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The retinoblastoma protein (RB) is historically known for its function in cell cycle control. However, mice carrying targeted Rb1 mutations have revealed that RB serves various non-cell cycle control roles. Notably, RB acts as a scaffold that recruits chromatin regulatory proteins, condensin II and enhancer of zeste homolog 2 (EZH2). These complexes protect the genome integrity through maintaining proper chromosome condensation, long range contacts, and transcriptionally repressive histone modification. This thesis explores the mechanistic links that regulate such RB-condensin II complex or that are leveraged upon pharmacological inhibition of the RB-EZH2 complex. First, I identified potential phosphorylation sites in the RB C-terminus (RBC) that may be downstream of T cell receptor (TCR) crosslinking in Jurkat leukemia T cells. I generated and validated phospho-specific antibodies that detect S838/T841 phosphorylation. Upon TCR crosslinking, S838/T841 phosphorylation was indeed detected. p38 mitogen activated protein kinase (MAPK), a known downstream target of TCR signaling, directly phosphorylated RB at S838/T841. Through Jurkat cells overexpressing a phospho-ablated mutant RB construct, I show that S838/T841 phosphorylation is required to dissociate condensin II from chromatin and facilitate chromosome decondensation. In the subsequent chapter, I investigated the consequence and mechanism of pharmacologically inhibiting EZH2. Upon EZH2 inhibition, splenic B cells specifically were eliminated in vivo, which was associated with increased transcription of repetitive elements. Using CRISPR-Cas9 to generate a novel mouse line with abrogated detection of repetitive element transcripts, named RIC mutant, I show that EZH2 inhibition signals inflammatory chemokine expression. This was mediated through reduced histone methylation by EZH2, followed by upregulation of repetitive elements and their detection by three cytosolic pattern recognition receptors (PRRs), RIG-I, MDA5 and cGAS. These PRRs were required to recruit pro-inflammatory immune cells to the spleen in vivo and eliminate B cells. Lastly, I show that RIG-I and cGAS are indispensable for activating anti-viral interferon gene expression in mouse melanoma cells upon EZH2 inhibition. Taken together, this work uncovers regulatory and molecular mechanisms that underlie a biological function or targeted inhibition of chromatin regulatory proteins recruited by RB in immune cells.

Centromeres are chromosomal regions that serve as sites for kinetochore formation and microtubule attachment, processes that are essential for chromosome segregation during mitosis. Centromeres are almost universally defined by the histone variant CENP-A. In the holocentric nematode C. elegans, CENP-A deposition depends on the loading factor KNL-2. Depletion of either CENP-A or KNL-2 results in defects in centromere maintenance, chromosome condensation and kinetochore formation, leading to chromosome segregation failure. Here, we show that KNL-2 is phosphorylated by CDK-1 in vitro, and that mutation of three C-terminal phosphorylation sites causes chromosome segregation defects and an increase in embryonic lethality. In strains expressing phosphodeficient KNL-2, CENP-A and kinetochore proteins are properly localised, indicating that the role of KNL-2 in centromere maintenance is not affected. Instead, the mutant embryos exhibit reduced mitotic levels of condensin II on chromosomes and significant chromosome condensation impairment. Our findings separate the functions of KNL-2 in CENP-A loading and chromosome condensation, and demonstrate that KNL-2 phosphorylation regulates the cooperation between centromeric regions and the condensation machinery in C. elegans. This article has an associated First Person interview with the first author of the paper., Summary: Phosphorylation of the essential centromere protein KNL-2 is required for mitotic chromosome condensation, but not for the role of KNL-2 in centromere maintenance and kinetochore formation.

The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. Then, after entering interphase, the positions of the scattered centromeres are stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWNs have little effect on chromatin organization involved in the control of gene expression, indicating the robustness of chromatin organization regardless of the type of centromere distribution.

Non-SMC condensin I complex subunit D2 and non-SMC condensin II complex subunit D3 induces inflammation via the IKK/NF-κB pathway in ulcerative colitis

Centromeres are regions of chromosomes specialised for tethering the spindle microtubules and withstanding the pulling forces during chromosome segregation. In most species the centromeric identity depends on the timely deposition of a histone H3 variant called CENP-A. This process is tightly regulated, in part by post-translational modifications of CENP-A, its chaperones and licensing factors. In the holocentric nematode Caenorhabditis elegans, KNL-2 (M18BP1) is the crucial factor for CENP-A recruitment, but the spatiotemporal regulation of its function remains unknown. In this thesis, we examine KNL-2 localisation and post-translational modifications. We find that a pool of KNL-2 is sequestered in nucleoli in interphase and re locates to chromatin in prophase thanks to CDK¬1 activity. We show that KNL-2 phosphorylation regulates condensin II levels on chromatin and that KNL-2 functions in CENP-A loading and chromosome condensation are independent. Finally, we optimise a proteomic approach (APEX2), to discover new interactors of KNL-2 and CENP-A.

