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. 2023 Mar;27(1):63-71.
doi: 10.18699/VJGB-23-09.

Expression of the NUP153 and YWHAB genes from their canonical promoters and alternative promoters of the LINE-1 retrotransposon in the placenta of the first trimester of pregnancy

V V Demeneva  1 , E N Tolmacheva  1 , T V Nikitina  1 , E A Sazhenova  1 , S Yu Yuriev  2 , A Sh Makhmutkhodzhaev  2 , A S Zuev  1 , S A Filatova  3 , A E Dmitriev  3 , Ya A Darkova  3 , L P Nazarenko  1 , I N Lebedev  4 , S A Vasilyev  5
Affiliations

Expression of the NUP153 and YWHAB genes from their canonical promoters and alternative promoters of the LINE-1 retrotransposon in the placenta of the first trimester of pregnancy

V V Demeneva et al. Vavilovskii Zhurnal Genet Selektsii. 2023 Mar.

Abstract
in English, Russian

The placenta has a unique hypomethylated genome. Due to this feature of the placenta, there is a potential possibility of using regulatory elements derived from retroviruses and retrotransposons, which are suppressed by DNA methylation in the adult body. In addition, there is an abnormal increase in the level of methylation of the LINE-1 retrotransposon in the chorionic trophoblast in spontaneous abortions with both normal karyotype and aneuploidy on different chromosomes, which may be associated with impaired gene transcription using LINE-1 regulatory elements. To date, 988 genes that can be expressed from alternative LINE-1 promoters have been identified. Using the STRING tool, genes (NUP153 and YWHAB) were selected, the products of which have significant functional relationships with proteins highly expressed in the placenta and involved in trophoblast differentiation. This study aimed to analyze the expression of the NUP153 and YWHAB genes, highly active in the placenta, from canonical and alternative LINE-1 promoters in the germinal part of the placenta of spontaneous and induced abortions. Gene expression analysis was performed using real-time PCR in chorionic villi and extraembryonic mesoderm of induced abortions (n = 10), adult lymphocytes (n = 10), spontaneous abortions with normal karyotype (n = 10), and with the most frequent aneuploidies in the first trimester of pregnancy (trisomy 16 (n = 8) and monosomy X (n = 6)). The LINE-1 methylation index was assessed in the chorionic villi of spontaneous abortions using targeted bisulfite massive parallel sequencing. The level of expression of both genes from canonical promoters was higher in blood lymphocytes than in placental tissues (p < 0.05). However, the expression level of the NUP153 gene from the alternative LINE-1 promoter was 17 times higher in chorionic villi and 23 times higher in extraembryonic mesoderm than in lymphocytes (p < 0.05). The expression level of NUP153 and YWHAB from canonical promoters was higher in the group of spontaneous abortions with monosomy X compared to all other groups (p <0.05). The LINE-1 methylation index negatively correlated with the level of gene expression from both canonical (NUP153 - R = -0.59, YWHAB - R = -0.52, p < 0.05) and alternative LINE-1 promoters (NUP153 - R = -0.46, YWHAB - R = -0.66, p < 0.05). Thus, the observed increase in the LINE-1 methylation index in the placenta of spontaneous abortions is associated with the level of expression of the NUP153 and YWHAB genes not only from alternative but also from canonical promoters, which can subsequently lead to negative consequences for normal embryogenesis.

