The N-terminus of Stag1 is required to repress the 2C program by maintaining rRNA expression and nucleolar integrity

Our understanding of how STAG proteins contribute to cell identity and disease have largely been studied from the perspective of chromosome topology and protein-coding gene expression. Here, we show that STAG1 is the dominant paralog in mouse embryonic stem cells (mESCs) and is required for pluripotency. mESCs express a wide diversity of naturally occurring Stag1 isoforms, resulting in complex regulation of both the levels of STAG paralogs and the proportion of their unique terminal ends. Skewing the balance of these isoforms impacts cell identity. We define a novel role for STAG1, in particular its N-terminus, in regulating repeat expression, nucleolar integrity, and repression of the two-cell (2C) state to maintain mESC identity. Our results move beyond protein-coding gene regulation via chromatin loops to new roles for STAG1 in nucleolar structure and function, and offer fresh perspectives on how STAG proteins, known to be cancer targets, contribute to cell identity and disease

Enhancer accessibility and CTCF occupancy underlie asymmetric TAD architecture and cell type specific genome topology.

Cohesin and CTCF are master regulators of genome topology. How these ubiquitous proteins contribute to cell-type specific genome structure is poorly understood. Here, we explore quantitative aspects of topologically associated domains (TAD) between pluripotent embryonic stem cells (ESC) and lineage-committed cells. ESCs exhibit permissive topological configurations which manifest themselves as increased inter- TAD interactions, weaker intra-TAD interactions, and a unique intra-TAD connectivity whereby one border makes pervasive interactions throughout the domain. Such 'stripe' domains are associated with both poised and active chromatin landscapes and transcription is not a key determinant of their structure. By tracking the developmental dynamics of stripe domains, we show that stripe formation is linked to the functional state of the cell through cohesin loading at lineage-specific enhancers and developmental control of CTCF binding site occupancy. We propose that the unique topological configuration of stripe domains represents a permissive landscape facilitating both productive and opportunistic gene regulation and is important for cellular identity

Enhancer accessibility and CTCF occupancy underlie asymmetric TAD architecture and cell type specific genome topology.

Cohesin and CTCF are master regulators of genome topology. How these ubiquitous proteins contribute to cell-type specific genome structure is poorly understood. Here, we explore quantitative aspects of topologically associated domains (TAD) between pluripotent embryonic stem cells (ESC) and lineage-committed cells. ESCs exhibit permissive topological configurations which manifest themselves as increased inter- TAD interactions, weaker intra-TAD interactions, and a unique intra-TAD connectivity whereby one border makes pervasive interactions throughout the domain. Such 'stripe' domains are associated with both poised and active chromatin landscapes and transcription is not a key determinant of their structure. By tracking the developmental dynamics of stripe domains, we show that stripe formation is linked to the functional state of the cell through cohesin loading at lineage-specific enhancers and developmental control of CTCF binding site occupancy. We propose that the unique topological configuration of stripe domains represents a permissive landscape facilitating both productive and opportunistic gene regulation and is important for cellular identity

Cohesin-independent STAG proteins interact with RNA and localise to R-loops to promote complex loading

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA and are localised to R-loops where they contribute to R-loop regulation. Our results place SA1 within R-loop domains upstream of the cohesin complex and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease

Cohesin-independent STAG proteins interact with RNA and R-loops and promote complex loading

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, RNA binding proteins and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease

Runx proteins regulate Foxp3 expression

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes

Cohesin-mediated interactions organize chromosomal domain architecture

To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase-scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here, we show that very specific long-range interactions are anchored by cohesin/CTCF sites, but not cohesin-only or CTCF-only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post-mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin-dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin-bound and non-bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programmes within them. Chromosomal compartmentalization has been recognized as important for genome function. High-resolution techniques such as Hi-C, ChIP- and 4C-seq offer novel insights into cohesin's dynamic role in shaping the nuclear architecture

Ueber die Wasserstoffionenkonzentration an den Sekreten und Schleimhauten bei Nasen-Kieferhohlen- und Rachenerkrankungen

