Gene repression by minimal lac loops in vivo

Abstract Terminal erythroid maturation requires coordinated activation of erythroid marker genes and repression of genes associated with the undifferentiated state. These gene expression patterns are mediated by the concerted action of the erythroid transcription factor GATA-1 and its cofactor FOG-1 that can activate or repress transcription depending on promoter context. We and others showed previously that one mechanism by which FOG-1 functions is to facilitate GATA-1 association with certain DNA target sites in vivo. Using gene complementation studies of GATA-1-ablated erythroid cells, we show that at several GATA-1-repressed target genes (c-kit, c-myc and GATA-2) FOG-1 is dispensable for GATA-1 occupancy in vivo but essential for gene repression and histone deacetylation. To examine how FOG-1 functions as co-repressor we performed affinity chromatography, conventional protein purification and in vitro binding studies to identify proteins that bind FOG-1. We discovered that FOG-1 directly associates with the nucleosome remodeling and histone deacetylase complex NURD. This interaction is mediated by a small conserved domain at the N-terminus of FOG-1 and the MTA-1 subunit of NURD. Association of FOG-1 with NURD occurs in vivo and depends on an intact N-terminus of FOG-1. A series of point mutations across the N-terminus of FOG-1 revealed a tight correlation between NURD binding and transcriptional repression. In particular, a single point mutation at the N-terminus of FOG-1 that abrogated NURD binding also blocked gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing a mutant form of FOG-1 that is defective for NURD binding. Together, these studies show that FOG-1 and very likely other FOG proteins are bona fide co-repressors that link GATA proteins to histone deacetylation and nucleosome remodeling via a novel protein interaction module.

Download Full-text

Physical and Functional Association of PLZF with Histone Methyl Transferase and Histone Demethylase.

Blood ◽

10.1182/blood.v108.11.731.731 ◽

2006 ◽

Vol 108 (11) ◽

pp. 731-731

Author(s):

Itsaso Hormaeche ◽

Kim Rice ◽

Joti Marango ◽

Fabien Guidez ◽

Arthur Zelent ◽

...

Keyword(s):

Histone Methylation ◽

Transcriptional Repression ◽

Target Genes ◽

Histone H3 ◽

Promyelocytic Leukemia ◽

Gene Repression ◽

Histone Demethylases ◽

Methyl Transferase ◽

Histone Methyl Transferase

Abstract The promyelocytic leukemia zinc finger protein (PLZF) is a transcription factor fused to RARα in the t(11;17) translocation associated with retinoic acid resistant acute promyelocytic leukemia (APL). As a result of this chromosomal abnormality, two oncogenic proteins are produced, PLZF-RARα and RARα-PLZF. Wild type PLZF is expressed in CD34+ progenitor cells and declines during differentiation. PLZF is a tumor suppressor that causes cell cycle arrest, downregulating genes such as cyclinA2 and c-myc. We previously showed that transcriptional repression by PLZF is mediated by the recruitment of histone deacetylases to target genes, this being critical for its ability to control growth and affect RAR target genes. We now show that PLZF alters the methylation state of histones in its target genes. A biotinylated form of PLZF co-purified in cells along with a histone methyl transferase (HMT) activity for native histones. Using mutant histone H3 tail peptides, we showed that this activity methylated histone H3 on lysine 9 (H3K9me). Tagged forms of PLZF as well as endogenous PLZF co-precipitated in vivo with G9a histone methyl transferase, an enzyme that can mono and dimethylate H3K9 in euchromatin subject to gene repression. The interaction of PLZF with G9a required the presence of the N-terminal BTB/POZ domain as well as a second, more C-terminal, repression domain of PLZF. Given the newly found role of active histone demethylation in gene control we also tested the interaction of PLZF with LSD1, an enzyme associated with gene repression that demethylates H3K4. As in the case of G9a, the interaction of PLZF with LSD1 required both repression domains, suggesting, that these proteins may be part of a multi-protein complex containing multiple contact points with PLZF. Expression of G9a or LSD1 augmented transcriptional repression mediated by PLZF on reporter genes, indicating a functional interaction between histone methylation modifiers and PLZF. To determine the ability of PLZF to affect chromatin methylation in vivo, a Gal4-PLZF fusion protein was expressed in cells containing a chromatin-embedded Gal4-tk-Luciferase reporter gene. In the presence of PLZF, a chromatin immunoprecipitation experiment showed an increase in H3K9 methylation of the target gene while H3K4 methylation decreased, consistent with the ability of PLZF to interact with LSD1 and G9a. Lastly we compared the ability of the histone modifying proteins to interact with the APL fusion proteins PLZF-RARα, PML-RARα and NPM-RARα. Co-precipitation experiments showed a robust interaction between PLZF-RARα and G9a and LSD1 while the PML-RARα and NPM-RARα fusions bound these proteins significantly less avidly. Collectively all these data indicate that specific histone methylation is an important mode of action of PLZF in gene repression. The retinoic acid resistance of t(11;17)-APL may be related to its ability to interact with HMTs and histone demethylases. Hence therapeutic targeting of HMTs and histone demethylases might be considered as a novel mode of therapy in APL and other hematological malignancies.

