Page 27 - Hormel Report 2021

recreating these mutations occurring in cancer cells in chromosomally normal human cells using CRISPR Cas9 and Cre-LoxP to determine the effect of cen- tromere instability on centromere assembly and func- tion and chromosome segregation. These studies will help us to understand the role of centromere deletion in genome instability and aneuploidy seen in cancer. In addition, we study the function of centromeric proteins in Cancer.
Centromeres in Scleroderma
We have a special attention on the function of centromeres in Scleroderma. The contribution of centromere defects to Scleroderma remain unknown despite many of the centromere proteins were discovered due to these patient-derived antibodies. Using novel genetic and cytogenetic analysis we find that affected fibroblasts from SSc patients show marked alterations in centromeric DNA regardless
of receiving treatment. Strikingly, we observed “leak-
ing” of centromere proteins from the nucleus into
the cytoplasm in all limited cutaneous Scleroderma patients who have anti-centromere antibodies. Scleroderma fibroblasts showed abnormal chromo-
some segregation, with micronuclei that activates the cGAS-STIN pathway of autoimmunity. Surprisingly, several micronuclei no longer recruit the centromere identity protein CENPA, despite they still retain centromere
sequences. Our studies reveal that centromeric genomic instability and epigenetic defects may lead to the pathogenesis of Scleroderma. As both centromere genetics and epigenetics defects, together with an immune response to centromeres
is seen in Scleroderma patients, we are clarifying whether Scleroderma is a centromere disease with very promising find- ings. The projects are currently supported by the Scleroderma Foundation and the Rheumatology Research Foundation.
Figure 1: Generation of neurons from iPSCs. CHON-002 fibroblasts were reprogrammed
to iPSCs by transducing with Yamanaka factors (OKSM). iPSCs were grown on AggreWellTM
800 for 1 week in neuronal induction media to generate embryoid bodies. Embryoid bodies were replated and maintained for 2 weeks on poly-L-Ornithine/Laminin coated plate to generate neuronal rosettes. Selected neuronal rosettes were replated and maintained for 2 weeks on poly-L-Ornithine/Laminin coated plate with neuronal progenitor medium to generate neuronal precursor cell. Neuronal precursor cells were cultured and maintained for 2 weeks on neuronal differentiation media. Differentiated neurons were maintained on neuronal matura- tion media for 10 days to generate mature neurons. Mature neuronal cultures were stained with different antibodies specific to different types of neurons, astrocytes and oligodendrocytes.
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Publication:
• Saha AK, Contreras-Galindo R, Niknafs YS, Iyer M, Qin T, Padmanabhan K, Siddiqui J, Palande M, Wang C, Qian B, Ward E, Tang T, Tomlins SA, Gitlin SD, Sartor MA, Omenn GS, Chinnaiyan AM, Markovitz DM. The role of the histone H3 variant CENPA in prostate cancer. J Biol Chem. 2020 Jun 19;295(25):8537-8549. doi: 10.1074/jbc. RA119.010080. PMID: 32371391