The forkhead domain hinge-loop plays a pivotal role in DNA binding and transcriptional activity of FOXP2

Abstract Forkhead box (FOX) proteins are a ubiquitously expressed family of transcription factors that regulate the development and differentiation of a wide range of tissues in animals. The FOXP subfamily members are the only known FOX proteins capable of forming domain-swapped forkhead domain (FHD) dimers. This is proposed to be due to an evolutionary mutation (P539A) that lies in the FHD hinge loop, a key region thought to fine-tune DNA sequence specificity in the FOX transcription factors. Considering the importance of the hinge loop in both the dimerisation mechanism of the FOXP FHD and its role in tuning DNA binding, a detailed investigation into the implications of mutations within this region could provide important insight into the evolution of the FOX family. Isothermal titration calorimetry and hydrogen exchange mass spectroscopy were used to study the thermodynamic binding signature and changes in backbone dynamics of FOXP2 FHD DNA binding. Dual luciferase reporter assays were performed to study the effect that the hinge-loop mutation has on FOXP2 transcriptional activity in vivo. We demonstrate that the change in dynamics of the hinge-loop region of FOXP2 alters the energetics and mechanism of DNA binding highlighting the critical role of hinge loop mutations in regulating DNA binding characteristics of the FOX proteins.

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Biophysical investigation of the dual binding surfaces of human transcription factors FOXO4 and p53

10.1101/2021.01.11.425814 ◽

2021 ◽

Author(s):

Jinwoo Kim ◽

Dabin Ahn ◽

Chin-Ju Park

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Cellular Senescence ◽

Structural Information ◽

Titration Calorimetry ◽

Transactivation Domain ◽

Forkhead Box ◽

Binding Surface ◽

Forkhead Domain ◽

Oncogenic Stress

AbstractCellular senescence is protective against external oncogenic stress, but its accumulation causes aging-related diseases. Forkhead box O4 (FOXO4) and p53 are human transcription factors known to promote senescence by interacting in the promyelocytic leukemia bodies. Inhibiting their binding is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the interaction of FOXO4 and p53 remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by using NMR spectroscopy. NMR chemical shift perturbation analysis showed that the binding between FOXO4’s forkhead domain (FHD) and p53’s transactivation domain (TAD), and between FOXO4’s C-terminal transactivation domain (CR3) and p53’s DNA binding domain (DBD), mediate the FOXO4-p53 interaction. Also, we showed that the CR3-binding surface of FOXO4 FHD interacts with p53 TAD2, and four residues of FOXO4 CR3 interact with the DNA-binding surface of p53 DBD. Further isothermal titration calorimetry experiments showed that the FOXO4 FHD-p53 TAD interaction takes precedence with high affinity and that the FOXO4 CR3-p53 DBD interaction follows. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence.

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GATA-1, but Not GATA-2, Antagonizes PU.1 Mediated Transcriptional Activity at the CBFA2T3 (ETO2, MTG16) Promoter through a Mechanism Dependent on GATA DNA Binding.

Blood ◽

10.1182/blood.v106.11.1749.1749 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1749-1749

Author(s):

Yogenthiran Saunthararajah ◽

SiJun Yang ◽

ShriHari Kadkol ◽

Marie Baraoidan ◽

Vinzon Ibanez ◽

...

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Transcriptional Activity ◽

Hematopoietic Cells ◽

Chromosomal Translocation ◽

Erythroid Differentiation ◽

Proximal Promoter ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Dna Binding Sites

Abstract CBFA2T3 (ETO2, MTG16), a target of chromosomal translocation in acute myeloid leukemia, has its highest expression in hematopoietic cells compared to other tissues. This suggests that its expression is regulated by major hematopoietic transcription factors. The proximal promoter from −171 to −65 bp has greater than 90% identity between mouse and human and contains recognition sites for major hematopoietic transcription factors PU.1, GATA-1 and GATA-2. Using chromatin immuno-precipitation and the MPD hematopoietic cell-line, this segment was pulled down with endogenous PU.1, GATA-1 and GATA-2. In luciferase reporter gene assays, PU.1 and GATA-2, but not GATA-1, activated the promoter. As would be expected from these findings, CBFA2T3 levels declined during terminal erythroid differentiation of primary hematopoietic cells. GATA-1, but not GATA-2, antagonized PU.1 mediated activation but this effect of GATA-1 was abrogated by mutation of the GATA DNA binding sites. Both GATA-1 and GATA-2 have been reported to antagonize PU.1 transcriptional activity by antagonizing PU.1 interactions with c-Jun (Zhang et al, Proc Natl Acad Sci USA1999;96:8705–8710); however, the DNA binding dependent mechanism reported here allows GATA-2 and GATA-1 to have contrasting relationships with PU.1 and may be the basis for the co-operation of GATA proteins with PU.1 in some contexts yet antagonism of PU.1 activity in others.

