Vertebrate centromeres in mitosis are functionally bipartite structures stabilized by cohesin

Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation

Epigenetic Signatures of Genes and Their Correlations with Various Signaling Pathways During Tumorigenesis

During the past 15 years the epigenetic regulation of gene has been studied extensively. The genesis of cancer and epigenetic regulation of genes are deeply interconnected. DNA methylation, histone tail modification, nucleosome remodelling, and non-coding RNA regulate many biological processes that are elementary to the cancer development. This thesis evaluates the expression profile and epigenetic regulation (especially, DNA methylation and histone H3 modifications) of various genes, such as caveolin 1 (CAV1), clusterin (CLU), beta 1 integrin (β1 integrin), histone H3.3 and chromatin modifying enzymes like DNA methyltransferases (DNMTs), histone methyltransferases (HMTs) and histone acetyltransferases (HATs) in colon and breast cancers. Additionally, lipid raft, RAS/MEK/ERK and FAK signaling pathway mediated regulation of histone modifications in colon cancer is also examined. mRNA and protein level analysis of respective gene products in cancer tissue samples and cell lines demonstrated that CAV1 is expressed in a stage-specific manner whereas nuclear CLU (nCLU) is down-regulated; whereas, secretory CLU (sCLU), β1 integrin and H3.3 genes are up-regulated in both the cancers. Inhibition of cell growth in breast and colon cancer cell lines after treatment with epigenetic modulators is associated with up-regulation of CAV1 and nCLU; ultimately resulting in down-regulation of β1 integrin. On investigating the epigenetic regulatory mechanisms of CAV1 gene, it is observed that histone modifications (H3K4me3, H3K9me3, H3K9AcS10p) predominantly regulate CAV1 expression in both cancers whereas promoter DNA methylation is partially responsible in case of only breast cancer. Moreover, expression of CLU is associated with global histone marks in case of breast cancer. In colon cancer, promoter H3K4me, H3K9me3 and H3K9AcS10p enrichment is the predominant regulator of CLU gene expression. Along with expression pattern and epigenetic regulation this thesis also evaluated the involvement of different signaling responsible for histone modifications. The modulation of global and gene specific histone marks in colon cancer is demonstrated to be regulated by lipid raft, RAS/MEK/ERK and FAK signaling pathways. These pathways influence global H3K4me3, H3K9me3, H3K9AcS10p levels and in turn differentially regulate gene specific expression, such as that of CAV1

DNA methylation regulates Microtubule-associated tumor suppressor 1 in human non-small cell lung carcinoma

Microtubule associated tumor suppressor 1 (MTUS1) has been recognized as a tumor suppressor gene in multiple cancers. However, the molecular mechanisms underlying the regulation of MTUS1 are yet to be investigated. This study aimed to clarify the significance of DNA methylation in silencing MTUS1 expression. We report that MTUS1 acts as tumor suppressor in non-small cell lung carcinoma (NSCLC). Analysis of in silica database and subsequent knockdown of DNMT1 suggested an inverse correlation between DNMT1 and mars]. function. Interestingly, increased methylation at MTUS1 promoter is associated with low expression of MTUS1. Treatment with DNA methyltransferases (DNMTs) inhibitor, 5-aza-2'-deoxycytidine (AZA) leads to both reduced promoter methylation accompanied with enrichment of H3K9Ac and enhanced MTUS1 expression. Remarkably, knockdown of MTUS1 showed increased proliferation and migration of NSCLC cells in contrast to diminished proliferation and migration, upon treatment with AZA. We concluded that low expression of MTUS1 correlates to DNA methylation and histone deacetylation in human NSCLC

Myeloid Krüppel-Like Factor 2 Critically Regulates K/BxN Serum-Induced Arthritis

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease, and Krüppel-like factor 2 (KLF2) regulates immune cell activation and function. Herein, we show that in our experiments 50% global deficiency of KLF2 significantly elevated arthritic inflammation and pathogenesis, osteoclastic differentiation, matrix metalloproteinases (MMPs), and inflammatory cytokines in K/BxN serum-induced mice. The severities of RA pathogenesis, as well as the causative and resultant cellular and molecular factors, were further confirmed in monocyte-specific KLF2 deficient mice. In addition, induction of RA resulted in a decreased level of KLF2 in monocytes isolated from both mice and humans along with higher migration of activated monocytes to the RA sites in humans. Mechanistically, overexpression of KLF2 decreased the level of MMP9; conversely, knockdown of KLF2 increased MMP9 in monocytes along with enrichment of active histone marks and histone acetyltransferases on the MMP9 promoter region. These findings define the critical regulatory role of myeloid KLF2 in RA pathogenesis

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H2O2) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H2O2 treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H2O2 was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H2O2 induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDHL This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer