Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1)

Abstract

SPI1 (also known as PU.1) is a dominant but transient regulator in early T-cell precursors and a potent transcriptional controller of developmentally important pro-T-cell genes. Before T-lineage commitment, open chromatin is frequently occupied by PU.1, and many PU.1 sites lose accessibility when PU.1 is later down-regulated. Pioneering activity of PU.1 was tested in this developmentally dynamic context by quantitating the relationships between PU.1 occupancy and site quality and accessibility as PU.1 levels naturally declined in pro-T-cell development and by using stage-specific gain- and loss-of-function perturbations to relate binding to effects on target genes. PU.1 could bind closed genomic sites, but rapidly opened many of them, despite the absence of its frequent collaborator, CEBPA. RUNX motifs and RUNX1 binding were often linked to PU.1 at open sites, but highly expressed PU.1 could bind its sites without RUNX1. The dynamic properties of PU.1 engagements implied that PU.1 binding affinity and concentration determine its occupancy choices, but with quantitative trade-offs for occupancy between site sequence quality and stage-dependent site accessibility in chromatin. At nonpromoter sites, PU.1 binding criteria were more stringent than at promoters, and PU.1 was also much more effective as a transcriptional regulator at nonpromoter sites where local chromatin accessibility depended on the presence of PU.1. Notably, closed chromatin presented a qualitative barrier to occupancy by the PU.1 DNA-binding domain alone. Thus, effective pioneering at closed chromatin sites also depends on requirements beyond site recognition, served by non-DNA-binding domains of PU.1.

Requirement of DNMT1 to orchestrate epigenomic reprogramming during NPM-ALK driven T cell lymphomagenesis

“…Epigenetic switching from polycomb repressive complex (PRC) marks to DNA methylation is a well-described phenomenon in human tumor cells reducing epigenetic plasticity of tumor cells (9), (63). Furthermore, T lymphocyte specific binding of the tumor suppressor MEN1, which has a role for Th2 cell function and prevents CD8+ T cell dysfunction (64,65), SPI1, a master regulator of early T cell transcription (66) and BATF, a AP-1 family member, which has been implicated in human ALCL growth and survival (67) were significantly overlapping with hypermethylated sites in ALK mouse tumors. For hypomethylated regions, we detected an overlap of ESC specific TFE3 binding sites.…”

Causal Gene Regulatory Network Modeling and Genomics: Second-Generation Challenges

“…The excitement about ''epigenetic states'' relative to developmental gene network models comes from evidence that these features may also persist and constrain future transcription factor activity (Arvey et al, 2012;Spitz and Furlong, 2012;Zhang et al, 2012;Chen and Dent, 2014;Bintu et al, 2016;Kribelbauer et al, 2017;Liu et al, 2018;Ungerbäck et al, 2018). However, the key questions about each of these chromatin-modifying mechanisms, with respect to their contributions to developmental gene regulation, have to do with their rates of establishment and their reversibilities or resistances to being reversed, when transcription factor activities change.…”

“…Importantly, the high-level expression effects are not simple ''overexpression toxicity,'' but rather a shift in developmental preference of the cell to a different, but coherent, cell fate. PU.1 binding to genomic sites in vivo appears to follow an orderly relationship of factor concentration to site-recognition affinity, and it even occupies sites with low accessibilities in chromatin if the site sequences are more optimal or if its own factor levels are higher (Ungerbäck et al, 2018). Thus, increasing levels of this factor should directly result in occupancy of an expanded set of genomic target sites, but a well-defined set, and these would be expected to account for the appearance of new responses to PU.1 when its levels rise above certain thresholds.…”

“…As just summarized, it is likely to be a network property based on activation of different targets at higher factor doses that alter the expression or impacts targets activated at lower doses. However, the closely analyzed example of PU.1 indicates that a ''closed'' state of chromatin can also raise the (affinity · concentration) threshold required for binding (Ungerbäck et al, 2018), whereas histone modifications such as H3K27 trimethylation may exclude its local binding entirely (Zhang et al, 2012;Ungerbäck et al, 2018). Thus, a chromatin configuration inherited from a prior regulatory state is an effective way to change the dose range over which a newly expressed transcription factor can gain access to a particular subset of its potential target sites, as compared with other sites that would otherwise be recognized with the same affinity.…”

Multi-scale dynamical modelling of T-cell development from an early thymic progenitor state to lineage commitment

“…Potential drivers of commitment include TCF1 (Tcf7), Gata3, and Runx1, which are necessary positive inputs for Bcl11b expression 24 , while PU.1 (Spi1) antagonizes developmental progression through commitment 23,40 . To determine the normal ranges of expression of Spi1, Tcf7, Runx1 and Gata3 in individual T-cell precursors in developmental stages leading up to Bcl11b activation for the model, DN thymocytes were purified from 5-week-old mice and analysed by multiplex single molecule RNA-FISH (smFISH) ( Fig.…”