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Lethal(3) malignant brain tumour like 2 (L3MBTL2) is an integral component of the polycomb repressive complex 1.6 (PRC1.6) and has been implicated in transcriptional repression and chromatin compaction. Here, we show that L3MBTL2 is modified by SUMO2/3 at lysine residues 675 and 700 close to the C-terminus. SUMOylation of L3MBTL2 neither affected its repressive activity in reporter gene assays nor it's binding to histone tails in vitro. In order to analyse whether SUMOylation affects binding of L3MBTL2 to chromatin, we performed ChIP-Seq analysis with chromatin of wild-type HEK293 cells and with chromatin of HEK293 cells stably expressing either FLAG-tagged SUMOylation-competent or SUMOylation-defective L3MBTL2. Wild-type FLAG-L3MBTL2 and the SUMOylation-defective FLAG-L3MBTL2 K675/700R mutant essentially occupied the same sites as endogenous L3MBTL2 suggesting that SUMOylation of L3MBTL2 does not affect chromatin binding. However, a subset of L3MBTL2-target genes, particularly those with low L3MBTL2 occupancy including pro-inflammatory genes, was de-repressed in cells expressing the FLAG-L3MBTL2 K675/700R mutant. Finally, we provide evidence that SUMOylation of L3MBTL2 facilitates repression of these PRC1.6-target genes by balancing the local H2Aub1 levels established by the ubiquitinating enzyme RING2 and the de-ubiquitinating PR-DUB complex.

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Previous analyses showed that, unlike Sp1, Sp3 fails to activate transcription in certain promoter settings. This is due to the presence of an inhibitory domain located between the second glutamine-rich activation domain and the DNA-binding domain. To further analyze the transcriptional properties of Sp3, we have expressed and purified recombinant Sp3 and Sp1 as epitope-tagged proteins from stable transfected insect cells. We found that Sp3 does act as a strong activator similar to Sp1 in an in vitro transcription assay using Sp1/Sp3-depleted HeLa nuclear extract. However, on the same promoter Sp3 is almost inactive when transfected into cells. Mutational studies demonstrate that a single lysine residue is responsible for the low transcriptional activity of Sp3 in vivo. We show that Sp3, but not a mutant of Sp3 that lacks this lysine residue, is highly acetylated in vivo. Our results strongly suggest that the transcriptional activity of Sp3 is regulated by acetylation. The consequences of acetylation for the activity of Sp3 are discussed.

Recently, a family of transcription factors structurally related to Sp1 has been described; thus, more than one activator may bind to the GC boxes present in a number of viral and cellular promoters. We have compared the transactivation potentials of Sp1, Sp3 and Sp4 proteins on the human immunodeficiency virus type 1 (HIV-1) promoter. The long terminal repeat (LTR) of HIV-1 contains three binding sites for the transcription factor Sp1 (GC boxes) which are involved in both basal and Tat-mediated transcriptional activation. Moreover, a cooperative interaction between NF-kappa B and Sp1 is required for HIV enhancer activation. We now demonstrate that Sp4 is an activator, while the Sp3 protein represses basal expression of HIV promoter. Remarkably, we found that over-expression of the transcription factor Sp3 was able to suppress Tat-mediated transactivation. These inhibitory effects of Sp3 correlate with its DNA binding activity, suggesting that Sp3 inhibition involves competition with Sp1 for occupancy of the GC boxes. Next, we have analyzed the role of different Sp1-related proteins in the stimulation of HIV-1 promoter in response to mitogens. We found that the binding of NF-kappa B is not by itself sufficient to induce HIV gene expression. Instead, an interaction between NF-kappa B and the trans-acting domain (A domain) of Sp1 bound to an adjacent site must occur. We found that the cooperative interaction between NF-kappa B and Sp1 is highly specific, since neither Sp3 nor Sp4 is capable of cooperating with NF-kappa B.

Previous analyses of the uteroglobin gene promoter revealed a GT1 box which is also found in the SV40 enhancer. The GT1 element in the context of the uteroglobin promoter is active in Ishikawa cells, a human endometrial cell line, but not in HeLa cells. Here we report the cloning by recognition site screening of two factors (SPR-1 and SPR-2) which bind to this GT1 motif. SPR-1 and SPR-2 are homologues of the transcription factor Sp1. All three proteins are closely related members of a gene family encoding proteins with very similar structural features. Like Sp1, SPR-1 and SPR-2 contain glutamine and serine/threonine rich amino acid stretches. Most significantly, the DNA binding domains of all three proteins are highly conserved and they recognize GT as well as GC boxes identically. SPR-2 mRNA is expressed ubiquitously, whereas SPR-1 transcripts are abundant in the brain but barely detectable in other organs. The possible function of these factors for the activity of the uteroglobin promoter is discussed.

To understand the mechanisms responsible for endometrium-specific expression of the uteroglobin gene, we have compared transcription from the uteroglobin promoter in a human endometrial cell line (Ishikawa) and in HeLa cells. In transient transfection experiments and in nuclear extracts, sequences from -395 to +14 of the uteroglobin gene are able to promote transcription of a reporter gene more efficiently in Ishikawa cells than in HeLa cells relative to the RSV or the SV40 early promoter. Analysis of progressive 5'-deletion mutants identifies three promoter regions, -258/-220, -205/-177, and -96/-35, that are important for preferential transcription in endometrial cells. DNase I footprinting experiments with nuclear extracts from Ishikawa and HeLa cells reveal a series of defined protections overlapping these regions. The relative intensity of individual protections differs between the two cell lines. Oligonucleotide competition experiments suggest that similar factor(s) bind(s) to the two relevant upstream regions of the promoter that share no homology to known regulatory elements. A protection over the TATA-box is detected only with extracts from Ishikawa cells. Band shift experiments show that an Ishikawa-specific factor binds to sequences overlapping the TATA-box region that are partially conserved in other endometrium-expressed genes. We propose that novel transcription factors mediate endometrium-specific expression of the uteroglobin gene in conjunction with a tissue-specific factor that binds to the TATA-box region.

The amino acid sequence of rat Clara Cell 10 kDa secretory protein (CC10) shows 55% identity to rabbit uteroglobin. In order to define the relationship between rat CC10 and rabbit uteroglobin in detail, the tissue-specific expression and hormonal regulation of rat CC10 mRNA was analyzed. We report that like rabbit uteroglobin, rat CC10 mRNA is expressed in lung and esophagus, as well as in uteri of estrogen- and progesterone-treated females. Expression of CC10 mRNA in lung is regulated by glucocorticoids. The similarity in expression pattern of rat CC10 mRNA and rabbit uteroglobin mRNA is reflected by a striking similarity in the 5'-flanking regions of the two genes. Despite this overall similarity, two regions of 0.3 kb and 2.1 kb are absent in the rat CC10 upstream gene region. The larger region includes a cluster of hormone receptor binding sites, believed to be responsible for differential regulation of rabbit uteroglobin by glucocorticoids and progesterone. Thus, while the sequence identities in the coding and 5'-flanking regions point towards a common ancestor for the uteroglobin and CC10 gene, later events (deletions/insertions) might have caused species-specific differences in their regulation.