Transcriptional Repression Function Research Articles

The Hedgehog Regulated Transcription Factor and Tumor Suppressor, Gli3, Regulates Hematopoietic Stem Cell Number, Self-Renewal and Differentiation

The Hedgehog (Hh) signaling pathway plays a critical role in embryonic development and adult tissue homeostasis and has emerged as an important therapeutic target in many cancers, including leukemia and myeloproliferative diseases. Our mechanistic understanding of Hh pathway signaling and regulation comes primarily from developmental studies in neural and limb development. Studies of Hedgehog signaling in the hematopoietic system have produced contradictory results, and no clear consensus regarding Hh signaling in normal hematopoiesis is available to inform the role of Hedgehog signaling in hematologic malignancies. In our work we have focused on understanding the downstream effectors of Hedgehog signaling, the Gli transcription factors. The three Gli proteins, Gli1, Gli2 and Gli3 have both transcriptional activator and repressor functions, which allow for regulation and fine-tuning of Hedgehog pathway output. Previous studies from our group have revealed that Gli1null HSCs had no defects in self-renewal, however myeloid differentiation and stress hematopoiesis were severely impaired (Merchant, et al., Blood 2010). In normal tissues, Hh pathway activation via Ptch/Smo causes an increase in the downstream activating transcription factor GLI1 and a decrease in the transcriptional repressor Gli3R. Our recent studies demonstrated that GLI3R has a tumor suppressor role in human acute myeloid leukemia by directly repressing AKT expression (Chaudhry et al., AACR Annual Meeting 2015). To date nothing is known about the role of Gli3 in normal hematopoiesis. In the present study, we crossed Vav-Cre transgenic mice to Gli3fl/fl mice to generate mice with a conditional loss of Gli3 (Gli3null) in the hematopoietic system. HSC self-renewal was analyzed by serial transplant. In comparison to HSCs from Gli3 wild type (Gli3WT) mice bone marrow (BM), HSCs from Gli3null BM showed decreased long-term engraftment and self-renewal. In addition, quantification of long-term HSC (LT-HSC, CD34neg Flt3neg KSL), short-term HSC (ST-HSC, CD34+ Flt3neg KSL), and multi-potent progenitor (MPP, CD34+ Flt3+ KSL) revealed that the frequency of LT-HSCs in Gli3null BM (0.004-0.007%) was lower compared to Gli3 WT BM (0.008-0.02%). In mice transplanted with Gli3null BM, myeloid expansion was observed with a block in T and B cell lineage differentiation. Analysis of the c-Kit+ Sca1neg Linneg (KL) myeloid progenitor compartment revealed a two-fold increase in the FcRγhigh CD34+ KL granulocyte-monocyte progenitors (GMPs) in Gli3null BM, suggesting an expansion of granulocytic compartment. Since Gli3R is a key negative regulator of Gli1, these are consistent with decrease in GMP and myeloid differentiation previously seen in Gli1null mice. In summary, our studies reveal a previously unknown function for Gli3 in regulating HSCs and myeloid differentiation, and help to elucidate the complex regulation of Hh signaling in the hematopoietic system. DisclosuresNo relevant conflicts of interest to declare.

Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efficacy in high-risk leukemia

AbstractIkaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.

Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.

The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner.

Role of peroxisome proliferator-activated receptors alpha and gamma in gastric ulcer: An overview of experimental evidences.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. Three subtypes, PPARα, PPARβ/δ, and PPARγ, have been identified so far. PPARα is expressed in the liver, kidney, small intestine, heart, and muscle, where it activates the fatty acid catabolism and control lipoprotein assembly in response to long-chain unsaturated fatty acids, eicosanoids, and hypolipidemic drugs (e.g., fenofibrate). PPARβ/δ is more broadly expressed and is implicated in fatty acid oxidation, keratinocyte differentiation, wound healing, and macrophage response to very low density lipoprotein metabolism. This isoform has been implicated in transcriptional-repression functions and has been shown to repress the activity of PPARα or PPARγ target genes. PPARγ1 and γ2 are generated from a single-gene peroxisome proliferator-activated receptors gamma by differential promoter usage and alternative splicing. PPARγ1 is expressed in colon, immune system (e.g., monocytes and macrophages), and other tissues where it participates in the modulation of inflammation, cell proliferation, and differentiation. PPARs regulate gene expression through distinct mechanisms: Ligand-dependent transactivation, ligand-independent repression, and ligand-dependent transrepression. Studies in animals have demonstrated the gastric antisecretory activity of PPARα agonists like ciprofibrate, bezafibrate and clofibrate. Study by Pathak et al also demonstrated the effect of PPARα agonist, bezafibrate, on gastric secretion and gastric cytoprotection in various gastric ulcer models in rats. The majority of the experimental studies is on pioglitazone and rosiglitazone, which are PPARγ activators. In all the studies, both the PPARγ activators showed protection against the gastric ulcer and also accelerate the ulcer healing in gastric ulcer model in rats. Therefore, PPARα and PPARγ may be a target for gastric ulcer therapy. Finally, more studies are also needed to confirm the involvement of PPARs α and γ in gastric ulcer.

