Polycomb proteins are epigenetic regulators that localize to developmental loci in the early embryo where they mediate lineage-specific gene repression. In Drosophila, these repressors are recruited to sequence elements by DNA binding proteins associated with Polycomb repressive complex 2 (PRC2). However, the sequences that recruit PRC2 in mammalian cells have remained obscure. To address this, we integrated a series of engineered bacterial artificial chromosomes into embryonic stem (ES) cells and examined their chromatin. We found that a 44 kb region corresponding to the Zfpm2 locus initiates de novo recruitment of PRC2. We then pinpointed a CpG island within this locus as both necessary and sufficient for PRC2 recruitment. Based on this causal demonstration and prior genomic analyses, we hypothesized that large GC-rich elements depleted of activating transcription factor motifs mediate PRC2 recruitment in mammals. We validated this model in two ways. First, we showed that a constitutively active CpG island is able to recruit PRC2 after excision of a cluster of activating motifs. Second, we showed that two 1 kb sequence intervals from the Escherichia coli genome with GC-contents comparable to a mammalian CpG island are both capable of recruiting PRC2 when integrated into the ES cell genome. Our findings demonstrate a causal role for GC-rich sequences in PRC2 recruitment and implicate a specific subset of CpG islands depleted of activating motifs as instrumental for the initial localization of this key regulator in mammalian genomes. Key developmental genes are precisely turned on or off during development, thus creating a complex, multi-tissue embryo. The mechanism that keeps genes off, or repressed, is crucial to proper development. In embryonic stem cells, Polycomb repressive complex 2 (PRC2) is recruited to the promoters of these developmental genes and helps to maintain repression in the appropriate tissues through development. How PRC2 is initially recruited to these genes in the early embryo remains elusive. Here we experimentally demonstrate that stretches of GC-rich DNA, termed CpG islands, can initiate recruitment of PRC2 in embryonic stem cells when they are transcriptionally-inactive. Surprisingly, we find that GC-rich DNA from bacterial genomes can also initiate recruitment of PRC2 in embryonic stem cells. This supports a model where inactive GC-rich DNA can itself suffice to recruit PRC2 even in the absence of more complex DNA sequence motifs.

Motivation: Repetitive DNA sequences, besides having a variety of regulatory functions, are one of the principal causes of genomic instability. Understanding their origin and evolution is of fundamental importance for genome studies. The identification of repeats and their units helps in deducing the intra-genomic dynamics as an important feature of comparative genomics. A major difficulty in identification of repeats arises from the fact that the repeat units can be either exact or imperfect, in tandem or dispersed, and of unspecified length. Results: The Spectral Repeat Finder program circumvents these problems by using a discrete Fourier transformation to identify significant periodicities present in a sequence. The specific regions of the sequence that contribute to a given periodicity are located through a sliding window analysis, and an exact search method is then used to find the repetitive units. Efficient and complete detection of repeats is provided together with interactive and detailed visualization of the spectral analysis of input sequence. We demonstrate the utility of our method with various examples that contain previously unannotated repeats. A Web server has been developed for convenient access to the automated program. Availability: The Web server is available at http://www.imtech.res.in/raghava/srf and http://www2.imtech.res.in/raghava/srf

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-β, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.

Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) plays an essential role in regulating matrix remodeling, cell growth, differentiation, angiogenesis and apoptosis in vitro and in vivo. We have recently shown that TIMP-2-mediated inhibition of tumor growth is independent of matrix metalloproteinase-mediated mechanisms, and is a consequence of modulating both the tumor cells and the tumor microenvironment. In the current study we aim to identify the molecular pathways associated with these effects. We analyzed the transcriptional profile of the human lung cancer cell line A549 upon overexpression of TIMP-2 and Ala+TIMP-2 (mutant that does not inhibit MMP activity), and we found changes in gene expression predominantly related to decreased tumor development and metastasis. Increased E-cadherin expression in response to both TIMP-2 and Ala+TIMP-2 expression was confirmed by real time quantitative RT-PCR and immunoblotting. A549 cells treated with epidermal growth factor (EGF) displayed loss of cobblestone morphology and cell-cell contact, while cells overexpressing TIMP-2 or Ala+TIMP-2 were resistant to EGF-induced morphological changes. Moreover, exogenous treatment with recombinant Ala+TIMP-2 blocked EGF induced down-regulation of E-cadherin. In vivo, immunohistochemistry of A549 xenografts expressing either TIMP-2 or Ala+TIMP-2 demonstrated increased E-cadherin protein levels. More importantly, transcriptional profile analysis of tumor tissue revealed critical pathways associated with effects on tumor-host interaction and inhibition of tumor growth. In conclusion, we show that TIMP-2 promotes an anti-tumoral transcriptional profile in vitro and in vivo, including upregulation of E-cadherin, in A549 lung cancer cells.