RUNX1 has recently been shown to play an important role in determination of mammary epithelial cell identity. However, mechanisms by which loss of the RUNX1 transcription factor in mammary epithelial cells leads to epithelial-to-mesenchymal transition (EMT) are not known. Here, we report that interaction between RUNX1 and its heterodimeric partner CBFβ is essential for sustaining mammary epithelial cell identity. Disruption of RUNX1-CBFβ interaction, DNA binding, and association with mitotic chromosomes alters cell morphology, global protein synthesis, and phenotyperelated gene expression. During interphase, RUNX1 is organized as punctate, predominantly nuclear, foci that are dynamically redistributed during mitosis, with a subset localized to mitotic chromosomes. Genome-wide RUNX1 occupancy profiles for asynchronous, mitotically enriched, and early G1 breast epithelial cells reveal RUNX1 associates with RNA Pol II-transcribed protein coding and long non-coding RNA genes and RNA Pol I-transcribed ribosomal genes critical for mammary epithelial proliferation, growth, and phenotype maintenance. A subset of these genes remains occupied by the protein during the mitosis to G1 transition. Together, these findings establish that the RUNX1-CBFβ complex is required for maintenance of the normal mammary epithelial phenotype and its disruption leads to EMT. Importantly, our results suggest, for the first time, that RUNX1 mitotic bookmarking of a subset of epithelial-related genes may be an important epigenetic mechanism that contributes to stabilization of the mammary epithelial cell identity.

CT10 regulator of kinase (CRK) and CRK-like (CRKL) form a family of signaling adaptor proteins that serve important roles in the regulation of fundamental cellular processes, including cell motility and proliferation, in a variety of cell types. The Src Homology 2 (SH2) domain of CRK and CRKL interacts with proteins containing phosphorylated tyrosine-X-X-proline (pYXXP) motifs, facilitating complex formation during signaling events. A handful of CRK/CRKL-SH2-specific interactors have been identified to date, although in silico analyses suggest that many additional interactors remain to be found. To identify CRK/CRKL-SH2 interactors with potential involvement in neuronal development, we conducted a mass spectrometry-based proteomics screen using a neuronal cell line (Neuro2A, or N2A). This resulted in the identification of 132 (6 known and 126 novel) YXXP-containing CRK/CRKL-SH2 interactors, of which 77 were stimulated to bind to the CRK/CRKL-SH2 domain following tyrosine phosphatase inhibition. Approximately half of the proteins identified were common interactors of both the CRK- and CRKL-SH2 domains. However, both CRK family member SH2 domains exhibited unique binding partners across experimental replicates. These findings reveal an abundance of novel neuronal CRK/CRKL-SH2 domain binding partners and suggest that CRK family SH2 domains possess undescribed docking preferences beyond the canonical pYXXP motif. KEYWORDS: CRK; CRKL; SH2; LC-MS/MS; Proteomics; Neurodevelopment; Signal Transduction

Key Words: Mammary phenotype, epithelial phenotype, RUNX1, mitotic gene 23 bookmarking. 24 25 26 2Significance: 27 This study elucidates mitotic gene bookmarking as a potential epigenetic 28 mechanism that impacts breast epithelial cell growth and phenotype and has potential 29 implications in breast cancer onset. 30 3 ABSTRACT 31RUNX1 has recently been shown to play an important role in determination of 32 mammary epithelial cell identity. However, mechanisms by which loss of the RUNX1 33 transcription factor in mammary epithelial cells leads to epithelial-to-mesenchymal 34 transition (EMT) are not known. Here, we report mitotic bookmarking of genes by RUNX1 35 as a potential mechanism to convey regulatory information through successive cell 36 divisions for coordinate control of mammary cell proliferation, growth, and identity. 37 Genome-wide RUNX1 occupancy profiles for asynchronous, mitotically enriched, and 38 early G1 breast epithelial cells reveal RUNX1 is retained during the mitosis to G1 39 transition on protein coding and long non-coding RNA genes critical for mammary 40 epithelial proliferation, growth, and phenotype maintenance. Disruption of RUNX1 DNA 41 binding and association with mitotic chromosomes alters cell morphology, global protein 42 synthesis, and phenotype-related gene expression. Together, these findings show for the 43 first time that RUNX1 bookmarks a subset of epithelial-related genes during mitosis that 44 remain occupied as cells enter the next cell cycle. Compromising RUNX1 DNA binding 45 initiates EMT, an essential first step in the onset of breast cancer. 46 47 48Breast cancer arises from a series of acquired mutations and epigenetic changes 49 that disrupt normal mammary epithelial homeostasis and create multi-potent cells that 50 can differentiate into biologically unique and clinically distinct subtypes (1-6). Epithelial-51 to-mesenchymal transition (EMT)-a trans-differentiation process through which 52 mammary epithelial cells acquire the aggressive mesenchymal phenotype-is a key 53 driver of breast cancer progression, invasion and metastasis (7-12). Transcription factors 54 Snail, Slug, Twist, and Zeb1/2 contribute to EMT during early, normal development and 55 have also been implicated in invasion (13)(14)(15)(16). Despite accumulating evidence that 56 defines a broad understanding of EMT regulation and maintenance of the epithelial 57 phenotype (7-12), the mechanism(s) by which mammary cells maintain their epithelial 58 phenotype is unknown. 59Runt-Related Transcription Factor 1 (RUNX1/AML1) is required for hematopoietic 60 lineage specification during development and hematopoiesis throughout life (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). In 61 addition to the recognized role in hematological malignancies, RUNX1 has been recently 62 identified as a key player in breast cancer development and tumor progression (31-38). 63Findings from our group (39), reinforced by studies from others (40, 41), have shown that 64 RUNX1 plays a critical role in maintaini...