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During the development of the mammalian cerebral cortex, numerous neurons are arranged in a six-layer structure with an inside-out fashion to form the neocortex and wire neural circuits. This process includes cell proliferation, differentiation, migration, and maturation, supported by precise genetic regulation. To understand this sequence of processes at the cellular and molecular levels, it is necessary to characterize the fundamental anatomical structures by gene expression. However, markers established in the adult brain sometimes behave differently in the fetal brain, actively changing during development. Spatial transcriptomes yield genome-wide gene expression profiles from each spot patterned on tissue sections, capturing RNA molecules from fresh-frozen sections and enabling sequencing analysis while preserving spatial information. However, a deeper understanding of this data requires computational estimation, including integration with single-cell transcriptome data and aggregation of spots on the single-cell cluster level. The application of such analysis to biomarker discovery has only begun recently, and its application to the developing fetal brain is largely unexplored. In this study, we performed a spatial transcriptome analysis of the developing mouse brain to investigate the spatiotemporal regulation of gene expression during development. Using these data, we conducted an integrated study with publicly available mouse data sets, the adult brain’s spatial transcriptome, and the fetal brain’s single-cell transcriptome. Our data-driven analysis identified novel molecular markers of the choroid plexus, piriform cortex, thalamus, and claustrum. In addition, we revealed that the internal structure of the embryonic claustrum is composed of heterogeneous cell populations.

Posttranslational modifications of histone are intimately related to chromatin/chromosome-mediated cellular events. Among all, the roles of histone modifications including acetylation, methylation, ubiquitination, and SUMOylation of lysine or arginine residue of nucleosome core histones in gene expression have been intensively studied. Genome-wide profiles of histone modification marks revealed their combinatorial organization in the functional features of chromatin. Analysis of histone modification by chromatin immunoprecipitation (ChIP) is one of the standard assays to examine chromatin states. Although high-throughput sequencing analysis (ChIP-seq) is now widely conducted, classical ChIP-qPCR analysis has advantages in investigation of multiple histone modification marks at a target site simply through the use of relatively small numbers of cells. Since ChIP-qPCR is devoid of biases caused by overamplification and inaccurate mapping of sequencing reads, it is a more reliable quantification method than genome-wide ChIP-seq especially for analyses of the low-mappability regions, which harbor many repetitive sequences and/or highly homologous segmental multiplications as found in gene clusters. We have recently analyzed histone H3 and H4 modifications of the Zscan4 family gene loci in an 880 kb gene cluster and found that the atypical enhancer-like structure is formed upon derepression of Zscan4. In this chapter, we describe the detailed protocols for histone modification ChIP-assay of repeat-enriched gene cluster regions. The protocol here we applied to mouse ES cells, but the protocol is perfectly applicable to human cultured cells and specimens.

The ubiquitously transcribed tetratricopeptide repeat on X chromosome (UTX) is a major histone H3 lysine 27 (H3K27) demethylase and the mixed-lineage leukemia (MLL) proteins are the H3K4 methyltransferases. UTX is one of the major components of MLL3- and MLL4-containing (MlLL3/4) complexes and likely has functions within the complexes. Although UTX is frequently mutated in various types of cancer and is thought to play a crucial role as a tumor suppressor, the importance of UTX interaction with MLL3/4 complexes in cancer formation is poorly understood. Here, we analyzed the ability of cancer-derived UTX mutant proteins to interact with ASH2L, which is a common core component of all the MLL complexes, and MLL3/4-specific components PTIP and PA1, and found that several single-amino acid substitution mutations in the tetratricopeptide repeat (TPR) affect UTX interaction with these components. Interaction-compromised mutants G137V and D336G and a TPR-deleted mutant Δ80-397 were preferentially localized to the cytoplasm, suggesting that UTX is retained in the nucleus by MLL3/4 complexes through their interaction with the TPR. Intriguingly, WT UTX suppressed colony formation in soft agar, whereas G137V failed. This suggests that interaction of UTX with MLL3/4 complex plays a crucial role in their tumor suppressor function. Preferential cytoplasmic localization was also observed for endogenous proteins of G137V and another mutant G137VΔ138 in HCT116 created by CRISPR-Cas9 gene editing. Interestingly, expression levels of these mutants were low and MG312 stabilized both endogenous as well as exogenous G137V proteins. These results reveal a novel mechanism of UTX regulation and reinforce the importance of UTX interaction with MLL3/4 complexes in cancer formation.

Low-density lipoprotein (LDL)-mimetic lipid nanoparticles (LNPs), decorated with MRI contrast agents and fluorescent dyes, were prepared by the covalent attachment of apolipoprotein-mimetic peptide (P), Gd(iii)-chelate (Gd), and sulforhodamine B (R) moieties on the LNP surface. The functionalized LNPs were prepared using the amide-forming potassium acyltrifluoroborate (KAT) ligation reaction. The KAT groups on the surface of LNPs were allowed to react with the corresponding hydroxylamine (HA) derivatives of P and Gd to provide bi-functionalized LNPs (PGd-LNP). The reaction proceeded with excellent yields, as observed by ICP-MS (for B and Gd amounts) and MALDI-TOF-MS data, and did not alter the morphology of the LNPs (mean diameter: ca. 50 nm), as shown by DLS and cryoTEM analyses. With the help of the efficient KAT ligation, a high payload of Gd(iii)-chelate on the PGd-LNP surface (ca. 2800 Gd atoms per LNP) was successfully achieved and provided a high r1 relaxivity (r1 = 22.0 s−1 mM−1 at 1.4 T/60 MHz and 25 °C; r1 = 8.2 s−1 mM−1 at 9.4 T/400 MHz and 37 °C). This bi-functionalized PGd-LNP was administered to three atherosclerotic apoE−/− mice to reveal the clear enhancement of atherosclerotic plaques in the brachiocephalic artery (BA) by MRI, in good agreement with the high accumulation of Gd in the aortic arch as shown by ICP-MS. The parallel in vivo MRI and ex vivo studies of whole mouse cryo-imaging were performed using triply functionalized LNPs with P, Gd, and R (PGdR-LNP). The clear presence of atherosclerotic plaques in BA was observed by ex vivo bright field cryo-imaging, and they were also observed by high emission fluorescent imaging. These directly corresponded to the enhanced tissue in the in vivo MRI of the identical mouse., LDL-mimetic lipid nanoparticles, decorated with MRI contrast agents and fluorescent dyes, were prepared by the covalent attachments of an apoB100-mimetic peptide, Gd(iii)-chelate, and rhodamine to enhance atherosclerosis in the in vivo imaging.

Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together.

Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of 2-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2-cell embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction, and are likely non-functional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides, may be involved in 2C-specific transcription program.

Rif1, originally discovered as a telomere binding factor in yeast, is evolutionally conserved and regulates various aspects of chromosome reactions including repair, DNA replication, and transcription in addition to telomere regulation. In mammals, Rif1 suppresses homologous recombination-dependent repair and stimulates non-homologous end-joining repair of double-stranded DNA breaks. Rif1 plays a crucial role in regulating timing of genome replication during S phase. It also affects the transcription profiles. Rif1, composed of the N-terminal HEAT repeat domain and the C-terminal DNA binding/oligomerization domain, tightly binds to chromatin and may facilitate the formation of chromatin domains that may be repressive for initiation of replication as well as for transcription. Rif1 also binds to many factors including protein phosphatase 1, which plays a role in suppression of origin firing. Rif1 is expressed at a high level in mouse embryonic stem (ES) cells and is involved in regulation of differentiation processes.