of EWS/FLI, followed by re-expression of the EWS/FLI cDNA, to verify that BCL11B expression is modulated by the fusion protein. Anchorage independent growth assays assessed the oncogenic potential of patient derived Ewing's sarcoma cell lines in which BCL11B had been knocked-down with shRNA. Luciferase assays and chromatin immunoprecipitation techniques were used to interrogate BCL11B regulation by EWS/FLI. Results: Our results show that BCL11B is up-regulated by EWS/FLI and is necessary for oncogenic transformation in patient derived Ewing's sarcoma cell lines. Our work addressing the mechanism of BCL11B regulation by EWS/FLI is on-going, but preliminary data suggests that BCL11B may be a direct target of EWS/FLI. Conclusion: This work identified BCL11B as an EWS/FLI up-regulated target that contributes to transformation in Ewing's sarcoma. It also provides a setting in which we can broaden our knowledge regarding the mechanisms used by EWS/FLI to up-regulate critical target genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2204. doi:1538-7445.AM2012-2204

The role of BCL11B in regulating the proliferation of human naive T cells

The effect of the B-cell chronic lymphocytic leukemia/lymphoma 11B gene (BCL11B) on human T-cell regulation remains unclear. To characterize the functions of BCL11B, recombinant BCL11B and BCL11B siRNA were transfected into human naive T cells to overexpress or knock down BCL11B expression, respectively. After BCL11B overexpression, the proliferation ability and the T-helper (Th) subset were increased, whereas no significant alteration in the expression pattern and clonality of the T-cell receptor Vβ subfamilies was observed. After BCL11B knockdown, a similar distribution of Vβ subfamilies was detected in the naive T cells; however, the proliferation capacity substantially decreased. Global gene expression profiling revealed that the dysregulated genes were mainly involved in T-cell activation and proliferation. BCL11B could selectively promote Th-cell differentiation because of increased CXCL10 and CXCL11 expression. BCL11B suppression may inhibit proliferation and induce apoptosis, which may relate to changes in the expression of CFLAR-CASP8-CASP10 in the mitochondrial pathways. In conclusion, BCL11B is required for T-cell survival; its overexpression could effectively increase the T-cell activation and proliferation abilities and Th-cell differentiation as well.

Down regulation of BCL11B expression inhibits proliferation and induces apoptosis in malignant T cells by BCL11B-935-siRNA

To screen the highly efficient and specific B-cell chronic lymphocytic leukemia/lymphoma 11B (BCL11B) small interfering RNA (siRNA) which are able to downregulate the BCL11B gene expression in human T-cell acute lymphoblastic leukemia, thereby inhibiting the leukemic T-cell proliferation and inducing apoptosis, four BCL11B-siRNAs and the scrambled non-silencing siRNA control (sc) were designed and obtained by chemosynthesis. After nucleofection, BCL11B expression in the mRNA and the protein levels were measured by qRT–PCR and immunoblotting, respectively. The biological consequences based on the highly efficient and specific BCL11B-siRNA were demonstrated by CCK-8 kit, morphological changes (Hoechst 33258 staining), high-resolution imaging, and flow cytometry. Reduction in the BCL11B mRNA level was observed at 24 or 48 hours in molt-4 T cells with BCL11B-935-siRNA, BCL11B-434-siRNA, or BCL11B-748-siRNA, respectively. BCL11B protein expression levels were reduced by 34·77% and 41·73% in the BCL11B-935-siRNA- and BCL11B-434-siRNA-treated cells, compared with the control level at 72 hours. In comparison with BCL11B-434-siRNA treatment group, the Molt-4 cells transfected with the BCL11B-935-siRNA showed significantly inhibited proliferation and effectively induced apoptosis (P<0·05). When highly efficient and specific BCL11B-935-siRNA was used to analyze the inhibition of BCL11B mRNA level in primary T-cell acute lymphoblastic leukemia (T-ALL) cells, similar result was obtained. In conclusion, siRNAs targeting the different exon domains resulted in different silencing effects and biological consequences. Suppression of BCL11B by RNA interference could inhibit the proliferation and induce the apoptosis effectively in leukemic T cells, which might be considered as a new target therapeutic strategy in T-cell malignancies.

