Friday, May 10, 2024
5/10/2024
Project summary: The goal of this proposal is to reveal mechanisms by which gene regulatory proteins overcome chromosome structural barriers, to change cell fates. The work will provide foundational insight into gene regulation and more efficient ways to generate new cell types for therapy and disease modeling. The proposal extends from 38 years of research on my R01 grant, to dissect the interplay between pioneer transcription factors and initially targeted chromatin, combined with new directions from my recently expired P01 grant, where we revealed the functional complexity of mammalian heterochromatin states. Work on both grants revealed that after the initial closed chromatin binding by pioneer factors, a rate-limiting step of cell reprogramming is the inefficiency of activating genes in H3K9me3 heterochromatin. We defined pioneer transcription factors by their ability to target a DNA motif, or a partial motif, on a nucleosome, and thereby initiate cooperative events in DNase/ATAC-resistant, transcriptionally silent chromatin bound by linker histone. Yet despite the ability to target linker histone- compacted chromatin in vitro and in vivo, we and others found that some, but not all, pioneer factors are impeded from binding H3K9me3 heterochromatin, leading us to investigate how certain pioneer factors can target such heterochromatin. In the past grant period, our biochemical, structural, single molecule tracking, genomic, and long-read sequencing approaches revealed that pioneer factors interact with core histones to facilitate nucleosome binding and chromatin opening, that pioneer factors use unstructured domains for opening and linker histone displacement, and that a pioneer factor licenses a nucleosome remodeler to further open local chromatin. Having genetically identified amino acids on pioneer factors for binding nucleosomes, not free DNA, in vitro allows us to assess, in vivo, truly pioneering events by the factors. We discovered diverse proteins functionally bound to heterochromatin that repress genes and DNA repeats and are associated with varying H3K9me3 and H3K27me3 marks, thereby defining heterochromatin subtypes. We propose to categorize pioneer factors by the subtypes of heterochromatin that they target. We will use transcription factor binding to in vitro heterochromatin reconstituted on nucleosome arrays, pulldowns between transcription factors and biochemically fractionated heterochromatin, and genetic approaches in mice and cell cultures to define protein domains on pioneer factors that enable heterochromatin subtype targeting and local chromatin opening, and that can be transferred to other proteins to enhance reprogramming. We will use our dual-degron technology and knock- downs to reveal how mammalian H3K9me3 heterochromatic domains are established and can be disassembled selectively, to enhance reprogramming. We will determine the basis for, and utility of, our discovery of a transient global disruption of heterochromatin elicited by reprogramming pioneer factors. The work will be performed in a research environment that promotes diversity, equity, and inclusion. Our approaches will reveal basic genetic mechanisms and will be applied to improving directed cell fate changes for biomedical purposes.
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