Erythropoiesis requires dramatic changes in gene expression in a cell that is rapidly proliferating and undergoing progressive nuclear condensation in anticipation of enucleation. Disruption of this process is associated with myelodysplastic syndromes and congenital anemias. Our lab has demonstrated that Setd8, the sole histone methyltransferase that can generate H4K20me1, plays an essential role in this process (Malik 2019). H4K20me1 accumulates during terminal erythroid maturation (Murphy Blood 2021) and can regulate chromatin structure and gene expression through interaction with multiple partners, including the Condensin II Complex. The Condensin II complex is a ring-like structure composed of two conserved SMC components (SMC2 and SMC4), two HEAT subunits (NCAPG2 and NCAPD3), and a kleisin subunit NCAPH2. The Condensin II complex plays an important role in chromatin condensation during mitosis, and establishing higher-order chromatin interactions in interphase cells, with some studies suggesting it also regulates gene expression (Yuen Science Adv 2017; Iwasaki Nature Comm 2019). Similar to Setd8, many subunits of the Condensin II complex are highly expressed in erythroid cells compared to most other cell types (biogps.org). We hypothesized that the Condensin II complex, in conjunction with Setd8 and H4K20me1, is important for establishing appropriate patterns of chromatin architecture and gene expression in maturing erythroblasts. To address this hypothesis, we deleted the NCAPH2 subunit in erythroid cells by crossing mice with floxed alleles of NCAPH2 with mice expressing cre-recombinase under the direction of the Erythropoietin receptor promotor (EpoRCre). Homozygous disruption of NCAPH2 was embryonic lethal by E13.5. NCAPH2 mutant embryos were similar in appearance to littermate controls until E12.5 when they developed notable pallor and a dramatic decline in the number of benzidine positive cells. Cell cycle analyses demonstrated that an accumulation of cells in G2/M preceded the dramatic decline in erythroblast numbers at E12.5. In contrast to cells from littermate controls, the NCAPH2 mutant cells were very heterogenous in cell and nuclear size and morphology. Surpisingly, most NCAPH2 mutant cells appeared to be hemoglobinized, suggesting sufficient iron accumulation and heme synthesis. In vitro cultures derived from primitive erythroid progenitors replicated in vivo findings, including normal early erythropoeisis, with significant abnormalities during mid- to late- maturation. Western blot in cycloheximide treated primitive erythroid cultures revealed that NCAPH2 has a long half-life, which likely contributes to the relative normalicy of early primitive erythoproesis. NCAPH2 mutant embryos also had a dramatic failure of definitive erythropoiesis, as evidenced by loss of mature erythroblasts in the fetal liver at E13.5. To gain insights into the mechanisms underlying these findings, we performed RNA-seq of NCAPH2 mutant, NCAPH2 het, and NCAPH2 WT erythroblasts from E11.5 embryos. Comparing NCAPH2 mutant and NCAPH2 WT erythroblasts there were 1121 down regulated genes and 743 upregulated genes (adj p-value Disclosures No relevant conflicts of interest to declare.

Interphase chromosomes are organized into topologically associated domains in order to establish and maintain integrity of transcriptional programs that remain poorly understood. Here, we show that condensin II and TFIIIC are recruited to bidirectionally transcribed promoters by a mechanism that is dependent on the retinoblastoma (RB) protein. Long-range chromosome contacts are disrupted by loss of condensin II loading, which leads to altered expression at bidirectional gene pairs. This study demonstrates that mammalian condensin II functions to organize long-range chromosome contacts and regulate transcription at specific genes. In addition, RB dependence of condensin II suggests that widespread misregulation of chromosome contacts and transcriptional alterations are a consequence of RB mutation.

The formation and spatial arrangement of chromosome territories (CTs) in interphase has been posited to influence the outcome and frequency of genomic translocations. This is supported by correlations between the frequency of inter-chromosomal contacts and translocation events in myriad systems. However, it remains unclear if CT formation itself influences the translocation potential of cells. We address this question in Drosophila cells by modulating the level of Condensin II, which regulates CT organization. Using whole-chromosome Oligopaints to identify genomic rearrangements, we find that increased contact frequencies between chromosomes due to Condensin II knockdown leads to an increased propensity to form translocations following DNA damage. Moreover, Condensin II over-expression is sufficient to drive spatial separation of CTs and attenuate the translocation potential of cells. Together, these results provide the first causal evidence that proper CT formation can protect the genome from potentially deleterious translocations in the presence of DNA damage.