Download Full-text

PLZF-RARα Utilizes the Histone Methyl Transferase G9a/GLP and the Histone Demethylase LSD1 to Repress RARα Target Genes and Block Myeloid Differentiation

Blood ◽

10.1182/blood.v112.11.198.198 ◽

2008 ◽

Vol 112 (11) ◽

pp. 198-198

Author(s):

Itsaso Hormaeche ◽

Kim L. Rice ◽

Arthur Zelent ◽

Melanie J. McConnell ◽

Jonathan D. Licht

Keyword(s):

Fusion Protein ◽

Transcriptional Repression ◽

Histone Deacetylases ◽

Target Genes ◽

Myeloid Cell ◽

Gene Repression ◽

Methyl Transferase ◽

Histone 3 ◽

Histone Methyl Transferase

Abstract As a result of the t(11;17) translocation in retinoic acid resistant subtype of acute promyelocytic leukemia (APL), the transcriptional repression domains of the Promyelocytic Leukemia Zinc Finger protein (PLZF) are fused to the ligand binding and DNA binding domains of the Retinoic Acid Receptor α (RARα). The expression of PLZF-RARα as well as the reciprocal RARα-PLZF protein both appear to contribute to leukemogenesis. While the mode of action of PML-RARα has been studied in detail, less is known about transcriptional repression mediated by PLZF-RARα. We and others previously showed an important role of histone deacetylases in PLZF and PLZF-RARα mediated gene repression. We now find that expression of PLZF-RARα also modulates gene expression through changes in the state of histone methylation at target promoters. PLZF-RARα co-precipitated in vivo with endogenous G9a, a histone methyl transferase responsible for the mono and di-methylation of euchromatic histone 3 lysine tail residue 9 (H3K9me1/2), a covalent modification associated with gene repression. Deletion analysis of the PLZF-RARα fusion protein showed that the BTB/POZ domain of PLZF fused to RARα was sufficient to mediate this interaction. PLZF-RARα also bound in vivo to LSD1, a histone demethylase that removes methyl groups from mono or di-methylated Histone 3 lysine 4 (H3K4me1/2), a change generally associated with gene repression. As with G9a the BTB/POZ domain of PLZF was implicated in binding to LSD1. Co-precipitation experiments showed a robust interaction between PLZF-RARα and G9a and LSD1 while RARα, PML-RARα and NPM-RARα bound much more weakly, suggesting that the interaction with these histone modifying enzymes may be a mechanism relatively specific to t(11;17)-associated APL. To identify genes modulated by PLZF-RARα and determine how PLZF-RARα affects the chromatin of such genes we induced expression of PLZF-RARα in a U937 tetracycline-regulated system. PLZF-RARα directly repressed known RARα target genes such as NFE2, PRAM1 and C/EBPε. As a result of PLZF-RARα expression, U937T cells were blocked in differentiation characterized by decreased expression of the myeloid cell surface markers CD11b, CD14 and CD33. Chromatin immunoprecipitation experiments in this cell line showed that PLZF-RARα expression was associated with an increase in H3K9me1/me2 at the NFE2, PRAM1 and C/EBPε promoters. Knockdown of endogenous G9a by shRNA transduction reversed transcriptional repression mediated by the fusion protein on all three promoters. Both results are consistent with the presence of G9a in PLZF-RARα transcriptional complex. By contrast, the H3K4 methylation changes in response to PLZF-RARα were promoter specific and complex: while NFE2 exhibited a decrease in H3K4me1/2, consistent with the recruitment of LSD1 and demethylation, PRAM1 and C/EBPε showed an increase in these two modifications. Inhibition of LSD1 by tranylcypromine treatment as well as knockdown of LSD1 by shRNA only reverted PLZF-RARα repression of NFE2. PLZF-RARα recruitment to all three genes was associated with a decrease in H3K4trimethylation, a modification only accomplished by jumanji-class histone demethylases. Consistent with the biochemical information, knockdown of G9a or its heterodimeric partner GLP, showed a strong biological phenotype, reverting the block in myeloid differentiation caused by PLZF-RARα as measured by the expression of the myeloid cell surface markers CD11b and CD14. Depletion of LSD1 only modestly interfered with the differentiation block mediated by the fusion protein. Gene regulation by PLZF-RARα is associated with a complex set of chromatin changes mediated by a combination of histone deacetylases, methyl transferase and demethylases. All three classes of enzymes may represent therapeutic targets in t(11;17)-APL.

