DNA compaction project - Coggle Diagram
DNA compaction project
Validate DNA compaction phenotype in Mycobacteria
PBS + tylaxopol starvation, PBS + T80 starvation, cell-cycle time-course, NO, H2O2, SDS, macrophage infection
In particular NO, H2O2, or iron deficiency, copper excess, acid
Imaging + quantitative analysis using JH's pipeline
Try different fixation protocols when dealing w/ Mtb to check not a fixation artifact?
Test known gene candidates
Lsr2, HupB, WhiB4, EspR
Lsr2 is not essential in Mtb, b/ KO has severe O2 sensitivity that may be a downstream effect of genes suppressed by Lsr2, similar to WhiB4 KO, which grow slower in vitro likely due to over-production of anti-oxidant systems...
Try DNA-marked strains: mark w/ dCRISPR, lacO/cI, etc... do the proteins get displaced?
Figure out ATAC-seq protocol
Make our own protein
Identify regions that selectively open/close under specific conditions
Find other proteins that may control global DNA regulation in Mycobacteria
Minch 2015, global ChIP data: identified many DNA-binding proteins that seem to bind everywhere, including those that are known independently to do so (Lsr2, EspR, WhiB4)
Expression data: Betts 2003 (starvation), Schnappinger 2003 (BMDM infection, +/- IFNg, +/- NOS2), Pisu 2020 (BMDM, AM, IM), Viskuli 2011 (H2O2, NO), Rachman 2006 (resected human lungs), Rustad 2008 (DosR, EHR); Rubin et al, FLUTE data 2016;
Need to add more
Single mutant screen to look for 1) global defect in DNA compaction or 2) defect in compacting specific loci identified from ATAC-seq
DNA condensation is toxic at long run, but may not find what we want (see EIP-K paper, PMID 22232273)
The Fundamental Questions of DNA
Need to fit inside of the cell
Need access for gene expression/repair/replication
Need to protect DNA from hostile conditions
Inventory of proteins considered NAPs is very different in Mycobacteria compared to Gram Negatives, where most of the work has been done: suggest taken a different evolutionary path to deal with the same questions
NAPs vs. TR?
Many DNA binding proteins in Mycobacteria genome that has many binding sites and unknown function
How are the known proteins regulated?
Is there a phosphatase(s) for Lsr2?
How to differentiate direct vs. indirect effects on DNA/transcription?
Inducible KD by degradation? Some of these are super abundant proteins...
Distinguish structural versus global gene regulation functions? Is there a distinction?
Crystal structure, disrupt dimerization functions
Can we do this w/ Lsr2?