Lysine-Specific Demethylase 1 Has Dual Functions as a Major Regulator of Androgen Receptor Transcriptional Activity

Lysine-Specific Demethylase 1 (LSD1, KDM1A) functions as a transcriptional corepressor through demethylation of histone 3 lysine 4 (H3K4) but has a coactivator function on some genes through mechanisms that are unclear. We show that LSD1, interacting with CoREST, associates with and coactivates androgen receptor (AR) on a large fraction of androgen-stimulated genes. A subset of these AR/LSD1-associated enhancer sites have histone 3 threonine 6 phosphorylation (H3T6ph), and these sites are further enriched for androgen-stimulated genes. Significantly, despite its coactivator activity, LSD1 still mediates H3K4me2 demethylation at these androgen-stimulated enhancers. FOXA1 is also associated with LSD1 at AR-regulated enhancer sites, and a FOXA1 interaction with LSD1 enhances binding of both proteins at thesesites. These findings show that LSD1 functions broadly as a regulator of AR function, that it maintains a transcriptional repression function at AR-regulated enhancers through H3K4 demethylation, and that it has a distinct AR-linked coactivator function mediated by demethylation of other substrates.

Abstract 1124: ETS1 regulates epithelial-to-mesenchymal transition (EMT) in prostate cancer cells

Abstract Background: The transcription factor ETS1 is overexpressed in up to 70% of prostate tumors. Three ETS1 isoforms which arise due to alternative splicing have been reported, ETS1p51, ETS1p42 and ETS1p27, each with distinct transcriptional activator or repressor functions. Elevated ETS1 levels in prostate tumors are correlated with more rapid progression to castrate-resistant disease and a poor prognosis, while ETS1 overexpression in vitro is correlated with increased migration and invasion of prostate cancer cells. Aim: To characterise ETS1 isoform expression in prostate tumors and to determine the effects of ETS1 overexpression on the regulation of epithelial-to-mesenchymal transition (EMT). Methods: ETS1 isoform expression was characterised in 45 malignant prostate and adjacent non-malignant prostatic tissues using isoform-specific RT-qPCR and western blotting. Expression of genes encoding regulators of EMT was screened using a Human EMT PCR array (SABiosciences) in the human prostate cancer cell line, LNCaP that had been transiently transfected to overexpress ETS1p51. RT-qPCR and western blotting were used to confirm the ETS1-induced levels of differentially expressed genes/proteins and western blotting for phospho-SMAD2 (pSMAD2)/total SMAD2 (tSMAD2) was used to investigate activation of TGF-β signalling. Results: ETS1p51 was the predominant ETS1 isoform expressed in both the normal prostate and prostate tumors, with significantly decreased mRNA (p<0.001), but significantly elevated (p<0.05) protein levels in the majority of malignant tissues. ETS1p51 overexpression increased (>1.5 fold) levels of pro-EMT regulators including members of the WNT (WNT5B, β-catenin) and TGF-β (TGF-β1/2/3) signalling pathways. Phosphorylation of the TGF-β signalling intermediate, SMAD2 was increased and mRNA/protein levels of the EMT transcriptional regulators, TWIST1, SNAIL1, SLUG and ZEB1 were also elevated. In ETS1p51 overexpressing cells, expression of the mesenchymal marker vimentin was increased, while expression of the epithelial tight-junction molecule, Claudin-1 was decreased. Together with increased expression of effectors of migration and invasion (e.g. SPARC, MMP2/9), the findings indicate induction of an EMT programme. Conclusions: Levels of ETS1p51, the predominant ETS1 isoform expressed in the prostate are upregulated by post-transcriptional mechanisms in prostate tumors. Increased ETS1p51 expression promotes TGF-β signalling and EMT, processes that are likely to contribute to the accelerated disease progression and poorer outcomes associated with ETS1 overexpressing prostate tumors. Citation Format: Jamie J. Rodgers, Michael Epis, Ronald Cohen, Peter Leedman, Jennet Harvey, Marc Thomas, Jacqueline Bentel. ETS1 regulates epithelial-to-mesenchymal transition (EMT) in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1124. doi:10.1158/1538-7445.AM2014-1124

