Study of acetylated histone h3k9 – an active chromatin mark – in chromosomes from adult and fetal human lymphocytes

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STUDY OF ACETYLATED HISTONE H3K9, AN ACTIVE CHROMATIN MARK, IN CHROMOSOMES FROM ADULT AND FETAL HUMAN LYMPHOCYTES

Olga A. Efimova1, Anna A. Pendina1,2, Yuliia G. Lezhnina2, Andrei V. Tikhonov1,2, Olga G. Chiryaeva1, Lyubov I. Petrova1, Vera S. Dudkina1, Alla S. Koltsova1,3, Mikhail I. Krapivin1,3, Anastasiia V. Petrovskaia-Kaminskaia1,3, Olga E. Talantova1, Tatiana V. Kuznetzova1, V. S. Baranov1,3

1 – Federal State Budgetary Scientific Institution "The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O.Ott", 3, Mendeleevskaya line, St. Petersburg, 199034, Russia

2 – Center for Medical Genetics, 5, Tobolskaya ul., St. Petersburg, 194044, Russia

3 – St Petersburg State University, 7-9, Universitetskaya nab., St Petersburg, 199034, Russia

BACKGROUND

Recently, the interest to study chromatin arrangement has grown due to the identification of a special group of diseases—epigenetic or chromatic human diseases. The term “epigenotype” was proposed for the first time in 1942 to describe the modification of gene expression in the process of development [1]. Currently, epigenetic means the modification of expression of individual genes without any structural changes in the sequence of their nucleotides. Epigenetic modifications ensure the establishment and monitoring of the differential activity of genes in the cells of different tissues and at different stages of ontogenesis. Thanks to epigenetic mechanisms, cell variety occurs with different phenotypes and functions in the conditions of identity of the genetic set in all cells of the same organisms [2]. Disorders of the genome epigenetic mark, including the ones caused by the effect of external factors, are the reasons for its abnormal functioning, which in turn results in epigenetic diseases, including genome imprinting diseases, in particular Silver–Russell, Beckwith–Wiedemann, Prader–Willi, and Angelman syndromes as well as ICF^[1], Rett, Rubinstein–Taybi, and Coffin–Lowry syndromes [3–5].

Chromatin epigenetic modifications include the methylation of DNA cytosine and posttranslational modifications of histone proteins. DNA methylation means the reversal of enzymatic reaction, which results in the connection of the methyl group to the fifth position of cytosine residues, mostly in dinucleotide 5’­CpG‑3’ [6]. Methylated residues of cytosine are seldom observed in the 5’­CрА‑3’ and 5’­CрТ‑3’ sequences [7]. Modifications of histone proteins, which include acetylation, methylation, ubiquitination, and phosphorylation, take place mostly in the N­terminal areas. DNA methylation and modifications of histones result in specific structural and functional status of chromatin and in the epigenetic regulation of its transcriptional activity specific for different cell types and/or stages of ontogenesis [8].

Previously conducted studies demonstrated that the distribution of methylated areas along the metaphase chromosome length coincides with their transverse banding detected by differential staining methods in different tissues: in lymphocytes of peripheral blood of individuals with normal karyotype [9–12], in lymphocytes of umbilical blood of the human fetus [13], in cells of chorion cytotrophoblasts [14, 15], and in embryonic lungs [15]. The pattern of methylation of metaphase chromosomes is characterized by a certain time and tissue specificity—different degrees of methylation of the same segments of chromosomes at different stages of human ontogenesis [13, 15]. In contrast to the detailed examined pattern of DNA methylation, the data of the distribution of the modified histone proteins along the length of metaphase chromosomes are still incomplete.

In this regard, the goal of this study is the comparative analysis of the distribution of acetylated histone Н3 (AcH3K9), an active chromatin mark, on the metaphase chromosomes from lymphocytes of adult peripheral blood and umbilical blood of the human fetus.

MATERIALS AND METHODS

Formulations of metaphase chromosomes stimulated with phytohemagglutinin of lymphocytes of peripheral blood of 13 adults and lymphocytes of umbilical blood of 10 human fetuses at 20 to 22 weeks of development served as the materials of research. Samples of peripheral and umbilical blood were taken at the Ott Institute of Obstetrics, Gynecology and Reproductive Medicine in connection with the necessity of karyotypical analysis. Normal karyotype was defined for all adults and fetuses.

