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The functional engagement between an enhancer and its target promoter ensures precise gene transcription1. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).

The increasing prevalence of non-tuberculous mycobacterium (NTM) infections alongside tuberculosis (TB) underscores a pressing public health challenge. Yet, the mechanisms governing their infection within the lung remain poorly understood. Here, we integrate metagenomic sequencing, metabolomic sequencing, machine learning classifiers, SparCC, and MetOrigin methods to profile bronchoalveolar lavage fluid (BALF) samples from NTM/TB patients. Our aim is to unravel the intricate interplay between lung microbial communities and NTM/ Mycobacterium tuberculosis infections. Our investigation reveals a discernible reduction in the compositional diversity of the lung microbiota and a diminished degree of mutual interaction concomitant with NTM/TB infections. Notably, NTM patients exhibit a distinct microbial community characterized by marked specialization and notable enrichment of Pseudomonas aeruginosa and Staphylococcus aureus , driving pronounced niche specialization for NTM infection. Simultaneously, these microbial shifts significantly disrupt tryptophan metabolism in NTM infection, leading to an elevation of kynurenine. Mycobacterium intracellulare , Mycobacterium paraintracellulare , Mycobacterium abscessus , and Pseudomonas aeruginosa have been implicated in the metabolic pathways associated with the conversion of indole to tryptophan via tryptophan synthase within NTM patients. Additionally, indoleamine-2,3-dioxygenase converts tryptophan into kynurenine, fostering an immunosuppressive milieu during NTM infection. This strategic modulation supports microbial persistence, enabling evasion from immune surveillance and perpetuating a protracted state of NTM infection. The elucidation of these nuanced microbial and metabolic dynamics provides a profound understanding of the intricate processes underlying NTM and TB infections, offering potential avenues for therapeutic intervention and management. [ABSTRACT FROM AUTHOR]

Abstract Background SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells. SOX2 appears to re-activate in several human cancers including glioblastoma multiforme (GBM), however, the detailed response program of SOX2 in GBM has not yet been defined. Results We show that knockdown of the SOX2 gene in LN229 GBM cells reduces cell proliferation and colony formation. We then comprehensively characterize the SOX2 response program by an integrated analysis using several advanced genomic technologies including ChIP-seq, microarray profiling, and microRNA sequencing. Using ChIP-seq technology, we identified 4883 SOX2 binding regions in the GBM cancer genome. SOX2 binding regions contain the consensus sequence wwTGnwTw that occurred 3931 instances in 2312 SOX2 binding regions. Microarray analysis identified 489 genes whose expression altered in response to SOX2 knockdown. Interesting findings include that SOX2 regulates the expression of SOX family proteins SOX1 and SOX18, and that SOX2 down regulates BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2), two genes with tumor suppressor activity in GBM. Using next generation sequencing, we identified 105 precursor microRNAs (corresponding to 95 mature miRNAs) regulated by SOX2, including down regulation of miR-143, -145, -253-5p and miR-452. We also show that miR-145 and SOX2 form a double negative feedback loop in GBM cells, potentially creating a bistable system in GBM cells. Conclusions We present an integrated dataset of ChIP-seq, expression microarrays and microRNA sequencing representing the SOX2 response program in LN229 GBM cells. The insights gained from our integrated analysis further our understanding of the potential actions of SOX2 in carcinogenesis and serves as a useful resource for the research community.

Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely 'hijacked' by cytoplasmic Malat1 to the cytoplasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention, potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HNRNPK. Losing these RNA-protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: To identify SNPs in the miRNA genes in colorectal cancer (CRC) patients and to investigate their association with CRC., Methods: DNAs were isolated from 30 CRC tumor tissues and 30 tumor-adjacent tissues, and subjected to target capture using a custom miRNA chip covering 685 miRNA genes from NimbleGen. The captured DNAs were then sequenced using the Illumina's sequencing technology, and the data were analyzed., Results: We identified 64 SNPs in 43 miRNA genes and most of these SNPs are novel SNPs not reported previously. Prediction of functional consequences of the SNPs using TargetScan and miRSNP showed that SNPs of hsa-mir-1273-G/A, hsa-mir-548h-3-C/U, hsa-mir-1290-A/G, and hsa-mir-1273-C/U resulted in reduction of their mature miRNA abundance. SNPs of hsa-mir-376b-C/G, hsa-mir-604-T/C, hsa-mir-1268-T/G and hsa-mir-146a-C/G resulted in changes in their targeted genes. Finally, we focused on the analysis of SNPs in mir-146a and we found that mir-146a rs1052918 C>G was predicted to promote tumorigenesis via the Wnt signaling pathway., Conclusions: SNPs in the miRNA genes are important for tumorigenesis. The changes by hsa-mir-146a rs1052918 C>G may result in loss of Wnt, constant activation of the Wnt signaling pathway, and uncontrolled cell proliferation and tumor progression.

