Protein Receptors Evolved from Homologous Cohesion Modules That Self-Associated and Are Encoded by Interactive Networked Genes

Previously, it was proposed that protein receptors evolved from self-binding peptides that were encoded by self-interacting gene segments (inverted repeats) widely dispersed in the genome. In addition, self-association of the peptides was thought to be mediated by regions of amino acid sequence similarity. To extend these ideas, special features of receptors have been explored, such as their degree of homology to other proteins, and the arrangement of their genes for clues about their evolutionary origins and dynamics in the genome. As predicted, BLASTP searches for homologous proteins detected a greater number of unique hits for queries with receptor sequences than for sequences of randomly-selected, non-receptor proteins. This suggested that the building blocks (cohesion modules) for receptors were duplicated, dispersed, and maintained in the genome, due to structure/function relationships discussed here. Furthermore, the genes coding for a representative panel of receptors participated in a larger number of gene–gene interactions than for randomly-selected genes. This could conceivably reflect a greater evolutionary conservation of the receptor genes, with their more extensive integration into networks along with inherent properties of the genes themselves. In support of the latter possibility, some receptor genes were located in active areas of adaptive gene relocation/amalgamation to form functional blocks of related genes. It is suggested that adaptive relocation might allow for their joint regulation by common promoters and enhancers, and affect local chromatin structural domains to facilitate or repress gene expression. Speculation is included about the nature of the coordinated communication between receptors and the genes that encode them.

Nuclear Opening and Histone Release Are Essential for Nuclear but Not Chromatin Condensation during Terminal Erythropoiesis

Nuclear condensation and enucleation are characteristic processes in mammalian terminal erythropoiesis. These processes are associated with the transient nuclear opening formation that mediates partial histone release to the cytoplasm. Our previous report showed that caspases are involved in the cleavage of nuclear lamina to enable histone release. However, it remains unclear whether nuclear opening formation and histone release regulate the genomic three-dimensional organization during nuclear condensation. To answer this question, we cultured E13.5 mouse fetal liver Ter119 negative erythroid progenitor cells in erythropoietin (EPO) containing medium for 48 h with or without the presence of caspase inhibitor. As expected, caspase inhibitor blocked nuclear opening formation and histone release, and significantly reduced nuclear condensation and enucleation. We next performed a Hi-C sequencing to investigate chromatin structural change during terminal differentiation and nuclear condensation. To this end, the cultured fetal liver erythroid cells with or without caspase inhibitor were harvested at 30 h right before enucleation for Hi-C sequencing. The sequencing results showed that cells at 30 h contain significantly more interactions than freshly isolated erythroid progenitors, which is consistent with chromatin condensation during terminal erythropoiesis. Further analysis showed that increased interactions mainly accumulate as inter-chromosomal interactions, suggesting inter-chromosome interaction is the dominant structural force driving erythrocyte chromatin condensation. Surprisingly, there were no significant chromatin structural changes between caspase inhibitor treated and mock-treated cells when compared at 30 h. We also performed ATAC-seq and RNA-seq with the same experiment settings, both corresponded to Hi-C sequencing and showed little difference under caspase inhibitor treatment. These results indicate that although histone release and nuclear condensation are compromised with the inhibition of caspases, chromatin stays condensed with well-organized three-dimensional structure and appropriate gene expression regulations. To further confirm this phenomenon, we generated caspase-3 and -7 double knock out (cas3cas7-/-) mice. Cas3cas7-/- mice are embryonically lethal due to defective cardiac development. The hematopoietic tissues in these mice have not been well studied. We harvested fetal liver Ter119 negative erythroid progenitor cells from E13.5 cas3cas7-/- mice and the cells from the littermate (cas7-/-, cas3+/-cas7-/-) mice were used as controls. We first cultured Ter119 negative fetal liver erythroid progenitors in EPO containing medium for 48 h. Immunofluorescence analysis showed that the nuclear opening was significantly inhibited, and the nuclear size significantly increased in the erythroid cells from cas3cas7-/- mice due to failure of histone release into cytoplasm. Flow cytometry analysis showed that enucleation was significantly impaired in cas3cas7-/- cells, but the cells could still differentiate although with lower efficiency. We further performed an in vivo assay in which E13.5 cas3cas7-/- fetal liver cells were transplanted into wild type lethally irradiated recipient mice. EPO medium cultured bone marrow lineage negative cells from these transplanted mice showed significant reduction in nuclear opening and histone release, and enlargement of nuclear size. However, these mice survived well despite anemia. These results indicate a portion of orthochromatic erythroblasts managed to enucleate even with the less condensed nuclei. Overall, our study demonstrates that nuclear opening and histone release are essential for nuclear condensation but have minimal effects on chromatin condensation or the regulation of gene expression in terminal erythropoiesis. Appropriate nuclear condensation is important for efficient enucleation. However, orthochromatic erythroblasts could still manage to enucleate although with low efficacies. Disclosures No relevant conflicts of interest to declare.

