Transcription Factor CTCF Inhibits Effects of 5-Azacitidine in MDS/AML Cells
Authors: Kapalova Martina , Burda Pavel , Vargova Karin , Savvulidi Filipp , Zikmund Tomas , Curik Nikola , Jonasova Anna , Stopka Tomas 
Published: October 2019

Abstract
Abstract 3848

Introduction:
5-azacitidine (AZA) represents very promising albeit not fully efficient therapy for int-2 and high risk MDS patients. Molecules that interfere with AZA therapy are not known. In significant proportion of MDS patients, PU.1 gene is methylated at −17-kb-located upstream regulatory element (URE) where several key transcription factors regulate PU.1 expression. PU.1 represents major factor that controls normal myeloid differentiation. Methylated URE in MDS progenitors can be efficiently demethylated by AZA leading to restoration of cell differentiation capacity (Curik et al 2012). PU.1 gene contains several binding sites for transcription factor CTCF. CTCF represents very important modulator of gene expression, whose binding to DNA can be prevented by DNA methylation. We herein asked if CTCF regulates PU.1 and if so, whether its association with PU.1 gene coincides with DNA methylation status of MDS blasts.

Methods:
Human high risk MDS patient CD34+ progenitors and MDS-derived erytroleukaemia OCI-M2 and murine erythroleukaemia cell (MEL) lines were studied by RT-PCR, immunoblotting, and chromatin immunoprecipitation (ChIP) assays. Manipulation of gene expression was done by transfection of cDNA or siRNA.

Results:
We herein show that CTCF binding sites at PU.1 gene similarly to URE are severely methylated in CD34+ progenitors from high risk MDS patients and MDS-derived erytroleukaemia cell line, and as expected, AZA induced their rapid demethylation. Methylated CTCF binding sites are not occupied by CTCF. However upon AZA-mediated demethylation, CTCF is recruited to the binding sites at PU.1 gene as determined by ChIP. Our other data provided evidence that CTCF interacts with the ISWI ATPse SNF2H (SMARCA5). Indeed, the recruitment of CTCF at PU.1 gene in MDS/AML cells was coincident with recruitment of its interacting partner SMARCA5. In addition, SMARCA5 facilitates CTCF binding to the DNA as demonstrated at ICR locus (near H19 and Igf2 genes) upon siRNA-mediated downregulation of SMARCA5. To understand role of CTCF-SMARCA5 recruitment to the PU.1 gene and its effects on PU.1 expression we upregulated CTCF expression by transfecting an expression plasmid encoding CTCF cDNA and observed that upon increasing CTCF levels the PU.1 protein level was downregulated. Conversely, downregulation of SMARCA5 by siRNA caused upregulation of PU.1 levels. These data indicated that PU.1 is negatively regulated by CTCF and SMARCA5. Furthermore, inhibitory effects of CTCF and SMARCA5 on PU.1 expression were also demonstrated in presence of AZA in MDS cells following DNA demethylation of PU.1 gene.

Conclusion:
Our results indicate that CTCF and SMARCA5 are cooperating inhibitory factors to downregulate PU.1 and that AZA-mediated demethylation facilitates the CTCF-SMARCA5 binding to PU.1 gene in MDS patients. CTCF and SMARCA5 are novel factors that interfere with positive prodifferentiation effects of AZA. (Grant support: P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012–264507, P301/12/P380, GAUK 251070 45410 and 251135 82210).

Disclosures:
No relevant conflicts of interest to declare.