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Study of expression analysis of SIRT4 and the coordinate regulation of bovine adipocyte differentiation by SIRT4 and its transcription factors

Bioscience Reports ◽

10.1042/bsr20181705 ◽

2018 ◽

Vol 38 (6) ◽

Cited By ~ 6

Author(s):

Jieyun Hong ◽

Shijun Li ◽

Xiaoyu Wang ◽

Chugang Mei ◽

Linsen Zan

Keyword(s):

Transcriptional Regulation ◽

Transcription Factors ◽

Adipocyte Differentiation ◽

Binding Protein ◽

Core Promoter ◽

Critical Role ◽

Luciferase Reporter ◽

Marker Genes ◽

Gene Assay ◽

Bovine Adipocytes

Sirtuins, NAD+-dependent deacylases and ADP-ribosyltransferases, are critical regulators of metabolism involved in many biological processes, and are involved in mediating adaptive responses to the cellular environment. SIRT4 is a mitochondrial sirtuin and has been shown to play a critical role in maintaining insulin secretion and glucose homeostasis. As a regulator of lipid homeostasis, SIRT4 can repress fatty acid oxidation and promote lipid anabolism in nutrient-replete conditions. Using real-time quantitative PCR (qPCR) to explore the molecular mechanisms of transcriptional regulation of bovine SIRT4 during adipocyte differentiation, we found that bovine SIRT4 is expressed at high levels in bovine subcutaneous adipose tissue. SIRT4 knockdown led to decreased expression of adipogenic differentiation marker genes during adipocyte differentiation. The core promoter of bovine SIRT4 was identified in the −402/−60 bp region of the cloned 2-kb fragment containing the 5′-regulatory region. Binding sites were identified in this region for E2F transcription factor-1 (E2F1), CCAAT/enhancer-binding protein β (CEBPβ), homeobox A5 (HOXA5), interferon regulatory factor 4 (IRF4), paired box 4 (PAX4), and cAMP responsive element-binding protein 1 (CREB1) by using Electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assay. We also found that E2F1, CEBPβ, and HOXA5 transcriptionally activate SIRT4 expression, whereas, IRF4, PAX4, and CREB1 transcriptionally repress SIRT4 expression. We further verified that SIRT4 knockdown could affect the ability of these transcription factors (TFs) to regulate the differentiation of bovine adipocytes. In conclusion, our results shed light on the mechanisms underlying the transcriptional regulation of SIRT4 expression in bovine adipocytes.

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Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1314 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1314-1323 ◽

Cited By ~ 20

Author(s):

E Parra ◽

M Varga ◽

G Hedlund ◽

T Kalland ◽

M Dohlsten

Keyword(s):

T Cells ◽

Dna Binding ◽

Transcriptional Activity ◽

Interleukin 2 ◽

Luciferase Reporter ◽

Jurkat T Cells ◽

Response Elements ◽

Nuclear Factors ◽

Hla Dr ◽

Nf Kappab

We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively.

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A Novel TWIST2-ID2 Axis That Modulates The Growth and Imatinib Sensitivity Of CML CD34+ Cells

Blood ◽

10.1182/blood.v122.21.511.511 ◽

2013 ◽

Vol 122 (21) ◽

pp. 511-511

Author(s):

Yun Zhao ◽

Wenjuan Ma ◽

Xiuyan Zhang ◽

Jiangxia Cui ◽

Ivan Sloma ◽

...

