Immunolabelling of human metaphase chromosomes reveals the same banded distribution of histone H3 isoforms methylated at lysine 4 in primary lymphocytes and cultured cell lines
© Terrenoire et al.; licensee BioMed Central. 2015
Received: 11 March 2015
Accepted: 14 April 2015
Published: 29 April 2015
Using metaphase spreads from human lymphoblastoid cell lines, we previously showed how immunofluorescence microscopy could define the distribution of histone modifications across metaphase chromosomes. We showed that different histone modifications gave consistent and clearly defined immunofluorescent banding patterns. However, it was not clear to what extent these higher level distributions were influenced by long-term growth in culture, or by the specific functional associations of individual histone modifications.
Metaphase chromosome spreads from human lymphocytes stimulated to grow in short-term culture, were immunostained with antibodies to histone H3 mono- or tri-methylated at lysine 4 (H3K4me1, H3K4me3). Chromosomes were identified on the basis of morphology and reverse DAPI (rDAPI) banding. Both antisera gave the same distinctive immunofluorescent staining pattern, with unstained heterochromatic regions and a banded distribution along the chromosome arms. Karyotypes were prepared, showing the reproducibility of banding between sister chromatids, homologue pairs and from one metaphase spread to another. At the light microscope level, we detect no difference between the banding patterns along chromosomes from primary lymphocytes and lymphoblastoid cell lines adapted to long-term growth in culture.
The distribution of H3K4me3 is the same across metaphase chromosomes from human primary lymphocytes and LCL, showing that higher level distribution is not altered by immortalization or long-term culture. The two modifications H3K4me1 (enriched in gene enhancer regions) and H3K4me3 (enriched in gene promoter regions) show the same distributions across human metaphase chromosomes, showing that functional differences do not necessarily cause modifications to differ in their higher-level distributions.
KeywordsImmunolabelling Metaphase chromosome Histone methylation Human epigenome
Our previously published work described how immunofluorescence microscopy could be used to provide an overview of the distribution of histone modifications across human metaphase chromosomes. Using metaphase chromosome spreads from lymphoblastoid cell lines (LCL) of normal karyotype and antisera to some key histone modifications, we showed that different histone modifications gave consistent and clearly defined banding patterns . Various modifications linked to transcriptional activity, such as histone H3 tri-methylated at lysine 4 (H3K4me3), H3 acetylated at lysine 27 (H3K27ac) and H3 acetylated at lysine 9 (H3K9ac), gave the same staining patterns, with strongly staining regions distributed across the euchromatic chromosome arms. In contrast, the banding pattern was strikingly different for modifications associated with gene silencing such as H3 tri-methylated at lysine 27 (H3K27me3), which gave broad bands that often overlapped, but were not coincident with, the sharp bands containing modifications associated with transcriptionally active chromatin. H4 tri-methylated at lysine 20 (H4K20me3), a modification associated with heterochromatin formation , was largely centromeric . We found that the distribution of active modifications was closely related to the distribution of regions rich in genes, CpG Islands (CGI) and SINE elements . However, it is not clear to what extent the higher level distributions revealed by indirect immunofluorescence (IIF) microscopy reflect the specific functional associations of individual histone modifications, or how they are influenced by the differentiation status of the host cell, or by long-term growth in culture. Here, we address these issues by (i) defining the distribution of H3K4me3 across metaphase chromosomes from primary human lymphocytes stimulated to grow in short-term culture, and comparing this with our previous results in LCL, and (ii) comparing the distributions of H3K4me3, a modification associated with promoter regions [3,4] with that of H3K4me1, a modification also linked to transcriptionally active chromatin, but now known to be associated with enhancer regions [3,5].
Distribution of H3K4me3 in chromosomes from primary lymphocytes and comparison with LCL
Comparison of the distribution of H3K4me3 and H3K4me1 in primary lymphocytes
By immunofluorescence microscopy, different histone modifications show distinctive distributions across human metaphase chromosomes; H3K20me3 is associated primarily with centric heterochromatin [1,7], while H3K27me3, a modification closely linked to gene silencing through the Polycomb complex , is distributed as broad bands, sometimes incorporating gene-rich regions but not restricted to such regions , finally H3K4me1, H3K4me3, H3K9ac and H3K27ac are all associated with regions rich in genes, CGI and SINE elements (present results and ). H4 acetylation gives banding that corresponds to the more sharply defined H3K4me3 bands  and in early experiments, was associated with gene-rich T-bands . The explanation for these distinctive, high level banded distributions probably lies in the general functions with which the modifications are linked. H4K20me3 is required for chromatin condensation and heterochromatin compaction . The multiple modifications that highlight gene-rich regions are all involved, in one way or another, in transcriptional activation, and their overall enrichment in gene-rich regions, irrespective of their exact functional involvements, is understandable. Epigenomic analyses  show that H3K4me1 and H3K4me3 are differently distributed at the gene level and below, but their distribution is indistinguishable at the 1-10Mb level revealed by chromosome immunofluorescent banding. Polycomb-associated modification H3K27me3 is well known to spread over wide genomic regions , and a role in suppressing extra-genic transcription would explain why its immunostaining reveals bands extending beyond gene-rich regions.
