Project description:The forkhead transcription factor (TF) FoxN3 recognizes both the canonical forkhead DNA motif (RYAAAYA), as well as a novel alternate motif (GACGC). These two motifs are more distinct than the typically observed primary and secondary motifs of other TFs, as the two motifs cannot be aligned and are not clearly related to each other. Amino acids at the canonical base-contacting positions in the forkhead DNA binding domain are largely conserved throughout the forkhead family, so the ability of FoxN3 and other bispecific forkhead factors to recognize the alternate site cannot be explained by amino acid substitutions at these positions. Neither the mechanism of this bispecific DNA recognition nor its regulatory function are understood. Here we show that human FoxN3 does recognize both motifs in cells. We also report the crystal structures of the FoxN3 DNA binding domain bound to the canonical forkhead binding site, and separately to the alternate DNA binding site. FoxN3 adopts a remarkably similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is significantly altered between the two motifs, allowing the same domain to recognize two motifs of different lengths. Changes between the two structures in the conformation of segments of the forkhead domain outside of the direct DNA-contacting positions contribute to the ability to bind the alternate site. Swapping these segments of the domain between different forkhead proteins changes the ability of proteins to recognize the alternate site, highlighting the importance of non-base-contacting positions in determining TF-DNA binding specificity. The structures present a new mechanism by which a single DBD can recognize two divergent sequences.
Project description:Development of multicellular animals requires epigenetic repression by Polycomb group proteins. The latter assemble in multi-subunit complexes, of which two kinds, Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2), act together to effect the repression of key developmental genes. How PRC1 and PRC2 recognize specific genes remains an open question. Here we report that small adjustment in the next generation sequencing of the Immunoprecipitated Chromatin (ChIP-seq) allows identification of several hundreds of DNA elements that tether canonical PRC1 to human developmental genes. Their analysis indicates that sequence features associated with canonical PRC1 tethering differ from those that favour the retention of PRC2. Throughout the genome, the two kinds of sequence features mix in different proportions to yield a gamut of DNA elements that range from those tethering predominantly PRC1 to ones capable of tethering both PRC1 and PRC2 or the elements that retain exclusively PRC2. The emerging picture is similar to paradigmatic targeting of Polycomb complexes by Polycomb Response Elements (PREs) of Drosophila but providing for unexpected degree of plasticity.
Project description:Relative contribution of sequence and structural features to the mRNA-binding of Argonaute/miRNA complexes and the degradation of miRNA targets; How miRNAs recognize their target sites is a puzzle that many experimental and computational studies aimed to solve. Several features such as perfect pairing of the miRNA seed, additional pairing in the 3â region of the miRNA, relative position in the 3â UTR and the A/U content of the environment of the putative site have been found to be relevant. Here we have used a large number of previously published data sets to assess the power that various sequence and structure features have in distinguishing between putative sites that do and those that do not appear to be functional. We found that although different data sets can give widely different answers when it comes to ranking the relative importance of these features, the sites inferred from most transcriptomics experiments as well as from comparative genomics appear similar at this level. This suggests that miRNA target sites have been selected in evolution on their ability to trigger mRNA degradation. To understand at what step in the miRNA induced response individual features play a role, we further transfected human HEK293 cells with miRNAs and analyzed the association of Argonaute/miRNA complexes with target mRNAs and the degradation of these messages. We found that structural features are only important for Argonaute binding, while sequence features such as the A/U content of the 3â UTR are important for mRNA degradation. Experiment Overall Design: Ago2 IPs of mock, miRNA-7, and miR-124 transfected cells were analyzed for 2 biological replicates. IPs of the biological replicates were analyzed with 2 technical replicates.
Project description:Relative contribution of sequence and structural features to the mRNA-binding of Argonaute/miRNA complexes and the degradation of miRNA targets How miRNAs recognize their target sites is a puzzle that many experimental and computational studies aimed to solve. Several features such as perfect pairing of the miRNA seed, additional pairing in the 3’ region of the miRNA, relative position in the 3’ UTR and the A/U content of the environment of the putative site have been found to be relevant. Here we have used a large number of previously published data sets to assess the power that various sequence and structure features have in distinguishing between putative sites that do and those that do not appear to be functional. We found that although different data sets can give widely different answers when it comes to ranking the relative importance of these features, the sites inferred from most transcriptomics experiments as well as from comparative genomics appear similar at this level. This suggests that miRNA target sites have been selected in evolution on their ability to trigger mRNA degradation. To understand at what step in the miRNA induced response individual features play a role, we further transfected human HEK293 cells with miRNAs and analyzed the association of Argonaute/miRNA complexes with target mRNAs and the degradation of these messages. We found that structural features are only important for Argonaute binding, while sequence features such as the A/U content of the 3’ UTR are important for mRNA degradation. Keywords: IP-array analysis
Project description:Heritable gene silencing is essential for the development of multicellular organisms. Polycomb repressive complexes 1 and 2 (PRC1 and 2) catalyze and recognize histone H2A lysine 119 mono-ubiquitination (H2AK119ub1) and histone H3 lysine 27 trimethylation (H3K27me3), respectively, to mediate heritable gene silencing, but the mechanism of inheritance is not fully understood. Using an inducible gene silencing strategy, we show that the epigenetic inheritance of Polycomb silencing in human cells is strongly dependent on local DNA sequences and chromatin environment. In addition, we find that inheritance is not strictly dependent on H3K27me3 recognition and can be partially achieved by H2AK119ub1 catalysis and recognition, independently of H3K27me3. These findings demonstrate that locus specific features and H2AK119ub1 play key roles in inheritance of Polycomb silencing in human cells.
Project description:Major features of transcription by human RNA polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates.
Project description:In this study, we aim to identify common human host genes involved in pathogenesis of different rota virus strains as an attempt to recognize probable antiviral targets. We have compared the host gene regulation after infection of human intestinal cell line (HT29) with three different wild type RV strains i.e. SA11 (simian, G3, P2), A5-13 (bovine, G8, P1) and Wa (human, G1, P8). HT29 cells mock infected or infected with three rota virus strains (SA11, A5-13, Wa). At 5hpi total RNA was extracted and microarray was done using Affymetrix protocol.