Download Full-text

Recruitment of N-CoR/SMRT-TBLR1 Corepressor Complex by Unliganded Thyroid Hormone Receptor for Gene Repression during Frog Development

Molecular and Cellular Biology ◽

10.1128/mcb.24.8.3337-3346.2004 ◽

2004 ◽

Vol 24 (8) ◽

pp. 3337-3346 ◽

Cited By ~ 81

Author(s):

Akihiro Tomita ◽

Daniel R. Buchholz ◽

Yun-Bo Shi

Keyword(s):

Thyroid Hormone ◽

Transcriptional Repression ◽

Histone Deacetylases ◽

Hormone Receptors ◽

Thyroid Hormone Receptor ◽

Histone Deacetylation ◽

Gene Repression ◽

Vertebrate Development ◽

Inducible Promoters

ABSTRACT The corepressors N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors) interact with unliganded nuclear hormone receptors, including thyroid hormone (T3) receptor (TR). Several N-CoR/SMRT complexes containing histone deacetylases have been purified. The best studied among them are N-CoR/SMRT complexes containing TBL1 (transducin beta-like protein 1) or TBLR1 (TBL1-related protein). Despite extensive studies of these complexes, there has been no direct in vivo evidence for the interaction of TBL1 or TBLR1 with TR or the possible involvement of such complexes in gene repression by any nuclear receptors in any animals. Here, we used the frog oocyte system to demonstrate that unliganded TR interacts with TBLR1 and recruits TBLR1 to its chromatinized target promoter in vivo, accompanied by histone deacetylation and gene repression. We further provide evidence to show that the recruitment of TBLR1 or related proteins is important for repression by unliganded TR. To investigate the potential role for TBLR1 complexes during vertebrate development, we made use of T3-dependent amphibian metamorphosis as a model. We found that TBLR1, SMRT, and N-CoR are recruited to T3-inducible promoters in premetamorphic tadpoles and are released upon T3 treatment, which induces metamorphosis. More importantly, we demonstrate that the dissociation of N-CoR/SMRT-TBLR1 complexes from endogenous TR target promoters is correlated with the activation of these genes during spontaneous metamorphosis. Taken together, our studies provide in vivo evidence for targeted recruitment of N-CoR/SMRT-TBLR1 complexes by unliganded TR in transcriptional repression during vertebrate development.

Download Full-text

A STAT3-dependent transcriptional circuitry inhibits cytotoxic gene expression in T cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1711160114 ◽

2017 ◽

Vol 114 (50) ◽

pp. 13236-13241 ◽

Cited By ~ 17

Author(s):

Thomas Ciucci ◽

Melanie S. Vacchio ◽

Rémy Bosselut

Keyword(s):

Gene Expression ◽

T Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Viral Infection ◽

Gene Repression ◽

Inhibitory Mechanism

CD8+T cells are preprogrammed for cytotoxic differentiation in the thymus as they acquire expression of the transcription factor Runx3. However, a subset of effector CD8+T cells (Tc17) produce IL-17 and fail to express cytotoxic genes. Here, we show that the transcription factors directing IL-17 production, STAT3 and RORγt, inhibit cytotoxicity despite persistent Runx3 expression. Cytotoxic gene repression did not require the transcription factor Thpok, which in CD4+T cells restrains Runx3 functions and cytotoxicity; and STAT3 restrained cytotoxic gene expression in CD8+T cells responding to viral infection in vivo. STAT3-induced RORγt represses cytotoxic genes by inhibiting the functions but not the expression of the “cytotoxic” transcription factors T-bet and Eomesodermin. Thus, the transcriptional circuitry directing IL-17 expression inhibits cytotoxic functions. However, by allowing expression of activators of the cytotoxic program, this inhibitory mechanism contributes to the instability of IL-17–producing T cells.

Download Full-text

The adenoviral E1A protein displaces corepressors and relieves gene repression by unliganded thyroid hormone receptors in vivo

Cell Research ◽

10.1038/cr.2009.55 ◽

2009 ◽

Vol 19 (6) ◽

pp. 783-792 ◽

Cited By ~ 8

Author(s):

Yukiyasu Sato ◽

Andrew Ding ◽

Rachel A Heimeier ◽

Ahmed F Yousef ◽

Joe S Mymryk ◽

...