Для плаценты характерен уникальный гипометилированный геном. Благодаря этой особенности плаценты в первом триместре беременности наблюдается потенциальная возможность использования регуляторных элементов, полученных от ретровирусов и ретротранспозонов, которые во взрослом организме подавляются метилированием ДНК. Кроме того, у спонтанных абортусов и с нормальным кариотипом, и с анеуплоидиями отмечается аномальное повышение уровня метилирования ретротранспозона LINE-1 в трофобласте хориона, что может быть связано с нарушением транскрипции генов с использованием регуляторных элементов LINE-1. На сегодняшний день идентифицировано 988 генов, способных экспрессироваться с альтернативных промоторов LINE-1. Из них с помощью инструмента STRING были отобраны гены, продукты которых взаимодействуют с белками, экспрессирующимися на высоком уровне в плаценте и участвующими в дифференцировке трофобласта, NUP153 и YWHAB. Целью настоящего исследования являлся анализ экспрессии генов NUP153 и YWHAB с канонических и альтернативных промоторов ретротранспозона LINE-1 в зародышевой части плаценты спонтанных и медицинских абортусов первого триместра беременности. Определение уровня экспрессии генов проводили с помощью ПЦР в режиме реального времени в лимфоцитах взрослых индивидов (n = 10), в ворсинах хориона и экстраэмбриональной мезодерме медицинских абортусов (n = 10) и спонтанных абортусов с нормальным кариотипом (n = 10) и с наиболее частыми анеуплоидиями в I триместре беременности (трисомия 16 (n = 8) и моносомия X (n = 6)). Индекс метилирования LINE-1 оценивали в ворсинах хориона спонтанных абортусов с помощью таргетного бисульфитного массового параллельного секвенирования. Уровень экспрессии обоих генов с канонических промоторов был выше в лимфоцитах крови, чем в тканях плаценты (p < 0.05). Однако уровень экспрессии гена NUP153 с альтернативного промотора LINE-1 был выше в 17 раз в ворсинах хориона и в 23 раза – в экстраэмбриональной мезодерме по сравнению с лимфоцитами (p < 0.05). Между группами спонтанных абортусов с моносомией X и остальными группами были выявлены статистически значимые различия. Уровень экспрессии NUP153 и YWHAB с канонических промоторов был выше в группе спонтанных абортусов с моносомией X по сравнению со всеми другими группами (p < 0.05). Индекс метилирования LINE-1 отрицательно коррелировал с уровнем экспрессии генов как c канонических (NUP153 – R = –0.59, YWHAB – R = –0.52, p < 0.05), так и c альтернативных промоторов LINE-1 (NUP153 – R = –0.46, YWHAB – R = –0.66, p < 0.05). Таким образом, наблюдаемое нами повышение индекса метилирования LINE-1 в плаценте спонтанных абортусов связано с уровнем экспрессии генов NUP153 и YWHAB не только с альтернативных, но и с канонических промоторов, что в дальнейшем может приводить к негативным последствиям для нормального эмбриогенеза.

Keywords: DNA methylation; NUP153; YWHAB; miscarriage; placenta; retrotransposon LINE-1.

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Figures

Fig. 1.
Fig. 1.. Functionally significant connections of the proteins involved in the development of the placenta (GO:0061450, GO:0097360, GO:0001890) with the NUP153 and YWHAB genes according to STRING
Yellow shows proteins that have functional bonds (highlighted in red) with the NUP153 and YWHAB genes (marked in orange) (STRING score > 0.4).
Table 1.
Table 1.. Sequences of oligonucleotide primers for assessing the level of expression of the NUP153, YWHAB, and GAPDH genes using real-time PCR
Notе. F– forward primer; R – reverse primer.
Fig. 2.
Fig. 2.. Scheme of the location of alternative LINE-1 promoters for the NUP153 and YWHAB genes
The arrows schematically mark the hybridization sites of oligonucleotide primers. The arrow starting at the beginning of the LINE-1 element indicates the direction of transcription from the direct LINE-1 promoter, which is canonical. The second arrow pointing in the opposite direction marks the direction of expression from the alternative antisense LINE-1 promoter, which is also alternative for the studied genes.
Fig. 3.
Fig. 3.. Comparison of the NUP153 (a) and YWHAB (b) gene expression levels from canonical promoters and alternative LINE-1 promoters in blood lymphocytes, chorion, and placental mesoderm.
Values are given as fold differences relative to the level of gene expression from the canonical promoter in adult lymphocytes. Expression levels of two different transcripts from the canonical promoter (NM_003404, NM_139323) are shown for the YWHAB gene. The reference gene is GAPDH. Can is the canonical promoter, and Alt is the alternative LINE-1 promoter.
Fig. 4.
Fig. 4.. Comparison of the expression level of the NUP153 (a) and YWHAB (b) genes from canonical promoters and alternative LINE- 1 promoters between groups of spontaneous abortions and induced abortions.
Values are given as fold differences relative to the level of gene expression of the canonical promoter in the group of induced abortions. SANK – spontaneous abortions with normal karyotype; Tri 16 – spontaneous abortions with trisomy 16; Mono X – spontaneous abortions with monosomy X.
Fig. 5.
Fig. 5.. Correlation of the NUP153 and YWHAB gene expression with the LINE-1 methylation index in the chorionic trophoblast of spontaneous abortions with normal karyotype, trisomy 16, and monosomy X.
Correlations of the LINE-1 methylation index with various transcripts of the NUP153 and YWHAB genes: a – NUP153 gene expression from the canonical promoter; b – NUP153 gene expression from the alternative promoter; c – YWHAB (NM_003404) gene expression from the canonical promoter; d – YWHAB (NM_139323) gene expression from the canonical promoter; e – YWHAB gene expression from the alternative promoter. SANK – spontaneous abortions with normal karyotype; Tri 16 – spontaneous abortions with trisomy 16; Mono X – spontaneous abortions with monosomy X.
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