Abstract 2632: Casein kinase II inhibitors regulate cell cycle progression in leukemia

Abstract Casein Kinase II (CK2) is an oncogenic kinase that is overexpressed in leukemia. The molecular mechanism by which CK2 exerts its oncogenic function is largely unknown. We have previously demonstrated that CK2 directly phosphorylates the Ikaros tumor suppressor in vivo at multiple, evolutionarily conserved amino acids. CK2-mediated phosphorylation of Ikaros results in reduced DNA binding affinity, loss of pericentromeric localization and impaired Ikaros transcriptional repression. We hypothesize that the pro-oncogenic activity of CK2 in leukemia involves functional inactivation of Ikaros, and that CK2 inhibition will result in restoration of the tumor suppressor function of Ikaros and an anti-leukemia effect. To test this hypothesis, we have tested the effect of specific CK2 inhibitors on Ikaros function as a transcriptional regulator of genes that promote cell cycle progression. Using quantitative Chromatin Immunoprecipitation (qChIP), we have shown that Ikaros binds to the promoters of two genes that are essential for mitosis, ANAPC1 and ANAPC7, in leukemia cell lines and in primary leukemia cells. Increased expression of Ikaros via retroviral transduction was associated with increased binding of Ikaros to the promoters of ANAPC1 and ANAPC7 and their transcriptional repression. Luciferase reporter assay was used to confirm that Ikaros can directly repress transcription of both ANAPC1 and ANAPC7. These data suggest that Ikaros negatively regulates cell cycle progression during mitosis and that Ikaros function as a repressor of ANAPC1 and ANAPC7 is impaired in leukemia. We tested the ability of CK2 inhibition to restore Ikaros repressor activity in leukemia. We evaluated the pre-B acute lymphoblastic leukemia (B-ALL) cell line, Nalm6 treated with a specific CK2 inhibitor, TBB as well as in a murine preclinical xenograft model of B-ALL. CK2 treatment resulted in increased Ikaros binding to promoters of ANAPC1 and ANAPC7, along with transcriptional repression of both genes. This was associated with prolonged survival of B-ALL xenograft mice that were treated with CK2 inhibitors. These data suggest that CK2 inhibitors can regulate cell cycle progression in leukemia cells by restoring the transcriptional repressor function of Ikaros. Our results demonstrate efficacy of CK2 inhibitors as a novel treatment for leukemia. Citation Format: Chandrika Gowda, Chunhua Song, Mansi Sachdev, Xiaokang Pan, Yali Ding, Kimberley Payne, Sinisa Dovat. Casein kinase II inhibitors regulate cell cycle progression in leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2632. doi:10.1158/1538-7445.AM2014-2632

SMCX and components of the TIP60 complex contribute to E2 regulation of the HPV E6/E7 promoter

An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/TRRAP histone acetyltransferase complex, also contributes to the E2 repression function, and we extend our studies on SMCX. Di- and tri-methyl marks on histone H3K4 are reduced in the presence of E2 and SMCX, suggesting a mechanism by which SMCX contributes to E2-mediated repression of the HPV LCR. Together, these findings lead us to hypothesize that E2 recruits histone-modifying cellular proteins to the HPV LCR, resulting in transcriptional repression of E6 and E7.