Abstract Re-sequencing of target genes is a highly effective approach for identifying mutations in cancers. Mutations, including indels (insertions, deletions, and the combination of the two), play important roles in carcinogenesis. Combining genomic DNA capture using high-density oligonucleotide microarrays (NimbleGen, Inc.) with next-generation high-throughput sequencing, we identified approximately 1600 indels for colorectal cancers in the Chinese population. Among them, 5 indels were localized to exonic regions of genes, including the TFDP1 (transcription factor Dp-1) gene. TFDP1 is an important transcription factor that coordinates with E2F proteins, thereby promoting transcription of E2F target genes and regulating the cell cycle and differentiation. We report here the identification of a recurrent frame-shift indel mutation (named indel84) in the TFDP1 gene in colorectal cancers by next-generation sequencing. We found in a validation set that TFDP1 indel84 is present in 70% of colorectal cancer (CRC) tissues. Wild-type TFDP1 encodes a protein of 410 amino acids with a potential DNA binding site at its N-terminal followed by several functional protein domains. The TFDP1 indel cDNA would generate an alternative TFDP1 protein missing the first 120 amino acids and potentially affecting the DNA binding domain. We further demonstrated that the TFDP1 indel84 mutation generated a gain-of-function phenotype by increasing cell proliferation, migration, and invasion of CRC cells. Our study identified a key molecular event for CRC that might have great diagnostic and therapeutic potentials.

The fundamental step of learning transcriptional regulation mechanism is to identify the target genes regulated by transcription factors (TFs). Despite numerous target genes identified by chromatin immunoprecipitation followed by high-throughput sequencing technology (ChIP-seq) assays, it is not possible to infer function from binding alone in vivo. This is equally true in one of the best model systems, the nematode Caenorhabditis elegans (C. elegans), where regulation often occurs through diverse TF binding features of transcriptional networks identified in modENCODE. Here, we integrated ten ChIP-seq datasets with genome-wide expression data derived from tiling arrays, involved in six TFs (HLH-1, ELT-3, PQM-1, SKN-1, CEH-14 and LIN-11) with tissue-specific and four TFs (CEH-30, LIN-13, LIN-15B and MEP-1) with broad expression patterns. In common, TF bindings within 3 kb upstream of or within its target gene for these ten studies showed significantly elevated level of expression as opposed to that of non-target controls, indicated that these sites may be more likely to be functional through up-regulating its target genes. Intriguingly, expression of the target genes out of 5 kb upstream of their transcription start site also showed high levels, which was consistent with the results of following network component analysis. Our study has identified similar transcriptional regulation mechanisms of tissue-specific or broad expression TFs in C. elegans using ChIP-seq and gene expression data. It may also provide a novel insight into the mechanism of transcriptional regulation not only for simple organisms but also for more complex species.

Alternative polyadenylation (APA) is widely present in the human genome and plays a key role in carcinogenesis. We conducted a comprehensive analysis of the APA products in glioblastoma multiforme (GBM, one of the most lethal brain tumors) and normal brain tissues and further developed a computational pipeline, RNAelements (http://sysbio.zju.edu.cn/RNAelements/), using covariance model from known RNA binding protein (RBP) targets acquired by RNA Immunoprecipitation (RIP) analysis. We identified 4530 APA isoforms for 2733 genes in GBM, and found that 182 APA isoforms from 148 genes showed significant differential expression between normal and GBM brain tissues. We then focused on three genes with long and short APA isoforms that show inconsistent expression changes between normal and GBM brain tissues. These were myocyte enhancer factor 2D, heat shock factor binding protein 1, and polyhomeotic homolog 1 (Drosophila). Using the RNAelements program, we found that RBP binding sites were enriched in the alternative regions between the first and the last polyadenylation sites, which would result in the short APA forms escaping regulation from those RNA binding proteins. To the best of our knowledge, this report is the first comprehensive APA isoform dataset for GBM and normal brain tissues. Additionally, we demonstrated a putative novel APA-mediated mechanism for controlling RNA stability and translation for APA isoforms. These observations collectively lay a foundation for novel diagnostics and molecular mechanisms that can inform future therapeutic interventions for GBM.

Abstract SOX2 is a high mobility group (HMG) box containing transcription factor that has been implicated in various types of cancer, but its role in colorectal cancers (CRC) has not been studied. Here we show that SOX2 is overexpressed in CRC tissues compared with normal adjacent tissues using immunohistochemical staining and RT-PCR. We also observed an increased SOX2 expression in nucleus of colorectal cancer tissues (46%, 14/30 cases vs. 7%, 2/30 adjacent tissues). Furthermore, knockdown of SOX2 in SW620 colorectal cancer cells decreased their growth rates in vitro cell line, and in vivo in xenograft models. ChIP-seq analysis of SOX2 revealed a consensus sequence of wwTGywTT. An integrated expression profiling and ChIP-seq analysis show that SOX2 is involved in the BMP signaling pathway, steroid metabolic process, histone modifications, and many receptor-mediated signaling pathways such as IGF1R and ITPR2 (Inositol 1,4,5-triphosphate receptor, type 2).

We described an approach of identifying single nucleotide polymorphisms (SNPs) in complete genomic regions of key genes including promoters, exons, introns, and downstream sequences by combining long-range polymerase chain reaction (PCR) or NimbleGen sequence capture with next-generation sequencing. Using the adenomatous polyposis coli (APC) gene as an example, we identified 210 highly reliable SNPs by next-generation sequencing analysis program MAQ and Samtools, of which 69 were novel ones, in the 123-kb APC genomic region in 27 pair of colorectal cancers and normal adjacent tissues. We confirmed all of the eight randomly selected high-quality SNPs by allele-specific PCR, suggesting that our false discovery rate is negligible. We identified 11 SNPs in the exonic region, including one novel SNP that was not previously reported. Although 10 of them are synonymous, they were predicted to affect splicing by creating or removing exonic splicing enhancers or exonic splicing silencers. We also identified seven SNPs in the upstream region of the APC gene, three of which were only identified in the cancer tissues. Six of these upstream SNPs were predicted to affect transcription factor binding. We also observed that long-range PCR was better in capturing GC-rich regions than the NimbleGen sequence capture technique.