The protein sequence and spatial structure of DNA helicase HELQ are highly conserved, spanning from archaea to humans. Aside from its helicase activity, which is based on DNA binding and translocation, it has also been recently reconfirmed that human HELQ possesses DNA–strand–annealing activity, similar to that of the archaeal HELQ homolog StoHjm. These biochemical functions play an important role in regulating various double–strand break (DSB) repair pathways, as well as multiple steps in different DSB repair processes. HELQ primarily facilitates repair in end–resection–dependent DSB repair pathways, such as homologous recombination (HR), single–strand annealing (SSA), microhomology–mediated end joining (MMEJ), as well as the sub-pathways’ synthesis–dependent strand annealing (SDSA) and break–induced replication (BIR) within HR. The biochemical functions of HELQ are significant in end resection and its downstream pathways, such as strand invasion, DNA synthesis, and gene conversion. Different biochemical activities are required to support DSB repair at various stages. This review focuses on the functional studies of the biochemical roles of HELQ during different stages of diverse DSB repair pathways.
Abstract Metastasis-initiating cells are considered to originate from stem cell-like cancer cells. In hepatocellular carcinoma, liver progenitor-like cells are reported to be derived from hepatocytes, indicating the possible acquisition of metastatic potential during hepatocyte-to-cholangiocyte transdifferentiation. Consistent with the expression pattern observed during ductal plate formation, we revealed an LPC transition with Onecut1 accumulation both during hepatocyte-to-cholangiocyte transdifferentiation and in a cell model. This event may be associated with transient acquisition of metastatic potential.
The human specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal neocortex and plays a key role in the evolutionary expansion of the neocortex. Here, we generated a homozygous ARHGAP11B knockout human induced pluripotent stem cell (hiPSC) line through CRISPR/Cas9 gene editing system. ARHGAP11B deficient cell line maintained a normal karyotype (46, XX), expressed pluripotency markers, and showed the capability to spontaneously differentiate into all three germ layers in vivo. The ARHGAP11B knockout cell line can provide a new cell model for studying the evolution of human neocortex.
Predictable DNA off-target effect is one of the major safety concerns for the application of cytosine base editors (CBEs). To eliminate Cas9-dependent DNA off-target effects, we designed a novel effective CBE system with dual guiders by combining CRISPR with transcription activator-like effector (TALE). In this system, Cas9 nickase (nCas9) and cytosine deaminase are guided to the same target site to conduct base editing by single-guide RNA (sgRNA) and TALE, respectively. However, if nCas9 is guided to a wrong site by sgRNA, it will not generate base editing due to the absence of deaminase. Similarly, when deaminase is guided to a wrong site by TALE, base editing will not occur due to the absence of single-stranded DNA. In this way, Cas9- and TALE-dependent DNA off-target effects could be completely eliminated. Furthermore, by fusing TALE with YE1, a cytidine deaminase with minimal Cas9-independent off-target effect, we established a novel CBE that could induce efficient C-to-T conversion without detectable Cas9- or TALE-dependent DNA off-target mutations. Predictable DNA off-target effect is one of the major safety concerns for the application of cytosine base editors (CBEs). To eliminate Cas9-dependent DNA off-target effects, we designed a novel effective CBE system with dual guiders by combining CRISPR with transcription activator-like effector (TALE). In this system, Cas9 nickase (nCas9) and cytosine deaminase are guided to the same target site to conduct base editing by single-guide RNA (sgRNA) and TALE, respectively. However, if nCas9 is guided to a wrong site by sgRNA, it will not generate base editing due to the absence of deaminase. Similarly, when deaminase is guided to a wrong site by TALE, base editing will not occur due to the absence of single-stranded DNA. In this way, Cas9- and TALE-dependent DNA off-target effects could be completely eliminated. Furthermore, by fusing TALE with YE1, a cytidine deaminase with minimal Cas9-independent off-target effect, we established a novel CBE that could induce efficient C-to-T conversion without detectable Cas9- or TALE-dependent DNA off-target mutations.
Following a DNA double strand break (DSB), several nucleases and helicases coordinate to generate single-stranded DNA (ssDNA) with 3' free ends, facilitating precise DNA repair by homologous recombination (HR). The same nucleases can act on stalled replication forks, promoting nascent DNA degradation and fork instability. Interestingly, some HR factors, such as CtIP and BRCA1, have opposite regulatory effects on the two processes, promoting end resection at DSB but inhibiting the degradation of nascent DNA on stalled forks. However, the reason why nuclease actions are regulated by different mechanisms in two DNA metabolism is poorly understood. We show that human HELQ acts as a DNA end resection regulator, with opposing activities on DNA end resection at DSBs and on stalled forks as seen for other regulators. Mechanistically, HELQ helicase activity is required for EXO1-mediated DSB end resection, while ssDNA-binding capacity of HELQ is required for its recruitment to stalled forks, facilitating fork protection and preventing chromosome aberrations caused by replication stress. Here, HELQ synergizes with CtIP but not BRCA1 or BRCA2 to protect stalled forks. These findings reveal an unanticipated role of HELQ in regulating DNA end resection at DSB and stalled forks, which is important for maintaining genome stability.