Emerging Contributions of Solid-State NMR Spectroscopy to Chromatin Structural Biology

The eukaryotic genome is packaged into chromatin, a polymer of DNA and histone proteins that regulates gene expression and the spatial organization of nuclear content. The repetitive character of chromatin is diversified into rich layers of complexity that encompass DNA sequence, histone variants and post-translational modifications. Subtle molecular changes in these variables can often lead to global chromatin rearrangements that dictate entire gene programs with far reaching implications for development and disease. Decades of structural biology advances have revealed the complex relationship between chromatin structure, dynamics, interactions, and gene expression. Here, we focus on the emerging contributions of magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS NMR), a relative newcomer on the chromatin structural biology stage. Unique among structural biology techniques, MAS NMR is ideally suited to provide atomic level information regarding both the rigid and dynamic components of this complex and heterogenous biological polymer. In this review, we highlight the advantages MAS NMR can offer to chromatin structural biologists, discuss sample preparation strategies for structural analysis, summarize recent MAS NMR studies of chromatin structure and dynamics, and close by discussing how MAS NMR can be combined with state-of-the-art chemical biology tools to reconstitute and dissect complex chromatin environments.

Progressive Domain Segregation in Early Embryonic Development and Underlying Correlation to Genetic and Epigenetic Changes

Chromatin undergoes drastic structural organization and epigenetic reprogramming during embryonic development. We present here a consistent view of the chromatin structural change, epigenetic reprogramming, and the corresponding sequence-dependence in both mouse and human embryo development. The two types of domains, identified earlier as forests (CGI-rich domains) and prairies (CGI-poor domains) based on the uneven distribution of CGI in the genome, become spatially segregated during embryonic development, with the exception of zygotic genome activation (ZGA) and implantation, at which point significant domain mixing occurs. Structural segregation largely coincides with DNA methylation and gene expression changes. Genes located in mixed prairie domains show proliferation and ectoderm differentiation-related function in ZGA and implantation, respectively. The chromatin of the ectoderm shows the weakest and the endoderm the strongest domain segregation in germ layers. This chromatin structure difference between different germ layers generally enlarges upon further differentiation. The systematic chromatin structure establishment and its sequence-based segregation strongly suggest the DNA sequence as a possible driving force for the establishment of chromatin 3D structures that profoundly affect the expression profile. Other possible factors correlated with or influencing chromatin structures, including transcription, the germ layers, and the cell cycle, are discussed for an understanding of concerted chromatin structure and epigenetic changes in development.

