Distinct structural and functional heterochromatin partitioning of lamin B1 and lamin B2 revealed using genome-wide Nicking Enzyme Epitope targeted DNA sequencing.
Gene expression is regulated by chromatin DNA methylation and other features, including histone post-translational modifications (PTMs), chromatin remodelers, and transcription factor occupancy. A complete understanding of gene regulation will require the mapping of these chromatin features in small cell number samples. Here we describe a novel genome-wide chromatin profiling technology, named as Nicking Enzyme Epitope targeted DNA sequencing (NEED-seq). NEED-seq offers antibody-targeted controlled nicking by Nt.CviPII-pGL fusion to study specific protein-DNA complexes in formaldehyde fixed cells, allowing for both visual and genomic resolution of epitope bound chromatin. When applied to nuclei, NEED-seq yielded genome-wide profile of chromatin associated proteins and histone PTMs. Additionally, NEED-seq of lamin B1 and B2 demonstrated their association with heterochromatin. Lamin B1 and B2 associated domains (LAD) segregated to three different states, and states with stronger LAD correlated with heterochromatic marks. Hi-C analysis displayed A and B compartment with equal lamin B1 and B2 distribution, although methylated DNA remained high in B compartment. LAD clustering with Hi-C resulted in subcompartments, with lamin B1 and B2 partitioning to facultative and constitutive heterochromatin respectively and were associated with neuronal development. Thus, lamin B1 and B2 show structural and functional partitioning in mammalian nucleus. Overall design: HT1080, NIH/3T3 and HCT116 cells were grown in 6 well plates until 70-80% confluency. After one wash with 1X PBS, cells were crosslinked in 1X PBS containing 4% formaldehyde for 10 min at room temperature (RT). Crosslinked cells were quenched using 0.25 M Glycine for 5 min at RT. Cells were de-crosslinked overnight at 58°C in 1X PBS and cytoplasm was extracted for 10-30 min on ice using CSK buffer (Supplementary File 1). After 1X PBS wash, cells were incubated with blocking buffer (Supplementary File 1) for 1 hr at RT followed by overnight incubation with primary antibodies at 4°C with gentle rocking. Next, fluorophore conjugated secondary antibodies were added for 1hr at RT after 3 washes with 1X T-PBS for 5 min at RT.  After addition of secondary antibody, cells were washes 3 times with 1X T-PBS for 5 min at RT and incubated for 1 hr at RT with 1 µl of Nt.CviPII-pGL in 800 µl NEED-seq binding buffer (Supplementary File 1) per well. Plates were then washed 3 times with NEED-seq washing buffer (Supplementary File 1) for 15 min at RT and incubated with 10 units of DNA Polymerase I, 5 ?M of dNTP mix containing biotinylated-dCTP for 1 hr at 37°C in 800 µl of 1X NEBufferTM 2 per reaction to allow nick translation to occur. Plates were again washed 3 times with NEED-seq washing buffer for 5-15 mins at RT with 1X PBS additional wash. DNA was extracted using Monarch genomic DNA purification kit. After DNA purification, genomic DNA was cleaved (digested) using 2 units of Nt.CviPII (NEB # R0626S) in 500 µl of 1X NEBufferTM 2 at 37°C  for 4 hrs to overnight (This ensures the nick translated 5mC conjugated cytosine in DNA be protected against digestion). NaCl 5M stock solution was added to reach 1 M final concentration (Streptavidin Binding Buffer, Supplementary File 1) along with 30 µl of Streptavidin magnetic beads for 2 hrs with end-over-end mixing at 4°C to capture biotinylated DNA fragments. Streptavidin bound DNA beads were washed twice with Streptavidin washing buffer (Supplementary File 1). A third wash was performed with 1X TE buffer. Beads were resuspended in 50 µl of TE buffer and libraries were made on beads using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina. 8 to 12 PCR cycles were used to amplify barcoded DNA and DNA library was sequenced on Novaseq 6000. NEED-seq protocol described above was used with some modifications. Briefly, K562 crosslinked cells with 4% formaldehyde were de-crosslinked overnight at 58°C in 1X PBS. Cytoplasmic extraction was performed for 30 min with end-over-end mixing at 4°C. After 300 x g centrifugation at 4°C for 5 min, cell pellet (nuclei) was washed once with 1X PBS and resuspended in 1X PBS. Nuclei were then captured by adding 10 µl of preactivated Concanavalin A magnetic beads for 10 min with end-over-end mixing at RT. Bead-bound nuclei were incubated with blocking buffer (Supplementary File 1) for 1 hr at RT. Primary antibodies were added (1/400 final dilution) and incubated overnight with end-over-end mixing at 4°C. After 3 washes with 1X T-PBS (Supplementary File 1) for 5 min at RT, blocking buffer (Supplementary File 1) was added with fluorophore conjugated secondary antibody for 1 hr at RT. After 3 washes with 1X T-PBS (Supplementary File 1) for 5 min at RT, beads were resuspended in 1 ml NEED-seq binding buffer (Supplementary File 1) including fusion enzyme Nt.CviPII-pGL and incubated for 1 hr at RT. Beads were then washed 3 times with NEED-seq washing buffer (Supplementary File 1)  for 5-15 mins at RT. After 1 wash with 1X PBS, beads were incubated with DNA Polymerase I, 5 ?M of dNTP mix for 1 hr at 37°C in 800 µl of 1X NEBufferTM 2 with end-over-end mixing. After 3 washes with washing buffer (Supplementary File 1) for 15 min at RT, beads were resuspended in 50 mM Tris-Cl (pH 7.5), 1 % SDS, 200 mM NaCl and 2 µl of proteinase K and incubated overnight at 65°C to lyse nuclei bound to the beads. DNA was extracted using phenol-chloroform and precipitated using glycogen. 200 ng of DNA was fragmented using Nt.CviPII and used to make libraries as described above. For low number of cells, barcoded DNA was amplified using ~18 PCR cycles. To determine the dynamic range of NEED-seq, cells were serially diluted after the labeling reaction. 1 nM of size selected DNA library was sequenced on Novaseq 6000.