CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

Arrangement of nuclear structures is not transmitted through mitosis but is identical in sister cells.

Although it is well known that chromosomes are non-randomly organized during interphase, it is not completely clear whether higher-order chromatin structure is transmitted from mother to daughter cells. Therefore, we addressed the question of how chromatin is rearranged during interphase and whether heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during interphase, especially when histone hyperacetylation was induced or transcription was inhibited. This natural phenomenon can influence the analysis of nuclear arrangement. Using photoconversion of Dendra2-tagged core histone H4 we showed that the distribution of chromatin in daughter interphase nuclei differed from that in mother cells. Similarly, the nuclear distribution of heterochromatin protein 1ß (HP1ß) was not completely identical in mother and daughter cells. However, identity between mother and daughter cells was in many cases evidenced by nucleolar composition. Moreover, morphology of nucleoli, HP1ß protein, Cajal bodies, chromosome territories, and gene transcripts were identical in sister cell nuclei. We conclude that the arrangement of interphase chromatin is not transmitted through mitosis, but the nuclear pattern is identical in naturally synchronized sister cells. It is also necessary to take into account the possibility that cell rotation and the degree of chromatin condensation during functionally specific cell cycle phases might influence our view of nuclear architecture.

Differentiation-independent fluctuation of pluripotency-related transcription factors and other epigenetic markers in embryonic stem cell colonies.

Embryonic stem cells (ESCs) maintain their pluripotency through high expression of pluripotency-related genes. Here, we show that differing levels of Oct4, Nanog, and c-myc proteins among the individual cells of mouse ESC (mESC) colonies and fluctuations in these levels do not disturb mESC pluripotency. Cells with strong expression of Oct4 had low levels of Nanog and c-myc proteins and vice versa. In addition, cells with high levels of Nanog tended to occupy interior regions of mESC colonies. In contrast, peripherally positioned cells within colonies had dense H3K27-trimethylation, especially at the nuclear periphery. We also observed distinct levels of endogenous and exogenous Oct4 in particular cell cycle phases. The highest levels of Oct4 occurred in G2 phase, which correlated with the pKi-67 nuclear pattern. Moreover, the Oct4 protein resided on mitotic chromosomes. We suggest that there must be an endogenous mechanism that prevents the induction of spontaneous differentiation, despite fluctuations in protein levels within an mESC colony. Based on the results presented here, it is likely that cells within a colony support each other in the maintenance of pluripotency.

Acetylation-dependent nuclear arrangement and recruitment of BMI1 protein to UV-damaged chromatin.

Polycomb group (PcG) proteins, organized into Polycomb bodies, are important regulatory components of epigenetic processes involved in the heritable transcriptional repression of target genes. Here, we asked whether acetylation can influence the nuclear arrangement and function of the BMI1 protein, a core component of the Polycomb group complex, PRC1. We used time-lapse confocal microscopy, micro-irradiation by UV laser (355 nm) and GFP technology to study the dynamics and function of the BMI1 protein. We observed that BMI1 was recruited to UV-damaged chromatin simultaneously with decreased lysine acetylation, followed by the recruitment of heterochromatin protein HP1ß to micro-irradiated regions. Pronounced recruitment of BMI1 was rapid, with half-time τ = 15 sec; thus, BMI1 is likely involved in the initiation step leading to the recognition of UV-damaged sites. Histone hyperacetylation, stimulated by HDAC inhibitor TSA, suppression of transcription by actinomycin D, and ATP-depletion prevented increased accumulation of BMI1 to γH2AX-positive irradiated chromatin. Moreover, BMI1 had slight ability to recognize spontaneously occurring DNA breaks caused by other pathophysiological processes. Taken together, our data indicate that the dynamics of recognition of UV-damaged chromatin, and the nuclear arrangement of BMI1 protein can be influenced by acetylation and occur as an early event prior to the recruitment of HPß to UV-irradiated chromatin.

Recruitment of Oct4 protein to UV-damaged chromatin in embryonic stem cells.

BACKGROUND: Oct4 is a specific marker of embryonic stem cell (ESC) pluripotency. However, little is known regarding how Oct4 responds to DNA damage. Here, we investigated whether Oct4 recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct4. These experiments should contribute to the knowledge of how ESC genomic integrity is maintained, which is crucial for potential application of human ESCs in regenerative medicine. METHODOLOGY/PRINCIPAL FINDINGS: We used time-lapse confocal microscopy, microirradiation by UV laser (355 nm), induction of DNA lesions by specific agents, and GFP technology to study the Oct4 response to DNA damage. We found that Oct4 accumulates in UV-damaged regions immediately after irradiation in an adenosine triphosphate-dependent manner. Intriguingly, this event was not accompanied by pronounced Nanog and c-MYC recruitment to the UV-damaged sites. The accumulation of Oct4 to UV-damaged chromatin occurred simultaneously with H3K9 deacetylation and H2AX phosphorylation (γH2AX). Moreover, we observed an ESC-specific nuclear distribution of γH2AX after interference to cellular processes, including histone acetylation, transcription, and cell metabolism. Inhibition of histone deacetylases mostly prevented pronounced Oct4 accumulation at UV-irradiated chromatin. CONCLUSIONS/SIGNIFICANCE: Our studies demonstrate pluripotency-specific events that accompany DNA damage responses. Here, we discuss how ESCs might respond to DNA damage caused by genotoxic injury that might lead to unwanted genomic instability.

Effects of epigenetic-based anti-cancer drugs in leukaemia and multiple myeloma cells.

Here, we focus on epigenetic changes in leukaemia and MM (multiple myeloma) cells. We show how the histone signature, DNA methylation and levels of select tumour-suppressor proteins can be affected by inhibitors of HDACs (histone deacetylases) and Dnmts (DNA methyltransferases). Both inhibitors, TSA (trichostatin A) and 5-AZA (5-azacytidine), have the ability to change the histone signature in a tumour-specific manner. In MM cells, we observed changes in H3K4 methylation, while in leukaemia cells, H3K9 methylation was especially affected by select inhibitors. Compared with normal peripheral blood lymphocytes, tumour cell samples were characterized by increased H3K9 acetylation, increased H3K4me2, H3K9me2 and HP1α (heterochromatin protein 1α) levels and specific changes were also observed for DNA methylation. Additionally, we showed that the tumour suppressor pRb1 (retinoblastoma protein) is more sensitive to epigenetic-based anti-cancer stimuli than p53. We have found significant decrease in the levels of pRb1 and p53 in both myeloma and leukaemia cells after HDAC inhibition.