**5. Unusual chromatin structure of TGC**

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

developmental program.

development both in flies and in mice [45].

ROBO signaling achieved by UR of its genes.

the precise stages of development.

of genome regulation in placental TGCs.

**place in TGC**

Notably, 8-10th day of gestation in mice corresponds to the placenta formation whereas at 10-16th days well-developed placenta functioning takes place. The authors [45] note that the increase in UR domain size and degree of underrepresentation from

Besides, it should be mentionned that, during the late stages of TGC lifespan, new underreplicated regions are also formed but they are more stochastic, less reproducible, and significantly smaller than those conserved between all stages [45]. Notably, underreplication of TGC coincide with period of the most significant stages of TGC invasion and anchoring to endometrium and is integrated in its

The above-mentioned data also show that UR domains are formed from a specific class of late-replicating heterochromatic regions that contain mainly non-coding DNA, suggesting that UR domains are not simply a byproduct of latereplicating heterochromatin, but are a precisely regulated subset of DNA sequences. The authors come to conclusion that presence of UR domains in *Drosophila* endoreduplicated cells and in murine TGC is an example of convergent evolution. In this case UR contributes to accelerating the cell cycles that makes possible fast rate of

The underreplication in the endoreduplicated trophoblast cells not only may fasten the cell cycles but also be important for the TGC specific functions. Thus, Hannibal et al. [45] also note that UR domains are enriched for specific classes of genes involved in cell adhesion and neurogenesis. It is still difficult to find an explanation for the UR of specific genes and gene clusters. It can only be assumed that a certain number of gene copies is optimal in a given cell type. UR of separate genome regions at the background of its multiple duplication makes it possible to fine-tune the number of functioning copies necessary for performing specific functions.

In some cases, significance of UR was clearly demonstrated. Thus, downregulation of genes that regulate cell adhesion, junction and related cytoskeleton rearrangements is necessary for trophoblast EMT transition and invasion in both mice and humans [47, 48, 58]. Upregulation of genes in the SLIT/ROBO neuronal guidance system in the human placenta has been found to be bound with preeclampsia [49]. It is possible, placenta oxygenation requires precise specific function of SLIT/

Therefore, significance of endoreduplication is not only multifold genome duplication itself and enlargement of the cell that may be of significance for TGC barrier function but also a possibility of a fine regulation of a number of functional gene copies to provide cells capabilities to accomplish some functions necessary at

**4. Amplification of some genes significant for the pregnancy also takes** 

The mammalian polytene chromosomes may also undergo amplification of specific gene cluster. In the murine placenta TGC, five amplified regions were found using whole-genome sequencing and digital droplet PCR [50]. All the gene clusters are known to play key roles in mammalian placenta development and maintenance: the prolactins that regulate trophoblast cell lineage differentiations [51], serpins [52] and cathepsins [53] that promote trophoblast invasion, as well as (NK)/C-type lectin complex that play a crucial role in the feto-maternal cross-talk [54–56].

Therefore, amplification at selective genomic regions is another important mode

8.0th to 9.5th day is linked to the "robust" endocycles of early gestation [46].

**64**

Besides the non-classic polytene chromosomes in rodent placenta TGC [5, 26], some details of unusual chromosome structure have been revealed recently in the endoreduplicated TGC of mice. In the course of differentiation of TSC into TGC, expression of most genes encoding canonical histone were downregulated [1] By contrast, genes encoding non-canonical histones - H2AX, H2AZ and H3.3 did not show downregulation. The micrococcal nuclease digesion assay as well as nucleosome stability assay using a microfluidic devise showed that chromatin progressive loosening of chromatin in the course of TSC differentiated. Experiments combining H3.3 knockdown and overexpression showed that variant H3.3 resulted in formation of the loose nucleosomes in the murine TGC [1].

The presence of H2AZ and H3.3 in the genome potentially correlated with actively transcribed genes, indicating that H2AZ and H3.3 were necessary for creating relaxed and transcriptionally active chromatin structures [57–59]. Therefore, H2AX, H2AZ, and H3.3 histone variant may be responsible for the formation of a loose nucleosome structure that was unique to TGCs [1].

Interestingly, knockdown of H3.3 variant in the differentiationg TSCs significantly decreased the number of cells containing more than 4n DNA content compared to the control cells [1]. Therefore, switch to the non-canonic histone variants seems to be a prereqisite of the trophoblast cell endoreduplication. On the other hand, loose chromatin organization may, like underreplication, promote fastening the modified cell cycle that allow reach multifold (up to 512c and higher) genome multiplication and formation a giant trophoblast cell layer at the border with semiallogenic maternal tissue.