The Regulation of Differentiation and Proliferation of the BCL11B Gene In T Cells

AbstractAbstract 2769In order to analyze the regulation of differentiation and proliferation of the BCL11B gene in human T cells, as well as the related molecular mechanism, BCL11B (B cell lymphoma/leukemia11B, BCL11B) recombinant plasmid and small interfering RNA (siRNA) were transferred into naïve T cells (CD3+CD45RA+ T cells) from healthy individual respectively by gene transfer and RNA interference techniques. The eukaryotic expression plasmid pIRES2-BCL11B-EGFP (pBCL11B) and BCL11B-siRNA-935(si-935), obtained by chemosynthesis, were transferred into different cell lines by using transfection techniques. The transfection efficiencies were examined by fluorescence microscopy and FCM. The expression of the mRNA and the corresponding protein levels of BCL11B were detected by real-time quantitative PCR with TaqMan technique and Western blotting. The pBCL11B plasmid and si-935 were transferred into naïve T cells from healthy individual using the Nucleofector™ technique. The microscopic morphologies were detected by atomic force microscopy (AFM), the expression pattern and clonality of TCR Vβ subfamily genes were analyzed by RT-PCR and genescan techniques, the effect of BCL11B in T cells proliferation in vitro were evaluated by the capability of CFU-T, the changes of T cells immune phenotypes (CD3, CD4, CD8, CD45RA, CD45RO, CD25 and CD86) were examined by FCM. The expression patterns of BCL11B were up-regulated or down-regulated respectively, then, the global gene expression profiling was analyzed by the Affymetrix HG U133 Plus 2.0 Gene Chips. Some of the differentially expressed genes were verified by various techniques. The up- or down-regulated BCL11B expression of mRNA and protein levels could be confirmed in naïve T cells. The AFM images revealed that the surface of naïve T cells treated with pBCL11B showed more signs of expansion than others, and were coated with a larger number of extracellular polymers on the outer layer. Seventeen Vβ subfamily T cells displayed polyclonality could be detected in naïve T cells in up-regulated BCL11B manner. The more effective of capability of CFU-T was also confirmed in the same naïve T cells group. The Th subset was obviously changed after up-regulated of BCL11B in naïve T cells, while the ratio of CD4/CD8 was doubled increased. By contrast, in si-935 group, all of the 24 Vβ subfamilies were unable to detect, the number of CFU-T decreased substantially, and the ratio of CD4/CD8 showed no significant difference in compared with control group. Global gene expression profiling analysis showed that up-regulated genes were found in 95 probe sets, while 124 genes were down-regulated in naïve T cells transfected with BCL11B gene, while up-regulated 302 genes and down-regulated 209 genes were detected in naïve T cells from si-935 group. The up- or down-regulation genes were mainly involved in T cells activation and proliferation, which were associated with the high levels of CD3 and IL-2. BCL11B could promote the Th cells differentiation selectively owing to the increased expression of CXCL10 and CXCL11. Suppression BCL11B expression might inhibit the proliferation and effectively induce the apoptosis, which related to the changes of CFLAR-CASP8-CASP10 in mitochondrial pathways. The changes of expression levels of these genes were confirmed by real-time PCR and ELISA. In conclusions, This is the first report providing a detailed analysis of the regulation of the BCL11B gene in T cell differentiation and proliferation, and the possible mechanism of this gene. It was shown that BCL11B was required for the survival of T cells. Up-regulated BCL11B expression could increase the abilities of T cells activation, proliferation, and Th cells differentiation effectively. However, suppression BCL11B expression might inhibit the proliferation and induce the apoptosis. Disclosures:Li:The study was supported by grants from the National Natural Science Foundation (30771980) and the Guangdong Science & Technology Project (2009B050700029): Research Funding.

Inhibition of BCL11B Expression Leads to Apoptosis of Malignant T Cells but Not CD34+ Cells.

AbstractAbstract 3755Altered expression, mutation, disruption, or rearrangement of the B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) has been associated with T-cell malignancies. BCL11B overexpression occurs primarily in T-cell malignancies, suggesting that it may be a target gene for cancer therapy. Our previous study showed that the inhibition of BCL11B expression by siRNA led to apoptosis in human T-cell acute lymphoblastic leukemia cell lines (Jurkat, huT78, and Molt-4), but not in normal, mature T cells. To confirm the potential of BCL11B siRNA as a therapeutic agent and to determine its safety, the present study further analyzed the effects of BCL11B suppression on hemopoietic stem/progenitor cells, CD34+ cells were collected from three samples of umbilical cord blood using magnetic-activated cell sorting. BCL11B expressions in mRNA level of umbilical cord blood CD34+ cells were analyzed by the real-time quantitative PCR with TaqMan technique. For nucleofection of CD34+ cells, BCL11B-siRNA935 and the U-001 program were used of a Nucleofection Device II. Mock-transfected cells nucleofected without siRNA were used as a negative control. To evaluate the role of differentiation and proliferation in CD34+ cells after BCL11B-siRNA935 transfection, erythroid burst-forming units (BFU-E), granulocyte/macrophage colony-forming units (CFU-GM), and megakaryocyte colony-forming units (CFU-Meg) from BCL11B siRNA935-, mock-CD34+ cells were performed using methylcellulose assays. Colonies were assessed 14 days after plating. The BCL11B mRNA expression level in the CD34+ cells was significantly lower than that in Molt-4 cells and in peripheral blood mononuclear cells from eight healthy individuals. There was no significant difference between BCL11B siRNA935-transfected and mock-transfected CD34+ cells with respect to the formation of BFU-E, CFU-GM, or CFU-Meg (P > 0.05). We speculate that the BCL11B gene, because of its very low expression level, may not play a major role in proliferation and differentiation of hematopoietic stem/progenitor cells, but we cannot rule out another anti-apoptotic mechanism. Nevertheless, BCL11B gene silencing alone does not affect the proliferation and differentiation of hematopoietic stem/progenitor cells in vitro. In conclusion, our findings provide evidence for an anti-apoptotic function of BCL11B in T-cell malignancies, but not in hemopoietic stem/progenitor cells, suggesting that BCL11B siRNA is safe and may be considered a new targeted therapeutic strategy for T-cell malignancies.* National Natural Science Foundation(30771980)& Guangdong Science and Technology Project (2007B030703008) Grants Disclosures:Li:National Natural Science Foundation (30771980) and Guangdong Science and Technology Project (2007B030703008, 2009B050700029) Grants: Research Funding.

