Banp regulates DNA damage response and chromosome segregation during the cell cycle in zebrafish retina

Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated region (MAR)-binding protein and it functions as a tumor suppressor. At the molecular level, Banp regulates transcription of metabolic genes via a CGCG-containing motif called the Banp motif. However, its physiological roles in embryonic development are unknown. Here, we report that Banp is indispensable for the DNA damage response and chromosome segregation during mitosis. Zebrafish banp mutants show mitotic cell accumulation and apoptosis in developing retina. We found that DNA replication stress and tp53-dependent DNA damage responses were activated to induce apoptosis in banp mutants, suggesting that Banp is required for regulation of DNA replication and DNA damage repair. Furthermore, consistent with mitotic cell accumulation, chromosome segregation was not smoothly processed from prometaphase to anaphase in banp morphants, leading to a prolonged M-phase. Our RNA- and ATAC-sequencing identified 31 candidates for direct Banp target genes that carry the Banp motif. Interestingly, a DNA replication fork regulator, wrnip1, and two chromosome segregation regulators, cenpt and ncapg, are included in this list. Thus, Banp directly regulates transcription of wrnip1 for recovery from DNA replication stress, and cenpt and ncapg for chromosome segregation during mitosis. Our findings provide the first in vivo evidence that Banp is required for cell-cycle progression and cell survival by regulating DNA damage responses and chromosome segregation during mitosis

Banp Regulates DNA Damage Response and Chromosome Segregation to Promote Cell-cycle Progression and Cell Survival in Zebrafish Retina

Btg3 associated nuclear protein (Banp) was initially identified as a nuclear matrix associated protein and is a tumor suppressor. Recently it was reported that Banp binds to the CGCG element containing motif enriched near the transcription initiation site of CpG island promoters, namely Banp motif, promotes the transcription in a DNA methylation dependent manner, and controls metabolic genes in pluripotent stem and differentiated neuronal cells. However, cellular roles of Banp in embryonic development remains to be elucidated. Here we report a novel role of Banp in cell-cycle progression and cell survival of zebrafish retinal progenitor cells (RPCs). In zebrafish banprw337 mutants, retinal progenitor cells showed mitotic cell accumulation and subsequent apoptosis. DNA replication stress and tp53-dependent DNA damage response were activated in banprw337 mutants. Inhibition of Tp53 significantly rescued apoptosis but not mitotic defect and DNA double strand break accumulation, suggesting that Banp is required for maintaining integrity of DNA during segregation and replication. Furthermore, live imaging of mitosis in banp morphant retinas revealed that chromosome segregation was not smoothly processed from prometaphase to anaphase, leading to prolonged M-phase. Bulk RNA-seq analysis show that mRNA expression of two chromosomal segregation regulators, cenpt and ncapg, were decreased in banprw337 mutants. Furthermore, ATAC-seq analysis showed that chromatin near their transcription start site was closed in banprw337 mutants and indeed Banp motif was found in this chromatin-closed region, suggesting that Banp directly regulates cenpt and ncapg transcription via Banp motif to promote chromosome segregation during mitosis. Our findings reveal that Banp is required for cell-cycle progression and cell survival by regulating replicative DNA damage response and mitotic chromosome segregation.Okinawa Institute of Science and Technology Graduate Universit

Haloperidol induces pharmacoepigenetic response by modulating miRNA expression, global DNA methylation and expression profiles of methylation maintenance genes and genes involved in neurotransmission in neuronal cells