The unusual chromatin status is revealed, in particular, in the organization of the inactive X-chromosome of the murine TGC [60]. Thus, investigation of the precise temporal and lineage-specific X-inactivation status of several genes in postimplantation mouse embryos showed stable gene silencing in most lineages, with significant levels of escape from XCI mainly in one extra-embryonic cell type - TGCs. It has been found that the *Xist* RNA-coated X chromosome has a highly unusual chromatin content in TGCs, presenting both heterochromatic marks such as H3K27me3 and euchromatic marks such as histone H4 acetylation and H3K4 methylation. This unusual combination of silent and active features is likely to reflect, and might underlie, the partial activity of the X chromosome in TGCs. However, some key loci seem to require dosage compensation in TGC that probably points out to combination of the relaxed and silenced gene expression as a specific mode of gene activity regulation in a condition of chromatin unusual organization in TGC.

## **6. Hypomethylation of human and rodent placenta**

Methylation status provides some new insight in the understanding of the trophoblast cell organization that underly their unique features. The human placental trophoblast shows general global hypomethylation [61]. It is possible that the loose-nucleosome structure of TGC in murine placenta and global hypomethylation in human placenta are similar phenomena. In human placenta, genome-wide hypomethylation, coupled with gene-specific hypermethylation of tumor-suppressor genes, is a common feature of human cancers [64]. Interestingly, the placenta parallels human cancers in both the overall decreased level of genomic DNA methylation and the specific hypermethylation of several tumor suppressor genes [61–64].

Such a parallel with carcinogenesis may be connected with the trophoblast invasive pathways. Inhibition of DNA methylation by 5-azacytidine treatment disrupts trophoblast invasive and migratory potential *in vitro* [65] and proper placental development *in vivo* [66]. Thus, threatment of BeWo cell with DNA methyltransferase inhibitor, 5′-aza-2′-deoxycytidine (AZA) resulted in conversion to non-migratory phenotype. AZA was found to increase mRNA level of E-cadherin and plakoglobin, components of cell junction structures - zonula adherens and desmosomes [65]. AZA treatment also resulted in decrease their gene promoter activity and protein levels. Increases in plakoglobin and E-cadherin promoter activity and inhibition of BeWo cell migration was also achieved with small interfering RNA-mediated depletion of both *DNMT-3a* and *DNMT-3b* [65].

Meantime, beside the trophoblast invasion, some DNA methylation (locusspecific and/or repeat-based) is important for differentiation of the functionally different trophoblast lineages forming the placental barrier and performing other placental specific functions. Thus, most homeobox genes were hypomethylated in the human placenta throughout gestation**.** Nevertheless, three homeobox genes, *TLX1*, *HOX10* and *DLX5* showed progressive methylation and decrease of their expression in the course of pregnancy – from first to third trimester. Using siRNA treatment the key role of *TLX1, HOXA10* and *DLX5* in trophoblast proliferation, differentiation and apoptosis was demonstrated [67]. It cannot be ruled out that progressive methylation of some homeobox genes promotes trophoblast differentiation into highly proliferative ones that, in turn, gives rise to villous cyto- and syncytiotrophoblast and the invasive trophoblast lineages. The data suggest an important role of several homeobox gene methylation in gene expression in the course of placenta formation.

In mice, *Dnmt3L* is expressed at high levels in the chorion, containing a multipotent trophoblast stem cell population. Disruption of *Dnmt3L* disturbs placental development including spongiotrophoblast and labyrinth malformation, leads to excess of TGC and defective attachment of the chorion to the ectoplacental cone. Excessive TGC development indicates that such a phenotype is bound to the hypomethylation. This is associated with an arrest of proliferation of the extraembryonic tissue [68]. It may be suggested that *Dnmt3L*-mediated *de novo* methylation is connected with initiation of differentiation of trophoblast into multiple lineages that imply maintenance of high level of mitotic proliferation regulated by Mash2 expression as well as syncytiotrophoblast formation demonstrated by *GCM1* expression [68]. It suggests that *Dnmt3L*-mediated *de novo* methylation is critical for proper placental development in mice [61].

Demonstration of placenta-specific hypomethylation of the *DNMT3L* gene supports a role in human placental development [61] probably bound to the start of differentiation of trophoblast into a range of lineages with different proliferative capacity.