The BCL11B Tumor Suppressor Is Mutated In Human T-Cell Acute Lymphoblastic Leukemia

AbstractAbstract 4177The BCL11B transcription factor, previously identified as a central player in normal α/β T-cell development, has recently been found to play critical roles in the maintenance of T-cell identity, with biallelic Bcl11b inactivation in T-cell precursors severely impairing their continued development into the T-cell lineage. Previous work has demonstrated that Bcl11b haploinsufficiency accelerates the onset of thymic lymphomas in p53-mutant mice, as well as T-lymphoid blast crisis in a mouse model of chronic myeloid leukemia induced by BCR-ABL. Furthermore, we have recently identified recurrent monoallelic Bcl11b deletions in 91% of T-cell acute lymphoblastic leukemias (T-ALL) arising in Atm-deficient mice, further supporting a role for Bcl11b haploinsufficiency in T-cell leukemogenesis. In order to determine whether BCL11B inactivation plays a role in the molecular pathogenesis of human T-ALL, we analyzed BCL11B status in primary T-ALL patient samples by array CGH and sequencing analysis. Monoallelic BCL11B deletions were identified in 6% of cases (n = 3 of 47) analyzed by array CGH, including one microdeletion within the BCL11B locus, one small deletion involving BCL11B and 6 additional genes, and one large 26 Mbp deletion of the distal arm of chromosome 14. BCL11B sequencing revealed heterozygous missense mutations in an additional 9% (n = 4 of 43) of primary T-ALL patient samples and in 19% (n = 3 of 16) of T-ALL cell lines. Structural homology modeling revealed that many of the mutations identified, including 3 of the 4 in the primary patient samples, disrupted key amino acids within BCL11B zinc finger domains that are involved in DNA recognition or structural stabilization required for zinc finger domain-mediated transcriptional activity. Analysis of TCRγ rearrangement status and gene expression data revealed that most cases with BCL11B inactivation were characterized by biallelic TCRγ rearrangements together with an early thymocyte precursor (ETP) gene expression signature, indicating developmental arrest at a prethymocyte stage of T-cell development, which we and others have shown confers an increased risk of treatment failure (Coustan-Smith et al. Lancet Oncol 2009; Gutierrez et al. J Clin Oncol 2010). Given that BCL11B expression during normal T-cell development increases markedly at the prethymocyte stage, our findings suggest that BCL11B inactivation may be directly responsible for developmental arrest at a prethymocyte stage during thymocyte transformation. Our findings provide compelling evidence that BCL11B is a tumor suppressor in a subset of human T-ALLs with a high risk of treatment failure. Disclosures:No relevant conflicts of interest to declare.

Analysis of the expression pattern of the BCL11B gene and its relatives in patients with T-cell acute lymphoblastic leukemia

BackgroundIn a human T-cell acute lymphoblastic leukemia (T-ALL) cell line (Molt-4), siRNA-mediated suppression of BCL11B expression was shown to inhibit proliferation and induce apoptosis, functions which may be related to genes involved in apoptosis (such as TNFSF10 and BCL2L1) and TGF-β pathways (such as SPP1and CREBBP).MethodsThe expression levels of the above mentioned genes and their correlation with the BCL11B gene were analyzed in patients with T-ALL using the TaqMan and SYBR Green I real-time polymerase chain reaction technique.ResultsExpression levels of BCL11B, BCL2L1, and CREBBP mRNA in T-ALL patients were significantly higher than those from healthy controls (P <0.05). In T-ALL patients, the BCL11B expression level was negatively correlated with the BCL2L1 expression level (rs = -0.700; P <0.05), and positively correlated with the SPP1 expression level (rs = 0.683; P <0.05). In healthy controls, the BCL11B expression level did not correlate with the TNFSF10, BCL2L1, SPP1, or CREBBP expression levels.ConclusionsOver-expression of BCL11B might play a role in anti-apoptosis in T-ALL cells through up-regulation of its downstream genes BCL2L1 and CREBBP.