Leukocyte fraction was taken for culturing. For visualization, glass tubes with blood were by centrifuged for 1.5 to 2 min at 1000 rpm. Culturing was conducted according to the standard protocol [16]. Metaphase chromosomes were fixed using an alcohol solution of 2% glacial acetic acid. Formulations, without drying, were put in solution of 1× phosphate-buffered saline for 5 to 10 min.

Areas of chromosomes enriched with AcH3K9 were detected using immunocytochemical staining with antibodies to AcH3K9 (АТ­AcH3K9; Abcam, USA) and the second antibodies conjugated with Cy3 (Amersham, UK) according to the previously used protocol with own modifications [12]. Culturing of antibodies was done according to the manufacturers’ recommendations. The AT-specific stain 4′,6-diamidino-2-phenylindole (DAPI) was used for chromosome identification.

Formulations were analyzed using a Leica DM LS microscope equipped with Fluotar ×20/0.40 and ×100/1.30 to 0.60 objectives, automatic camera adapter, Leica DFC320 color camera, and a set of color filters. Leica DFC Twain software was used to obtain the photo images. The distribution of chromatin enriched with AcH3K9 was assessed using the availability and intensity of immunofluorescent signals. The distribution and intensity of immunofluorescent signals were assessed semiquantitatively (visually) and quantitatively using ImageJ 1.34s software. Tools of ImageJ 1.34s software allowed to measure the signal intensity in the dedicated area of the digital image. Every pixel in the dedicated area of the image was automatically assigned the value of 0 to 255 depending on its shade of gray in 8-bit mode. The average intensity of illumination of the dedicated area was calculated automatically, summing up the values assigned to all pixels and dividing the obtained sum by the number of pixels. Using measurements in the ImageJ 1.34s software, the absolute values were obtained for the average intensity of the segment fluorescence. The obtained values were standardized based on the ratio of the absolute value of the average intensity of the segment fluorescence and that of the benchmark. The segment on the analyzed chromosome, in which the intensity was not changed in the process of visual assessment, was assumed as the benchmark. The obtained average relative values of fluorescence intensity were compared to that in the Statistica version 8.0 program using Mann–Whitney U criterion.

To prevent the extraction of histone proteins from chromatin, the content of acetic acid in the fixing solution was reduced to 2%. A large amount of chromosome overlaps was observed on the formulations, due to which the analysis of the full chromosome set of the cell was impossible. The distribution of AcH3K9 along the chromosome arms was analyzed on fragments of metaphase bands containing 5–15 well-identified chromosomes not overlapping each other. A total of 300 fragments of metaphase bands were analyzed: 180 from lymphocytes of adults and 120 from those of fetuses.

RESULTS

An irregular distribution of the fluorescent signals of АТ­AcH3K9 along the length of metaphase chromosomes was detected in both adults and human fetuses. Signals were localized in certain areas with clear boundaries and formed a specific banding pattern of each chromosome (Fig. 1а and b). The nature of the banding of the individual chromosomes was repeated on all metaphase bands.

The boundaries of the areas enriched with AcH3K9 coincided with the boundaries of segments detected using DAPI staining. The major acetylated sites were localized in DAPI­negative areas of chromosomes corresponding to R­segments (Fig. 1c).

To determine the degree of segment enrichment with acetylated histone H3, semiquantitative assessment of АТ­AcH3K9 fluorescence intensity was assessed using a four-point scale. Conditionally, four types of fluorescent signals were detected:0 = absence of fluorescent signal1 = very weak fluorescent signal2 = weak fluorescent signal3 = intense fluorescent signal.

In accordance with the classification, idiograms were plotted for all karyotype autosomes, which reflect the intensity and segment localization of acetylated histone Н3 (Fig. 2).