<div><p>Introduction</p><p>Haloperidol has been extensively used in various psychiatric conditions. It has also been reported to induce severe side effects. We aimed to evaluate whether haloperidol can influence host methylome, and if so what are the possible mechanisms for it in neuronal cells. Impact on host methylome and miRNAs can have wide spread alterations in gene expression, which might possibly help in understanding how haloperidol may impact treatment response or induce side effects.</p><p>Methods</p><p>SK-N-SH, a neuroblasoma cell line was treated with haloperidol at 10μm concentration for 24 hours and global DNA methylation was evaluated. Methylation at global level is maintained by methylation maintenance machinery and certain miRNAs. Therefore, the expression of methylation maintenance genes and their putative miRNA expression profiles were assessed. These global methylation alterations could result in gene expression changes. Therefore genes expressions for neurotransmitter receptors, regulators, ion channels and transporters were determined. Subsequently, we were also keen to identify a strong candidate miRNA based on biological and in-silico approach which can reflect on the pharmacoepigenetic trait of haloperidol and can also target the altered neuroscience panel of genes used in the study.</p><p>Results</p><p>Haloperidol induced increase in global DNA methylation which was found to be associated with corresponding increase in expression of various epigenetic modifiers that include <i>DNMT1</i>, <i>DNMT3A</i>, <i>DNMT3B</i> and <i>MBD2</i>. The expression of miR-29b that is known to putatively regulate the global methylation by modulating the expression of epigenetic modifiers was observed to be down regulated by haloperidol. In addition to miR-29b, miR-22 was also found to be downregulated by haloperidol treatment. Both these miRNA are known to putatively target several genes associated with various epigenetic modifiers, pharmacogenes and neurotransmission. Interestingly some of these putative target genes involved in neurotransmission were observed to be upregulated while CHRM2 gene expression was down regulated.</p><p>Conclusions</p><p>Haloperidol can influence methylation traits thereby inducing a pharmacoepigenomic response, which seems to be regulated by DNMTs and their putative miRNA expression. Increased methylation seems to influence CHRM2 gene expression while microRNA could influence neurotransmission, pharmacogene expression and methylation events. Altered expression of various therapeutically relevant genes and miRNA expression, could account for their role in therapeutic response or side effects.</p></div

Effect of HLP on global DNA methylation in SK-N-SH cells.

<p>Effect of HLP on global DNA methylation in SK-N-SH cells.</p

Effect of HLP on miR-22 expression in SK-N-SH cells.

<p>Effect of HLP on miR-22 expression in SK-N-SH cells.</p

Effect of HLP on gene expression of epigenetic genes in SK-N-SH cells.

<p>Effect of HLP on gene expression of epigenetic genes in SK-N-SH cells.</p

Effect of HLP on expression of neurotransmitter receptors, regulators, neuroscience related ion channels and transporters.

<p>Effect of HLP on expression of neurotransmitter receptors, regulators, neuroscience related ion channels and transporters.</p

Effect of HLP on miR-29b expression in SK-N-SH cells.

<p>Effect of HLP on miR-29b expression in SK-N-SH cells.</p

Interaction of arsenic with gap junction protein connexin 43 alters gap junctional intercellular communication

Chronic exposure to Arsenic pollution in ground water is one of the largest environmental health disasters in the world. The toxicity of trivalent Arsenicals primarily happens due to its interaction with sulfhydryl groups in proteins. Arsenic binding to the protein can change the conformation of the protein and alter its interactions with other proteins leading to tissue damage. Therefore, much importance has been given to the studies of Arsenic bound proteins, for the purpose of understanding the origins of toxicity and to explore therapeutics. Here we study the dynamic effect of Arsenic on Connexin 43 (Cx43), a protein that forms the gap junctions, whose alteration deeply perturbs the cell-to-cell communication vital for maintaining tissue homeostasis. In silico molecular modelling and in vitro studies comparing Arsenic treated and untreated conditions show distinct results. Gap junction communication is severely disrupted by Arsenic due to reduced availability of unaltered Cx43 in the membrane bound form. In silico and Inductively Coupled Plasma Mass Spectrometry studies revealed the interaction of Arsenic to the Cx43 preferably occurs through surface exposed cysteines, thereby capping the thiol groups that form disulfide bonds in the tertiary structure. This leads to disruption of Cx43 oligomerization, and altered Cx43 is incompetent for transportation to the membrane surface, often forming aggregates primarily localizing in the endoplasmic reticulum. Loss of functional Cx43 on the cell surface have a deleterious effect on cellular homeostasis leading to selective vulnerability to cell death and tissue damage