Increased Expression of Bcl11b Leads to Chemoresistance Accompanied by G1 Accumulation

BackgroundThe expression of BCL11B was reported in T-cells, neurons and keratinocytes. Aberrations of BCL11B locus leading to abnormal gene transcription were identified in human hematological disorders and corresponding animal models. Recently, the elevated levels of Bcl11b protein have been described in a subset of squameous cell carcinoma cases. Despite the rapidly accumulating knowledge concerning Bcl11b biology, the contribution of this protein to normal or transformed cell homeostasis remains open.Methodology/Principal FindingsHere, by employing an overexpression strategy we revealed formerly unidentified features of Bcl11b. Two different T-cell lines were forced to express BCL11B at levels similar to those observed in primary T-cell leukemias. This resulted in markedly increased resistance to radiomimetic drugs while no influence on death-receptor apoptotic pathway was observed. Apoptosis resistance triggered by BCL11B overexpression was accompanied by a cell cycle delay caused by accumulation of cells at G1. This cell cycle restriction was associated with upregulation of CDKN1C (p57) and CDKN2C (p18) cyclin dependent kinase inhibitors. Moreover, p27 and p130 proteins accumulated and the SKP2 gene encoding a protein of the ubiquitin-binding complex responsible for their degradation was repressed. Furthermore, the expression of the MYCN oncogene was silenced which resulted in significant depletion of the protein in cells expressing high BCL11B levels. Both cell cycle restriction and resistance to DNA-damage-induced apoptosis coincided and required the histone deacetylase binding N-terminal domain of Bcl11b. The sensitivity to genotoxic stress could be restored by the histone deacetylase inhibitor trichostatine A.ConclusionsThe data presented here suggest a potential role of BCL11B in tumor survival and encourage developing Bcl11b-inhibitory approaches as a potential tool to specifically target chemoresistant tumor cells.

Effects of Down-Regulating BCL11B Expression On the Proliferation, Apoptosis and Global Gene Expression Profiling of Molt-4 Cells.

Abstract 4505The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B) gene plays a crucial role in T-cell development, differentiation, and proliferation. BCL11B disturbed expression, mutation, disruption, or rearrangement has been associated with T-cell malignancies. Here, we described the effects of BCL11B target-specific small interfering RNA (BCL11B-siRNA) on the proliferation, apoptosis, and global gene expression profiling of Molt-4 cells. The BCL11B-siRNA beginning with the 924bp of mRNA sequence, and the scrambled non-silencing siRNA control (sc) were obtained by chemosynthesis. Non-treated, mock-transfected, and sc-treated cells were used as controls. BCL11B expression in mRNA levels were analyzed at 24 h, 48 h and 72 h after siRNA delivered by nucleofection using the real-time quantitative PCR with Taqman technique. At the same time points, cell proliferations were assayed by the cell count kit-8 method. The morphology and the percentage of apoptosis were revealed by Hoechst33258 stain and flow cytometry at 72 h, respectively. An obvious reduction in mRNA level was observed in BCL11B-siRNA treated cells at 24 h, compared to the controls (P< 0.05). The proliferation rates of BCL11B–siRNA treated cells were significantly decreased from 24 h to 72 h (P< 0.05). BCL11B–siRNA treated cells showed a large increase in PI-positive cells, reaching to 84.6%. However, the percentage of each cell phases did not reveal significantly difference. Thus, the apoptosis could be induced in all phases of the cell cycle. The Hoechst33258 stain was used to verify the apoptosis. To elucidate the molecular mechanisms accounting for BCL11B-siRNA-mediated cell death, global gene expression profiling were performed on the BCL11B–siRNA treated cells and non-treated control by the Affymetrix HG U133 Plus 2.0 GeneChip at 24 h after nucleofection. The BCL11B showed 10.1 fold reductions in mRNA levels. Upregulated genes was found in 1982 probe sets, while 1884 genes down-regulated, at least two folds upon Bcl11b suppression. Involvement of the T-cell activation or differentiation as TRAT1, CD3G, CD247, LCK, PTGER4, or CD1D, all revealed at least 7 folds down-regulated, which may contribute to the inhibition of proliferation. Six genes of the TNF receptor family were up-regulated at least 2 folds as well as the BCL-2 gene down-regulated 3.5 folds. The apoptosis was most likely affected by the simultaneous activation of TNF receptor family genes and suppression of the Bcl-2 gene. Those related to the autophagy and the cell cycle did not find significant change. In conclusion, we demonstrated that the survival of Molt-4 cells was critically dependent on BCL11B gene. Suppression of BCL11B by RNA interference selectively inhibited the proliferation and induced the apoptosis effectively in transformed Molt-4 cells. Therefore, down-regulation of Bcl11b might be considered as a new target therapeutic strategy in T-cell malignancies. Disclosures:Huang:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Chen:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Yang:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Chen:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Zhou:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Li:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding.