Cold-inducible RNA-binding protein (CIRP), RNA-binding motif protein 3 (RBM3) and serine and arginine rich splicing factor 5 (SRSF5) are RNA-binding proteins that are transcriptionally upregulated in response to moderately low temperatures and a variety of cellular stresses in mammalian cells. Induction of these cold-inducible proteins (CIPs) is dependent on transient receptor potential (TRP) V4 channel protein, but seems independent of its ion channel activity. We herein report that in addition to TRPV4, TRPV3 and TRPM8 are necessary for the induction of CIPs. We established cell lines from the lung of TRPV4-knockout (KO) mouse, and observed induction of CIPs in them by western blot analysis. A TRPV4 antagonist RN1734 suppressed the induction in wild-type mouse cells, but not in TRPV4-KO cells. A TRPV3 channel blocker S408271 and a TRPM8 channel blocker AMTB as well as siRNAs against TRPV3 and TRPM8 suppressed the CIP induction in mouse TRPV4-KO cells and human U-2 OS cells. A TRPV3 channel agonist 2-APB induced CIP expression, but camphor did not. Neither did a TRPM8 channel agonist WS-12. These results suggest that TRPV4, TRPV3 and TRPM8 proteins, but not their ion channel activities are necessary for the induction of CIPs at 32 °C. Identification of proteins that differentially interact with these TRP channels at 37 °C and 32 °C would help elucidate the underlying mechanisms of CIP induction by hypothermia.
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. HCC cells consume large amounts of glutamine to survive, but can adapt to glutamine depletion in the presence of an exogenous asparagine. L-asparaginase (ASNase) converts glutamine and asparagine to glutamate and aspartate, respectively, and has been used to treat leukemia. Here we examined the effects of ASNase treatment on HCC cells and explored the potential impact of combining ASNase with the tyrosine kinase inhibitor lenvatinib (Len) for HCC treatment. Cell viability and death of HCC cell lines treated with either Len or ASNase alone or with Len and ASNase combined were determined. We assessed mRNA and protein expression levels of glutamine synthetase (GS) and asparagine synthetase (ASNS) by real-time quantitative PCR and immunoblotting. The antitumor effect of the combination therapy relative to Len or ASNase monotherapy was also evaluated in a xenograft tumor mouse model. ASNase treatment inhibited growth of SNU387 and SNU398 HCC cells, which have low GS and high ASNS expression levels, respectively, but did not clearly inhibit growth of the other cell lines. Len plus ASNase combination therapy synergistically inhibited proliferation and induced oxidative stress leading to cell death of some HCC cells lines. However, cell death of Huh7 cells, which express ASCT2, an important glutamine transporter for cancer cells, was not affected by the combination treatment. In a xenograft model, Len combined with ASNase significantly attenuated tumor development relative to mice treated with Len or ASNase alone. ASNase-mediated targeting of two amino acids, glutamine and asparagine, which are indispensable for HCC survival, induces oxidative stress and can be a novel cancer treatment option that exerts a synergistic effect when used in combination with Len.
Abstract We explored the beneficial effects of GW7647, a peroxisome proliferator activated receptor α (PPARα) agonist, and metformin, an anti-diabetic drug on an advanced nonalcoholic steatohepatitis (NASH) model in rodents and investigated the possible mechanisms involved. Mice were fed control chow or a choline-deficient l -amino acid-defined diet containing 45% fat (HF-CDAA). The mice fed HF-CDAA diets for 16 weeks were divided into four groups: the no treatment (HF-CDAA), HF-CDAA containing 1000 mg/kg metformin, HF-CDAA containing 10 mg/kg GW7647, and HF-CDAA with both metformin and GW7647 groups. Metformin alone slightly deteriorated the aspartate and alanine aminotransferase (AST/ALT) values, whereas co-treatment with GW7647 and metformin greatly suppressed liver injury and fibrosis via activation of the AMP-activated protein kinase (AMPK) pathway. Further study revealed that co-treatment decreased the expression of inflammatory-, fibrogenesis-, and endoplasmic reticulum (ER) stress-related genes and increased the oxidized nicotinamide adenine dinucleotide (NAD)/reduced nicotinamide adenine dinucleotide (NADH) ratio, suggesting the superiority of co-treatment due to restoration of mitochondrial function. The additive benefits of a PPARα agonist and metformin in a HF-CDAA diet-induced advanced NASH model was firstly demonstrated, possibly through restoration of mitochondrial function and AMPK activation, which finally resulted in suppression of hepatic inflammation, ER stress, then, fibrosis.
VOLUME 282 (2007) PAGES 13716–13725 There was an error in Fig. 5B. Lanes 7–10 did not show the experimental data obtained. This error has now been corrected by removing these lanes. Accordingly, the legend and the text referring to Fig. 5B should be changed from “TSA or nicotinamide” to “TSA” and from “but not nicotinamide” to “but not nicotinamide (data not shown),” respectively. This correction does not affect the results or conclusions of this work. The authors sincerely apologize for the mistake that occurred during figure preparation and for any inconvenience this error may have caused readers.
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