Keyword(s):

Thyroid Hormone ◽

Hormone Receptors ◽

Gene Repression ◽

Thyroid Hormone Receptors ◽

E1a Protein

Download Full-text

The Mammalian Sin3 Proteins Are Required for Muscle Development and Sarcomere Specification

Molecular and Cellular Biology ◽

10.1128/mcb.00975-10 ◽

2010 ◽

Vol 30 (24) ◽

pp. 5686-5697 ◽

Cited By ~ 43

Author(s):

Chris van Oevelen ◽

Christopher Bowman ◽

Jessica Pellegrino ◽

Patrik Asp ◽

Jemmie Cheng ◽

...

Keyword(s):

Muscle Development ◽

Target Genes ◽

Massively Parallel Sequencing ◽

Muscle Cells ◽

Gene Repression ◽

Massively Parallel ◽

Parallel Sequencing ◽

Number Of Genes

ABSTRACT The highly related mammalian Sin3A and Sin3B proteins provide a versatile platform for chromatin-modifying activities. Sin3-containing complexes play a role in gene repression through deacetylation of nucleosomes. Here, we explore a role for Sin3 in myogenesis by examining the phenotypes resulting from acute somatic deletion of both isoforms in vivo and from primary myotubes in vitro. Myotubes ablated for Sin3A alone, but not Sin3B, displayed gross defects in sarcomere structure that were considerably enhanced upon simultaneous ablation of both isoforms. Massively parallel sequencing of Sin3A- and Sin3B-bound genomic loci revealed a subset of target genes directly involved in sarcomere function that are positively regulated by Sin3A and Sin3B proteins. Both proteins were coordinately recruited to a substantial number of genes. Interestingly, depletion of Sin3B led to compensatory increases in Sin3A recruitment at certain target loci, but Sin3B was never found to compensate for Sin3A loss. Thus, our analyses describe a novel transcriptional role for Sin3A and Sin3B proteins associated with maintenance of differentiated muscle cells.

Download Full-text

Ezh2 Requires PHF1 To Efficiently Catalyze H3 Lysine 27 Trimethylation In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.02017-07 ◽

2008 ◽

Vol 28 (8) ◽

pp. 2718-2731 ◽

Cited By ~ 196

Author(s):

Kavitha Sarma ◽

Raphael Margueron ◽

Alexey Ivanov ◽

Vincenzo Pirrotta ◽

Danny Reinberg

Keyword(s):

Chromatin Immunoprecipitation ◽

Polycomb Group ◽

Gene Repression ◽

Hoxa Gene Expression ◽

Polycomb Repressive Complexes ◽

Repressive H3k27me3 Mark ◽

Hoxa Gene

ABSTRACT The mammalian Polycomblike protein PHF1 was previously shown to interact with the Polycomb group (PcG) protein Ezh2, a histone methyltransferase whose activity is pivotal in sustaining gene repression during development and in adulthood. As Ezh2 is active only when part of the Polycomb Repressive Complexes (PRC2-PRC4), we examined the functional role of its interaction with PHF1. Chromatin immunoprecipitation experiments revealed that PHF1 resides along with Ezh2 at Ezh2-regulated genes such as the HoxA loci and the non-Hox MYT1 and WNT1 genes. Knockdown of PHF1 or of Ezh2 led to up-regulated HoxA gene expression. Interestingly, depletion of PHF1 did correlate with reduced occupancy of Bmi-1, a PRC1 component. As expected, knockdown of Ezh2 led to reduced levels of its catalytic products H3K27me2/H3K27me3. However, reduced levels of PHF1 also led to decreased global levels of H3K27me3. Notably, the levels of H3K27me3 decreased while those of H3K27me2 increased at the up-regulated HoxA loci tested. Consistent with this, the addition of PHF1 specifically stimulated the ability of Ezh2 to catalyze H3K27me3 but not H3K27me1/H3K27me2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favor of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression.

Download Full-text

A Novel Approach in Expanding CD34+CD90+ and CD34+CD38-CD90+ Cells Associated with Enhanced in Vivo Repopulating potential.

