Resetting Of H3K4me2 During Mouse Parental-to-zygote Transition

BackgroundIt is well known that nucleosomes are composed of DNA and histones,Serving as the basic functional unit of chromatin.A nucleosome has a 147bp fragment of DNA,with an additional 50bp of connective DNA attached to the histone H1.Among the numerous epigenetic markers,histone modification is an important one,and there are many different kinds of modification.Different kinds of modification affect the activity of gene expression and promote or inhibit the transcription activity.Histones with free amino acids at the N-terminal,such as arginine or lysine,can undergo different modifications.There are many kinds of histone modifications with different functions,among which acetylation and methylation are the two most common forms of histone modifications.The methylation at arginine promoted transcription,while the methylation at lysine activated or inhibited transcription.Most acetylation promotes gene expression.In addition,there is cooperation or antagonism among different histone modifications.The N-terminal lysine residues of histone H3 exhibit mono-methylation(H3K4me1),di-methylation(H3K4me2),or tri-methylation(H3K4me3),catalyzed by a family of proteins containing a SET domain.Previous studies have shown that the distribution pattern of H3K4me2 in plants is similar to H3K4me3.What’s more,Previous studies have shown that H3K4me2 enrichment in Oryza sativa and Arabidopsis is negatively correlated with gene transcription,which is an inhibitory epigenetic marker.However,Some works have shown that H3K4me2 exists in male gametes of mammals and is highly conserved.In addition,H3K4me2 is expressed in mouse embryonic stem cells,and its distribution is dynamic and progressive during mouse brain differentiation and tissue development.H3K4me2 has been found to be a key epigenetic factor in the specific expression of genes and signaling pathways in Primordial Germ cells(PGC).At present,the distribution and function of H3K4me3 in mammalian embryo development have been described.However,the global map of pre-implantation embryonic development of H3K4me2 in mammals and whether there is a reprogramming process of signals are still missing.Therefore,how is the genome-wide distribution of H3K4me2 at various stages of early embryonic development in mammals?What is the relationship between histone modification of H3K4me2 and chromatin accessibility and gene expression during early embryonic development in mammals?With these questions in mind,we aim to unravel the mystery of H3K4me2 during early embryonic development in mammals.ObjectiveMapping the whole genome of H3K4me2 during early embryonic development in mammals;To explore the relationship between H3K4me2 and chromatin accessibility;To elucidate the effect of methylation status of H3K4me2 on gene expression during early embryonic development in mammals.To analyze the effect and regulation of histone modification of H3K4me2 on transcription during early embryonic development in mammals.MethodsChronic myeloid leukemia cell line K562 and Mouse embryonic stem cells(ESC)were cultured in a thermostatic electric incubator at 37℃,5%CO2 and saturated humidity.H3K4me2 specific antibody suitable for immunofluorescence(IF)detection was applied to obtain the expression signal and corresponding location of H3K4me2 in mouse gametes and early embryos before implantation.Male mice of DBA/2 strain and female mice of C57BL/6N strain(purchased from Charles River Co.,Ltd)were used to obtain GV oocytes,the second meiotic metaphase(MII)oocytes and early embryos at various stages.Mouse oocytes and early embryos were collected,and after zona pellucida was removed,Improved Use low input Cleavage Under Targets and Release Using Nuclease(Uli CUT&RUN)technology was applied for targeted capture and segmentation of oocytes and early embryos to construct DNA libraries.The nextgeneration sequencing was carried out to map the whole genome of H3K4me2.At the stage of blastocyst development,the inner cell mass(ICMs)and trophoblast cell(TEs)of blastocysts with excellent morphology were separated manually by means of embryo biopsy.Finally,we conducted bioinformatics processing on the raw experimental data,and combined the transcriptome data(RNA-seq),DNA methylation data and chromatin accessibility(ATAC-seq)data of mouse early embryonic development for analysis.Results1.CUT&RUN experimental data of mouse oocytes and early embryos showed that H3K4me2 signal was detected in GV oocytes,almost all H3K4me2 signals were erased in MII oocytes,PN5 zygote and early 2-cell stages.In late 2-cell phase after zygotic genome activation,H3K4me2 signal was observed.2.Immunofluorescence experiments showed that no obvious signal was detected in MII oocytes,PN5 zygotes,Early 2-cell and Late 2-cell stages,while obvious signal could be observed in other stages of early embryonic development.3.H3K4me2 showed an atypical pattern in GV oocytes and mouse sperm,while there are differences in the distribution characteristics between male and female gametes.4.During early embryonic development after fertilization in mammals,H3K4me2 signal distribution showed a non-classical pattern,and H3K4me2 signal was mainly enriched in the high CpG density region in 4-cell and 8-cell embryos.5.The CpG-rich H3K4me2 promoter regulatory region is decomposed into active or inhibitory state in ICM,and the active promoters is often accompanied by H3K4me3.6.H3K4me2 is positively correlated with chromatin accessibility.it is an epigenetic modification marker that promotes gene expression.ConclusionsWe utilized an optimized low-input CUT&RUN method to study the epigenetic markers of H3K4me2 during early embryonic development in mammals.In this study,we found that H3K4me2 signal was erased in mouse metaphase oocytes of meiotic second phase and then reconstructed in late 2-cell after zygotic genome activation.Combined with our research results,we can draw the following conclusions:H3K4me2 expression is detected in mouse gametes and early embryos,which is a positive regulator of gene expression;The expression of H3K4me2 was dynamically reprogrammed,and its reconstruction appeared with the end of ZGA;H3K4me2 signals are enriched in promoter and distal region.Except for the hypermethylated state of the whole DNA at sperm stage,H3K4me2 is enriched in hypomethylated region at other stages and often occurs with high CpG density;4/8 cells were regarded as the priming stage,during which chromatin regions were largely open,and activated gene promoters at ICM stage were partially degraded,with the remaining active promoters accompanied by H3K4me3;Histone modification of H3K4me2 is closely related to transcription and is involved in tissue-specific production.Our study will contribute to the further exploration of epigenetic regulatory networks during early mammalian embryonic development and provide new insights into the function and significance of histone modifications in epigenetics.