Mechanism of REST/NRSF regulation of clustered protocadherin α genes

Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5–1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDHα genes. Thus, REST/NRSF represses PCDHα gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

Mechanism of REST/NRSF Regulation of Clustered Protocadherin α Genes

ABSTRACTRepressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5-1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDHα genes. Thus, REST/NRSF represses PCDHα gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

Conserved structure of the NPR1 gene distal promoter isolated from a chili pepper (Capsicum annuum L.) in West Sumatera

Background: The non-expressor of pathogenesis related gene 1 (NPR1) protein is one of the key regulators in the systemic acquired resistance plant defense system. The cis-acting elements of its distal promoter gene are characterized by salicylic acid inducing elements such as the W-box, RAV1AAT and ASF1, accompanied by enhancer and silencer elements. This study was aimed to isolate and characterize the distal promoter sequence of the NPR1 gene (PD_CbNPR1) from the chili pepper (Capsicum annuum L.) genotype Berangkai, a local genotype known to produce large yields, but is susceptible to viral infection. Elucidating its sequence structure will open a broad range of possibilities to engineer the NPR1 gene expression which is important to improve chili pepper resistant. Methods: PCR-based cloning combined with a primer walking strategy was applied in this study. The BioEdit tool was used to edit the sequence and verify sequence integrity, while homology analysis was conducted with BLASTn searching. Identification of a cis-acting element was detected by PLACE, PlantCare, and PlantPAN. Results: Isolation of the complete distal promoter sequence of PD_CbNPR1 produced a fragment 5,950 bp in size. BLASTn search analysis indicated that PD_CbNPR1 sequence is highly conserved (99% identity) showing only a single nucleotide polymorphism (SNP) (base substitution) compared with its reference sequence. Analysis using PLACE tools successfully identified nine cis-acting elements containing a W-box, WLE1, RAV1AAT, TATA-box, CAAT-box, GARE and GT1 with multi repeats and diverse motives, as well as enhancer and silencer elements, which is characterized by a CCAAT-box and GAGAAATT pattern, respectively. Conclusion: The distal promoter of the NPR1 gene is highly conserved, showing only one SNP caused by one base substitution event.

Conserved structure of the NPR1 gene distal promoter isolated from a chili pepper (Capsicum annuum L.) in West Sumatera

Background: The non-expressor of pathogenesis related gene 1 (NPR1) protein is one of the key regulators in the systemic acquired resistance plant defence system. The cis-acting elements of its distal promoter gene are characterized by salicylic acid inducing elements such as the W-box, RAV1AAT and ASF1, accompanied with enhancer and silencer elements. This study was aimed to isolate and characterize the distal promoter sequence of the NPR1 gene (PD_CbNPR1) from the chili pepper (Capsicum annuum L.) genotype Berangkai, a local genotype known to produce large yields, but is susceptible to viral infection. Elucidating its sequence structure will open a broad range of possibilities to engineer the NPR1 gene expression which is important to improve chili pepper resistant. Methods: PCR-based cloning combined with a primer walking strategy was applied in this study. The BioEdit tool was used to edit the sequence and verify sequence integrity, while homology analysis was conducted with BLASTn searching. Identification of a cis-acting element was detected by PLACE. Results: Isolation of the complete distal promoter sequence of PD_CbNPR1 produced a fragment 5,950 bp in size. BLASTn search analysis indicated that PD_CbNPR1 sequence is highly conserved (99% homology) showing only a single nucleotide polymorphism (SNP) (base substitution) compared with its reference sequence. Analysis using PLACE tools successfully identified nine cis-acting elements containing a W-box, WLE1, RAV1AAT, TATA-box, CAAT-box, GARE and GT1 with multi repeats and diverse motives, as well as enhancer and silencer elements, which is characterized by a CCAAT-box and GAGAAATT pattern, respectively. Conclusion: The distal promoter of the NPR1 gene is highly conserved, showing only one SNP caused by one base substitution event.

An atlas of silencer elements for the human and mouse genomes

ABSTRACTThe study of gene regulation is dominated by a focus on the control of gene activation or controlling an increase in the level of expression. Just as critical is the process of gene repression or silencing. Chromatin signatures have allowed for the global mapping of enhancer cis-regulatory elements, however, the identification of silencer elements by computational or experimental approaches in a genome-wide manner are lacking. We present a simple but powerful computational approach to identify putative silencers genome-wide. We used a series of consortia data to predict silencers in over 100 human and mouse cell or tissue types. We performed several analyses to determine if these elements exhibited characteristics expected of a silencers. Motif enrichment analyses on putative silencers determined that motifs belonging to known transcriptional repressors are enriched, as well as overlapping known transcription repressor binding sites. Leveraging promoter capture HiC data from several human and mouse cell types, we found that over 50% of putative silencer elements are interacting with gene promoters having very low to no expression. Next, to validate our silencer predictions, we quantified silencer activity using massively parallel reporter assays (MPRAs) on 7500 selected elements in K562 cells. We trained a support vector machine model classifier on MPRA data and used it to refine potential silencers in other cell types. We also show that similar to enhancer elements, silencer elements are enriched in disease-associated variants. Our results suggest a general strategy for genome-wide identification and characterization of silencer elements.

Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7

HnRNP A1 regulates many alternative splicing events by the recognition of splicing silencer elements. Here, we provide the solution structures of its two RNA recognition motifs (RRMs) in complex with short RNA. In addition, we show by NMR that both RRMs of hnRNP A1 can bind simultaneously to a single bipartite motif of the human intronic splicing silencer ISS-N1, which controls survival of motor neuron exon 7 splicing. RRM2 binds to the upstream motif and RRM1 to the downstream motif. Combining the insights from the structure with in cell splicing assays we show that the architecture and organization of the two RRMs is essential to hnRNP A1 function. The disruption of the inter-RRM interaction or the loss of RNA binding capacity of either RRM impairs splicing repression by hnRNP A1. Furthermore, both binding sites within the ISS-N1 are important for splicing repression and their contributions are cumulative rather than synergistic.