Haploinsufficiency and acquired loss of Bcl11b and H2AX induces blast crisis of chronic myelogenous leukemia in a transgenic mouse model

Chronic myelogenous leukemia (CML) is a hematological malignancy that begins as indolent chronic phase (CP) but inevitably progresses to fatal blast crisis (BC). p210BCR/ABL, a chimeric protein with enhanced kinase activity, initiates CML CP, and additional genetic alterations account for progression to BC, but the precise mechanisms underlying disease evolution are not fully understood. In the present study, we investigated the possible contribution of dysfunction of Bcl11b, a zinc-finger protein required for thymocyte differentiation, and of H2AX, a histone protein involved in DNA repair, to the transition from CML CP to BC. For this purpose, we crossed CML CP-exhibiting p210BCR/ABL transgenic (BA(tg/-)) mice with Bcl11b heterozygous (Bcl11b(+/-)) mice and H2AX heterozygous (H2AX(+/-)) mice. Interestingly, p210BCR/ABL transgenic, Bcl11b heterozygous (BA(tg/-)Bcl11b(+/-)) mice and p210BCR/ABL transgenic, H2AX heterozygous (BA(tg/-)H2AX(+/-)) mice frequently developed CML BC with T-cell phenotype and died in a short period. In addition, whereas p210BCR/ABL was expressed in all of the leukemic tissues, the expression of Bcl11b and H2AX was undetectable in several tumors, which was attributed to the loss of the residual normal allele or the lack of mRNA expression. These results indicate that Bcl11b and H2AX function as tumor suppressor and that haploinsufficiency and acquired loss of these gene products cooperate with p210BCR/ABL to develop CML BC.

BCL11B enhances TCR/CD28-triggered NF-κB activation through up-regulation of Cot kinase gene expression in T-lymphocytes

BCL11B is a transcriptional regulator with an important role in T-cell development and leukaemogenesis. We demonstrated recently that BCL11B controls expression from the IL (interleukin)-2 promoter through direct binding to the US1 (upstream site 1). In the present study, we provide evidence that BCL11B also participates in the activation of IL-2 gene expression by enhancing NF-kappaB (nuclear factor kappaB) activity in the context of TCR (T-cell receptor)/CD28-triggered T-cell activation. Enhanced NF-kappaB activation is not a consequence of BCL11B binding to the NF-kappaB response elements or association with the NF-kappaB-DNA complexes, but rather the result of higher translocation of NF-kappaB to the nucleus caused by enhanced degradation of IkappaB (inhibitor of NF-kappaB). The enhanced IkappaB degradation in cells with increased levels of BCL11B was specific for T-cells activated through the TCR, but not for cells activated through TNFalpha (tumour necrosis factor alpha) or UV light, and was caused by increased activity of IkappaB kinase, as indicated by its increase in phosphorylation. As BCL11B is a transcription factor, we investigated whether the expression of genes upstream of IkappaB kinase in the TCR/CD28 signalling pathway was affected by increased BCL11B expression, and found that Cot (cancer Osaka thyroid oncogene) kinase mRNA levels were elevated. Cot kinase is known to promote enhanced IkappaB kinase activity, which results in the phosphorylation and degradation of IkappaB and activation of NF-kappaB. The implied involvement of Cot kinase in BCL11B-mediated NF-kappaB activation in response to TCR activation is supported by the fact that a Cot kinase dominant-negative mutant or Cot kinase siRNA (small interfering RNA) knockdown blocked BCL11B-mediated NF-kappaB activation. In support of our observations, in the present study we report that BCL11B enhances the expression of several other NF-kappaB target genes, in addition to IL-2. In addition, we provide evidence that BCL11B associates with intron 2 of the Cot kinase gene to regulate its expression.