The BCL6 RD2 Domain Governs Commitment of Activated B Cells to Form Germinal Centers

To understand how the Bcl6 transcriptional repressor functions in the immune system, we disrupted its RD2 repression domain in mice. Bcl6RD2(MUT) mice exhibit a complete loss of germinal center (GC) formation but retain normal extrafollicular responses. Bcl6RD2(MUT) antigen-engaged B cells migrate to the interfollicular zone and interact with cognate T helper cells. However, these cells fail to complete early GC-commitment differentiation and coalesce as nascent GC aggregates. Bcl6 directly binds and represses trafficking receptors S1pr1 and Gpr183 by recruiting Hdac2 through the RD2 domain. Deregulation of these genes impairs B cell migration and may contribute to GC failure in Bcl6RD2(MUT) mice. The development of functional GC-TFH cells was partially impaired in Bcl6RD2(MUT) mice. In contrast to Bcl6(-/-) mice, Bcl6RD2(MUT) animals experience no inflammatory disease or macrophage deregulation. These results reveal an essential role for RD2 repression in early GC commitment and striking biochemical specificity in Bcl6 control of humoral and innate immune-cell phenotypes.

Probing phosphorylation-dependent protein interactions within functional domains of histone deacetylase 5 (HDAC5).

Class IIa histone deacetylases (HDACs) are critical transcriptional regulators, shuttling between nuclear and cytoplasmic cellular compartments. Within the nucleus, these HDACs repress transcription as components of multiprotein complexes, such as the nuclear corepressor and beclin-6 corepressor (BCoR) complexes. Cytoplasmic relocalization relieves this transcriptional repressive function. Class IIa HDAC shuttling is controlled, in part, by phosphorylations flanking the nuclear localization signal (NLS). Furthermore, we have reported that phosphorylation within the NLS by the kinase Aurora B modulates the localization and function of the class IIa HDAC5 during mitosis. While we identified numerous additional HDAC5 phosphorylations, their regulatory functions remain unknown. Here, we studied phosphorylation sites within functional HDAC5 domains, including the deacetylation domain (DAC, Ser755), nuclear export signal (NES, Ser1108), and an acidic domain (AD, Ser611). We have generated phosphomutant cell lines to investigate how absence of phosphorylation at these sites impacts HDAC5 localization, enzymatic activity, and protein interactions. Combining molecular biology and quantitative MS, we have defined the interactions and HDAC5-containing complexes mediated by site-specific phosphorylation and quantified selected changes using parallel reaction monitoring. These results expand the current understanding of HDAC regulation, and the functions of this critical family of proteins within human cells.

The six conserved serine/threonine sites of REPRESSOR OF ga1-3 protein are important for its functionality and stability in gibberellin signaling in Arabidopsis.

Our results provide further insight into the regulation of DELLA proteins in Arabidopsis . We clarified that phosphorylation modification of the six conserved sites is important for RGA functions and stability. The DELLA proteins, important plant growth and development repressors mediate the gibberellin (GA) signaling pathway. Although these proteins exhibit phosphorylation and de-phosphorylation states at the molecular level, little is known regarding the effects of different modifications of DELLA proteins on the regulation of their bioactivity and stability at the genetic level. In this study, six conserved serine (Ser)/threonine (Thr) sites of REPRESSOR OF ga1-3 (RGA) were substituted with alanine (RGA6A) or aspartic acid (RGA6D) to mimic the states of constitutive de-phosphorylation and phosphorylation, respectively. We found that the overexpression of de-phosphomimic RGA in Col-0 plants caused GA-overdose phenotypes, which were similar to DELLA-deficient mutant. These phenotypes were probably attributed to de-phosphomimic RGA, which retained its transcriptional activation activity that induces GA biosynthetic genes, but lost the transcription repressor function that inhibits GA-responsive genes. Further, de-phosphomimic RGA was unstable and easily degradable unlike the wild-type RGA, suggesting that the de-phosphorylated form is necessary for its degradation. In contrast, phosphomimic RGA overexpression caused GA-deficient phenotypes with non-degradable RGA. These phenotypes were probably due to phosphomimic RGA, which represses GA-responsive gene expression instead of inducing GA biosynthetic genes. In addition, phosphomimic RGA was stable and hardly degradable, which aggravated the RGA-inhibiting function in GA signaling. In conclusion, we show that the six conserved Ser/Thr sites are important for the different bioactivities of the RGA protein that regulate the GA response, and also for RGA stability via the mimicking of phosphorylation/de-phosphorylation.

EZH2: novel therapeutic target for human cancer.

