DynamicRunx1chromatin boundaries affect gene expression in hematopoietic development

Abstract

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increased in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells was associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletions of promoter-proximal CTCF sites at the sub-TAD boundaries had no obvious effects on E-P interactions but led to partial loss of domain structure, mildly affected gene expression, and delayed hematopoietic development. Together, our analyses of gene regulation at a large multi-promoter developmental gene revealed that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.

Transcriptional Regulation of RUNX1: An Informatics Analysis

“…Upon differentiation of mesoderm to HPC, contacts were lost with the more upstream R1REs, while contacts to both P1 and P2 were increased with R1REs 11 and 21. Concomitantly, the Runx1 gene was expressed from the P2 promoter upon differentiation to mesoderm, and both P1 and P2 upon adopting HPC identity [66]. This study therefore reinforces the assumption that conserved REs for Runx1 are important for expression of Runx1 during haematopoietic differentiation.…”

“…Of the identified R1REs, 8 interact with the RUNX1 promoters; R1REs 1, 2, 4, 10, 15, 18 and 21 have differing interactions with Runx1 P1, P2 and R1RE1 in different mouse or human cell lines (Table 1) [23,25,27,28]. A recent preprint describes a study in which enhancer-promoter contacts at the mouse Runx1 gene were determined step-wise during differentiation of mouse embryonic stem cells (ESC) to mesoderm and then on to HPC [66]. During mesoderm specification, the P2 promoter increased interactions with R1REs 1, 4, 6, 13, and 18 (and additional mouse enhancers not conserved in human).…”

“…We first sought to identify enhancers for Runx1 that are conserved between human and mouse. In mice, previously studies identified 29 possible regulators of Runx1 (−371, −368, −354, −328, −327, −322, −321, −303, −181, −171, −101, −59, −58, −48, −43, −42, +1, +3, +24, +32, +59, +64, +87, +99, +110, +171, +181, +199, +204) [21,24,25,[31][32][33]66]. Several studies have focused on a strong Runx1 enhancer in mice: Runx1 +23/+24/eR1/RE1 [19][20][21].…”

“…Mouse ATAC-seq [66] highlighted altered accessibility at R1REs in different cell types. R1REs 1, 4, 6, 10, 13 and 18 were accessible in mesoderm cells; whereas R1REs 1, 10, 13, 18, and 21 were accessible in haematopoietic progenitor cells (HPC).…”