Exploring chromatin structural roles of non-coding RNAs at imprinted domains

Different classes of non-coding RNA (ncRNA) influence the organization of chromatin. Imprinted gene domains constitute a paradigm for exploring functional long ncRNAs (lncRNAs). Almost all express an lncRNA in a parent-of-origin dependent manner. The mono-allelic expression of these lncRNAs represses close by and distant protein-coding genes, through diverse mechanisms. Some control genes on other chromosomes as well. Interestingly, several imprinted chromosomal domains show a developmentally regulated, chromatin-based mechanism of imprinting with apparent similarities to X-chromosome inactivation. At these domains, the mono-allelic lncRNAs show a relatively stable, focal accumulation in cis. This facilitates the recruitment of Polycomb repressive complexes, lysine methyltranferases and other nuclear proteins — in part through direct RNA–protein interactions. Recent chromosome conformation capture and microscopy studies indicate that the focal aggregation of lncRNA and interacting proteins could play an architectural role as well, and correlates with close positioning of target genes. Higher-order chromatin structure is strongly influenced by CTCF/cohesin complexes, whose allelic association patterns and actions may be influenced by lncRNAs as well. Here, we review the gene-repressive roles of imprinted non-coding RNAs, particularly of lncRNAs, and discuss emerging links with chromatin architecture.

Unraveling three-dimensional chromatin structural dynamics during spermatogonial differentiation

Spermatogonial stem cells (SSCs) are able to undergo self-renewal and differentiation. Unlike the self-renewal that replenishes the SSC and progenitor pool, the differentiation is an irreversible process committed to meiosis. While the preparations for meiotic events in differentiating spermatogonia (Di-SG) are likely to be accompanied by alterations in chromatin structure, the three-dimensional (3D) chromatin architectural difference between SSCs and Di-SG, and the higher-order chromatin dynamics during spermatogonial differentiation, have not been systematically investigated. Here, we performed in situ high throughput chromosome conformation capture (Hi-C), RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses on porcine undifferentiated spermatogonia (Un-SG, which consist of SSCs and progenitors) and Di-SG. By integrating and analyzing these data, we identified that Di-SG exhibited increased disorder but weakened compartmentalization and topologically associating domains (TADs) in comparison with Un-SG, suggesting that diminished higher-order chromatin architecture in meiotic cells, as shown by recent reports, is preprogramed in Di-SG. Our data also revealed that A/B compartments and TADs were related to dynamic gene expression during spermatogonial differentiation. We further unraveled the contribution of promoter-enhancer interactions (PEIs) to pre-meiotic transcriptional regulation, which has not been accomplished in previous studies due to limited cell input and resolution. Together, our study uncovered the 3D chromatin structure of SSCs/progenitors and Di-SG, as well as the interplay between higher-order chromatin architecture and dynamic gene expression during spermatogonial differentiation, providing novel insights into the mechanisms for SSC self-renewal and differentiation and having implications for diagnosis and treatment of male sub-/infertility.

Transcriptionally Active Chromatin—Lessons Learned from the Chicken Erythrocyte Chromatin Fractionation

The chicken erythrocyte model system has been valuable to the study of chromatin structure and function, specifically for genes involved in oxygen transport and the innate immune response. Several seminal features of transcriptionally active chromatin were discovered in this system. Davie and colleagues capitalized on the unique features of the chicken erythrocyte to separate and isolate transcriptionally active chromatin and silenced chromatin, using a powerful native fractionation procedure. Histone modifications, histone variants, atypical nucleosomes (U-shaped nucleosomes) and other chromatin structural features (open chromatin) were identified in these studies. More recently, the transcriptionally active chromosomal domains in the chicken erythrocyte genome were mapped by combining this chromatin fractionation method with next-generation DNA and RNA sequencing. The landscape of histone modifications relative to chromatin structural features in the chicken erythrocyte genome was reported in detail, including the first ever mapping of histone H4 asymmetrically dimethylated at Arg 3 (H4R3me2a) and histone H3 symmetrically dimethylated at Arg 2 (H3R2me2s), which are products of protein arginine methyltransferases (PRMTs) 1 and 5, respectively. PRMT1 is important in the establishment and maintenance of chicken erythrocyte transcriptionally active chromatin.