Keyword(s):

Dna Binding ◽

Progenitor Cells ◽

Lentiviral Vector ◽

Critical Role ◽

K562 Cells ◽

Luciferase Reporter ◽

Control Group ◽

Wild Type ◽

Mesenchymal Transition ◽

Cd34 Cells

Abstract TWIST is a basic helix-loop-helix transcription factor that specifies Drosophila mesoderm development. In mammals there are 2 members, TWIST1 and TWIST2. TWIST2 is a regulator of osteoblasts and muscle development and plays a critical role in the epithelial-mesenchymal transition process, as well as in cancer initiation and metastasis. Twist2-deficient mice develop a myeloproliferative disease. These findings led us to query a potential role of TWIST2 in normal and leukemic (CML) human hematopoietic cells. RT-PCR and immuno-fluorescence analysis of CD34+ bone marrow (BM) cells obtained from healthy donors demonstrated their expression of TWIST2 transcripts and protein. Lentiviral vector-mediated knockdown of TWIST2 with 2 independent shRNA sequences enhanced the erythroid and granulopoietic colony-forming activity of transduced normal BM cells ∼2-fold compared with control transduced cells (n=3, p<0.05). Interestingly, ChIP studies showed that TWIST2 can bind directly to the DNA promoter for ID2 in CD34-enriched BM cells and knockdown of TWIST2 reduced ID2 expression by 50%. In lin-CD34+ cells from 14 chronic myeloid leukemia (CML) patients, we found both TWIST2 and ID2 transcripts to be 5 and 6 fold lower than those from 6 healthy BM donors (p<0.05), with similar findings for TWIST2 and ID2 protein in the same cells. BCR-ABL1-transduced Baf/3 cells also showed a reduction in Twist2 expression. Conversely, TWIST2 expression became elevated when K562 cells were treated with Imatinib mesylate (IM). We then generated a lentiviral vector encoding TWIST2 which proved capable of inhibiting the growth of K562 and MEG-01 cells as well as CFC production from CML CD34+ cells (n=11, p<0.05). Overexpression of TWIST2 in MEG-01 cells also reduced their tumorigenic ability in subcutaneously injected nude mice (0/8 for TWIST2 group, 7/8 for control group). In addition, increased TWIST2 sensitized the IM response of K562 cells and IM-resistant CD34+ cells from CML patients (2 in chronic phase and 2 in blast crisis). Correspondingly, knockdown of TWIST2 in K562 cells enhanced their cloning efficiency by 15% and made them IM-resistant. To obtain further insight into these biological effects of TWIST2, we generated several TWIST2 mutant cDNAs, including ones with a N-terminal truncation (ΔN), a C-terminal truncation (ΔC), a F86P dimerization mutant and a b- DNA binding mutant. Analysis of the effects of these mutants when overexpressed in CML cells and cell lines showed TWIST2 dimer formation was critical for the effects obtained with wild-type TWIST2, whereas the DNA binding domain could modulate these effects but was not essential, and the N-terminal and C-terminal domains were dispensable. We also found that overexpression of TWIST2 enhanced ID2 expression in CML CD34+ cells (n=3), as well as K562 and MEG-01 cells, and ChIP analyses confirmed the binding of TWIST2 to ID2 promoter DNA from K562 and MEG-01 cells. Using ID2 promoter-driven luciferase reporter and a mutant derivative (with only the E-box sequence altered), we found that TWIST2 could activate the wild-type promoter but not the mutated one in both K562 and MEG-01 cells. Finally, we co-transduced CML cells from 3 patients with TWIST2 and shRNA against ID2 and found that this reversed the suppressed production of CFC obtained with TWIST2 alone. Similarly in K562 cells this treatment partially restored their growth rate and IM resistance. Taken together, we report a novel TWIST2-ID2 regulatory axis in normal hematopoietic progenitor cells, which can also modulate the growth and IM response of CML progenitor cells. These findings provide a baseline for the future development of more effective therapy of CML. Disclosures: No relevant conflicts of interest to declare.

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Hemin-Mediated Regulation of an Antioxidant-Responsive Element of the Human Ferritin H Gene and Role of Ref-1 during Erythroid Differentiation of K562 Cells

Molecular and Cellular Biology ◽

10.1128/mcb.26.7.2845-2856.2006 ◽

2006 ◽

Vol 26 (7) ◽

pp. 2845-2856 ◽

Cited By ~ 82

Author(s):