Auf dem Oto-Rhino-und Laryngologischen Gebiet sind bisher physikalisch-chemische Untersuchungen sehr wenig erforscht worden, besonders liegen hier nur sparliche Studien uber das Verhalten der Wasserstoffionenkonzentration vor. Ich habe mit Hilfe der Chinhydronelektrode das PH in den Sekreten bei Rhinitis chronica, Sinuitis maxillaris chronica gemessen, sowie an den Hohlenschleimhauten bei Sinuitis maxillaris chronica, gleichfalls in den Gewebssaften bei Gaumen- und Rachen-tonsillitiden bestimmt. Aus den erhaltenen PH-Werten in Zusammenhang mit dem Krankheitsverlauf lassen sich folgende Schlusse ziehen: 1. PH-Werte: In Nasensekreten bei Rhinitis Katarrhalis chronica 7.68-8.10, wahrend als normales PH bei Gesunden 8.10-8.33 festgestellt wurde. An den Krusten bei Rhinitis atrophicans chronica betrug das PH 6.79-7.35, dagegen an den unterhalb der Krusten sich befindlichen weichen Sekreten PH 7.52-8.0.2. 2. Die Staungssekrete bei Sinuitis maxillaris chronica zeigten ein PH 6.19-8.09. In rein eitrigen Sekreten bei eitriger Form der Erkrankung fand eine Verschiebung nach der sauren, in den schleimigen bei katarrhalischer Form nach der alkalischen Seite hin statt, und bei den ersteren befanden sich die Eiterzellen in hochgradigen Regressionszu-standen, welche Prozesse bei den letzteren verhaltnissmassig geringfugig waren. An den klar gelblichen, flussigen Sekreten bei Zysten und Hydrops bekam man PH=7.39-7.45, ein Alkalitatsgrad, welcher dem PH des Blutes bei normalen Menschen entspricht. Es ist also anzunehmen, dass der PH-Wert der Sekrete das Ergebnis der Mischung der einzelnen Komponenten der Absonderung (Eiter, Schleim, Exsudat) ist und gleichzeitig von der Starke der regressiven Degeneration der Eiterzellen abhangt. 3. Mit der Erleichterung der Symptome durch geeignete Hohlenspulung bei Sinuitis maxillaris chronica gehen die regressiven Prozesse an den Eiterzellen auch zuruck, steigen die PH-Werte bis etwas 8.0 an, dann pflegt das Leiden allmahich zur Heilung zu kommen. Die PH-Betrage und die Starke der regressive

The role of reactive oxygen and nitrogen species in the development of Fanconi Anemia, an inherited bone marrow failure disorder

The objective of this thesis was to investigate the role of endogenous reactive oxygen and nitrogen species in the pathogenesis of Fanconi's Anemia (FA) using Fanconi Anemia Complementation Group C (Fancc) - deficient mice. This objective was examined in two distinct ways. First, through the generation and characterization of a novel mouse model for FA with intrinsic oxidant stress and secondly, through the observation that nitric oxide (NO) may have a role in cytokine - mediated inhibition of hematopoiesis in FA. FA is an autosomal recessive disorder, which primarily affects children and young adults, resulting in morbidity and mortality due to B M failure or acute myelogenous leukemia (AML). Currently, eight complementation groups have been identified and six FA genes have been cloned. Although knockout mice have been generated for each of the genes cloned, they do not exhibit the primary hematopoietic defect of FA. Thus, no spontaneously occurring mouse model for FA exists with which to better define the pathogenesis of this disease. Several lines of evidence have pointed to abnormal regulation of intracellular reactive oxygen species (ROS) in individuals with FA. To investigate the possible role of the Fancc protein in the regulation of an in vivo redox state, mice were generated having combined deficiencies in the genes encoding the cytoplasmic antioxidant, Cu/Zn superoxide dismutase (Sod1) and Fancc. Fancc⁻[sup /]⁻Sod1⁻[sup /]⁻ mice developed hepatic lipid accumulation, peripheral blood bicytopenia, marrow hypocellularity, little to no growth of committed progenitor cells in vitro, and decreased frequencies of long-term progenitors. This novel murine model of FA partially replicates the hematopoietic defect of this disease and may be useful in defining novel therapies. The second theme of this thesis involved the observation that NO may have a role in FA BM failure. Cytokine inhibition of hematopoietic progenitor colony growth from Fancc⁻[sup /]⁻ mice was completely rescued in the presence of an iNOS inhibitor, L-NMMA. Fancc⁻[sup /]⁻ progenitor cells were hypersensitive to NO generating drugs in vitro while primary macrophages had elevated expression of iNOS and NO production in response to IFNγ and IFNγ/LPS. To date, no information exists regarding FA and NO and these studies have opened a new avenue of investigation in FA research.Medicine, Faculty ofMedical Genetics, Department ofGraduat