Effects of down-Regulating BCL11B Expression on the Proliferation and Apoptosis of Molt-4 Cells by RNA Interference

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B /CTIP2/ RIT1/hRitα) gene, which encodes C2H2 zinc-finger transcription factors, plays critical roles during development of T lymphocytes and the central nervous system. Recently, BCL11B gene has been identified to be associated with hematopoietic malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL). However, little is known regarding the molecular mechanism by which BCL11B may contribute as a tumor-suppressor gene. In order to clarify the role of BCL11B gene in T-cell malignancies, we analyzed the effects of BCL11B target-specific small interfering RNA (siRNA) on the proliferation and apoptosis of Molt-4 cells, which is the naturally BCL11B overexpressing cell line. BCL11B-specific siRNA and scrambled non-silencing siRNA control were designed and synthesized by Invitrogen. The siRNAs were transfected into MOLT-4 cells by nucleofector techique. The level of BCL11B mRNA expression was analyzed at 48 and 72 hours after nucleofection by Real-time quantitative PCR with Taqman technique. The cells proliferation rate was assayed by MTT method at 24 and 48 hours after transfection, respectively. Cell morphology was showed by Liu's Stain. The changes of cell cycle and apoptosis rate were detected by flow cytometry. The results showed that the level of BCL11B mRNA expression was inhibited in siRNA transfected cells at 72 hours after transfection, as compared with scrambled controls. The survival rates of siRNA-transfected cells were significantly decreased at both 24 and 48 hours (P < 0.01). Apoptotic cells could be observed morphologically. The apoptosis rates of Molt-4 cells treated with BCL11BsiRNA were highly increased compared with scrambled control ( P < 0.05). However, the percentage of different phases did not show significantly difference between siRNAtransfected cells and control cells, which was suggested that the apoptosis could be induced all phases of cell cycle. In conclusion, our data indicated that, the survival of Molt-4 cells might be critically dependent on BCL11B gene. Downregulation of BCL11B expression by RNA interference selectively can inhibit the proliferation and induced the apoptosis effectively in transformed Molt-4 cells, farther investigation will be performed in primary T-ALL cells to confirm the effect of presented BCL11B-siRNA on T-ALL.

Functional roles for the striatal-enriched transcription factor, Bcl11b, in the control of striatal gene expression and transcriptional dysregulation in Huntington's disease

Transcriptional dysregulation has emerged as a central pathogenic mechanism in Huntington's disease (HD), which is associated with neuropathological changes predominantly in the striatum. Here we demonstrate that expression of Bcl11b (a.k.a. CTIP2), a transcription factor exhibiting highly-enriched localization in adult striatum, is significantly decreased in HD cells, mouse models and human subjects and that overexpression of Bcl11b attenuates toxic effects of mutant huntingtin in cultured striatal neurons. We show that Bcl11b directly activates the proximal promoter regions of striatal-enriched genes and can increase mRNA levels of striatal-expressing genes. We further demonstrate an interaction between Bcl11b and huntingtin protein in cultured cells and brain homogenates from HD R6/1 and YAC72 transgenic mice. We propose that sequestration and/or decreased expression of Bcl11b in HD is responsible, at least in part, for the dysregulation of striatal gene expression observed in HD and may contribute to the specificity of pathology observed in this disease.

Transcriptional Profiling of Mammalian Spermatogonial Stem Cells: Insights into Mechanisms Regulating Their Self-Renewal.

Spermatogenesis is a stem cell dependent process in which the self-renewal and differentiation of spermatogonial stem cells (SSCs) provides the foundation for continual spermatozoa production. Even though crucial for male fertility, knowledge of the mechanisms regulating SSC fate decisions is limited. Rarity in the testis and lack of specific phenotypic or molecular markers to distinguish SSCs from other testis cell populations has posed major challenges in studying their functions. Currently, the only unequivocal means to identify SSCs and examine their biological activity is by functional transplantation. SSC function is controlled by both extrinsic niche stimuli and intrinsic expression of specific genes. The testis somatic cell population is believed to supply extrinsic niche growth factors that influence SSC functions, of which Sertoli cells are thought to be the main contributors but input from other cell populations is likely. The growth factor glial cell line-derived neurotrophic factor (GDNF) is a niche factor produced by Sertoli cells that influences the self-renewal of rodent SSCs. We have identified GDNF-regulated genes in cultures of germ cells proven to be enriched for self-renewing SSCs by functional transplantation. From those studies we discovered that GDNF stimulates expression of the transcription regulators bcl6b, etv5, and lhx1. Our subsequent experiments determined that expression of these molecules is important for SSC self-renewal in vitro. Additionally, disruption of both Bcl6b and Etv5 expression in vivo results in impaired spermatogenesis, with progressive loss of germ cells as a male ages resulting in the appearance of seminiferous tubules with a Sertoli cell only phenotype and male fertility defects. More recently we have examined the transcriptional profile of an SSC-enriched cell fraction isolated form mouse testes and compared gene expression levels to those in an SSC-depleted mixed testis cell population. We isolated the Thy1-expressing cell fraction from mouse pup testes and corresponding Thy1-depeleted cell fractions, and using functional transplantation determined that the Thy1+ fraction was approximately 25-fold enriched for SSCs compared to the Thy1-depelted fraction. Global gene expression profiles were measured in both populations using Affymterix oligonucleotide microarrays, producing a database of gene expression information that serves as a mining tool for identifying potential novel mechanisms regulating SSC functions. From this information we identified abundant expression of colony stimulating factor 1 receptor (CSF-1R) by the SSC-enriched Thy1+ cell fraction. To determine a biological significance of this information, we supplemented culture media with recombinant soluble CSF-1 in addition to GDNF. Using functional transplantation to assay SSC expansion rates, significant enhancement of SSC self-renewal was measured in CSF-1 supplemented cultures compared to GDNF treatment alone. Importantly, examination of testis cross sections using immunofluoresence revealed expression of CSF-1 by clusters of Leydig cells and some peritubular myoid cells. These results implicate Leydig cells and possibly myoid cells as contributors to the SSC niche microenvironment and add CSF-1 to the list of niche factors influencing SSC self-renewal.