Kenta Iwasaki ◽

Elizabeth L. MacKenzie ◽

Kiros Hailemariam ◽

Kensuke Sakamoto ◽

Yoshiaki Tsuji

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

K562 Cells ◽

Erythroid Differentiation ◽

Luciferase Reporter ◽

H Gene ◽

Human Ferritin ◽

Responsive Element ◽

Ferritin H ◽

Antioxidant Responsive Element

ABSTRACT An effective utilization of intracellular iron is a prerequisite for erythroid differentiation and hemoglobinization. Ferritin, consisting of 24 subunits of H and L, plays a crucial role in iron homeostasis. Here, we have found that the H subunit of the ferritin gene is activated at the transcriptional level during hemin-induced differentiation of K562 human erythroleukemic cells. Transfection of various 5′ regions of the human ferritin H gene fused to a luciferase reporter into K562 cells demonstrated that hemin activates ferritin H transcription through an antioxidant-responsive element (ARE) that is responsible for induction of a battery of phase II detoxification genes by oxidative stress. Gel retardation and chromatin immunoprecipitation assays demonstrated that hemin induced binding of cJun, JunD, FosB, and Nrf2 b-zip transcription factors to AP1 motifs of the ferritin H ARE, despite no significant change in expression levels or nuclear localization of these transcription factors. A Gal4-luciferase reporter assay did not show activation of these b-zip transcription factors after hemin treatment; however, redox factor 1 (Ref-1), which increases DNA binding of Jun/Fos family members via reduction of a conserved cysteine in their DNA binding domains, showed induced nuclear translocation after hemin treatment in K562 cells. Consistently, Ref-1 enhanced Nrf2 binding to the ARE and ferritin H transcription. Hemin also activated ARE sequences of other phase II genes, such as GSTpi and NQO1. Collectively, these results suggest that hemin activates the transcription of the ferritin H gene during K562 erythroid differentiation by Ref-1-mediated activation of these b-zip transcription factors to the ARE.

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Construction and Functional Analysis of Luciferase Reporter Plasmid Containing PCNA Gene Promoter

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.556-562.257 ◽

2014 ◽

Vol 556-562 ◽

pp. 257-260

Author(s):

Tong Cun Zhang ◽

Yue Wang ◽

Xing Hua Liao ◽

Nan Wang ◽

Hao Zhou

Keyword(s):

Breast Cancer ◽

Cell Proliferation ◽

Transcription Factors ◽

Transcriptional Activity ◽

Nuclear Antigen ◽

Luciferase Reporter ◽

Reporter Plasmid ◽

Luciferase Reporter Construct ◽

Luciferase Reporter Plasmid ◽

Mcf 7

PCNA (proliferating cell nuclear antigen) is a protein related to tumor development, which has been used extensively in breast cancer diagnosis and prognosis. PCNA has proven to be a useful marker to evaluate cell proliferation and prognosis when combined with other breast cancer markers. Construction of PCNA promoter luciferase reporter plasmid will provide the theory basis for researching the effect of other transcription factors on regulating PCNA transcription. In this study, a human PCNA promoter luciferase reporter construct was generated by PCR amplification of PCNA promoter. The PCR fragment was digested and cloned into pGL3 vector. The promoter sequence was verified by sequencing. The results showed that luciferase reporter plasmids of PCNA promoter were successfully constructed. Then the effects of some key transcription factors, which play important roles in breast cancer cell proliferation, were investigated by luciferase reporter assays in MCF-7 cells. The results showed that ERα can enhance transcriptional activity of PCNA. Furthermore, 17-β-estradiol (E2) also shows an obvious impact in activating PCNA transcription. Our data illuminated that E2 enhances ERα-induced proliferation potential of MCF-7 cells by stimulating the transcriptional activity of PCNA. Our research will provide a model to screen some novel factors in regulating proliferation marker transcription.

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The Muscle Transcription Factor MyoD Promotes Osteoblast Differentiation by Stimulation of the Osterix Promoter

Endocrinology ◽

10.1210/en.2007-1556 ◽

2008 ◽

Vol 149 (7) ◽

pp. 3698-3707 ◽

Cited By ~ 12

Author(s):

Jocelyn Hewitt ◽

Xiaghuai Lu ◽

Linda Gilbert ◽

Mark S. Nanes

Keyword(s):