Enhancer of Zeste homlog 2 (EZH2) is a catalytic subunit of epigenetic regulator Polycomb repressive complex 2 (PRC2), which trimethylates Lys 27 of histone H3, leading to silencing of the target genes that are involved in a variety of biological processes including tumor progression and stem cell maintenance. However, in addition to its canonical PRC2-dependent transcriptional repression function, EZH2 also acts as a gene activator in a noncanonical PRC2-independent manner. Overexpression of EZH2 has been detected in diverse cancers, and is associated with tumor malignancy. Moreover, activating mutations and inactivating mutations of EZH2 are also associated with certain types of cancer. Given EZH2’s multi-faceted function and role in cancer, context-specific strategy for targeting EZH2/EZH2-mediated signaling could serve as future targeted therapy/personalized medicine for human cancer.

The Fanconi anemia pathway has a dual function in Dickkopf-1 transcriptional repression

Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with a progressive decline in hematopoietic stem cells, developmental defects, and predisposition to cancer. These various phenotypic features imply a role of FA proteins in molecular events regulating cellular homeostasis. Interestingly, we previously found that the Fanconi C protein (FANCC) interacts with the C-terminal-binding protein-1 (CtBP1) involved in transcriptional regulation. Here we report that FANCC with CtBP1 forms a complex with β-catenin, and that β-catenin activation through glycogen synthase kinase 3β inhibition leads to FANCC nuclear accumulation and FA pathway activation, as measured by the Fanconi D2 protein (FANCD2) monoubiquitination. β-catenin and FANCC nuclear entry is defective in FA mutant cells and in cells depleted of the Fanconi A protein or FANCD2, suggesting that integrity of the FA pathway is required for FANCC nuclear activity. We also report that FANCC with CtBP1 acts as a negative regulator of Dickkopf-1 (DKK1) expression, and that a FA disease-causing mutation in FANCC abrogates this function. Our findings reveal that a defective FA pathway leads to up-regulation of DKK1, a molecule involved in hematopoietic malignancies.

SUMOylation of MeCP2 is essential for transcriptional repression and hippocampal synapse development

Methyl CpG binding protein 2 (MeCP2) binds to methylated DNA and acts as a transcriptional repressor. Mutations of human MECP2 gene lead to Rett syndrome, a severe neural developmental disorder. Here, we report that the MeCP2 protein can be modified by covalent linkage to small ubiquitin-like modifier (SUMO) and SUMOylation at lysine 223 is necessary for its transcriptional repression function. SUMOylation of MeCP2 is required for the recruitment of histone deacetylase complexes 1/2 complex. Mutation of MeCP2 lysine 223 to arginine abolishes its suppression of gene expression in mouse primary cortical neurons. Significantly, mutation of MeCP2 K223 site leads to developmental deficiency of rat hippocampal synapses in vitro and in vivo. Thus, the SUMOylation of MeCP2 at K223 is a critical switch for transcriptional repression and plays a crucial function in regulating synaptic development in the central nervous system.

Notch Alters Sumoylation To Govern GFI1 Protein Stability and Support Its Transcriptional Repression Function