Glial Cell Line-derived Neurotrophic Factor Regulation of Genes Essential for Self-renewal of Mouse Spermatogonial Stem Cells Is Dependent on Src Family Kinase Signaling

Self-renewal and differentiation by spermatogonial stem cells (SSCs) is the foundation for continual spermatogenesis. SSC self-renewal is dependent on glial cell line-derived neurotrophic factor (GDNF); however, intracellular mechanisms stimulated by GDNF in SSCs are unknown. To investigate these mechanisms we utilized a culture system that maintains a mouse undifferentiated germ cell population enriched for self-renewing SSCs. In these cultures mRNA for the transcription factors Bcl6b, Erm, and Lhx1 are up-regulated by GDNF and decreased in its absence. The expression of all three molecules was further identified in undifferentiated spermatogonia in vivo. Using small interfering RNA to reduce expression and transplantation to quantify stem cell numbers, Bcl6b, Erm, and Lhx1 were shown to be important for SSC maintenance in vitro. Next, GDNF was shown to activate both Akt and Src family kinase (SFK) signaling in SSCs, and culture of SSCs with inhibitors to Akt or SFKs followed by transplantation analysis showed significant impairment of SSC maintenance in vitro. Apoptosis analysis revealed a significant increase in the percentage of apoptotic cells when Akt, but not SFK, signaling was impaired, indicating that multiple signaling pathways are responsible for SSC self-renewal and survival. Biochemical and gene expression experiments revealed that GDNF up-regulated expression of Bcl6b, Erm, and Lhx1 transcripts is dependent on SFK signaling. Overall, these data demonstrate that GDNF up-regulation of Bcl6b, Erm, and Lhx1 expression through SFK signaling is a key component of the intracellular mechanism for SSC self-renewal.

FUNCTIONAL DECIPHERING OF THE CORE SIGNALING MECHANISM FOR SPERMATOGONIAL STEM CELL SELF-RENEWAL AND SURVIVAL

The biological fate decisions of self-renewal and differentiation by spermatogonial stem cells (SSCs) provide the foundation for continual spermatogenesis in the mammalian testis. Since SSCs are rare in the testis, it is extremely difficult to study the mechanisms that regulate their biological functions. The SSC self-renewal fate decision has been shown to be dependent on glial cell line-derived neurotrophic factor (GDNF) influence. However, the intracellular mechanisms utilized by GDNF in SSCs are unknown. Recently we identified GDNF-regulated genes in SSCs, which likely encode for molecules that regulate self-renewal. From these data significant up-regulated expression of the transcription factors Bcl6b, Erm, and Lhx1 was identified and Bcl6b was subsequently shown to be important for SSC self-renewal in vitro and in vivo. The objective of this study was to begin deciphering the intracellular signaling mechanisms utilized by GDNF to regulate SSC self-renewal and survival. We utilized a mouse GDNF-dependent culture system that maintains an undifferentiated germ cell population enriched for self-renewing SSCs. Continuous in vitro culture and transplantation analysis are critical elements to define the presence of SSCs in any population of cells subject to examination for factors regulating SSC function. Using siRNA to reduce expression in cultured SSCs and transplantation to quantify stem cell numbers, Bcl6b, Erm, and Lhx1 were all shown to be important for SSC self-renewal in vitro. Next, GDNF was shown to activate AKT in SSCs and this activation could be blocked by inhibition of both PI3K and Src Family Kinases (SFKs). Culture of SSCs with inhibitors to AKT (AKT inhibitor IV), PI3K (LY294002), or SFKs (SU6656) followed by transplantation analysis showed significant impairment of SSC maintenance in vitro compared to control cultures. Apoptosis analysis revealed a significant increase in the percentage of apoptotic cells when both AKT and PI3K inhibitors, but not SFK inhibitor, was added to culture media indicating that multiple signaling pathways are responsible for SSC selfrenewal and survival. Up-regulation of Bcl6b, Erm, and Lhx1 gene expression by GDNF in self-renewing SSCs could be blocked by inhibition of AKT. However, expression of both Plzf and Oct4, which are expressed in SSCs but not GDNF-regulated, were also significantly reduced when AKT was inhibited. GDNF was further shown to phosphorylate SFKs in cultured SSCs and inhibition of SFK function could specifically block GDNF-regulated expression of Bcl6b, Erm, and Lhx1 without effecting either Plzf or Oct4 expression. These data demonstrate that GDNFactivated AKT signaling is essential for SSC survival, but not a specific pathway used for GDNF-regulated gene expression. Importantly, these data also demonstrate that SFK stimulation is an essential intracellular signaling mechanism utilized by GDNF to specifically regulate SSC selfrenewal. (platform)