Genome instability is an enabling characteristic of cancerous cells. It has recently been discovered that the retinoblastoma protein (pRB), typically known for its role in cell cycle regulation, also aids in the maintenance of genome stability. Intriguingly, mutations to the pRB gene, RB1, can arise late in tumorigenesis in cancer cells whose cell cycle regulation is already compromised by another mutation. This suggests that pRB’s functions in genome stability could underlie cancer relevant characteristics that are independent of its ability to negatively regulate proliferation. The overall aim of this thesis is to characterize the different means through which pRB contributes to the preservation of genome integrity. Using CRISPR/Cas9, isogenic RB1 mutant genotypes were created in a number of cancer cell lines. Cells with at least one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. When the underlying origins of these phenotypes were investigated further, I discovered elevated levels of reactive oxygen species as well as impaired homologous recombination repair in cells with RB1 mutations. When xenografted into immune compromised mice, RB1 mutation also results in an increased capacity to seed new tumors in the lungs. This thesis also investigates the functions of the pRB-condensin II complex in maintaining genome stability, specifically in interphase cells. Using a gene-targeted mouse model that disrupts the ability of pRB to recruit condensin II, Rb1L, locations of pRB-dependent condensin II recruitment were investigated. I found that both condensin II and another architectural protein complex, TFIIIC, are recruited to promoters between bidirectional genes by a mechanism that is reliant on pRB. Recruitment of these architectural proteins at bidirectional promoters is required to establish long-range chromosome interactions and transcriptional insulation between gene pairs. In addition, pRB deletion in cancer causes similar misregulation at divergent promoters, indicating that loss of insulation impacts the transcriptome of cancer cells. Overall, this work demonstrates that beyond altered proliferative control, loss of pRB can also contribute to cancer progression through enhanced DNA damage and altered chromosome topology.

Summary Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells., Graphical Abstract, Highlights • Architecture of ATPγS-bound human condensin II • Human condensin II possesses two putative DNA binding compartments • Human condensins drive robust ATP-dependent compaction of nucleosome-bound DNA • Loop extrusion by human condensins can be symmetric or asymmetric, Kong and Cutts et al. present the general architecture of ATPγS-bound human condensin I and II complexes. They demonstrate that both human condensins are ATP-dependent motors that drive robust compaction of nucleosome-bound DNA, in either a symmetric or asymmetric manner, supporting the loop extrusion model under physiological conditions in higher eukaryotes.

This thesis investigates mechanistic links between genome integrity and the recruitment of chromatin organizing proteins to repetitive DNA sequences mediated by the retinoblastoma tumor suppressor protein (pRB). I demonstrate that a CDK-resistant interaction between the pRB C-terminus and the E2F1 coiled-coil marked box domain establishes a scaffold that facilitates recruitment of multiple chromatin-organizing proteins to repetitive sequences across the genome throughout the cell cycle. Specifically, pRB recruits the enhancer-of-zeste-homologue 2 (EZH2) histone methyltransferase to establish repressive facultative heterochromatin at repetitive sequences, and the Condensin II complex to ensure proper DNA replication and mitotic progression. To disrupt the CDK-resistant pRB-E2F1 interaction in vivo, a gene-targeted mutant mouse strain bearing a germline F832A substitution (Rb1S) is generated. Viable homozygous mutants permit exploration of CDK-resistant pRB-E2F1 functions in cell culture and in vivo. Rb1S/S MEFs and adult splenocytes exhibit pronounced misregulation of endogenous retroviruses (ERVs), long interspersed nuclear elements (LINEs) and major satellites (MaSats). Misexpression is associated with reduced co-occupancy of pRB and EZH2, along with reduced H3K27me3 at repetitive genomic regions but not developmental H3K27me3 targets. Furthermore, Rb1S/S MEFs exhibit increased γH2AX, aneuploidy, ppRPA32, and chromosome segregation errors. γH2AX accumulates specifically at major satellites that exhibit reduced co-occupancy of pRB and Condensin II. Collectively, the consequences of perturbed EZH2 and Condensin II recruitment contribute to a state of genomic instability in Rb1S/S cells that likely underlie the onset of spontaneous lymphomas that arise from the spleen or mesenteric lymph nodes of aged homozygous mutant mice. Finally, I explore whether the pRB-E2F1 scaffold provides an opportunity for therapeutic exploitation, and whether these properties direct alter tumor phenotypes in combination with p53 inactivation. Overall, this work suggests that chromatin-organization mediated through the CDK-resistant pRB-E2F1 complex underscores a previously unknown facet of pRB-mediated tumor suppression.

