Where does this DNA come from?
The source of extracellular DNA in blood plasma can be determined by the proteins associated with it
Ekaterina Gracheva, "Elements"
Fig. 1. Scheme of an experiment on DNA sequencing from extracellular chromatin. The cells of the body differ in the activity of genes. For example, the ALB gene encoding the albumin protein is active in liver cells (liver cell, yellow), while in heart muscle cells (heart cell, purple) it remains inactive. Blood samples were collected from healthy people and patients with various diseases and plasma was isolated. The blood plasma contains extracellular chromatin (cell-free chromatin), consisting of extracellular DNA and histone proteins with appropriate modifications (red dots – modifications of inactive genes, green dots – modifications of active genes). This chromatin belongs to cells from different sources. Further, chromatin fragments containing histone modifications of interest were isolated using the chromatin immunoprecipitation (ChIP) method. Then the DNA sequences in the isolated chromatin were determined using Next-gen sequencing and, finally, the results obtained were compared with published data on the results of DNA sequencing of immunoprecipitated chromatin in various cell types. An illustration from the article under discussion in Nature Biotechnology.
A small amount of extracellular DNA is present in the blood plasma of each person. Its number increases in patients with various diseases, such as tumors or infections. The main source of extracellular DNA is decaying cells. However, reliable methods for determining exactly which cells this DNA used to belong to did not exist for a long time. Researchers from Israel took advantage of the fact that extracellular DNA is linked to histones and other chromatin proteins, and developed such a method based on a thorough analysis of epigenetic labels. The method was tested on blood plasma samples from more than 250 volunteers. It turned out that in healthy people, the main source of extracellular DNA is bone marrow megakaryocytes, and in patients – different groups of cells from damaged organs (and in the case of oncological diseases – tumor cells). In addition, the method shows which parts of the DNA of the original cells were working at the time of the appearance of extracellular DNA in blood plasma. The developed approach can become the basis of new minimally invasive methods of diagnosis of diseases.
Approximately 1-10 ng of extracellular DNA (Circulating free DNA) can be detected in a milliliter of blood plasma of a healthy person. Its main source is human cells that have completed their work and have been destroyed through apoptosis, necrosis or other cell death processes. In patients with oncological diseases, the concentration of extracellular DNA in blood plasma is higher than in healthy patients due to the contribution of tumor cells.
New DNA sequencing techniques have turned extracellular DNA into a diagnostic tool. This is how non-invasive diagnostics of oncological diseases can be carried out. To do this, extracellular DNA is isolated from blood plasma in patients, and then it is tested for the presence of mutations specific to various types of tumors (for more information about these methods, see the review by S. Fiala, E. P. Diamantis, 2018. Utility of circulating tumor DNA in cancer diagnostics with emphasis on early detection). However, the most well-known diagnostic method based on the study of extracellular DNA is noninvasive prenatal tests. They are based on the analysis of the extracellular DNA of the fetus circulating in the mother's blood.
But sequencing is not the only way to study extracellular DNA. In addition to the actual sequence of nucleotides, DNA fragments also have epigenetic modifications that reflect the work of genes or in general all processes related to DNA and do not affect the sequence of nucleotides..
The most studied of such modifications is the methylation of cytosine bases of DNA, that is, the addition of a methyl group to cytosine located next to guanine (CpG, where C is cytosine, p is a phosphodiester bond, G is guanine). DNA methylation most often "turns off" genes if the CpG sections of DNA associated with the "triggering" of genes (promoters) are methylated.
Cells of different types have different methylation patterns, because in addition to the "household" genes in each cell type genes that are important for the function of these cells work. For example, in extracellular DNA from liver cells, the methylation pattern of extracellular DNA will differ from extracellular DNA from cardiomyocytes. But such patterns do not reflect well what happened to the cell recently. The fact is that various enzymes circulate in the blood, including DNases that destroy DNA molecules and proteases that destroy histones. Because of them, the half-life of extracellular DNA ranges from two minutes to two hours, depending on the method of determination and the conditions under which extracellular DNA was collected (for more information, see in a review by S. Khier, L. Lohan, 2018. Kinetics of circulating cell-free DNA for biomedical applications: critical appraisal of the literature).
In this case, the methylation pattern is established in the cell during development and is transmitted from the mother cell to the daughter cell. In the case of cancer cells, you can see some obvious changes in the methylation patterns, but this does not happen in all cases.
Fig. 2. The origin of extracellular DNA and the changes occurring in it. Cells release extracellular DNA during apoptosis (apoptosis), necrosis (necrosis) or by active secretion (secretion). The source of extracellular DNA is both ordinary cells of the body, as well as cancer cells and cells of the tumor microenvironment. Extracellular DNA may be present in the bloodstream as part of apoptotic bodies, in free form, or as part of exosomes (exosomal DNA). Extracellular DNA analysis can be used to study point mutations, changes in the number of copies of genes (copy number alterations), chromosomal rearrangements (rearragements) and changes in methylation (methylation changes). Illustration from the article by J. Wan et al., 2017. Liquid biopsies come of age: towards implementation of circulating tumour DNA.