Inhibition of BCL11B expression leads to apoptosis of malignant but not normal mature T cells

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Krüppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.

Effect on the Expression Level of BCL11b in Jurkat Cells During Transfer Process by Nucleofection

目的 了解核转染(Nucleofection)过程对Jurkat细胞中BCL11b基因表达水平的影响情况.方法利用实时定量PCR方法检测分析经不同程序Nucleofection过程和不同时间培养的Jurkat细胞的BCL11b基因的表达水平.结果经Nucleofector的C-16、S-18和G-10+siRNA程序处理后,C-16与S-18组细胞培养2h的BCL11b水平明显升高(p<0.05),C-16、S-18和G-10+siRNA三组细胞培养10h后BCL11b水平均高于对照组(p<0.05).对照组、C-16和S-18组细胞随着培养时间延长(在2～24h之间),BCL11b水平降低(p<0.05),而在G-10+siRNA处理组,直到培养48h BCL11b水平才有所下降.结论 Nucleofection处理过程对Jurkat细胞BCL11b表达有一定的影响,但有一定的时效性。

Identification of Subtype-Specific Genomic Alterations of Acute and Lymphoma Types of Adult T-Cell Leukemia/Lymphoma.

Background: Adult T cell leukemia/lymphoma (ATLL) is a human T-lymphotropic virus type-I mediated neoplasm and four major clinical subtypes are recognized; i.e., smoldering, chronic, lymphoma and acute types. Among these subtypes, acute and lymphoma types are fetal disease with poor prognosis. Although these two subtypes represent different clinical features, detailed analysis for genomic/genetic differences has not been sufficiently investigated.Purpose: To investigate genomic aberrations of these subtypes, we performed array-based comparative genomic hybridization (array CGH: 2308 BAC clones spotted in duplicate) for 66 cases of ATLL (17 cases of acute and 49 cases of lymphoma subtypes). To determine target genes in recurrently amplified regions, quantitative real-time PCR (RQ-PCR) was conducted.Results: Array CGH: Comparison of genome profiles revealed that lymphoma type shows more frequent gain and loss than those of acute type. The numbers of respective genomic gain and loss regions of lymphoma type were 9.7 and 9.8 on average. The numbers of genomic gain and loss regions of acute type were 4.2 and 4.5 on average. Gain of 1q, 2p, 4q, 7p and 7q, and loss of 10p, 13q, 16q and 18p were characteristic to lymphoma type whereas gain of 3p and 3q was specific to acute type. Recurrent high-level amplifications were found at 1p35, 6p25, 7p22.2, 7q21–q22, 7q31–q36 and 14q32 in lymphoma type. However, such genomic amplifications were not found in acute type.RQ-PCR: CARMA1 (7p22 locus), which encodes signaling proteins associated with development of B and T cells, was a possible target of the 7p22 amplification in lymphoma type. The extremely higher expression of BCL11B (14q32 locus) which is a key regulator of both differentiation and survival during thymocyte development, was found in acute type irrespective of the 14q32 amplification. However, no overexpression of BCL11B was found in lymphoma type with high copy number gain, indicating that BCL11B is not the target gene for 14q32 amplification in the lymphoma type.Conclusion: Array-CGH revealed that patterns of genomic aberrations between acute and lymphoma types were different. Quantitative gene expression analysis also implicated distinct mechanism of aberrant gene expressions between acute and lymphoma types. Combination of array-CGH and quantitative gene expression analysis provided evidence that the acute and lymphoma types are developed via distinct genomic/genetic pathways.