Alkaline Phosphatase ◽

Transcription Factor ◽

Transcription Factors ◽

Critical Role ◽

C2c12 Cells ◽

Bone Morphogenetic Protein 2 ◽

Luciferase Reporter ◽

Upstream Sequence ◽

Bhlh Transcription Factors ◽

Stimulation Of

Transcription factors regulate tissue-specific differentiation of pluripotent mesenchyme to osteoblast (OB), myoblast (MB), and other lineages. Osterix (Osx) is an essential transcription factor for bone development because knockout results in lack of a mineralized skeleton. The proximal Osx promoter contains numerous binding sequences for MyoD and 14 repeats of a binding sequence for Myf5. These basic helix-loop-helix (bHLH) transcription factors have a critical role in MB differentiation and muscle development. We tested the hypothesis that bHLH transcription factors also support OB differentiation through regulation of Osx. Transfection of a MyoD expression vector into two primitive mesenchymal cell lines, C3H/10T1/2 and C2C12, stimulated a 1.2-kb Osx promoter-luciferase reporter 70-fold. Myf5 stimulated the Osx promoter 6-fold. Deletion analysis of the promoter revealed that one of three proximal bHLH sites is essential for MyoD activity. The Myf5 repeat conferred 60% of Myf5 activity with additional upstream sequence required for full activity. MyoD bound the active bHLH sequence and its 3′-flanking region, as shown by EMSA and chromatin immunoprecipitation assays. Real-time PCR revealed that primitive C2C12 and C3H/10T1/2 cells, pre-osteoblastic MC3T3 cells, and undifferentiated primary marrow stromal cells express the muscle transcription factors. C2C12 cells, which differentiate to MB spontaneously and form myotubules, were treated with bone morphogenetic protein 2 (BMP-2) to induce OB differentiation. BMP-2 stimulated expression of Osx and the differentiation marker alkaline phosphatase and blocked myotubule development. BMP-2 suppressed the muscle transcription factor myogenin, but expression of MyoD and Myf5 persisted. Silencing of MyoD inhibited BMP-2 stimulation of Osx and blocked the later appearance of bone alkaline phosphatase. MyoD support of Osx transcription contributes to early OB differentiation.

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Mechanisms of Leukemogenesis by MLL-CALM: Characterization of a CALM-Derived Transcriptional Regulatory Domain.

Blood ◽

10.1182/blood.v104.11.3387.3387 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3387-3387

Author(s):

Mwe Mwe Chao ◽

Emily J. Fox ◽

Daniel S. Wechsler

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Nuclear Export ◽

Transcriptional Activity ◽

Hox Genes ◽

Luciferase Activity ◽

Expression Vectors ◽

Luciferase Reporter ◽

Fusion Partner ◽

Cytoplasmic Localization

Abstract Background: MLL translocations are common in infant leukemias, and >50 distinct translocation partners have been described. We recently identified the CALM gene as a novel MLL partner in an infant with aggressive AML. Interestingly, CALM was first discovered as a translocation partner for AF10, which had previously been identified as an MLL fusion partner in aggressive leukemias and lymphomas. The native CALM protein exhibits predominantly cytoplasmic localization, and participates in clathrin-dependent endocytosis and intracellular vesicle transport. We have previously shown that expression of MLL-CALM immortalizes murine hematopoietic progenitors, and that fusion of the carboxy terminus of CALM to MLL alters MLL transcriptional activity. We hypothesize that CALM possesses a specific transcriptional activation domain (TAD) which modulates MLL transcriptional activity of HOX genes, thereby contributing to leukemogenesis. Objectives: 1) To determine whether native CALM localizes to the nucleus, 2) To delineate specific CALM domains which constitute the CALM TAD, and 3) To determine whether MLL-CALM activates transcription through the murine HOXA7 promoter. Methods: Human fibroblast cells were treated with Leptomycin B (an antifungal antibiotic which specifically inhibits nuclear export) and stained with an anti-CALM antibody. We prepared a set of expression vectors in which various portions of CALM are fused to a GAL4 DNA-binding domain. These vectors were co-transfected with a GAL4-luciferase reporter plasmid into COS7 cells, and luciferase activity was measured 48 hours after transient transfection. Luciferase assays were also performed using MSCV-MLL-CALM or MSCV-CALM plasmids co-transfected with a HOXA7 promoter-luciferase reporter construct. Results: After inhibition of nuclear export, native CALM localized to both the nucleus and cytoplasm. Significant luciferase activity was only observed with constructs containing distal CALM carboxy amino acids (aa 436–660). Mutation of an NR (Nuclear Receptor) Box motif (aa 510–514) did not affect CALM-dependent transcription. We found that two endocytosis-related NPF domains play opposite roles: deletion of NPF#1 (aa 437–439) dramatically reduced, while mutation of NPF#2 (aa 639–641) increased transcriptional activity. Expression constructs lacking GAL4 DNA binding domains had no effect on transcription, and GAL4 binding sites were required for luciferase activity in this system. Finally, MLL-CALM activated transcription of the murine HOXA7 promoter in comparison with native CALM or empty vector. Conclusions: We have confirmed that native CALM is able to localize to the nucleus, and we have begun to identify specific critical residues in the CALM TAD. The presence of a CALM TAD in MLL-CALM suggests that altered transcriptional regulation of MLL-dependent HOX genes may play an important role in MLL-CALM dependent transformation. Our observations raise the possibility that other MLL partners with native cytoplasmic localization may possess unrecognized transcriptional activity, and provide new insight into both MLL-CALM and CALM-AF10 mediated leukemogenesis.

