MCF7 cells acquire estrogen-independent proliferation after long-term estrogen deprivation (LTED), which recapitulates endocrine therapy resistance. LTED cells can become primed for apoptosis, but the underlying mechanism is largely unknown. We previously reported that
Eleanor
non-coding RNAs (ncRNAs) upregulate the
ESR1
gene in LTED cells. Here, we show that
Eleanors
delineate the topologically associating domain (TAD) of the
ESR1
locus in the active nuclear compartment of LTED cells. The TAD interacts with another transcriptionally active TAD, which is 42.9 Mb away from
ESR1
and contains a gene encoding the apoptotic transcription factor FOXO3. Inhibition of a promoter-associated
Eleanor
suppresses all genes inside the
Eleanor
TAD and the long-range interaction between the two TADs, but keeps
FOXO3
active to facilitate apoptosis in LTED cells. These data indicate a role of ncRNAs in chromatin domain regulation, which may underlie the apoptosis-prone nature of therapy-resistant breast cancer cells and could be good therapeutic targets.
Chromosome higher order structure has been an enigma for over a century. The most important structural finding has been the presence of a chromosome scaffold composed of non-histone proteins; so-called scaffold proteins. However, the organization and function of the scaffold are still controversial. Here, we use three dimensional-structured illumination microscopy (3D-SIM) and focused ion beam/scanning electron microscopy (FIB/SEM) to reveal the axial distributions of scaffold proteins in metaphase chromosomes comprising two strands. We also find that scaffold protein can adaptably recover its original localization after chromosome reversion in the presence of cations. This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes. We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity. Our model provides new insights into chromosome higher order structure.
Recent advances in next-generation sequencing (NGS) and chromosome conformation capture (3C) analysis have led to the development of Hi-C, a genome-wide version of the 3C method. Hi-C has identified new levels of chromosome organization such as A/B compartments, topologically associating domains (TADs) as well as large megadomains on the inactive X chromosome, while allowing the identification of chromatin loops at the genome scale. Despite its powerfulness, Hi-C data analysis is much more involved compared to conventional NGS applications such as RNA-seq or ChIP-seq and requires many more steps. This presents a significant hurdle for those who wish to implement Hi-C technology into their laboratory. On the other hand, genomics data repository sites sometimes contain processed Hi-C data sets, allowing researchers to perform further analysis without the need for high-spec workstations and servers. In this chapter, we provide a detailed description on how to calculate A/B compartment profiles from processed Hi-C data on the autosomes and the active/inactive X chromosomes.
Kinesin family member 4 (KIF4) and condensins I and II are essential chromosomal proteins for chromosome organization by locating primarily to the chromosome scaffold. However, the mechanism of how KIF4 and condensins localize to the chromosome scaffold is poorly understood. Here, we demonstrate a close relationship between the chromosome localization of KIF4 and condensin I, but not condensin II, and show that KIF4 and condensin I assist each other for stable scaffold formation by forming a stable complex. Moreover, phosphorylation of KIF4 and condensin I by Aurora B and polo-like kinase 1 (Plk1) is important for KIF4 and condensin I localization to the chromosome. Aurora B activity facilitates the targeting of KIF4 and condensin I to the chromosome, whereas Plk1 activity promotes the dissociation of these proteins from the chromosome. Thus, the interdependency between KIF4 and condensin I, and their phosphorylation states play important roles in chromosome scaffold organization during mitosis.
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