Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: Corrupted CIZ1–Xi assemblies in breast cancer cells. (A) Female primary human breast epithelial cells (HMECs) stained for CIZ1 via its C-terminal anchor domain (AD, green), and co-localization with H2AK119ub1 (red) as a marker of Xi chromatin. DNA is blue. Inset, example nucleus with CIZ1–AD and H2AK119ub1 shown individually in grayscale. Bar is 10 μm. (B) Frequency of cells with discrete nuclear CIZ1 assemblies, detected via CIZ1-AD (blue) or CIZ1-RD (red) in cycling populations of the indicated breast-derived cell types. Error bars show SEM. A reduced frequency of CIZ1–Xi assemblies is observed in non-cancer breast cell line MCF-10A and cancer cell line MCF7, while in the more aggressive BT-474 and MDA-MB-231 cancer cells, large CIZ1 SMACs are rare for both RD and AD epitopes, and in SK-BR-3 populations only detectable via the AD epitope. In all four of the cancer lines, the appearance of those assemblies that are detected is less compact and coherent (see part D). (C) Model showing multivalent interaction between CIZ1 N- and C-terminal RNA interaction domains, and RNAs including Xist in the vicinity of the inactive X chromosome . (D) Example immunofluorescence images of CIZ1-RD (red) and CIZ1-AD (green) in HMEC and the indicated breast cancer cell lines, after pre-fixation wash with detergent-containing buffer (Det. only), or after high-salt extraction (Det./high salt). Right, nuclei in which RD and AD are shown individually in grayscale. Bar is 10 μm. The RD and AD epitopes were differentially detected or extracted in some cases, indicating that they are not always part of the same polypeptide (for example compare nucleus-wide RD in SK-BR-3 cells, in detergent-treated cells to detergent/high-salt treated cells). (E) CIZ1 exon-specific TPMs from four breast cancer (MCF7, BT-474, SK-BR-3, and MDA-MB-231) and one normal breast-tissue derived cell model (MCF10A), normalized to the first translated exon (exon 2), showing imbalanced domain expression, favoring the C-terminal anchor domain (AD). Exon map is aligned with protein domains (see also ), and the location of epitopes used to report on CIZ1-AD (green, Ab87) or CIZ1 replication domain (RD, red, Ab1793) are shown. Below, the relative frequency of reads aligning to human CIZ1 exon 10, demonstrating consistent coverage in the normal MCF10A line, and a transition in the cancer cell lines within exon 10, at the location of an alternative transcription start site (see also ).

Article Snippet: All cell lines used are of female origin and were authenticated for this study by Eurofins Genomics human cell line authentication service (Eurofins Medigenomix Forensik GmbH), which returned the expected identities with 92–100% confidence in all cases.

Techniques: Staining, Marker, Derivative Assay, Immunofluorescence, Extraction, Expressing

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: (Related to and ). CIZ1 domains, transcript levels , and domain expression in common solid tumors. (A) Protein domain map aligning human ( NP_001124488.1 ) and mouse ( NP_082688.1 ) CIZ1. Numbers correspond to amino acids encoded at exon boundaries. The domains highlighted are: Prion-like domains 1 and 2 (PLD1 and PLD2, purple) at positions 1–78 and 360–451, respectively (human), and positions 1–67 and 361–399, respectively (mouse), 10 three zinc fingers (ZnF_C2H2 SM00355, ZF_C2H2 sd00020, and ZF_C2H2 sd00020, blue) at positions 593–617, 656–676, and 687–709, respectively (human), and 537–561, 600–620, and 631–653, respectively (mouse), an acidic domain (red) containing a concentrated area of aspartates and glutamates at position 741–761 (human) and 689–709 (mouse), and a matrin-3 homology domain (ZnF_U1 smart0045, yellow) at position 796–831 (human) and 746–770 (mouse). Box shows % identity at the amino acid level across these domains. Human and mouse CIZ1 are 65% identical at the protein level, with identity concentrated in the conserved domains (up to 96%). (B) Bright-field images of breast-derived cell types ordered based on phenotype, with corresponding hormone and growth factor receptor status. The bar is 100 μm. (C) CIZ1 locus in Homo sapiens with corresponding exon numbers. Potential CIZ1 alternative transcription start sites (TSSs) in exons 10 and 11 predicted in the FANTOM5 project are indicated (red stars). The coding sequence would be expected to begin at a methionine in exon 11. The chromatin landscape in human mammary epithelial cells (HMEC), a cervical cancer cell line (HeLa) and a breast cancer cell line (MCF7) is shown below. Diagram generated using UCSC genome browser . (D) Total CIZ1 TPM derived from the indicated number of cancer (C) and normal (N) tissues in TCGA compared using GEPIA for the indicated disease types. No significant difference is detected (where log 2 FC was >1, and P value <0.05) when comparing all amalgamated transcripts that map to the CIZ1 gene (unresolved by exon). (E) Relative expression of exons 7 (red) and 16 (blue), normalized to the average of three unmatched control samples for each of six common solid tumor types in multi-tissue cDNA array CSRT101. Individual patient data plus the average of the controls calibrated to 1 (Av.C, left) and data aggregated by disease stage (0–IV, right) are shown. 0 represents histologically normal tissue. Individual sample information for all arrays is given in .

Article Snippet: All cell lines used are of female origin and were authenticated for this study by Eurofins Genomics human cell line authentication service (Eurofins Medigenomix Forensik GmbH), which returned the expected identities with 92–100% confidence in all cases.

Techniques: Expressing, Zinc-Fingers, Derivative Assay, Sequencing, Generated, Control