Areas of chromatin with intense signal (3 points) were located in 33 R­segments of adults and fetuses. A weak signal (2 points) was detected in 62 R­segments of chromosomes of adults and 59 R­segments of chromosomes of the human fetus. Areas with very weak fluorescent signal (1 point), less enriched with acetylated histone H3, were detected in all G­segments in 38 R­segments of chromosomes of adults and 42 R­segments of chromosomes of fetuses. Fluorescent signal was not detected (0 point) in the centromeric heterochromatin of chromosomes 1, 9, and 16 in both adults and human fetuses.

The segment semiquantitative and quantitative analyses of the fluorescence intensity of АТ­AcH3K9 allowed the detection of differences in the intensity of signals of chromosomes of adults and fetuses only in the single segments: 2q31, 5p13, 5p15, 9q13, and 16p13. In segments 2q31, 5p13, 5p15, and 16p13, a higher intensity of fluorescence was detected in adults in comparison to the human fetus, whereas, in segment 9q13, on the contrary, it was lower (Mann–Whitney U criterion, p ≤ 0.05; Fig. 3).

Thus, the distribution of AcH3K9 along the metaphase chromosome arms from lymphocytes was characterized with the segment specificity and was equal in adults and human fetuses, except for the single segments, in which the level of content of AcH3K9 was different.

DISCUSSION

The acetylation of histone Н3 is a mark of transcritionally active chromatin [17, 18]. As the research was conducted using metaphase chromosomes, for which transcription activity is not typical, the urgent issue was whether epigenetic patterns were changed in the cell cycle. For a number of posttranslational modifications of histones—acetylation of H2AK4, H2BK12, H2BK15, H2BK20, H3K19, H3K23, H4K5, H4K8, H4K12, and H4K16 and methylation of H3K27 and H3K36—the changes due to the switch of phases of the cell cycle were demonstrated [19]. However, the level of acetylation of Н3K9 was stable within the cell cycle [20]. Thus, the level of AcН3K9 detected on metaphase chromosomes can define the potential functional activity of chromatin in the interphase period.

Gene density was different along the chromosome length: it was higher in R­segments than in G­segments [21, 22]. A high level of gene content in R­segments and a low level in G­segments can be potentially correlated with the different levels of their transcriptional activity, which explains the specificity of AcH3K9 localization observed in our study. According to the obtained data, the degree of enrichment of the R­ and Т­segments with AcН3K9 was different. With regard to the fact that the deacetylation of histone Н3K9 results in chromatic repression [23–25], it can be supposed that hypoacetylated R­segments contain chromatic with low trasncriptional activity.

Segment 2q31 stands out with five segments, in which the basic differences in the degree of acetylation between the chromosomes of adults and fetuses were detected and the differences in the degree in DNA methylation were detected previously [13]. According to the obtained results, segment 2q31 was characterized with a high level of histone H3 acetylation and a low level of DNA methylation in lymphocytes of adults and with a low level of histone H3K9 acetylation and a high level of DNA methylation in the fetus lymphocytes [13]. This indicated a potentially lower transcriptional activity of chromatin in segment 2q31 of human fetus at 20 to 22 weeks of development in comparison to that of adults. Segment 2q31 localized genes of the immunoglobulin superfamily CD51 (ITGAV) and CD49D (ITGA4). The detected features of methylation and acetylation of segment 2q31 can be explained by the active work of the immune system of adults rather than the human fetus [26].

Thus, in accordance with the obtained results, there is a special type of linear banding of metaphase chromosomes of lymphocytes stipulated by the differential distribution of AcН3K9. The increase and reduction of the degree of acetylation of the individual segments of chromosomes from fetus lymphocytes in comparison to that of adults demonstrate that the described pattern of acetylation reflects both the structural and functional features of chromosome segments. It should be noted that the disturbance of genome functioning is the most difficult for diagnostics, although they can serve as the reason for the number of pathological conditions. Further research on the nature of acetylation of histones of metaphase chromosomes together with an analysis of transcriptional activity can become the basis of the test systems for the assessment of epigenetic status of chromatin, including in the prenatal period of human ontogenesis. It is important that the property of epigenome plasticity —the ability to regulate epigenetic processes using external effects, including with medicines, hormones, and diet—provides prospects for the development of approaches for the target-specific therapeutic correction of abnormal epigenetic profiles.

