Chromosome structure and antisense transcription in leukemia

Blood development is regulated by levels of transcription factor PU.1 (encoded as SPI1/Sfpi1). During myeloid differentiation PU.1 levels need to increase to avoid a differentiation block, which would lead to leukemia. In contrast PU.1 expression needs to stop completely to develop T-cells. So far the mechanisms of PU.1 suppression, physiologic as for T-cell differentiation or pathologic as for leukemia development are unknown. We here hypothesize that expression of a long noncoding antisense RNA plays a central role in silencing the expression of PU.1. We further hypothesize that specific 3-dimensional chromosome architectures facilitate expression of either PU.1 mRNA or PU.1 antisense transcription by locating distal enhancer- or modifier- segments either to the proximal or the antisense promoter. Our preliminary data suggest that the tight interplay between Runx factors and PU.1 directly influences PU.1 antisense expression. Runx function is frequently affected in various leukemias such as the core-binding factor (CBF) leukemias t(8;21) and inv(16) and, importantly, PU.1 is functionally deficient in these diseases. We thus hypothesize that fusion oncoproteins of CBF leukemias hijack a mechanism, which is required for normal T-cell development. They establish a higher-order chromatin structure leading to PU.1 antisense transcription and active PU.1 silencing. Former studies have mainly focused on mechanism how transcription factors are up-regulated and regulation of PU.1 is particularly well studied in this respect. Here, we aim to study the opposite suggesting that silencing transcription factor PU.1 is an active process that requires a specific chromosome formation and transcription of a non-coding antisense transcript. We propose to pursue the following specific aims: Aim 1. PU.1 mRNA / antisense expression and spatial chromosome organization. Within this Aim we will provide a comprehensive picture of coding/non-coding transcription and spatial chromosome organization which will serve as blueprint for aim 2 and 3. (A) We first will perform a genome-wide chromatin interaction screen to capture distal genetic regions spatially associated with the PU.1 antisense and proximal promoter. (B) Then we will evaluate PU.1 mRNA / antisense expression and spatial chromosome organization in normal hematopoiesis and leukemia patients. Aim 2. Competitive promoter model: Mechanism of spatial chromosome regulation and antisense transcription. Here we aim to establish mechanistic model systems that will allow us to investigate the role of PU.1 antisense transcription in respect to chromosome conformation changes and vice versa. Aim 3. Function of PU.1 antisense transcription in hematopoietic differentiation and leukemia. Using the murine models developed in aim2 (conditional PU.1 antisense promoter knockouts, mutated Runx site knockins) we will reveal (A) the functional role of PU.1 antisense expression during hematopoiesis and (B), whether and how the driving mutations of core-binding factor (CBF) leukemias, AML1-ETO (t8;21) and CBFb-MYH11 (inv16) mediate leukemogenesis through PU.1 antisense expression.