Genome-Wide Mapping of Histone Modifications and Transcription Factor Binding Sites in Neuroendocrine Small Cell Lung Cancer Cell Lines Using CUT&RUN

So the goal of our research is to identify the interactions between lineage defining transcription factors and chromatin remodeling proteins in the different types of small cell lung cancer, in regulating the transcription landscapes, and how these interactions can contribute to disease processes, including cancer initiation and progression. We aim to achieve this by conducting experiments like CUT&RUN, which is a next generation sequencing technique that can profile or identify at a global scale the genomic locations of where these differentially modified histones and DNA-bound transcription factors. Ultimately, we hope to identify new pathways that are epigenetically regulated that may be therapeutically actionable in the development of new treatments for patients with this very aggressive form of lung cancer.

To begin, remove the cultured small cell lung cancer neuroendocrine cells from the 37 degrees Celsius incubator and examine them under a microscope to ensure their quality. Then transfer the cell suspension to a 50 milliliter conical tube and centrifuge the tube at 1, 200 g for five minutes at room temperature. Aspirate the medium from the conical tube by using the vacuum in the hood and rinse the resulting cell pellet with 20 milliliters of PBS.

Centrifuge the tube again at 1, 200 g for five minutes, and aspirate the PBS. Add Accutase based on pellet size and incubate for three to five minutes at 37 degrees Celsius to digest the cell pellet. Next, terminate the digestion by adding five milliliters of PBS with 10%FBS and mixing to obtain a single cell suspension.

After centrifuging the cells again as demonstrated earlier, resuspend the pellet in five milliliters of PBS. Count the starting cells and confirm their viability and integrity using the automatic cell counter. Calculate the total number of cells required and add 20%excess to account for pipetting errors.

Transfer the calculated total number of cells to 1.5 milliliter tubes and centrifuge the tubes at 600 g for three minutes at room temperature. resuspend the cells in wash buffer at a density of five times 10 to the power of five cells per reaction for a total of six reactions. Use 100 microliters of buffer per reaction and pipette gently but thoroughly to ensure a uniform suspension.

Centrifuge the cells again, remove the supernatant, and repeat the wash. Aliquot 100 microliters of the washed cells into each tube of an eight strip set that contains 10 microliters of activated Concanavalin A beads. Gently pipette to mix the contents.

Incubate the bead cell slurry for 10 minutes at room temperature to allow cell binding to the beads, and quickly spin the tubes in a mini centrifuge to collect the slurry without allowing the beads to settle. Place the tubes on a 0.2 milliliter tube magnet rack. Allow the slurry to clear and pipette to remove the supernatant.

Then remove the tubes from the magnet. Immediately add 50 microliters of ice cold antibody buffer to each reaction and gently pipette to resuspend the beads. Label the eight strip tubes appropriately.

Add 0.5 micrograms each of H3K4me3, H3K4me1, and immunoglobulin G antibodies and 0.6 micrograms of E2F7 polyclonal antibody from different sources to each reaction tube. Gently pipette to mix the contents of each reaction tube thoroughly and incubate them overnight on a nutator at four degrees Celsius with tube caps elevated. Remove the eight strip tubes from the nutator and perform a quick spin to collect liquid at the bottom of each tube.

Now place the tubes on the magnet stand until the slurry clears. Then carefully pipette to remove and discard the supernatant. Keeping the tubes on the magnet stand, wash each tube twice with 200 microliters of cold cell permeabilization buffer and use a multiple channel pipette to remove the supernatant after each wash.

Next, remove the tubes from the magnet stand. Immediately add 50 microliters of cold cell permeable buffer to each reaction and gently pipette to resuspend the cells and beads. Then add 2.5 microliters of protein AG-micrococcal nuclease to each reaction and mix well by gently pipetting.

After a 10 minute incubation at room temperature, perform a quick spin of the tubes. Place them back on the magnet stand until the slurry clears, and then remove the supernatant. After washing the beads twice with cold cell permeabilization buffer, gently resuspend them in 50 microliters of the cell permeable buffer.

While keeping the tubes on ice, add one microliter of 100 millimolar calcium chloride to each reaction in the eight tube strip from the previous step. Gently pipette to fully resuspend the beads and promote efficient digestion. Incubate the tubes on a nutator at four degrees Celsius for two hours, keeping the tube caps elevated throughout the incubation.

Now in a new 1.5 milliliter tube, prepare the stop buffer mix by combining 33 microliters of stop buffer with one microliter of escherichia coli spike in DNA at 0.5 nanograms per reaction. Gently vortex to mix the solution. At the end of the two hour incubation, add 33 microliters of the prepared stop buffer mixture to each reaction tube and mix thoroughly.

Incubate the reaction tubes in a thermocycler at 37 degrees Celsius for 10 minutes. After a quick spin, place the tubes on a magnet rack until the solution becomes clear. Carefully transfer approximately 85 microliters of the supernatant containing the CUT&RUN release DNA into fresh 1.5 milliliter tubes.

Purify the recovered DNA, assess its quantity, and perform fragment size distribution for quality control. Then proceed with CUT&RUN library preparation, quantification, indexed library pooling, and paired-end next generation sequencing. Achaete-scute homolog-1 high cell lines, DMS79 and H146, and Neurogenic Differentiation Factor 1 high cell lines, H82 grew as non-adherent aggregates or compact spheroids in suspension, while H446 exhibited mixed morphology, with approximately 80%inherent cells and 20%in suspension.

CUT&RUN DNA fragment analysis revealed that the enhancer histone mark H3K4me1 yielded the highest DNA amounts across all cell lines, followed by H3K4me3, with E2F7 and IgG yielding the lowest amounts. For extracted CUT&RUN DNA, the fragment size distribution for H3K4me1 and H3K4me3 showed clear peaks for mono-nucleosome and di-nucleosome along with genomic DNA. In contrast, no distinct peaks were detected for E2F7 or the IgG control.

Despite low DNA input, next generation sequencing libraries were successfully constructed for all CUT&RUN targets, including E2F7 and IgG. Heat map analysis in DMS79 wild type and H146 heterozygous mutant cells showed that H3K4me3 and E2F7 signals were enriched near transcription start sites, whereas H3K4me1 signals were more broadly distributed. Genome browser visualization at a randomly selected chromatin region showed distinct peaks for H3K4me3, H3K4me1, and E2F7, while IgG showed no enrichment.

And so traditionally, scientists and researchers have used chromatin immunoprecipitation followed by sequencing or ChIP-seq to address questions like the ones that we are asking. However, ChIP-seq requires a rather high amount of input materials, which is not always feasible, depending on which model is being used. CUT&RUN, on the other hand, requires significantly less input material, as low as 50, 000 cells, and requires less high throughput sequencing reads, which means it's less costly, and produces far less background when it comes to data generation.

CUT&RUN is a technique that can be applied to not only cells in 2D culture, but it can be adapted for other preclinical models, such as patient derived organoids and xenografts. It can also be used to profile the chromatin landscape in tissues, including both normal and diseased tissue. So this is an incredibly useful technology or technique for cancer researchers, but can be very useful as well for non-cancer researchers that are focused on studying different human diseases.

This protocol allows a mapping of histone modifications and the abiding profiles of the diverse proteins in situ in the cellular genome. Providing histone PTMs and the TF binding across SCLC subtypes may reveal subtype specific targetable genes and regulatory elements, such as enhancers and the super-enhancers.