Characterization of a set of antibodies specific for three human histone H1 subtypes

Abstract

A series of human histone H1 subtype-specific antibodies are described that were generated for localization and functional studies. Since our previous attempts to produce such antibodies against intact subtypes met with limited success, resulting in one antibody against a subtype we have designated H1-3, the approach used in the work presented is based on the production of antibodies against synthetic peptides or peptide fragments encompassing the variant NH2-terminal region of each protein. Subtype-specific antibodies were obtained against synthetic peptides derived from subtypes designated H1-1 and H1-2 and the NH2-terminal fragment from an N-bromosuccinimide digest of H1-4. Antibody specificities were documented in all cases by enzyme-linked immunosorbent and protein immunoblot assays against the purified subtypes as well as immunoblots against whole cell and nuclear extracts. In addition, the in vivo distribution of each antibody was determined by indirect immunofluorescence. H1-1 appears to be distributed in parallel with DNA concentration, similar to the results with an antibody that recognizes all subtypes. However, H1-2 and H1-4 are non-uniformly distributed, exhibiting similar punctate staining patterns. The staining patterns described are different from the pattern described for the distribution of H1-3, suggesting that several subtypes are concentrated in distinct regions of the nucleus and, therefore, may be associated with distinct regions of the genome.

Distribution of somatic H1 subtypes is non‐random on active vs. inactive chromatin II: Distribution in human adult fibroblasts

“…After 36–72 h of incubation, the tritium‐labelled chromatin was crosslinked and sonicated then immunoprecipitated with polyclonal antibodies generated against one of four somatic H1 subtypes or with an antibody against all of the subtypes (designated anti‐total H1). Production and characterization of the rabbit polyclonal antibodies against the H1s has been described previously [Parseghian et al, 1993, 1994b].…”

G1 phase‐dependent nucleolar accumulation of human histone H1x

“…Microscopy analysis revealed that the H1 subtype H1.2 is uniformly distributed, reflecting the DNA distribution within the cell nucleus. In contrast to this, the distributions of H1.3 and H1.4 are non‐uniform, but exhibit punctate staining patterns (Parseghian et al, 1994). A preferential localization at the nuclear periphery was shown for H1.5 (Parseghian et al, 1993), and H1° was observed in condensed chromatin areas and in perinucleolar regions (Breneman et al, 1993; Gorka et al, 1993).…”

Development of H1e histone linker‐specific antibodies by means of synthetic peptides

“…After semi-preparative RP-HPLC ( Fig. 1B) Immunization of rabbits with these conjugates elicited antipeptide sera, which were first tested using ELISA It has been pointed out that for different H1 histone subtypes, peptides encompassing the NH 2 -terminal region are good candidate for antibody recognition (21,24). We observed that Pep h1e3 presenting with a globular domain near the C-terminal end also gives rise to a specific antibody against H1e.…”

“…To dispose of any individual H1 subtype antibody could be very useful in developmental biology as it has been shown recently that in H1 0 null mice the level H1c, H1d, and H1e was increased so as to maintain normal ratio of H1 to nucleosomes in H1 0 deficient chromatin (20). Despite the fact that polyclonal or monoclonal antibodies have already been obtained against H1 and H1 0 (21, 22, 23, 24), no mouse‐specific anti‐H1e antibodies have been obtained so far.…”

“…It has been pointed out that for different H1 histone subtypes, peptides encompassing the NH 2 ‐terminal region are good candidate for antibody recognition (21,24). We observed that Pep h1e3 presenting with a globular domain near the C‐terminal end also gives rise to a specific antibody against H1e.…”