Signal-dependent control of cell differentiation by E protein transcription factors

点击翻译按钮获取中文摘要

The generation of different cell types during animal development is regulated by the interplay between cell signaling cascades and transcription factor activity. E protein transcription factors  are encoded in vertebrates by three gene loci: E2A (Tcf3), HEB (Tcf12) and E2--2 (Tcf4). E proteins function as dimers with themselves, each other, or other basic helix--loop--helix (bHLH) factors. We and others have found that the loci encoding HEB and E2-2 generate both canonical (Can) and alternative (Alt) E proteins, whereas E2A exists only in the canonical form. Our studies have shown that HEBAlt and HEBCan play distinct roles in T cell development, but otherwise very little is known about E-Alt proteins. During the last NSERC Discovery grant, we discovered that the unique Alt domain of E--Alt proteins contains a tyrosine phosphorylation motif that is required for optimal function, revealing a new node of interaction between E proteins and signaling pathways. We also discovered that E--Alt proteins are present in animals that lack T cells. This discovery prompted us to conduct an in--silico screen for open chromatin marks near the Alt exon, and found them in pre-adipocytes, neural progenitors, and muscle precursors, suggesting a role for HEBAlt in development of these cell types. We hypothesized that E--Alt proteins may be able to prime E-protein responsive genes for expression while holding them in check until a differentiation signal is received. Even more exciting, we have discovered that "master regulators" of distinct lineages bind to the Alt promoters, suggesting that E--Alt mRNAs can be induced as part of an early wave of lineage--specific genes. Here we propose to identify and characterize interaction partners of E--Alt proteins, evaluate the requirements for the Alt domain in muscle, neural, and adipocyte lineages, and establish the relationship between E--Alt binding of DNA and chromatin accessibility. In Aim 1, we will use cells expressing HA-tagged HEBAlt WT or mutant proteins to perform co-precipitation, followed by mass spectrometry sequencing and verification of partners by Western blots. In Aim 2, we will isolate precursors from knockout (loss of function) or inducible transgenic mice (gain of function) and evaluate their differentiation in vitro. Aim 3 will focus on acquiring ChIP-seq and RNA-seq data to determine the sites of E-Alt binding in the precursors of different lineages, the chromatin configuration at those sites, and the status of mRNA expression from those loci. Due to our prior studies, we have abundant tools and the track record to study HEBAlt, and we have also assembled a team of collaborators with diverse expertise to investigate its role outside the immune system. These studies will lead to a new understanding of how the multi-step process of lineage specification and differentiation occurs, and may provide a new paradigm in developmental biology.