Role of nuclear matrix protein CIZ1 in X chromosome inactivation

X chromosome inactivation (XCI) is a gene dosage compensation mechanism that results in transcriptional silencing of one of the two X chromosomes in female cells. Nuclear matrix protein CIZ1, which has established links with diseases such as cancer, plays a role in initiation of mammalian DNA replication through interaction with cyclin-dependent kinases. Recently it has also been observed to localize to the inactive X chromosome and loss of the protein results in disruption of the long non-coding RNA Xist. In mice, loss of CIZ1 leads to development of a lymphoma-like phenotype in female mice. My project has used RNAseq analysis, immune-detection techniques, and transfections to investigate the relationship between CIZ1 and X inactivation. I present evidence that implicates the C-terminus end of CIZ1 in binding at the Xi region and the N-terminus in recruitment of the protein to the Xi. I have also shown that loss of CIZ1 leads to disruption of the H3K27me3 at the Xi. At the gene expression level, absence of CIZ1 does not lead to any evident gene reactivation from the inactive X or changes in expression of genes at the X inactivation centre (Xic). However, whole-genome analysis showed that loss of CIZ1 causes widespread changes in gene expression, which could underlie the lymphoma-like phenotype in female mice, and begins to suggest a mechanism of action for CIZ1

Estudio anatomopatológico de la presencia de calcificaciones en tejido ovárico. Valoración diagnóstica.

INTRODUCCIÓN: El ovario presenta una heterogeneidad celular que hace complejo el diagnóstico de imagen e histológico, sigue siendo un reto diagnosticar precozmente la patología tumoral de ovario. Los focos hiperecogénicos no se describen en la exploración ecográfica transvaginal, este ha sido el motivo por el que hemos empezado este trabajo. OBJETIVO: Estudiar la distribución de la patología ovárica según grupos de edad, así como valorar la presencia de calcificaciones en la histopatología de los ovarios analizados. MATERIAL Y MÉTODOS: Se ha realizado un estudio observacional descriptivo retrospectivo de ovarios recibidos en el servicio de Anatomía Patológica (AP) entre noviembre 2018 y septiembre 2019. Se analizaron 189 casos. RESULTADOS: El 12,3% de los ovarios presentan calcificaciones (19/155). Se han considerado los teratomas quísticos maduros (TQM) como un grupo independiente. De 11 casos con calcificaciones en pacientes mayores de 50 años, 7 casos correspondían a cáncer de ovario (63,6%). De 8 calcificaciones que se observaron en pacientes de entre 21 y 50 años, 5 se asocian con patología ovárica benigna (62,5%). DISCUSIÓN: Existen pocas publicaciones que valoren la presencia de calcificaciones en patología ovárica. La mayor parte de estas pertenecen a pruebas de TAC y PET, y en algunos se ha relacionado su presencia con patología benigna. En este trabajo hemos observado que la relación entre el resultado de Anatomía Patológica y la presencia de calcificaciones depende especialmente de la edad, relacionando calcificaciones en pacientes mayores de 50 años con cáncer de ovarioINTRODUCTION: The ovary presents a cellular heterogeneity that makes imaging and histological diagnosis complex, it remains a challenge to diagnose early ovarian tumor pathology. Hyperechogenic foci are not usually described in the transvaginal ultrasound scan, this has been the reason why we have started this work. OBJECTIVE: To study the distribution of ovarian pathology according to age groups as well as to assess the presence of calcifications in the histopathology of the analysed ovaries. MATERIAL AND METHODS: A retrospective descriptive observational study of the ovaries received in the Anatomical Pathological Service (AP) between November 2018 and September 2019 has been carried out. A total of 189 cases of women over 16 years old of age were analyzed. RESULTS: 12,3% of the ovaries showed calcifications (19/155). Mature cystic teratomas (MCTs) have been considered as an independent group. Of the 11 cases with calcifications in patients over 50 years old, 7 cases corresponded to ovarian cancer (63.6%). Of the 8 calcifications observed in patients between 21 and 50 years old, 5 were associated with benign ovarian pathology (62.5%). DISCUSSION: There are few publications that assess the presence of calcifications in ovarian pathology. Most of these belong to CT and PET tests, and in some cases their presence has been related to benign pathology. In this work we have observed that the relationship between the result of Anatomical Phatology and the presence of calcifications depends especially on age, relating calcifications in patients over 50 years with ovarian cancer