Another important group of epigenetic characteristics is the modification of histone proteins with which DNA is associated. The main repeating unit of chromatin (DNA and associated proteins) – the nucleosome. It consists of two histone dimers H3 and H4 and two histone dimers H2A and H2B. DNA is wound onto the nucleosome like a coil, and chromatin is obtained from nucleosomes organized into more complex structures. Histones perform not only a structural role, but also participate in the regulation of genes. Like many other proteins, they undergo post-translational modifications – the addition of various functional groups (methyl, phosphate, acetyl) or even small proteins (ubiquitin, SUMO and others). Such modifications control the work of histones and affect gene expression. For example, the addition of two or three methyl groups to histones H3 by lysine 4 (H3K4me2 or H3K4me3), on which the promoter DNA is wound, activates the start of transcription. And H3 histones with three methyl groups on lysine 9 (H3K9me3) are present in the regions of the genome corresponding to permanently switched-off genes. The location of histone modifications more accurately reflects what is happening here and now with DNA: whether the gene is active or not.
In blood plasma, extracellular DNA is bound to histone proteins: when a cell is destroyed, chromatin fragments consisting of one or more nucleosomes with bound DNA enter the extracellular space. The method developed by Israeli scientists who decided to study the chromatin of extracellular DNA and analyze the data that can be obtained through this approach is based on this observation.
The authors collected 268 blood plasma samples from more than a hundred people. Of these, 61 people were healthy, four had acute myocardial infarction, and 29 patients had various liver diseases. In addition, the extracellular DNA of 56 patients with metastatic colon cancer was examined.
First, from blood samples, scientists isolated fragments of chromatin containing extracellular DNA. Next, they used antibodies to various histone modifications: the previously mentioned H3K4me2 and H3K4me3, histone H3 with one methyl group on lysine 4 (H3K4me1), a marker of enhancers (DNA sites that activate promoters that trigger transcription), as well as histone H3 with three methyl groups on lysine 36 (H3K36me3), which corresponds to the transcribed parts of active genes.
The antibodies were used to isolate from the many fragments of extracellular chromatin those fragments that contain the listed modifications. Then, sequencing was used to study which extracellular DNA sequences they contain (see ChIP-seq).
3. Methylation of histone 3 by lysine 4 and its contribution to gene expression.
a – in the composition of chromatin, DNA wraps around nucleosomes, which consist of two copies of histones H2A, H2B, H3 and H4. The N-end of histones sticks out of the nucleosomes. Histone modifications affect amino acids from this site. One (orange), two (green) or three (blue) methyl groups (H3K4me1, H3K4me2 and H3K4me3, respectively) can be attached to histone H3 lysine 4.
b – modifications H3K4me1, H3K4me2 and H3K4me3 mark different parts of the genome. All three modifications with different intensity can be found in the area of the transcription start point (TSS, transcription start site) in the promoter region of active genes (active gene). In addition, H3K4me1 is found in the area of enhancers – DNA sites with which transcription factors bind to enhance gene transcription. ChIR-seq read density is the frequency of occurrence of a DNA site during sequencing after chromatin immunoprecipitation. An illustration from an article by B. Collins et al., 2019. Histone H3 lysine K4 methylation and its role in learning and memory.
Chromatin immunoprecipitation (ChIP, chromatin immunoprecipitation) is a common method of studying the work of genes. At the same time, working with chromatin, which is in the blood plasma, is a non–trivial task. There are already a lot of antibodies in the blood plasma that interfere with this process, and the concentration of chromatin requires very sensitive methods.
According to the results of ChIP experiments, a set of different DNA sites and the corresponding status of their activity in the form of histone modifications were obtained. By analyzing this set, the authors studied the main sources of extracellular DNA, as well as how they differ in healthy and sick people.
The results obtained from healthy volunteers were generally very similar. The main source of extracellular DNA in them were megakaryocytes – bone marrow cells, which are the precursors of platelets. This conclusion was made by identifying among the active genes detected using ChIP, genes specific to these cells (for example, GP6 and PF4). This result differs from previous observations. Earlier studies using extracellular DNA methylation analysis showed that the main source of extracellular DNA in blood plasma is erythroblasts (J. Lam et al., 2017. DNA of Erythroid Origin Is Present in Human Plasma and Informs the Types of Anemia). Such differences can be explained by the fact that both megakaryocytes and erythroblasts originate from the same type of cells – myelopoiesis progenitor cells. Methylation analysis does not distinguish the DNA of these cells accurately enough. But the analysis of chromatin and, consequently, the work of various genes can do this. A signal was also detected from the genes of neutrophils and monocytes, as well as liver cells. This result corresponds to previously published data obtained using methylation analysis (J. Moss et al, 2018. Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease).
As for patients with metastatic colon cancer, as expected, areas were found among extracellular DNA indicating active genes of colon tumors. The corresponding modifications were found both on promoters and enhancers, and on transcribed DNA sequences of the genes themselves. This was not observed in healthy study participants.
