ABSTRACT: By 4C-seq protocol we investigated DNA contacts across the genome by the FLC gene in the model plant Arabidopsis thaliana in order to explore a potential role of long-distance chromosomal interactions in the regulation of flowering.
Project description:Using a transcriptional network derived from 2000 breast cancer gene expression profiles we identify the master regulators (MRs) of FGFR2 signalling. To validate the identified regulons, we examined whether there was enrichment of TF binding near the transcription start sites (TSS) of genes found in the regulons of a particular MR. For ESR1 and SPDEF, ChIP-seq experiments were performed in MCF-7 cells, while existing data was analysed for FOXA1 (Hurtado et al. Nature Genetics, 43:27–33, 2010) and GATA3 (Theodorou, et al., Genome Res 23: 12-22, 2013). ChIP-seq experiments were performed on three biological replicates per each transcription factor. For each sample, 36bp single-end reads were obtained. Peak regions were identified in all ChIP-seq TF data sets using the peak caller algorithm MACS (Zhang et al., Genome Biology, 9(9):R137, 2008) with default parameters.
Project description:In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3M-NM-^T cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements at the borders of subtelomeres and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and components of the nuclear envelope by maintaining chromatin structure required for anchoring DNA insulators to nuclear pores. For MNase samples, duplicate mutant mononucleosome fractions were compared with duplicate WT mononucleosomes.
Project description:About half of all human and mouse miRNA genes are located within introns of protein-coding genes. Despite this, little is known about functional interactions between miRNAs and their host genes. The intronic miRNA miR-128 regulates neuronal excitability and controls dendrite outgrowth of projection neurons during development of the mouse cerebral cortex. Its host genes R3hdm1 and Arpp21 encode highly conserved, putative RNA-binding proteins. Here we use iCLIP to describe the RNA-binding activity of ARPP21, which recognizes uridine-rich sequences with exquisite sensitivity for 3UTRs. Surprisingly, ARPP21 antagonizes miR-128 activity by co-regulating a subset of miR-128 target mRNAs enriched for neurodevelopmental functions. In contrast to miR-128, we show that ARPP21 acts as a positive post-transcriptional regulator, at least in part through interaction with the eukaryotic translation initiation complex eIF4F. This molecular antagonism is also reflected in inverse activities during dendritogenesis: miR-128 overexpression or knockdown of ARPP21 reduces dendritic complexity; ectopic ARPP21 leads to an increase. The regulatory interaction between ARPP21 and miR-128 is a unique example of convergent function by two products of a single gene.
Project description:The experiment was performed to test the hypothesis that the chromosomal interaction pattern of FLOWERING LOCUS C (FLC) Arabidopsis thaliana gene (At5g10140) changes its epigenetic state throughout vernalization.
Project description:Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cancer types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 megabases downstream of Myc that are occupied by SWI/SNF, as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in 3% of acute myeloid leukemia. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs Enhancer usually regulates its targets through physical contact/interaction. In order to study chromosome conformation of Myc locus and potential distal enhancer E1-E5 region in murine AML cells, we utilize the high resolution 4C-seq and analysis pipeline to search cis elements that physical interact with Myc and E1-E5 region through setting up two individual viewpoints in these two regions.
Project description:We explored the RNA binding properties of LINE-1 ORF1p, both free and in the L1 RNP, using a recently developed photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation technique (PAR-CLIP) to comprehensively identify ORF1p binding sites in the transcriptome of human cells (HEK293T). Our results show that ORF1p binds to a wide range of cellular mRNAs, with an enrichment for binding at the 3’ UTR. Our data also show that ORF1p binds very strongly with retrotransposable RNA, i.e., L1, Alu and SVA. PAR-CLIP analysis of L1 RNPs and free ORF1p RNA binding profiles, comparison to HuR RNA binding profile
Project description:The aim of the experiment is to determine the sequences of binding sites on RNA transcripts that are interacting with the protein KIFC1 during cell division (here, pro metaphase). In addition to the sequence of the binding sites, we are interested in their genomic localisation (annotation) and in which region of the transcripts they are found (CDS, introns, UTRs, etc ...).
Project description:Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol_II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adapter protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1/COMPASS H3K4 methyltransferase and the nuclear Protein Phosphatase 1 (PP1) complexes to the initiating Pol_II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1 or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes, active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs. 4C-seq was performed on two different viewpoints (TSSs of the genes Ccl2 and Jun) in BMDMs.