Maintenance of epigenetic landscape requires CIZ1 and is corrupted in differentiated fibroblasts in long-term culture

The inactive X chromosome (Xi) serves as a model for establishment and maintenance of repressed chromatin and the function of polycomb repressive complexes (PRC1/2). Here we show that Xi transiently relocates from the nuclear periphery towards the interior during its replication, in a process dependent on CIZ1. Compromised relocation of Xi in CIZ1-null primary mouse embryonic fibroblasts is accompanied by loss of PRC-mediated H2AK119Ub1 and H3K27me3, increased solubility of PRC2 catalytic subunit EZH2, and genome-wide deregulation of polycomb-regulated genes. Xi position in S phase is also corrupted in cells adapted to long-term culture (WT or CIZ1-null), and also accompanied by specific changes in EZH2 and its targets. The data are consistent with the idea that chromatin relocation during S phase contributes to maintenance of epigenetic landscape in primary cells, and that elevated soluble EZH2 is part of an error-prone mechanism by which modifying enzyme meets template when chromatin relocation is compromised

The nuclear matrix protein CIZ1 facilitates localization of Xist RNA to the inactive X-chromosome territory

The nuclear matrix protein Cip1-interacting zinc finger protein 1 (CIZ1) promotes DNA replication in association with cyclins and has been linked to adult and pediatric cancers. Here we show that CIZ1 is highly enriched on the inactive X chromosome (Xi) in mouse and human female cells and is retained by interaction with the RNA-de-pendent nuclear matrix. CIZ1 is recruited to Xi in response to expression of X inactive-specific transcript (Xist) RNA during the earliest stages of X inactivation in embryonic stem cells and is dependent on the C-terminal nuclear matrix anchor domain of CIZ1 and the E repeats of Xist. CIZ1-null mice, although viable, display fully penetrant female-specific lymphoproliferative disorder. Interestingly, in mouse embryonic fibroblast cells derived from CIZ1-null embryos, Xist RNA localization is disrupted, being highly dispersed through the nucleoplasm rather than focal. Focal localization is reinstated following re-expression of CIZ1. Focal localization of Xist RNA is also disrupted in activated B and T cells isolated from CIZ1-null animals, suggesting a possible explanation for female-specific lymphoproliferative disorder. Together, these findings suggest that CIZ1 has an essential role in anchoring Xist to the nuclear matrix in specific somatic lineages

Identification of DHX9 as a cell cycle regulated nucleolar recruitment factor for CIZ1

CIP1-interacting zinc finger protein 1 (CIZ1) is a nuclear matrix associated protein that facilitates a number of nuclear functions including initiation of DNA replication, epigenetic maintenance and associates with the inactive X-chromosome. Here, to gain more insight into the protein networks that underpin this diverse functionality, molecular panning and mass spectrometry are used to identify protein interaction partners of CIZ1, and CIZ1 replication domain (CIZ1-RD). STRING analysis of CIZ1 interaction partners identified 2 functional clusters: ribosomal subunits and nucleolar proteins including the DEAD box helicases, DHX9, DDX5 and DDX17. DHX9 shares common functions with CIZ1, including interaction with XIST long-non-coding RNA, epigenetic maintenance and regulation of DNA replication. Functional characterisation of the CIZ1-DHX9 complex showed that CIZ1-DHX9 interact in vitro and dynamically colocalise within the nucleolus from early to mid S-phase. CIZ1-DHX9 nucleolar colocalisation is dependent upon RNA polymerase I activity and is abolished by depletion of DHX9. In addition, depletion of DHX9 reduced cell cycle progression from G1 to S-phase in mouse fibroblasts. The data suggest that DHX9-CIZ1 are required for efficient cell cycle progression at the G1/S transition and that nucleolar recruitment is integral to their mechanism of action