Another group of participants were patients with acute myocardial infarction. As expected, extracellular DNA from cardiomyocytes was found in them, which die in significant quantities during a heart attack. Moreover, the sensitivity of the technique for determining the source of DNA turned out to be so high that it was possible to determine the presence of extracellular DNA of cardiomyocytes in a patient with very minor damage to the heart muscle, determined by measuring troponin in blood plasma.
In addition, the authors examined a group of patients with various liver injuries. Increased levels of the enzyme alanine aminotransferase (ALT) – a standard biochemical marker of such damage. It turned out that extracellular DNA may even be the best marker of liver problems. For example, in a patient who had part of the liver removed, ALT levels slowly returned to normal after surgery. At the same time, the level of extracellular DNA related to liver cells returned to normal much earlier (Fig. 4). This is due to the fact that the half-life of extracellular DNA is much shorter than that of protein markers. The authors of the study under discussion hope that the use of extracellular DNA as a marker of diseases can be transferred to the clinic. At the same time, a complete analysis of all possible sources of extracellular DNA is possible, which means an unbiased approach to diagnosis.
Fig. 4. After partial removal of the liver (operation), the level of various markers of damage to this organ increases. The ALT level (ALT, black line) and the level of extracellular DNA related to liver cells (brown line) increase immediately after surgery. But, as can be seen, the extracellular DNA of liver cells returns to a healthy level faster than ALT. For comparison, the levels of extracellular DNA related to T-lymphocytes (T cells, green line) and NK cells (blue line) are presented. On the horizontal axis – time (in days), on the vertical axis – the level in comparison with the norm. An illustration from the article under discussion in Nature Biotechnology.
Another possibility of the published method is the study of working genes in cells that have become the source of extracellular DNA. The standard approach – the determination of RNA sequences in cells – is no longer suitable here, since the cells themselves are no longer there, and the RNA in the blood plasma is preserved much worse than DNA. But the presence of extracellular chromatin corresponding to active promoters allows us to get a picture of what was happening in these cells. The results of chromatin analysis correspond well to the already published data of RNA sequencing of various cells. Including data from RNA sequencing of single cells. Such data, for example, for the liver, are combined into "atlases" that allow you to distinguish between different groups of cells in organs.
Thus, in patients with liver diseases, it was possible to determine not only the fact of liver damage itself, but also which specific hepatocytes were affected (in particular, their location in the lobule of the liver).
Finally, the authors investigated whether it is possible to apply the developed method in order to track the effectiveness of cancer treatment. Some of the studied blood plasma samples were obtained in a long-term study of patients with metastatic colon cancer. Blood plasma was taken from patients before and after treatment, the sample included patients whose manifestations of the disease were minimal.
Oncological diseases are very multifaceted, and each tumor has its own molecular features. These features were also tracked using extracellular chromatin analysis: it differed more strongly between oncological patients than between healthy ones. In addition, the method reflects the course of therapy: by the number of active genes associated with the vital activity of the tumor, it is possible to track the response of the tumor to treatment.
The occurrence of malignant tumors is often associated with excessive activity of any genes that occur for various reasons. One of them is the amplification of oncogenes. It occurs when the normal process of DNA doubling is disrupted. As a result, instead of one copy of the DNA section, several appear on the chromosome, which allows to produce factors in excess that accelerate cell division and lead to cancer degeneration. A large amount of DNA reflecting the so-called amplicon HER2 was found in the samples studied in patients with colon cancer. Amplification of this DNA region containing the ERBB2 gene – the epidermal growth factor receptor gene – is observed mainly in breast cancer, although it also occurs in other oncological diseases, for example, in 4% of patients with colon cancer. Extracellular chromatin analysis reflected not only the presence of this amplification (such changes can be found by simple sequencing and sequencing of methylated DNA, see Chan et al., 2013. Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing). Amplicon HER2 was indeed active in tumor cells due to the presence of H3K4me3, a marker of active promoters.
Thus, researchers now have a powerful tool in their hands not only for studying extracellular DNA, but also for diagnostics that can be transferred to the clinic in the future. Extracellular chromatin analysis can answer many questions: where is the source of extracellular DNA, which genes are active in this source, and how does this activity change over time. In addition, this is an objective approach to assessing the state of the body: if we study a sample of available extracellular DNA, then there is a greater chance of finding abnormalities throughout the body. And for this, you just need to collect a small amount of blood from the patient.
But will doctors soon have such technology? It is still difficult to assess this for sure. The chromatin immunoprecipitation method is still most often used in research laboratories that study the causes of diseases and ways to treat them. This method requires a rather complex preparation of the material, good antibodies to histone modifications, as well as the ability to sequence and analyze a large amount of material. Such studies take a lot of time – valuable when it comes to the patient's life.
Nevertheless, over time, the technique may become more accessible, because until recently it was difficult to imagine that even small sections of patients' DNA could be studied in detail.
Source: Sadeh et al., ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin // Nature Biotechnologies. 2021.
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