Thread by @HironoriFunabi1, Delighted to share our review article, "Regulation and Consequences of cGAS Activation [...]

Delighted to share our review article, "Regulation and Consequences of cGAS Activation by Self-DNA", by Christian Zierhut @ChriZieri.
Free article available until Sep 4.
https://authors.elsevier.com/c/1bPjB3QxxSgq3f

Here">https://authors.elsevier.com/c/1bPjB3Q... is a thread on how mitosis researchers bumped into the innate immunity.
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Mitosis is a process to distribute chromosomes to dividing cells. As the nucleosome is the basic structural unit of chromosomes, we wanted to understand function and regulation of nucleosomes in mitosis. Addressing this question in vivo was difficult, however.
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As nucleosomes regulate transcription, even if any mitotic processes are affected upon manipulating nucleosomes, this could be due to a change in mRNA levels of mitotic regulators. Xenopus egg extract is a unique system that can circumvent this problem. 3/

In Xenopus egg extracts, cell cycle regulation, DNA replication, mitotic chromosome condensation and spindle formation can be recapitulated in the absence of de novo transcription, as eggs have a stockpile of RNAs and proteins for early embryonic cell divisions. 4/

If we could specifically deplete histones from egg extracts and complement with recombinant histones, we should be able to assess transcription-independent roles of histones. Due to their high abundance, however, it has been technically difficult to deplete histones
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Luckily, Hiroshi Kimura @Cell_Tokyo_Tech gave us a valuable tool. Collaborating with Naohito Nozaki, Hiroshi generated many mAbs for histone modifications. Hiroshi kindly share these mAbs with us to see if any of them can effectively deplete histones from Xenopus egg extracts. 6/

This approach worked! Christian showed that mAb against histone H4K12ac depleted >95% of the H3-H4 complex from Xenopus egg extracts. Christian made a heroic effort to optimize this method, which is now reproduced in other labs, like @brugueslab! 7/

Using this method, Christian demonstrated that nucleosomes are essential for nuclear pore complex (NPC) formation and spindle assembly, while DNA was sufficient to recruit nuclear membrane.
https://pubmed.ncbi.nlm.nih.gov/24952593/
8/">https://pubmed.ncbi.nlm.nih.gov/24952593/...

Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion...

Nucleosomes are the fundamental unit of chromatin, but analysis of transcription-independent nucleosome functions has been complicated by the gene-expression changes resulting from histone manipula...

https://pubmed.ncbi.nlm.nih.gov/24952593/

Christian was even able to bypass the requirement of nucleosomes for NPC formation by targeting two critical histone binding proteins (RCC1 and ELYS) to DNA. This suggested that somehow nucleosome-dependent system, rather than DNA-dependent system, was selected by evolution.
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What was the benefit of nucleosome-dependent NPC formation?

In Discussion, we speculated that the nucleosome acts as a hallmark of self-DNA, distinguishing from foreign and aberrant DNAs.
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How can we test this hypothesis?
A novel cytoplasmic DNA sensor cGAS, discovered by Zhijian Chen in 2013, caught our eyes.
https://pubmed.ncbi.nlm.nih.gov/23258413/
11/">https://pubmed.ncbi.nlm.nih.gov/23258413/...

Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway -...

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through...

https://pubmed.ncbi.nlm.nih.gov/23258413/

Upon binding to foreign DNA (e.g., viruses and bacteria) in the cytoplasm, cGAS becomes activated and stimulates inflammation. Genomic DNAs and mitochondrial DNAs are protected from cytoplasmic cGAS by the nuclear envelope and the mitochondrial envelope, respectively.
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When the nuclear envelope breaks down, cytoplasmic cGAS can access to chromosomes. cGAMP, the cyclic nucleotide that is generated by cGAS, is stable in the cytoplasm, so even transient activation of cGAS can be a problem.
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So, what is the mechanism by which cGAS is not normally activated by chromosomal DNA?
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Although we initially predicted that cGAS does not efficiently bind to nucleosomes, Christian showed that cGAS binds to nucleosomes with higher affinity than to naked DNA. However, nucleosomes inhibit DNA-dependent cGAS activation by a competitive mechanism.
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This mechanism explains why cGAS cannot readily be activated by chromosomes, which also have nucleosome-free DNA regions; we assumed that abundant nucleosomes can interfere with binding of cGAS to nucleosome-free DNA.
https://pubmed.ncbi.nlm.nih.gov/31299200/
16/">https://pubmed.ncbi.nlm.nih.gov/31299200/...

The Cytoplasmic DNA Sensor cGAS Promotes Mitotic Cell Death - PubMed

Pathogenic and other cytoplasmic DNAs activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to induce inflammation via transcriptional activation by IRF3 and...

https://pubmed.ncbi.nlm.nih.gov/31299200/

Andrew Jackson, Nicolas Manel @NicolasManelLab and their colleagues also independently reported that nucleosomes are not good activator of cGAS.
https://pubmed.ncbi.nlm.nih.gov/28738408/
https://pubmed.ncbi.nlm.nih.gov/28738408/... href=" https://pubmed.ncbi.nlm.nih.gov/30270045/
17/">https://pubmed.ncbi.nlm.nih.gov/30270045/...

NONO Detects the Nuclear HIV Capsid to Promote cGAS-Mediated Innate Immune Activation - PubMed

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and...

https://pubmed.ncbi.nlm.nih.gov/28738408/

Nucleosome-based protection is not perfect, though. Christian’s review classifies the processes that make cGAS sense "self-DNA", and how distinct consequences (inflammation, senescences, apoptosis, etc) are indued. Many unsolved questions are highlighted as well.
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Christian will carry on this project in his own lab @ICR_London, opening in this fall.
Christian is a fantastic person with extensive experience on training a number of PhD students and technicians. He will be a great PI in London!
https://www.icr.ac.uk/our-research/researchers-and-teams/dr-christian-zierhut
19/">https://www.icr.ac.uk/our-resea...

Dr Christian Zierhut - The Institute of Cancer Research, London

Dr Christian Zierhut leads the Genome Stability and Innate Immunity Team. His research focuses on how chromosomal aberrations that frequently occur in cancer promote intracellular immune responses,...

https://www.icr.ac.uk/our-research/researchers-and-teams/dr-christian-zierhut

Christian& #39;s departure will generate a new postdoc position in the Funabiki lab. Please check out our homepage http://funabikilab.com"> http://funabikilab.com for details. Ad at jobRxiv is here.
https://twitter.com/jobRxiv/status/1283416318281121793?s=20
Thanks">https://twitter.com/jobRxiv/s... for reading this!
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