Background: Radioresistance is the main reason for the failure of radiotherapy in non-small-cell lung cancer (NSCLC); however, the molecular mechanism of radioresistance is still unclear.
Methods: An RNA-Seq assay was used to screen differentially expressed long non-coding RNAs (lncRNAs) and genes in irradiation-resistant NSCLC cells. RT-PCR and Western blotting assays were performed to analyze the expressions of lncRNAs and genes. The chromosome conformation capture (3C) assay was performed to measure chromatin interactions. Cell cytotoxicity, cell apoptosis, sphere formation and Transwell assays were performed to assess cellular function.
Results: In this study, it was found that LINC01224 increased during the induction of radioresistance in NSCLC cells. LINC01224 was located within the enhancer of ZNF91, and LINC01224 could affect the transcription of ZNF91 by regulating the long-range interactions between the ZNF91 enhancer and promoter. Moreover, upregulation of LINC01224 and ZNF91 could promote irradiation resistance by regulating the stem cell-like properties of NSCLC cells.
In addition, high expression levels of LINC01224 and ZNF91 in tissue samples were associated with radioresistance in NSCLC patients.
Conclusion: Our findings demonstrated that LINC01224/ZNF91 drove radioresistance regulation by promoting the stem cell-like properties in NSCLC.
Keywords: LINC01224; ZNF91; non-small cell lung cancer; radioresistance; stem cell-like properties.
ZNF91 deletion in human embryonic stem cells leads to ectopic activation of SVA retrotransposons and up-regulation of KRAB zinc finger gene clusters
Transposable element (TE) invasions have shaped vertebrate genomes over the course of evolution. They have contributed an extra layer of species-specific gene regulation by providing novel transcription factor binding sites. In humans, SINE-VNTR-Alu (SVA) elements are one of three still active TE families; approximately 2800 SVA insertions exist in the human genome, half of which are human-specific.
TEs are often silenced by KRAB zinc finger (KZNF) proteins recruiting corepressor proteins that establish a repressive chromatin state.
A number of KZNFs have been reported to bind SVAs, but their individual contribution to repressing SVAs and their roles in suppressing SVA-mediated gene-regulatory effects remains elusive.
We analyzed the genome-wide binding profile for ZNF91 in human cells and found that ZNF91 interacts with the VNTR region of SVAs. Through CRISPR-Cas9-mediated deletion of ZNF91 in human embryonic stem cells, we established that loss of ZNF91 results in increased transcriptional activity of SVAs.
In contrast, SVA activation was not observed upon genetic deletion of the ZNF611 gene encoding another strong SVA interactor.
Epigenetic profiling confirmed the loss of SVA repression in the absence of ZNF91 and revealed that mainly evolutionary young SVAs gain gene activation-associated epigenetic modifications.
Genes close to activated SVAs showed a mild up-regulation, indicating SVAs adopt properties of cis-regulatory elements in the absence of repression.
Notably, genome-wide derepression of SVAs elicited the communal up-regulation of KZNFs that reside in KZNF clusters. This phenomenon may provide new insights into the potential mechanisms used by the host genome to sense and counteract TE invasions.
An evolutionary arms race between KRAB zinc-finger genes ZNF91/93 and SVA/L1 retrotransposons.
Throughout evolution primate genomes have been modified by waves of retrotransposon insertions. For each wave, the host eventually finds a way to repress retrotransposon transcription and prevent further insertions. In mouse embryonic stem cells, transcriptional silencing of retrotransposons requires KAP1 (also known as TRIM28) and its repressive complex, which can be recruited to target sites by KRAB zinc-finger (KZNF) proteins such as murine-specific ZFP809 which binds to integrated murine leukaemia virus DNA elements and recruits KAP1 to repress them.
KZNF genes are one of the fastest growing gene families in primates and this expansion is hypothesized to enable primates to respond to newly emerged retrotransposons.
However, the identity of KZNF genes battling retrotransposons currently active in the human genome, such as SINE-VNTR-Alu (SVA) and long interspersed nuclear element 1 (L1), is unknown. Here we show that two primate-specific KZNF genes rapidly evolved to repress these two distinct retrotransposon families shortly after they began to spread in our ancestral genome.
ZNF91 underwent a series of structural changes 8-12 million years ago that enabled it to repress SVA elements. ZNF93 evolved earlier to repress the primate L1 lineage until ∼12.5 million years ago when the L1PA3-subfamily of retrotransposons escaped ZNF93’s restriction through the removal of the ZNF93-binding site.
Our data support a model where KZNF gene expansion limits the activity of newly emerged retrotransposon classes, and this is followed by mutations in these retrotransposons to evade repression, a cycle of events that could explain the rapid expansion of lineage-specific KZNF genes.
Evolutionary expansion and divergence in the ZNF91 subfamily of primate-specific zinc finger genes.
Most genes are conserved in mammals, but certain gene families have acquired large numbers of lineage-specific loci through repeated rounds of gene duplication, divergence, and loss that have continued in each mammalian group. One such family encodes KRAB-zinc finger (KRAB-ZNF) proteins, which function as transcriptional repressors.