A modified protocol for inducing reversible assembly of mitotic chromosomes in situ is developed. As judged by this assay, which is combined with quantitative morphological analyses using a supervised machine-learning algorithm, condensin II plays a crucial role in both the recovery of chromatin shapes and the reorganization of chromosome axes., Condensins I and II are multisubunit complexes that play a central role in mitotic chromosome assembly. Although both complexes become concentrated along the axial region of each chromatid by metaphase, it remains unclear exactly how such axes might assemble and contribute to chromosome shaping. To address these questions from a physico-chemical point of view, we have established a set of two-step protocols for inducing reversible assembly of chromosome structure in situ, namely within a whole cell. In this assay, mitotic chromosomes are first expanded in a hypotonic buffer containing a Mg2+-chelating agent and then converted into different shapes in a NaCl concentration-dependent manner. Both chromatin and condensin-positive chromosome axes are converted into near-original shapes at 100 mM NaCl. This assay combined with small interfering RNA depletion demonstrates that the recovery of chromatin shapes and the reorganization of axes are highly sensitive to depletion of condensin II but less sensitive to depletion of condensin I or topoisomerase IIα. Furthermore, quantitative morphological analyses using the machine-learning algorithm wndchrm support the notion that chromosome shaping is tightly coupled to the reorganization of condensin II-based axes. We propose that condensin II makes a primary contribution to mitotic chromosome architecture and maintenance in human cells.

Condensin II interacts with human CENP-A chaperone HJURP and is present at centromeres in early G1. Condensin II, but not condensin I, is required for efficient CENP-A deposition in human cells. HJURP-induced chromatin decondensation at de novo centromeres is counteracted by the activity of condensin II., Centromeric chromatin is required for kinetochore assembly during mitosis and accurate chromosome segregation. A unique nucleosome containing the histone H3–specific variant CENP-A is the defining feature of centromeric chromatin. In humans, CENP-A nucleosome deposition occurs in early G1 just after mitotic exit at the time when the CENP-A deposition machinery localizes to centromeres. The mechanism by which CENP-A is deposited onto an existing, condensed chromatin template is not understood. Here we identify the selective association of the CENP-A chaperone HJURP with the condensin II complex and not condensin I. We show CAPH2 is present at centromeres during early G1 at the time when CENP-A deposition is occurring. CAPH2 localization to early G1 centromeres is dependent on HJURP. The CENP-A chaperone and assembly factor HJURP induces decondensation of a noncentromeric LacO array, and this decondensation is modulated by the condensin II complex. We show that condensin II function at the centromere is required for new CENP-A deposition in human cells. These data demonstrate that HJURP selectively recruits the condensin II chromatin-remodeling complex to facilitate CENP-A deposition in human cells.

Condensins are pentameric complexes that were initially described as being involved in the dynamics of chromosomes during mitosis. It has been recently established that two related complexes (Condensin I and Condensin II) contribute to this process. An increasing sum of studies, using different approaches in various organisms, leads to the paradigm that Condensins are required for the correct segregation of replicated chromosomes by cooperating somehow with Topoisomerase II in sister chromatid resolution. Depending on species and/or experimental studies, these complexes also contribute to some aspects of the assembly and compaction of mitotic chromosomes. Recent studiesprovidedevidencesthatCondensinsandrelatedcomplexesalsofunctioninnon-mitoticprocessessuchasreplicationandtranscription. Biochemical studies have highlighted mechanistic aspects of Condensin function and initiated a fine functional dissection of core and regulatory subunits. However, the exact contribution of each subunit remains largely elusive as well as the functional interplay between Condensin I and Condensin II. © 2004 Elsevier SAS. All rights reserved.

[Figure][1] More CAP-H2 (green) attaches to chromatin (blue) in a control cell (left) than in a cell depleted of Mps1 (right). The kinase Mps1 has a long to-do list during mitosis, and [Kagami et al.][2] identify yet another responsibility: promoting chromosome condensation. The

Neuroblastoma is a disease in which malignant cells form in nerve tissue. Amplification of MYCN, a member of the MYC proto-oncogene family, is frequently observed in neuroblastoma, and it is associated with malignant phenotype and poor prognosis.1 In the April 1, 2014 issue of Cell Cycle, Murakami-Tonami et al.2 reported that downregulation of SMC2, a subunit of condensin, leads to cell death through dysfunction of DNA repair specifically in MYCN-amplified neuroblastoma cells, suggesting that SMC2 can be an effective anti-cancer target in this type of tumor.

[Figure][1] Late S phase sister chromatids (blue) are distinct in a control cell (left) but unresolved in a cell (right) missing a key portion of condensin II (red). The condensin II complex gives cells a head start on mitosis by helping to separate sister chromatids during S phase, [Ono

[Figure][1] The N terminus of MCPH1 (green) and condensin II (red) occupy mutually exclusive positions on a chromosome. A protein that’s defective in patients with reduced brain size helps prevent untimely chromosome condensation, [Yamashita et al.][2] show. Babies born with autosomal