GSE136264
Histone H1 distribution and epitope exposure within chromatin high-order structure
Within the cell nucleus, histone H1 is the most mobile histone.  Yet, it is regarded as key to the establishment and stability of chromatin higher-order structure.  The implication is that chromatin higher-order structure is dynamic, in part due to the mobility of H1.  Defining the positions of H1 on chromatin in situ, therefore, represents a challenge.  Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq) is traditionally the method for mapping histones onto DNA elements.  But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higher-order structure.  A second formaldehyde fixation after antibody binding to intact in situ chromatin, but before sonication (xxChIP-seq), preserves this information and reveals which histone epitopes are inaccessible in situ.  In this study, the distribution of two histone H1 variants H1.2 and H1.5 is defined and compared by both xChIP-seq and xxChIP-seq in undifferentiated HL-60/S4 cells, illustrating the influences of preserved chromatin higher-order structure. We have found that xChIP and xxChIP signals are anticorrelated. H1.2 versus H1.5-enrichments were particularly distinct near bound Pol II, SMC3 proteins, as well as domains marked by H3K4me1, H3K9ac, H3K27ac and H3K36me3. H1.5-enriched regions have an average nucleosome repeat length NRL=184 bp, as opposed to 190 bp for H1.2-enriched regions. We have also studied the distribution of H1 variants with respect to DNA methylation. Most changes of DNA methylation during differentiation appear within ~60bp from the nucleosome entry/exit and are preferably associated with H1.2-bound nucleosomes, whereas H1.5 histones are depleted from CpGs. Our results suggest that different physical packing of nucleosomes may exist in H1.2- versus H1.5-enriched areas, characterized by differential H1 epitope exposure as assessed by xChIP and xxChIP. Overall design: All ChIP-Seq experiments were performed on undifferentiated HL-60/S4 cells that were fixed, permeabilized and stored in cryovials (containing ~107 cells/cryovial) in liquid Nitrogen.  Cells had been harvested from growth medium at ~106 cells/ml, centrifuged and washed with PBS, fixed in 1% HCHO/PBS for 10 min at RT, stopped with 0.125 M glycine for 5 min, washed with PBS, followed by PBS + 0.1 M PMSF.  The fixed cells were permeabilized for 10 min at 4oC in a lysis buffer containing 25 mM HEPES buffer (pH 7.8), 1 mM MgCl2, 10mM KCl, 0.1% NP40, 1 mM DTT and 0.5 mM PMSF.  Following centrifugation and removal of supernatants, cell pellets were frozen in residual lysis buffer at liquid Nitrogen temperature. For both single-fixation and double-fixation ChIP (xChIP and xxChIP), chromatin was disrupted with a Covaris Focused Ultrasonicator M220.  In xChIP, each frozen cell pellet (1 cryovial) was dispersed in 130 µl of Covaris Sonication Buffer (1 mM EDTA, 10 mM Tris [pH 7.6], 0.1 % SDS), followed by sonication (20 min, 200 cycles, 75 Watts, Duty Cycle 20%, 7oC).  The sonicates were centrifuged at 18,000xg, 10 min, 40C and the supernatants recovered.  SDS was reduced in the supernatants to ~0.003% and replaced with 0.05% Tween-20, employing repeated dilution with PBST (PBS+0.05% Tween-20) and centrifugal concentration using a Centricon YM-50.  Six centrifugations of ~1/2 dilutions with PBST at 1000xg, 10 min resulted in ~0.5 ml of the final retentate with reduced SDS.  IgG-free BSA (Sigma A3294) was added to a final BSA concentration of 5%. In xxChIP, the once-fixed frozen cell pellets were dispersed in a buffer reminiscent of the permeabilizing buffer used in immunostaining reactions (0.1% Triton X-100, 0.1 mM PMSF plus Sigma Protease Inhibitor Cocktail [P8340]) for 20 min at RT.  After PBS washes, the permeabilized cells were suspended in PBST+5% IgG-free BSA (PBSTB) and rotated for 90 min at RT.  To 300 µl aliquots containing ~6x107 cells, the primary antibody was added: 30 µl anti-histone H1.2 (1 mg/ml) or 60 µl anti-histone H1.5 (0.5 mg/ml).  The cells plus antibody were rotated for 4 hours at RT.  Following antibody incubation, the cells were washed several times with PBS to remove unbound antibody.  They were made 1% HCHO/PBS for the second fixation and rotated 2.5 minutes at RT.  Fixation was stopped with 0.125 M glycine for 5 min, cells washed with PBS and dispersed in 1.0 ml of Covaris Sonication Buffer (1 mM EDTA, 10 mM Tris [pH 7.6], 0.1 % SDS), followed by sonication at optimized conditions (40 min, 400 cycles, 75 Watts, Duty Cycle 26%, 7oC).  The sonication buffer was replaced with PBST, employing centrifugal concentration, as described above. Of necessity, the immunoprecipitation (IP) protocols differed slightly, comparing xChIP to xxChIP.  The xChIP preparations in PBSTB buffer were incubated with control agarose (1 hour, with rotation) and recovered from the minicolumns by centrifugation at 4oC.  Samples of the “cleaned” sonicates were retained as “Input”.  Simultaneously, Protein A/G agarose minicolumns, equilibrated in PBSTB, were incubated 4-5 hours with 4 µg of rabbit anti-histone H1.2 or H1.5, rotating at RT, followed by washing with PBSTB.  The equilibrated and “cleaned” sonicates were incubated with the antibody-bound Protein A/G agarose minicolumns overnight, rotating at 4oC.  Subsequently, the sonicate-bound columns were washed 5 times with PBSTB and 2 times with PBST, to remove unbound chromatin.  Elution of the bound chromatin fragments was accomplished by addition of 50 µl of 100 mM NaHCO3 + 1% SDS, tumbling for 15 min at RT.  After centrifugal recovery, a second elution with 50 µl was performed, yielding ~100 µl of pooled eluate.  The IP eluates were digested with RNAse and proteinase K overnight at 65oC.  DNA was purified employing Sigma Gene Elute (NA1020-1KT).  By contrast, xxChIP preparations, having the anti-H1 antibodies already bound and crosslinked to the chromatin fragments and in PBSTB buffer, were “cleaned” on the control agarose minicolumns, an aliquot removed for “Input” and the remainder incubated with Protein A/G agarose minicolumns overnight, rotating at 4oC, followed by washing to remove unbound chromatin.  Elution of the bound chromatin fragments and DNA purification were similar to the xChIP procedures.