The zinc-finger (ZF) transcriptional repressors, Growth factor independence (GFI)-1 and -1B are master regulators of lineage allocation in hematopoietic, aerodigestive tract and central nervous system development. GFI family members share near invariant SNAG and ZF domains at their N- and C-termini, respectively. Their SNAG domains recruit Lysine Specific Demethylase (LSD)-1, a dominant effector of transcriptional repression by GFI proteins, while ZFs 3-5 of both GFI1 and GFI1B recognize a common consensus element in target promoters. A non-homologous linker separates the SNAG and ZF domains. Despite near identical DNA binding and co-factor recruitment characteristics, GFI family proteins direct distinct hematopoietic outcomes. Granulopoiesis requires Gfi1, and Gfi1 mutations cause severe congenital neutropenia (SCN) type II. Gfi1 is also necessary for T-lymphopoiesis and maintenance of the malignant phenotype in lymphoid leukemias and lymphomas. In contrast, Gfi1b specifies erythroid and megakaryocytic fates and is often abundantly expressed in myeloid leukemias. These distinct outcomes cannot be explained by patterns of expression alone, as Gfi1 and Gfi1B transcripts partially overlap during hematopoiesis. These findings suggest a need to distinguish between GFI family proteins as they integrate developmental signals to coordinate alternate hematopoietic fates. How this occurs is not understood. We hypothesized that functional differences between GFI1 and GFI1B may arise from interactions and post-translational modifications occurring uniquely within their linker regions. We show the GFI1 linker contains a type-I SUMOylation consensus motif centered on K239 that is absolutely conserved among mammalian GFI1 proteins but absent from GFI1B. K239 is required for robust GFI1 SUMOylation, as arginine substitution (K239R) significantly impairs formation of high molecular weight, SUMO-conjugated GFI1 derivatives. Coincidentally, K239R substitution also significantly impairs LSD1 recruitment by the GFI1 SNAG domain. The SUMOylation site in the GFI1 linker is contained within the binding site for PIAS3, an E3 SUMO ligase. PIAS3 co-expression antagonizes transcriptional repression by GFI1, but not GFI1B. The GFI1-K239R derivative represses transcription comparable to wild type GFI1, but resists the inhibitory effects of PIAS3. Moreover, GFI1-K239R preferentially co-purifies with chromatin in fractionated cells, displaying a prolonged half-life relative to wild type GFI1. These data suggest that GFI1 SUMOylation occurs on chromatin, facilitates LSD1 recruitment and serves as a prelude to GFI1 degradation. We surmised that the Notch1 intracellular domain (N1-ICD), which requires GFI1 for both normal and malignant T-lymphopoiesis, might alter GFI1 SUMOylation to reinforce its functions as a transcriptional regulator. We show that N1-ICD interacts directly with the GFI1 linker via the same motif that binds PIAS3. N1-ICD and PIAS3 compete for GFI1 binding and display an oppositional relationship in GFI1-mediated transcriptional repression. N1-ICD reinforces GFI1 as a transcriptional repressor, blocks GFI1 conjugation by ubiquitin family proteins and prevents GFI1 degradation. Our findings suggest a post-transcriptional mechanism by which Notch signaling endorses GFI1 as a transcriptional regulator and intimate GFI1 binding partners like LSD1 as candidate biological response modifiers in Notch-driven malignancies. Disclosures:No relevant conflicts of interest to declare.

Holocarboxylase synthetase acts as a biotin-independent transcriptional repressor interacting with HDAC1, HDAC2 and HDAC7

In human cells, HCS catalyzes the biotinylation of biotin-dependent carboxylases and mediates the transcriptional control of genes involved in biotin metabolism through the activation of a cGMP-dependent signal transduction pathway. HCS also targets to the cell nucleus in association with lamin-B suggesting additional gene regulatory functions. Studies from our laboratory in Drosophila melanogaster showed that nuclear HCS is associated with heterochromatin bands enriched with the transcriptionally repressive mark histone 3 trimethylated at lysine 9. Further, HCS was shown to be recruited to the core promoter of the transcriptionally inactive hsp70 gene suggesting that it may participate in the repression of gene expression, although the mechanism involved remained elusive. In this work, we expressed HCS as a fusion protein with the DNA-binding domain of GAL4 to evaluate its effect on the transcription of a luciferase reporter gene. We show that HCS possesses transcriptional repressor activity in HepG2 cells. The transcriptional function of HCS was shown by in vitro pull down and in vivo co-immunoprecipitation assays to depend on its interaction with the histone deacetylases HDAC1, HDAC2 and HDAC7. We show further that HCS interaction with HDACs and its function in transcriptional repression is not affected by mutations impairing its biotin-ligase activity. We propose that nuclear HCS mediates events of transcriptional repression through a biotin-independent mechanism that involves its interaction with chromatin-modifying protein complexes that include histone deacetylases.

BZIP transcription factor SmJLB1 regulates autophagy-related genes Smatg8 and Smatg4 and is required for fruiting-body development and vegetative growth in Sordaria macrospora

Autophagy is a precisely controlled degradation process in eukaryotic cells, during which the bulk of the cytoplasm is engulfed by a double membrane vesicle, the autophagosome. Fusion of the autophagosome with the vacuole leads to breakdown of its contents, such as proteins and organelles, and the recycling of nutrients. Earlier studies of autophagic genes of the core autophagic machinery in the filamentous ascomycete Sordaria macrospora elucidated the impact of autophagy on fungal viability, vegetative growth and fruiting-body development. To gain further knowledge about the regulation of autophagy in S. macrospora, we analyzed the function of the bZIP transcription factor SmJLB1, a homolog of the Podospora anserina basic zipper-type transcription factor induced during incompatibility 4 (IDI-4) and the Aspergillus nidulans transcription factor jun-like bZIP A (JlbA). Generation of the homokaryotic deletion mutant demonstrated S. macrospora Smjlb1 is associated with autophagy-dependent processes. Deletion of Smjlb1 abolished fruiting-body formation and impaired vegetative growth. SmJLB1 is localized to the cytoplasm and to nuclei. Quantitative real-time PCR experiments revealed an upregulated expression of autophagy-related genes Smatg8 and Smatg4 in the Smjlb1 deletion mutant, suggesting a transcriptional repression function of SmJLB1.