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Single-Stranded DNA-Binding Proteins (SSBPs) Regulate the Abundance of the LIM-Homeodomain Protein LHX2 and Augment Its Transcriptional Activity.

Blood ◽

10.1182/blood.v110.11.1239.1239 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1239-1239

Author(s):

Ying Cai ◽

Zhixiong Xu ◽

Lalitha Nagarajan ◽

Stephen J. Brandt

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Protein Turnover ◽

Transcriptional Activity ◽

Binding Activity ◽

Protein Abundance ◽

Single Stranded Dna ◽

Dna Binding Activity ◽

E Box ◽

Lim Homeodomain

Abstract A small family of proteins with putative single-stranded DNA-binding activity has been shown to augment the biological actions of LIM-homeodomain (LIM-HD) transcription factors through the mediation of the LIM domain-binding protein LDB1. We recently established that two of these SSBPs, Ssbp2 and Ssbp3, were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing transcription factors Tal1, E2A, and Gata-1 and LIM-only protein Lmo2 and showed that Ssbp2 stimulated E box-GATA DNA-binding activity by inhibiting Ldb1 ubiquitination and Ldb1 and Lmo2 turnover (Genes & Dev.21:942–955, 2007). Since LIM-HD proteins are substrates of different E3 ubiquitin ligases than LIM-only proteins and have the additional property of binding DNA, we sought to determine the effect of SSBPs on LIM-HD expression and function. Using the prototype LIM-HD protein Lhx2 and one of its best-characterized target genes, Cga, for analysis, we found that an Ssbp3-, Ldb1-, and Lhx2-containing complex associated with an Lhx2 binding element in the Cga promoter in vitro and in mouse pituitary cells (alphaT3-1 cell line) in vivo. We then showed that enforced expression of Ssbp2 and Ssbp3 in alphaT3-1 cells increased Lhx2 and Ldb1 protein abundance, Lhx2 DNA-binding activity, and Cga expression and augmented Lhx2 transcriptional activity in an Ldb1-dependent fashion. While Lhx2-Ldb1-Ssbp3 DNA-binding activity increased in Ssbp3- relative to vector-transfected cells, the affinity of this complex for DNA was unaltered. Similar to the effect of Ssbp2 on Lmo2 in murine erythroleukemia (MEL) cells, overexpressed Ssbp3 reduced Lhx2 protein turnover in cycloheximide-treated alphaT3-1 cells without affecting Lhx2 RNA levels. In contrast, knockdown of endogenous Ssbp3, but not Ssbp2 which is expressed at much lower levels in these cells, reduced Lhx2 and Ldb1 abundance, Lhx2 DNA-binding activity, Lhx2, Ldb1, and Ssbp3 loading onto the Cga promoter, Cga promoter activity, and endogenous Cga gene expression. Significantly, neither overexpression nor knockdown of Ssbp2 in MEL cells, which express both the LIM-only protein Lmo2 and LIM-HD protein Lhx2, affected Lhx2 protein abundance, and Lhx2 DNA-binding activity was undetectable in nuclear extracts from these cells despite the presence of immunoreactive Lhx2. These studies indicate that SSBP augmentation of LIM-HD function results from Ldb1-mediated inhibition of LIM-HD protein turnover and increased assembly of a LIM-HD/LDB1/SSBP DNA-binding complex. The much greater affinity for LDB1 of LIM-only compared to LIM-HD proteins is likely a major determinant of the SSBP effect on LIM-HD protein abundance. Finally, these findings are consistent with cell type-specific contributions of different SSBPs, even for similar LDB1-dependent actions.

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