Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function.

Sp1 and Sp3 belong to the specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed, and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell-cycle and growth control, metabolic pathways, and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice, conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, whereas the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double-knockout mice. We employed flow cytometry, cell culture, and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics, we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. Supporting this notion, selective Mylk inhibition by ML7 affected proplatelet formation and stabilization and resulted in defective ITAM receptor-mediated platelet aggregation.

Identification of SUMO-dependent chromatin-associated transcriptional repression components by a genome-wide RNAi screen.

SUMO modification of many transcription factors is linked to transcriptional repression. The molecular mechanisms by which SUMO attachment represses transcription are largely unknown. Here we report a genome-wide RNA interference screen in Drosophila melanogaster cells for components regulating and mediating SUMO-dependent transcriptional repression. Analysis of >21,000 double-stranded RNAs (dsRNAs) identified 120 genes whose dsRNA-mediated knockdowns impaired SUMO-dependent transcriptional repression. Several of these genes encode chromatin-associated proteins, including the ATP-dependent chromatin remodeler Mi-2, the D. melanogaster ortholog of the C. elegans protein MEP-1, and the polycomb protein Sfmbt. Knockdown of these proteins did not impair SUMO conjugation, demonstrating that they act downstream of SUMO attachment. Biochemical analyses revealed that MEP-1, Mi-2, and Sfmbt interact with each other, bind to SUMO, and are recruited to promoters in a SUMOylation-dependent manner. Our results suggest that MEP-1, Mi-2, and Sfmbt are part of a common repression complex established by DNA-bound SUMO-modified transcription factors.

Epigenetic silencing of spermatocyte-specific and neuronal genes by SUMO modification of the transcription factor Sp3.

SUMO modification of transcription factors is linked to repression of transcription. The physiological significance of SUMO attachment to a particular transcriptional regulator, however, is largely unknown. We have employed the ubiquitously expressed murine transcription factor Sp3 to analyze the role of SUMOylation in vivo. We generated mice and mouse embryonic fibroblasts (MEFs) carrying a subtle point mutation in the SUMO attachment sequence of Sp3 (IKEE(553)D mutation). The E(553)D mutation impedes SUMOylation of Sp3 at K(551)in vivo, without affecting Sp3 protein levels. Expression profiling revealed that spermatocyte-specific genes, such as Dmc1 and Dnahc8, and neuronal genes, including Paqr6, Rims3, and Robo3, are de-repressed in non-testicular and extra-neuronal mouse tissues and in mouse embryonic fibroblasts expressing the SUMOylation-deficient Sp3E(553)D mutant protein. Chromatin immunoprecipitation experiments show that transcriptional de-repression of these genes is accompanied by the loss of repressive heterochromatic marks such as H3K9 and H4K20 tri-methylation and impaired recruitment of repressive chromatin-modifying enzymes. Finally, analysis of the DNA methylation state of the Dmc1, Paqr6, and Rims3 promoters by bisulfite sequencing revealed that these genes are highly methylated in Sp3wt MEFs but are unmethylated in Sp3E(553)D MEFs linking SUMOylation of Sp3 to tissue-specific CpG methylation. Our results establish SUMO conjugation to Sp3 as a molecular beacon for the assembly of repression machineries to maintain tissue-specific transcriptional gene silencing.

SUMO-modified Sp3 represses transcription by provoking local heterochromatic gene silencing.

Modification of many transcription factors including Sp3 and steroidogenic factor 1 with the small ubiquitin-like modifier (SUMO) is associated with transcriptional repression. Here, we show that SUMOylation of transcription factors bound to DNA provokes the establishment of compacted repressive chromatin with characteristics of heterochromatin. Chromatin immunoprecipitation experiments revealed SUMO-dependent recruitment of the chromatin remodeller Mi-2, MBT-domain proteins, heterochromatic protein 1, and the histone methyltransferases SETDB1 and SUV4-20H, concomitant with the establishment of histone modifications associated with repressed genes, including H3K9 and H4K20 trimethylation. These results indicate that SUMOylation has a crucial role in regulating gene expression by initiating chromatin structure changes that render DNA inaccessible to the transcription machinery.

Differential regulation of sense and antisense promoter activity at the Csf1R locus in B cells by the transcription factor PAX5.

OBJECTIVE: The transcription factor PAX5 is essential for the activation of B-cell-specific genes and for the silencing of myeloid-specific genes. We previously determined the molecular mechanism by which PAX5 silences the myeloid-specific colony-stimulating-factor-receptor (Csf1R) gene and showed that PAX5 directly binds to the Csf1r promoter as well as to an intronic enhancer that generates an antisense transcript in B cells. Here we examine the role of PAX5 in the regulation of sense and antisense transcription in B cells. MATERIALS AND METHODS: We performed PAX5-specific chromatin immunoprecipitation analyses across the Csfr1 locus. We investigated the role of PAX5 in regulating Csf1r sense and antisense promoter activity by transient transfections and by employing a Pax5(-/-) pro-B-cell line expressing an inducible PAX5 protein. PAX5 interacting factors were identified by pull-down experiments. The role of the transcription factor Sp3 in driving antisense promoter expression was examined in B cells from Sp3 knockout mice. RESULTS: PAX5 differentially regulates the Csf1r promoter and the promoter of the antisense transcript. PAX5 interferes with PU.1 transactivation at the sense promoter by binding to a PAX5 consensus sequence. At the antisense promoter, PAX5 does not specifically recognize DNA, but interacts with Sp3 to upregulate antisense promoter activity. Antisense promoter activation by PAX5 is dependent on the presence of its partial homeo-domain. CONCLUSIONS: We demonstrate that PAX5 regulates Csf1r in B cells by reducing the frequency of binding of the basal transcription machinery to the promoter and by activating antisense RNA expression.

Specificity protein 2 (Sp2) is essential for mouse development and autonomous proliferation of mouse embryonic fibroblasts.

BACKGROUND: The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with the phenotypes of Sp1, Sp3 and Sp4 knockout strains shows that, despite their structural similarity and evolutionary relationship, all four glutamine-rich members of the Sp family of transcription factors have distinct non-redundant functions in vivo.

Sp1/Sp3 compound heterozygous mice are not viable: impaired erythropoiesis and severe placental defects.

The ubiquitously expressed zinc finger transcription factors Sp1 and Sp3 play critical roles in embryonic development. Sp1 knockout mice die around embryonic day 10.5. Mice lacking Sp3 are postnatal lethal. Mice heterozygous for either Sp1 or Sp3 are apparently normal, although slightly smaller. Here, we show that compound heterozygosity of Sp1 and Sp3 results in embryonic lethality accompanied by a spectrum of developmental abnormalities, including growth retardation, morphological alterations of the lung, impaired ossification, anemia, and placental defects. Anemia in Sp1/Sp3 compound heterozygous mutant embryos is associated with impaired maturation of erythrocytes. Analyses of the placenta revealed a markedly reduced spongiotrophoblast layer and a severe disorganization of the labyrinth layer in Sp1/Sp3 compound heterozygous as well as in Sp3-deficient mutant embryos. Our findings demonstrate that a threshold of Sp1 and Sp3 activity is required for normal embryonic development, suggesting that Sp1 and Sp3 act cooperatively to regulate downstream targets.