One particular subfamily of KRAB-ZNF genes, including ZNF91, has expanded specifically in primates to comprise more than 110 loci in the human genome.
Genes of the ZNF91 subfamily reside in large gene clusters near centromeric regions of human chromosomes 19 and 7 with smaller clusters or isolated copies in other locations.
Phylogenetic analysis indicates that many of these genes arose before the split between the New and Old World monkeys, but the ZNF91 subfamily has continued to expand and diversify throughout the evolution of apes and humans. Paralogous loci are distinguished by divergence within their zinc finger arrays, indicating selection for proteins with different regulatory targets.
In addition, many loci produce multiple alternatively spliced transcripts encoding proteins that may serve separate and perhaps even opposing regulatory roles because of the modular motif structure of KRAB-ZNF genes.
The tissue-specific expression patterns and rapid structural divergence of ZNF91 subfamily genes suggest a role in determining gene expression differences between species and the evolution of novel primate traits.
Characterization of the human Fc gamma RIIB gene promoter: human zinc-finger proteins (ZNF140 and ZNF91) that bind to different regions function as transcription repressors.
Expression of the human low-affinity Fc receptors for IgG (human Fc gamma RII) is differentially regulated. We report here the characterization of the promoter structure of the human Fc gamma RIIB gene and the isolation of the promoter region-binding proteins by a yeast one-hybrid assay.
The minimal 154-bp region upstream from the transcription start site of the human Fc gamma RIIB gene was shown to possess promoter activity in a variety of cells.
An electrophoretic mobility shift assay indicated that multiple nuclear factors in cell extracts bind to the two regions [F2-3 (-110 to -93) and F4-3 (-47 to -31)] of the human Fc gamma RIIB gene promoter.
ZNF91 siRNA |
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| 20-abx941160 | Abbexa |
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ZNF91, CT (Zinc finger protein 91,ZNF91,) |
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| MBS6006091-02mL | MyBiosource | 0.2(mL | 695 EUR |
ZNF91, CT (Zinc finger protein 91,ZNF91,) |
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| MBS6006091-5x02mL | MyBiosource | 5x0.2mL | 2975 EUR |
ZNF91 Antibody |
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| 1-CSB-PA02005A0Rb | Cusabio |
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ZNF91 antibody |
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| 70R-8707 | Fitzgerald | 50 ug | 467 EUR |
ZNF91 antibody |
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| 70R-8708 | Fitzgerald | 50 ug | 467 EUR |
ZNF91 Antibody |
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| GWB-MQ831C | GenWay Biotech | 50ug | Ask for price |
ZNF91 Antibody |
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| GWB-MQ832D | GenWay Biotech | 50ug | Ask for price |
ZNF91 antibody |
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| MBS5301340-01mL | MyBiosource | 0.1mL | 750 EUR |
ZNF91 antibody |
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| MBS5301340-5x01mL | MyBiosource | 5x0.1mL | 3215 EUR |
ZNF91 antibody |
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| MBS5301891-01mL | MyBiosource | 0.1mL | 750 EUR |
ZNF91 antibody |
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| MBS5301891-5x01mL | MyBiosource | 5x0.1mL | 3215 EUR |
ZNF91, CT (Zinc finger protein 91,ZNF91,) (AP) |
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| MBS6367988-02mL | MyBiosource | 0.2mL | 980 EUR |
ZNF91, CT (Zinc finger protein 91,ZNF91,) (AP) |
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| MBS6367988-5x02mL | MyBiosource | 5x0.2mL | 4250 EUR |
ZNF91, CT (Zinc finger protein 91,ZNF91,) (PE) |
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| MBS6367998-02mL | MyBiosource | 0.2mL | 980 EUR |
ZNF91, CT (Zinc finger protein 91,ZNF91,) (PE) |
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| MBS6367998-5x02mL | MyBiosource | 5x0.2mL | 4250 EUR |
ZNF91, CT (Zinc finger protein 91,ZNF91,) (APC) |
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| MBS6367989-02mL | MyBiosource | 0.2mL | 980 EUR |
ZNF91, CT (Zinc finger protein 91,ZNF91,) (APC) |
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| MBS6367989-5x02mL | MyBiosource | 5x0.2mL | 4250 EUR |
ZNF91, CT (Zinc finger protein 91,ZNF91,) (FITC) |
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| MBS6367991-02mL | MyBiosource | 0.2mL | 980 EUR |
Mutation analysis indicated that GGGAGGAGC (-105 to -97) and AATTTGTTTGCC (-47 to -36) sequences are responsible for binding to nuclear factors respectively. By using GGGAGGAGC and AATTTGTTTGCC as bait sequences, we cloned two zinc-finger proteins (ZNF140 and ZNF91) that bind to the F2-3 and F4-3 regions within the promoter of the human Fc gamma RIIB gene respectively.
When the ZNF140 and ZNF91 were transfected with reporter plasmid, both showed repressor activity with additive effects.
Thus, these results indicate that these cloned ZNF140 and ZNF91 proteins function as repressors for the human Fc gamma RIIB transcription.