Best wishes and next steps

[Alberto Diaspro]

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Friends
best wishes for a relaxing Xmas time… before ending the year lets start with the new year perspectives… I am interested in starting from optical studies of chromatin-DNA, any comment is welcome.
My starting point will be this one

https://www.researchgate.net/publication/20714688_Circular_intensity_differential_scattering_and_chromatin-DNA_structure_-_A_combined_theoretical_approach

Best
Alby

[George McNamara]

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Hi Alby,

I recommend exploring the use of your microscopes and nanoscopes and single molecule scopes with ATAC-see and NucleoATAC to see live human cell DNA. Instead of dense Tn5 integration sites, you could "go sparse" with Tn5. The Chen paper implies Tn5 can be used live in mammalian cells, though I did not see that in the ATAC-see paper. There are transposases that work in mammalian cells, including various versions of Sleeping Beauty and PiggyBac. There are also endogenous integrases/recombinases/transposases, including PGBD5, THAP9 and most famously Rag1/Rag2.

Chen X, Shen Y, Draper W, Buenrostro JD, Litzenburger U, Cho SW, Satpathy AT,  Carter AC, Ghosh RP, East-Seletsky A, Doudna JA, Greenleaf WJ, Liphardt JT, Chang
HY. 
ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing. 
Nat Methods. 2016 Dec;13(12):1013-1020. 
doi: 10.1038/nmeth.4031.
PubMed PMID: 27749837.

Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.

PMID:

 

27749837

 

DOI:

 

10.1038/nmeth.4031

Schep AN, Buenrostro JD, Denny SK, Schwartz K, Sherlock G, Greenleaf WJ.
Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions. 
Genome Res. 2015 Nov;25(11):1757-70. 
doi: 10.1101/gr.192294.115. 
PubMed PMID: 26314830; PubMed Central PMCID: PMC4617971.

Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal "fingerprint" as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding.

© 2015 Schep et al.; Published by Cold Spring Harbor Laboratory Press.

PMID:

 

26314830

 

PMCID:

 

PMC4617971

 

DOI:

 

10.1101/gr.192294.115

You may also want to consider FabLEM and its GFP equivalents (using state of the art FPs, not 20 year old EGFP ... I hope you are not still driving a 20 year old VW love-bug):

Stasevich TJ, Hayashi-Takanaka Y, Sato Y, Maehara K, Ohkawa Y, Sakata-Sogawa K, Tokunaga M, Nagase T, Nozaki N, McNally JG, Kimura H. 
Regulation of RNA polymerase II activation by histone acetylation in single living cells. 
Nature. 2014 Dec 11;516(7530):272-5. 
doi: 10.1038/nature13714. 
PubMed PMID: 25252976.

In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

PMID:

 

25252976

 

DOI:

 

10.1038/nature13714

Hayashi-Takanaka Y, Yamagata K, Wakayama T, Stasevich TJ, Kainuma T, Tsurimoto T, Tachibana M, Shinkai Y, Kurumizaka H, Nozaki N, Kimura H. 
Tracking epigenetic  histone modifications in single cells using Fab-based live endogenous modification labeling. 
Nucleic Acids Res. 2011 Aug;39(15):6475-88. 
doi: 10.1093/nar/gkr343. 
PubMed PMID: 21576221; PubMed Central PMCID: PMC3159477.

Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

PMID:

 

21576221

 

PMCID:

 

PMC3159477

 

DOI:

 

10.1093/nar/gkr343

Enjoy,
George
p.s. You wrote chromatin, but comparison with mitochondrial nucleoid DNA may be useful (alternatively, you could use "rho" cells to avoid ATAC-see seeing mitochondrial DNA). For a way to manipulate human cell mitochondrial DNA, see, for examples, from former colleagues at U Miami:

Selective elimination of mitochondrial mutations in the germline by genome editing.
Reddy P, Ocampo A, Suzuki K, Luo J, Bacman SR, Williams SL, Sugawara A, Okamura D, Tsunekawa Y, Wu J, Lam D, Xiong X, Montserrat N, Esteban CR, Liu GH, Sancho-Martinez I, Manau D, Civico S, Cardellach F, Del Mar O'Callaghan M, Campistol J, Zhao H, Campistol JM, Moraes CT, Izpisua Belmonte JC.
Cell. 2015 Apr 23;161(3):459-69. doi: 10.1016/j.cell.2015.03.051.
PMID: 25910206

The use of mitochondria-targeted endonucleases to manipulate mtDNA.
Bacman SR, Williams SL, Pinto M, Moraes CT.
Methods Enzymol. 2014;547:373-97. doi: 10.1016/B978-0-12-801415-8.00018-7.
PMID: 25416366


Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs.
Bacman SR, Williams SL, Pinto M, Peralta S, Moraes CT.
Nat Med. 2013 Sep;19(9):1111-3. doi: 10.1038/nm.3261.
PMID: 23913125

On 12/26/2016 4:27 AM, Alberto Diaspro wrote:

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Friends
best wishes for a relaxing Xmas time… before ending the year lets start with the new year perspectives… I am interested in starting from optical studies of chromatin-DNA, any comment is welcome.
My starting point will be this one

https://www.researchgate.net/publication/20714688_Circular_intensity_differential_scattering_and_chromatin-DNA_structure_-_A_combined_theoretical_approach

Best
Alby