Blood ◽

10.1182/blood.v120.21.2337.2337 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2337-2337

Author(s):

Hiro Tatetsu ◽

Chong Gao ◽

Shikiko Ueno ◽

Daniel G Tenen ◽

Li Chai

Keyword(s):

Gene Therapy ◽

Culture Condition ◽

Ex Vivo ◽

Embryonic Stem ◽

Gene Repression ◽

Nurd Complex ◽

Human Cord Blood ◽

Cd34 Cells

Abstract Abstract 2337 The nucleosome remodeling and deacetylase (NuRD) complex is involved in gene repression and normal hematopoiesis. In embryonic stem (ES) cells, NuRD complex directly maintains the self-renewal from lineage commitment. Murine models with defects in NuRD complex present with increased hematopoietic stem/progenitor cells (HSPCs). Several transcriptional factors (TFs), such as SALL4, FOG1, BCL11A, BCL11B, ZNF521 use a conservative 12 amino acid domain to recruit NuRD components for targeted gene repression. In this study, we plan to test the hypothesis that by blocking the interaction between NuRD and its TFs, we can phenocopy its knockdown effects on human HSPC. Human cord blood (CB) CD34+ cells were purified, and treated with peptide with the NuRD interacting sequences (wt-pep) or its controls mutant peptide (mut-pep) or scramble peptide (scr-pep). In vitro and in vivo assays were performed to evaluate whether wt-pep treatment can maintain or expand human cord blood HSPCs, which were further characterized as CD34+CD90+ and CD34+CD38-CD90+ sub-population with known repopulating potential after ex vivo culture. First we observed wt-pep treated CB CD34+ (1×104) cultures with cytokines (SCF, Flt3 ligand, IL-3 and IL-6) yielded 10.6 times greater numbers of CD34+CD90+ cells (Fig 1) and 19.3 times greater numbers of CD34+CD38-CD90+ as compared to the cultures containing cytokines with scr-pep after 6 days of culture (p<0.05). Colony formation assay demonstrated wt-pep treated cells gave mainly rise to large colonies, while scr-pep treated cells generated high proportion of small colonies, 8 days after plating (p<0.05). Treatment of CB CD34+ cells with wt-pep resulted in an increase in total colony-forming cell (CFC) content compared to other control groups, 14 days after plating (p<0.005). Then, to determine the most contributed to populations, Lin-CD34+CD38+CD90±, Lin-CD34+CD38-CD90± populations were fractionally sorted and treated with wt-pep. While the expansion rate of Lin-CD34+CD38+CD90± were too low to evaluate, wt-pep treated Lin-CD34+CD38-CD90+ and Lin-CD34+CD38-CD90- cells gave rise to 79.3% and 52.7% of CD34+CD90+ cells, respectively. In addition, Lin-CD34+CD38-CD90 + cells gave rise to much more CD34+CD38- population in wt-peptide treated cells as compared with scr-peptide treated cells (44.0%, 16.2% respectively) and almost all of these cells co-expressed CD90. These data suggested that both Lin-CD34+CD38-CD90± cells treated with wt-peptide could give rise to CD34+CD90+ as well as CD34+CD38-CD90+ cells. In addition, CD34+CD38-CD90+ cells seemed more contributed than CD34+CD38-CD90- cells for expansion of CD34+38-CD90+ cells. Based on in vitro experiment results, we investigated immunodeficient (SCID) repopulating activity (SRA) to determine efficacy of wt-pep to HSPCs in vivo. We observed that mice transplanted the progeny of 1×104 CB CD34+ cells treated with wt-pep (n=5) had higher degree of human cell chimerism (0.38%) than that with scr-peptide (0%) (n=5) at 4 weeks in peripheral blood. The 8 weeks chimerism of wt-peptide treated progeny (13.6%; n=5) were greater than scr-peptide treated progeny (1.88%; n=5) (Fig 2; p < 0.005) and also greater than unmanipulated CD34+ cells (1×104) (0.09%; n=3) (Fig 2; p < 0.05). In addition, these mice (n=5) demonstrated that multilineage differentiation. We further analyzed transcription levels of several HSPC related genes, which were compared between CB CD34+ cells treated with wt-peptide and scramble peptide. Significantly higher transcript levels were detected for HoxB4 (p < 0.05), GATA 2 (p < 0.05) and Evi1 (p < 0.05) and lower levels were detected for PTEN (p < 0.05) by real time quantitative RT-PCR in wt-peptide expanded cells as compared with scr-pep cultures. Unlike embryonic stem cells, human HSPC can not be maintained ex vivo in a culture condition. The limit in source cells has greatly hampered the field of HSPC research. Optimizing ex vivo HSPC expansion culture condition not only can facilitate basic research on these cells, but also can be translated into clinical applications such as enhancement of CB engraftment and gene therapy. Our study demonstrates a novel peptide approach for ex vivo expansion of a unique CD34+CD90+ and CD34+CD38-CD90+ population with increased engraftment potential. Future studies will reveal whether the same condition can beapplied to HSPC culture for gene therapy. Disclosures: No relevant conflicts of interest to declare.

Download Full-text