Emerging from NF-κB’s Shadow, SUMOylated IκBα Represses Transcription

In this issue of Cancer Cell, Mulero and colleagues describe an NF-κB independent transcriptional repression function for SUMOylated IκBα. This compelling and provocative model links IκBα to the activity of the Polycomb repressors and provides a mechanism to link inflammatory signaling to skin homeostasis.

Zfp819, a novel KRAB-zinc finger protein, interacts with KAP1 and functions in genomic integrity maintenance of mouse embryonic stem cells

Pluripotency is maintained by both known and unknown transcriptional regulatory networks. In the present study, we have identified Zfp819, a KRAB-zinc finger protein, as a novel pluripotency-related factor and characterized its role in pluripotent stem cells. We show that Zfp819 is expressed highly in various types of pluripotent stem cells but not in their differentiated counterparts. We identified the presence of non-canonical nuclear localization signals in particular zinc finger motifs and identified them as responsible for the nuclear localization of Zfp819. Analysis of the Zfp819 promoter region revealed the presence of a transcriptionally active chromatin signature. Moreover, we confirmed the binding of pluripotency-related factors, Oct4, Sox2, and Nanog to the distal promoter region of Zfp819, indicating that the expression of this gene is regulated by a pluripotency transcription factor network. We found that the expression of endogenous retroviral elements (ERVs) such as Intracisternal A Particle (IAP) retrotransposons, Long Interspersed Nuclear Elements (LINE1), and Short Interspersed Nuclear Elements (SINE B1) is significantly upregulated in Zfp819-knockdown (Zfp819_KD) cells. In line with the activation of ERVs, we observed the occurrence of spontaneous DNA damage in Zfp819_KD cells. Furthermore, we tested whether Zfp819 can interact with KAP1, a KRAB-associated protein with a transcriptional repression function, and found the interaction between these two proteins in both in vitro and in vivo experiments. The challenging of Zfp819_KD cells with DNA damaging agent revealed that these cells are inefficient in repairing the damaged DNA, as cells showed presence of γH2A.X foci for a prolonged time. Collectively, our study identified Zfp819 as a novel pluripotency-related factor and unveiled its function in genomic integrity maintenance mechanisms of mouse embryonic stem cells.

More targets, more pathways and more clues for mutant p53

Mutations in the transcription factor p53 are among the most common genetic alterations in human cancer, and missense p53 mutations in cancer cells can lead to aggressive phenotypes. So far, only few studies investigated transcriptional reprogramming under mutant p53 expression as a means to identify deregulated targets and pathways. A review of the literature was carried out focusing on mutant p53-dependent transcriptome changes with the aims of (i) verifying whether different p53 mutations can be equivalent for their effects, or whether there is a mutation-specific transcriptional reprogramming of target genes, (ii) understanding what is the main mechanism at the basis of upregulation or downregulation of gene expression under the p53 mutant background, (iii) identifying novel candidate target genes of WT and/or mutant p53 and (iv) defining cellular pathways affected by the mutant p53-dependent gene expression reprogramming. Nearly 600 genes were consistently found upregulated or downregulated upon ectopic expression of mutant p53, regardless of the specific p53 mutation studied. Promoter analysis and the use of ChIP-seq data indicate that, for most genes, the expression changes could be ascribed to a loss both of WT p53 transcriptional activation and repressor functions. Pathway analysis indicated changes in the metabolism/catabolism of amino acids such as aspartate, glutamate, arginine and proline. Novel p53 candidate target genes were also identified, including ARID3B, ARNT2, CLMN, FADS1, FTH1, KPNA2, LPHN2, PARD6B, PDE4C, PIAS2, PRPF40A, PYGL and RHOBTB2, involved in the metabolism, xenobiotic responses and cell differentiation.