Epigenetic control during embryogenesis

Division Of Basic Sciences - Nci

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Abstract

We uncovered the molecular function of LSH using in vivo and in vitro approaches, and found that LSH promotes DNA methylation of nucleosomes, which form the smallest subunit of chromatin. Comparing the DNA methylation pattern of wild type cells with those in LSH depleted cells, we found that methylation of linker DNA is fairly independent of LSH, whereas the methylation of DNA wrapped around the core histone proteins (termed nucleosome DNA) is greatly reduced in the absence of LSH. Using reconstituted nucleosomes in vitro, we detected that DNA methyltransferase methylates only linker DNA and not nucleosome DNA. However, using recombinant LSH in combination with CDCA7 we found that LSH can move the position of DNA wrapped around the histone core proteins and can expose nucleosome DNA to methyltransferases. Consequently, nucleosome DNA can be successfully methylated. Recombinant mutant LSH (with a L801del mutation derived from ICF4 patients) showed intermediate activity on nucleosomes in vitro. An ICF4 disease mouse model, with the same L801 del mutation using CRISPR-Cas9 genetic engineering, confirmed this finding in vivo and showed intermediate DNA methylation levels at nucleosome DNA. Our results suggest that LSH provides access to nucleosome DNA for methyltransferases and explain why ICF4 patients suffer from severe loss of DNA methylation in their genome. To determine functional domains of LSH, several deletional LSH expression plasmids and patient mutations were generated and expressed in human cell lines and examined for interaction with chromatin or CDCA7 using chromatin fractionation and co-immunoprecipitation. We found that the N-terminal region (1aa-234aa) is sufficient for interaction with the co-factor CDCA7. Within this region aa30 to aa135 are essential for CDCA7 interaction and for association of LSH with chromatin. Since LSH and CDCA7 interact directly with each other in vitro, and we know that CDCA7 improves association with a nucleosome, it suggests that LSH may be in part recruited by CDCA7 to chromatin. Using the artificial intelligence system AlphFold, we predicted and verified that amino acid residues H70, K74, and K85 are critical for interaction with CDCA7 and for association with chromatin. To test for a role of posttranslational modifications, we utilized phosphatases and found that interactions with CDCA7 and with chromatin were diminished after treatment suggesting the possibility that LSH's molecular function can be modulated by drugs interfering with kinases or phosphatases. Our findings define the N-terminal region of LSH as critical for association with co-factors which modulate LSH association with chromatin and are known to modulate LSH activity. Revealing the molecular mechanisms of interaction can assist in developing targeted therapies, such as modulating LSH phosphorylation to mitigate ICF4 disease symptoms and improve patient outcomes. To study chromatin architecture under the control of LSH in immune cells we generated a unique mouse model. Using purified B lymphocytes, we examined DNA methylation level using whole genome bisulfite sequencing and we assessed 3D chromatin configuration using HIC-Seq. We detected a profound loss of DNA methylation, which occurs specifically in compartment B (repressed chromatin). Moreover, we uncovered a significant association of DNA methylation and chromatin structure, the greater the loss of DNA methylation the stronger chromatin decompaction and the more switches of compartment B (repressed chromatin) to compartment A (active chromatin). This is associated with a failure of long-range chromatin loop formation, some of which are critical for successful recombination of immunoglobulin genes and antibody secretion. In addition, chromatin expansion was linked to enhanced expression of repeat sequences normally silenced in the genome and associated with increased susceptibility to double strand DNA break formation as determined by End-Seq. Our findings suggest that LSH promotes DNA methylation which affects chromatin compaction in specific regions of the genome. The perturbed chromatin folding mechanism in ICF4 cells affects the regulation of several DNA based processes which likely contribute to immunodeficiency and genomic instability in the ICF4 syndrome. Studying the CRISPR-Cas9 genetically engineered ICF4 mutant mice, we detected infertility in male and female ICF4 mutant mice, a phenotype previously not yet reported in ICF4 patients, since the onset of the severe disease occurs in childhood. Studying male ICF4 germ cells, we found an arrest before the pachytene stage leading to a complete lack of mature sperm cells. Earlier germ cells showed reduced synapsis formation and an absence of XY bodies, indicating incomplete synapsis despite evidence of normal initiation of recombination, as evidenced by double-stranded break formation assessed by End-seq. In addition, we detected signs of non-homologous chromosome pairing and an increased distance of X and Y chromosomes during leptotene, suggesting impaired homolog alignment independent of the formation of double-stranded DNA breaks. Germ cells also showed reduced DNA methylation pattern associated with increases of usually silenced repeats. We propose that LSH maintains normal chromatin states through regulation of DNA methylation, and in its absence chromosome alignment and synapsis formation fail resulting in germ cell arrest and infertility. These findings provide novel insights into the ICF4 syndrome and highlight LSH as a potential target for correcting germ cell structural defects.