It remains uncertain whether the patterns of histone modification that define individual chromosome bands are a simple reflection of gene richness and/or ongoing transcription, or whether they play a determining role in chromatin packaging and intra-nuclear location at the Mb level. In this respect, it is of interest that the metaphase chromosome bands for H3K4me3 are indistinguishable between primary lymphocytes and lymphoblastoid cell lines. The lymphocyte metaphase spreads shown here are derived from short term culture and are likely to be from the first mitosis of these naturally post-mitotic cells. Our results show that banding is not noticeably influenced by the major epigenetic changes that must accompany establishment of lymphoblastoid cell line and adaptation to long-term growth in culture. It may be that at the highest level, the broad distribution of histone modifications (ie. banding) is determined by the need to adopt a specific, compacted chromosome structure at metaphase, and to maintain an established pattern of gene expression through mitosis, rather than the differentiation or growth status of the cell.
At the light microscope level, the banded distribution across human metaphase chromosomes of two modified histones associated with active chromatin, H3K4me1 and H3K4me3, is the same, even though they are enriched at enhancers and promoters respectively and play different roles in transcriptional regulation.
The epigenetic changes that accompany adaptation to long-term growth in culture do not alter the banded distribution of H3K4me3 across human metaphase chromosomes.
Peripheral blood was taken by venepuncture from healthy adult volunteers, with informed consent and ethical approval (National Research Ethics Committee, approval number Leeds East 07/Q1206/25). Mononuclear cells (PBMC) from 10ml aliquots of whole blood were isolated by LymphoPrep™ (Axis-shield). The white cell layer was aspirated, diluted to 50ml in PBS, spun down and washed twice in PBS and once in RPMI 1640 culture medium. Isolated PBMC were cultured and co-stimulated with PHA (5μg/ml ) and human interleukin-2 (30U/ml, both from Gibco ®) in RPMI1640 medium supplemented with 10% foetal bovine serum (Gibco) and 1% (v/v from Gibco stock solutions) L-glutamine and penicillin/streptomycin . After 24 hours, cells were treated with colcemid (0.05μg/ml, Biochrom, Berlin) overnight (16h), prior to being spun down, washed twice in ice cold PBS, swollen in 75mM KCl (10min, at room temperature 1x105 cells/ml) and spun onto glass slides using a Shandon Cytospin 4 (Thermo Electron corporation) . Unfixed chromosomes from primary lymphocytes proved to be more fragile than those from LCL and to mitigate this, solutions were kept ice-cold and centrifugation was reduced to 1,200 rpm (Shandon cytospin 4, Thermo Fisher) for 5 min.
Immunostaining of metaphase spreads from native unfixed chromosomes was carried out exactly as described previously  using rabbit antisera to H3K4me1 (R204) and H3K4me3 (R612) and fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit immunoglobulin (Sigma F1262) diluted x1000. Antisera were diluted in KCM buffer (120mM KCl, 20mM NaCl,10mM Tris/HCl pH 8.0, 0.5mM EDTA, 0.1% (v/v) Triton X-100) supplemented with 1% BSA (Sigma-Aldrich). Rabbit antisera were prepared -in-house and their specificities validated as previously described [1,10]. To stabilise labelled chromosomes, slides were fixed in 4% (v/v) formaldehyde in KCM buffer, before mounting in Vectorshield (Vector Lab, Peterborough, UK) supplemented with the DNA counterstain diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) at 2 μg/ml, all as described . Metaphase spreads were visualized on a Zeiss Axioplan 2 epifluorescence microscope under a x100 oil immersion lens. Metaphases chromosome capture and karyotyping were carried out with Smart Capture and Smart Type software (Digital Scientific, Cambridge, UK).
We thank Sara Dyer and Mike Griffiths of the West Midlands Regional Genetics Laboratory for support and encouragement, Peter Cockerill for help in obtaining ethical approval for blood collection and all the blood donors who kindly volunteered. This work was supported by Cancer Research UK.
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