Acknowledgements

A.S. Koltsova and M.I. Krapivin are recipients of the presidential scholarship.

 

Fig. 1. Fragments of metaphase bands from lymphocytes of an adult (a) and human fetus at 21/22 weeks of development (b) after immunofluorescent detection of AcH3K9. (c) Photo images of chromosomes 1 and 11 of an adult after DAPI staining and immunofluorescent detection of AcH3K9 and idiograms of G­segmentation (G).

Хромосома 1	Chromosome 1
Хромосома 11	Chromosome 11

 

Fig. 2. Scheme of the segment localization of AcH3K9 (left, color idiogram) on the chromosomes from lymphocytes of an adult and G-banding (right, black and white idiograms). Two shades of red demonstrate the different intensities of fluorescence of АТ­AcH3K9 in the R­segments—assessed in 2 and 3 points. A low level of luminescence is shown in black (1 point) typical for all G-segments; luminescence assessed in 1 point in the R-segments is shown in white. Gray color marks the segments where signal is absent (0 point).

 

 

Fig. 3. Distribution of АсН3K9 along the arms of chromosomes 2, 5, 9, and 16 from lymphocytes of adults (left) and human fetuses (right). Arrows show the segments with different levels of acetylation of Н3K9.

Хромосома 2	Chromosome 2
Хромосома 5	Chromosome 5
Хромосома 9	Chromosome 9
Хромосома 16	Chromosome 16

 

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^[1]ICF = immunodeficiency accompanied by centromeric instability and facial anomalies.

About the authors

Olga A. Efimova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Author for correspondence.
Email: efimova_o82@mail.ru
ORCID iD: 0000-0003-4495-0983
SPIN-code: 6959-5014
Scopus Author ID: 14013324600

PhD, researcher, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034

Anna A. Pendina

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology; Сenter for Medical Genetics

Email: pendina@mail.ru
SPIN-code: 3123-2133

PhD, researcher, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034; 5, Tobolskaya ul., St. Petersburg, 194044

Yuliia G. Lezhnina

Center for Medical Genetics

Email: avesfly@mail.ru

biologist

Russian Federation, 5, Tobolskaya ul., St. Petersburg, 194044

Andrei V. Tikhonov

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology; Center for Medical Genetics

Email: tixonov5790@gmail.com
SPIN-code: 3170-2629

PhD, researcher, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034; 5, Tobolskaya ul., St. Petersburg, 194044

Olga G. Chiryaeva

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: chiryaeva@mail.ru
SPIN-code: 4027-4908

PhD, researcher, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034

Lyubov I. Petrova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: petrovaluba@mail.ru
SPIN-code: 8599-6886

laboratory geneticist, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034

Vera S. Dudkina

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: dudkinavs@mail.ru
SPIN-code: 6286-7287

laboratory geneticist, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034

Alla S. Koltsova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology; Saint Petersburg State University

Email: rosenrot15@yandex.ru
SPIN-code: 3038-4096

assistant researcher, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034; 7/9, Universitetskaya emb., St. Petersburg, 199034

Mikhail I. Krapivin

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology; Saint Petersburg State University

Email: krapivin-mihail@mail.ru
SPIN-code: 4989-1932

assistant researcher, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034; 7/9, Universitetskaya emb., St. Petersburg, 199034

Anastasiia V. Petrovskaia-Kaminskaia

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology; Saint Petersburg State University

Email: tonx2012@yandex.ru

assistant researcher, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034; 7/9, Universitetskaya emb., St. Petersburg, 199034

Olga E. Talantova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: olga_talantova@mail.ru

MD, researcher, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034

Tatiana V. Kuznetzova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: tkuznetzova@mail.ru
SPIN-code: 1000-7522

PhD, researcher, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034

Vladislav S. Baranov

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology; Saint Petersburg State University

Email: baranov@vb2475.spb.edu
SPIN-code: 9196-7297

MD, Professor, laboratory head, laboratory for prenatal diagnosis of congenital and inherited diseases

Russian Federation, 3, Mendeleevskaya line, St. Petersburg, 199034; 7/9, Universitetskaya emb., St. Petersburg, 199034

References

Supplementary files