Consequences of Plk4 overexpression in mouse pancreatic organoids

Overexpression of the polo-like kinase 4 (Plk4), the master regulator of centrosome duplication, leads to centrosome amplification, a common feature of cancer, including pancreatic cancer. Previous studies have shown that overexpression of Plk4 in mice carrying a doxycycline-inducible Plk4 transgene (Plk4OE) causes centrosome amplification, a reduction in primary cilia number, and increased proliferation of pancreatic endocrine α-cells and β-cells. This project aimed to investigate the consequences of Plk4 overexpression on pancreas cell biology using mouse pancreatic ductal organoids. In accord with previous observations, immunofluorescence experiments confirmed that Plk4 overexpression leads to centrosome amplification and reduction in primary cilia number. I also investigated the consequences of long-term Plk4 overexpression on the differentiation status of pancreatic organoids. Immunofluorescence and RT-qPCR analyses revealed ductal-to-endocrine transdifferentiation, with insulin secretion and expression of β-cell markers in Plk4OE +dox organoids. I then used whole-transcriptome sequencing to investigate the consequences of Plk4 overexpression on gene expression. Results revealed differential gene expression between control and +dox Plk4OE organoids, with enrichment for genes with centrosome, cilia, cell cycle, cell death, cancer and endocrine function. Finally, I optimised and tested a protocol for the in vitro differentiation of pancreatic organoids, with induced insulin production in pancreatic organoids, irrespective of Plk4 overexpression. In summary, the findings of this thesis confirm the reported effects of Plk4 overexpression on centrosomes and primary cilia. They also reveal a previously unknown effect of Plk4 overexpression on the identity of pancreatic ductal cells. However, whether this effect is a direct consequence of Plk4 overexpression itself or a secondary consequence of some Plk4-mediated event, such as the downregulation of primary cilia formation, remains unclear. Finally, I present an in vitro differentiation protocol capable of driving ductal organoids into insulin-producing organoids without the need for genetic modification.Cancer Research UK studentshi

The nuclear matrix protein CIZ1 facilitates localisation of Xist RNA to the inactive X-chromosome territory

The nuclear matrix protein Cip1-interacting zinc finger protein 1 (CIZ1) promotes DNA replication in association with cyclins, and has been linked with adult and pediatric cancers. Here we show that CIZ1 is highly enriched on the inactive X chromosome (Xi) in mouse and human female cells, and is retained by interaction with the RNAdependent nuclear matrix. CIZ1 is recruited to Xi in response to expression of Xist RNA during the earliest stages of X-inactivation in embryonic stem cells, and is dependent on the C-terminal nuclear matrix anchor domain of CIZ1 and the E-repeats of Xist. CIZ1 null mice, although viable, display fully penetrant female specific lymphoproliferative disorder. Interestingly, in MEF cells derived from CIZ1 null embryos Xist RNA localisation is disrupted, being highly dispersed through the nucleoplasm rather than focal. Focal localisation is reinstated following re-expression of CIZ1. Focal localisation of Xist RNA is also disrupted in activated B and T cells isolated from CIZ1 null animals, suggesting a possible explanation for female specific lymphoproliferative disorder. Together, these findings suggest that CIZ1 has an essential role in anchoring Xist to the nuclear matrix in specific somatic lineages

The Xist Locus

Chromosome balance is critical for normal development. When a condition with extra or missing chromosome exists, the effects are damaging if the balance is not somehow restored. A classic example is Down’s syndrome in humans, which is characterized by trisomy 21. When the single-X male:double-X female system of sex determination evolved, survival was dependent on the co-evolution of a dosage compensation system. In Drosophila, the essence of dosage compensation is hypertranscription of the male single X chromosome, thereby producing equivalence of X-linked gene expression in males and females. In mammals, the unique feature of dosage compensation is silencing of one of the two Xs in the female to bring about equivalence of expression between male and female X chromosomes. A critical factor in the silencing of one X chromosome in the females of placental mammals is the noncoding RNA, XIST, which stands for ‘X inactive specific transcript’. XIST, the gene encoding this RNA, is the focus of this article